Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF IDENTIFYING INHIBITORS OF CDC25
RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application No.:
60/172,215, filed August 31, 1999, the contents of which are incorporated
herein by
reference in their entirety.
BACKGROUND OF THE INVENTION
The application of modem molecular genetics to the study of cancer has
established that changes in specific DNA sequences can lead to tumor
initiation,
growth and progression. Many of these changes occur in genes which alter
cellular
proliferation, either directly (growth stimulatory factors/receptors such as
Ras, ErbB2,
bcr/abl) or indirectly (transcriptional proteins such as Rb, myc and p53).
Recently,
1 ~ many of these oncogenic changes have been linked to alterations in cell
cycle
regulation. For example, many oncogenes encode components of the pathways by
which growth factor signals feed into the cell cycle to induce cell division.
Changes
in cell cycle proteins, such as cyclins D1 and E, have also been demonstrated
to be
oncogenic in some cell types.
Cdc25 is a family of dual specificity phosphatases which dephosphorylate
both protein phosphotyrosine and phosphothreonine residues. Cdc25 regulates
cell
cycle progression by controlling the phosphorylation state of the cyclin
dependent
kinases. When phosphorylated on Tyrts and Thrt~, the cyclin dependent kinases
(cdl:)
are inactive and cell cycle progression is prevented. Dephosphorylation by
Cdc25
activates cdk and the cell cycle progresses. The activity of Cdc25
phosphatases is
_5
clearly required for cell cycle transition, and these enzymes serve as the
rate-limiting
mitotic activators. For example, mutation of Cdc25 in yeast produces cells
that arrest
in G2 phase. Mutation of the Cdc25 homologue in Drosophila results in G3
arrest of
cells early in embryogenesis.
Three distinct mammalian Cdc25 homologues have been identified, denoted
3O
Cdc25A, Cdc25B and Cdc25C. Each of these appears to target a particular
cdkl'cyclin
complex. In mammalian cells microinjection of antibodies against Cdc25A or C
inhibits cell entry into S (Hoffinann et aL, E'~LIBO.I. 13: 4302-10 (1994))
and M
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(Millar et al., Proc. Nat. Acad, Sci. USA 88: 10500-4 (1991)) respectively.
Recent
publications have demonstrated that Cdc25C is central to DNA damage checkpoint
arrest, such as an arrest produced by a cytotoxic agent. Damage activates the
serine
kinase Chk-1 that phosphorylates Cdc25C on serine 216. This phosphorylation
makes
Cdc25C a binding partner for the family of 14-3-3 binding proteins. Binding to
14-3-
3 proteins is believed to prevent Cdc25C from activating the cdk/cyclin
complex
cdc2/cycB and results in G2 cell cycle arrest (Furnari et al., Science 277:
1495-1497
(1997); Sanchez et al., Science 277: 1497-1501 (1997), Peng et al., Science
277:
1501-1505 (1997)). Additional studies have demonstrated that both Cdc25A and B
can act as oncogenes and transform cells when overexpressed (Galaktionov et
al.,
Science 269: 1575-1577 (1995)).
Due to its role in regulating the cell cycle, Cdc25 is a potential target for
therapies aimed at controlling proliferative diseases, such as cancer. The
development of biochemical assays for Cdc25 has enabled drug discovery to
proceed
along the pathways of identifying lead Cdc25 inhibitors by high-throughput
screening
of compound libraries and by testing compounds that mimic substrate structure;
however, rational, structure-based design has not been possible up to this
point
because of the lack of accurate three-dimensional data.
SUMMARY OF THE INVENTION
The present invention relates to polypeptides which comprise the ligand
binding domain of Cdc25, crystalline forms of these polypeptides and the use
of these
crystalline forms to determine the three dimensional structure of the
catalytic domain
of Cdc25. The invention also relates to the use of the three dimensional
structure of
the Cdc25 catalytic domain in methods of designing and/or identifying
potential
inhibitors of Cdc25 activity, for example, compounds which inhibit the binding
of a
native substrate to the Cdc25 catalytic domain.
In one embodiment, the present invention provides polypeptides comprising
the ligand binding domain of Cdc25, crystalline forms of these polypeptides,
optionally complexed with a ligand, and the three dimensional structure of the
polypeptides, including the three dimensional structure of the Cdc25 catalytic
domain.
The polypeptide, preferably, has the catalytic activity of a Cdc25.
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In another embodiment, the invention provides a method of determining the
three dimensional structure of a crystalline polypeptide comprising the Cdc25
catalytic domain. The method comprises the steps of (1) obtaining a crystal of
the
polypeptide comprising the catalytic domain of Cdc25; (2) obtaining x-ray
diffraction
data for said crystal; and (3) solving the crystal structure of said crystal
by using said
x-ray diffraction data and the atomic coordinates for the Cdc25 binding domain
of a
second polypeptide. The method optionally comprises the additional step of
obtaining
the polypeptide prior to obtaining the crystal.
The invention further relates to a method of identifying a compound which is a
potential inhibitor of Cdc25. The method comprises the steps of (1) obtaining
a
crystal of a polypeptide comprising the catalytic domain of Cdc25; (2)
obtaining the
atomic coordinates of the polypeptide in said crystal; (3) using said atomic
coordinates to define the catalytic domain of Cdc25; and (4) identifying a
compound
which fits the catalytic domain. The method can further include the steps of
obtaining
or synthesizing the compound identified in step 4, and assessing the ability
of the
identified compound to inhibit at least one biological activity of Cdc25, such
as
enzymatic activity.
In another embodiment, the method of identifying a potential inhibitor of
Cdc25 comprises the step of determining the ability of one or more functional
groups
and/or moieties of the compound, when present in, or bound to, the Cdc25
catalytic
domain, to interact with one or more subsites of the Cdc25 catalytic domain.
Generally, the Cdc25 catalytic domain is defined by the atomic coordinates of
a
polypeptide comprising the Cdc25 catalytic domain. If the compound is able to
interact with a preselected number or set of subsites, or has a calculated
interaction
energy withn a desired or preselected range, the compound is identified as a
potential
inhibitor of Cdc25.
The invention further provides a method of designing a compound which is a
potential inhibitor of Cdc25. The method includes the steps of (1) identifying
one or
more functional groups capable of interacting with one or more subsites of the
Cdc25
catalytic domain; and (2) identifying a scaffold which presents the functional
group or
functional groups identified in step 1 in a suitable orientation for
interacting with one
or more subsites of the Cdc25 catalytic domain. The compound which results
from
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attachment of the identified functional groups or moieties to the identified
scaffold is
a potential inhibitor of Cdc25. The Cdc25 catalytic domain is, generally,
defined by
the atomic coordinates of a polypeptide comprising the Cdc25 catalytic domain.
In yet another embodiment, the invention provides compounds which are
inhibitors of Cdc25 and which fit, or bind to, the Cdc25 catalytic domain.
Such
compounds typically comprise one or more functional groups which, when the
compound is bound in the Cdc25 catalytic domain, interact with one or more
subsites
of the catalytic domain. Generally, the Cdc25 catalytic domain is defined by
the
atomic coordinates of a polypeptide comprising the Cdc25 catalytic domain. In
a
particular embodiment, the Cdc25 inhibitor is a compound which is identified
or
designed by a method of the presnt invention.
The present invention further provides a method for treating a condition
mediated by Cdc25 in a patient. The method comprises administering to the
patient a
therapeutically or prophylactically effective amount of a Cdc25 inhibitor,
such as a
Cdc25 inhibitor of the invention, for example, a compound identified as a
Cdc25
inhibitor or designed to inhibit Cdc25 by a method of the present invention.
The present invention provides several advantages. For example, the
invention provides the first detailed three dimensional structure of the
catalytic
domain of a Cdc25 protein. This structure enables the rational development of
inhibitors of Cdc25 by permitting the design and/or identification of
molecular
structures having features which facilitate binding to the Cdc catalytic
domain. The
methods of use of this structure disclosed herein, thus, permit more rapid
discovery of
compounds which are potentially usefixl for the treatment of conditions which
are
mediated, at least in part, by Cdc25 activity.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 presents the amino acid sequence of human Cdc25A (SEQ ID NO: 1).
Fig. 2 presents the amino acid sequence of human Cdc25B (SEQ ID NO: 2).
Fig. 3 presents the amino acid sequence of human Cdc25C (SEQ ID NO: 3).
Fig. 4 presents the amino acid sequence of polypeptide Cdc25A ON1A (SEQ
ID NO: 4).
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Fig. S presents the amino acid sequence ofpolypeptide Cdc25B ON1B (SEQ
ID NO: 5).
Fig. 6 presents the amino acid sequence of polypeptide Cdc25A ONSA (SEQ
ID NO: 6).
Fig. 7 presents the amino acid sequence of polypeptide Cdc25C ON1C (SEQ
ID NO: 7).
Fig. 8 presents the amino acid sequence of polypeptide Cdc25A ON8A (SEQ
ID NO: 8).
Fig. 9 presents the amino acid sequence ofpolypeptide Cdc25A ONBA-c17
(SEQ ID NO: 9).
Fig. 10 presents the amino acid sequence of polypeptide Cdc25B ONSB (SEQ
ID NO: 10).
Fig. 11 presents the amino acid sequence of polypeptide Cdc25B ONBB (SEQ
ID NO: 11 ).
Fig. 12 presents the amino acid sequence of polypeptide Cdc25B ONBB-c 17
(SEQ ID NO: 12).
Fig. 13 presents the amino acid sequence of polypeptide Cdc25B ONBB-c18
(SEQ ID NO: 13).
Fig. 14 presents the amino acid sequence of polypeptide Cdc25C ON9C (SEQ
ID NO: 14).
Fig. 15A to 15PPP present the atomic coordinates for dc25B(DIV8B)/cdc1249
complex (crystal 19).
Fig. 16A to 16I present the atomic coordinates for Cdc25A(ON1A).
Fig. 17A to 17EE present the atomic coordinates for Cdc25B(ON1B)/cdc1249
complex (crystal 5).
Fig. 18A to 18X present the atomic coordinates for Cdc25A(ONBA) (crystal
3).
Fig. 19A to 19I present the atomic coordinates for Cdc25B(~N1B)/cdc1671
complex (crystal 15).
Fig. 20 illustrates the complex of the Cdc25B catalytic domain and the
pentapeptide cdc 1249 showing the protein secondary structure, the ligand
bound at
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the catalytic loop (thick bonds), and the ligand bound at the distal site
(thin bonds)
Fig. 21 shows the complex of the Cdc25B catalytic domain and the
pentapeptide cdc 1249 showing two symmetry related protein molecules
interacting
with the ligand bound at the catalytic site; water molecules and ions are not
shown
Fig. 22 shows the complex of the Cdc25B catalytic domain and the
pentapeptide cdc 1249 showing a top view of the molecular surface around the
ligand
binding area.
Fig. 23 shows a side view of the complex of the Cdc25B catalytic domain and
the pentapeptide cdc 1249.
Fig. 24 shows a top view of the complex of the Cdc25B catalytic domain and
the pentapeptide cdc1249 with protein residues labeled.
Fig. 25 shows a side view of the complex of the Cdc25B catalytic domain and
the pentapeptide cdc1249.
Fig. 26 illustrates the complex of the Cdc25B catalytic domain and the
pentapeptide cdc 1249 showing a top view of the molecular surface around the
ligand
binding area, with each subsite labeled.
Fig. 27 shows the complex of the Cdc25B catalytic domain and the
pentapeptide cdc1249 showing a side view with subsites 1-6 labeled
Fig. 28 is a side view of a potential tight-binding inhibitor complexed to the
Cdc25B catalytic domain.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to the x-ray crystallographic study of
polypeptides comprising the catalytic domains of Cdc25. The atomic coordinates
which result from the study are of use in identifying compounds which fit in
the
catalytic domain and are, therefore, potential inhibitors of Cdc25. These
Cdc25
inhibitors are of use in methods of treating a patient having a condition
which is
modulated by Cdc25 activity, for example, a condition characterized by
excessive,
inappropriate or undesirable cellular proliferation. Recent evidence indicates
that
Cdc25 plays a role in the development of cancer. For example, studies have
suggested that overexpression of Cdc25B in transgenic mice under the MMTV
promoter make them more susceptible to DMBA induced mammary tumors (Slosberg
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et al., Proc. Am. Assoc. Cancer Res. 39: 255 (1998)). Both Cdc25A and Cdc25B
are
frequently overexpressed in breast cancer (Galaktionov et al., Science 269:
1575-1577
(1995)), head and neck cancer (Gasparotto et al., Cancer Res 57: 2366-2368
(1997)),
gastric carcinoma (Kudo et al., Jpn .l. Cancer Res 88: 947-952 ( 1997)), and
Non
Hodgkin's lymphomas (Hernandez et al., Cancer Res 58: 1762-1767 (1998)).
Studies in cell lines lacking the cdk inhibitor p 15 indicate TGF-(3 can
inhibit
cell progression by modulating levels of Cdc25A (Iavarone et al., Nature 387:
417-
422 (1997)). Similarly, levels of Cdc25A and growth of a tumor cell line has
been
shown to be modulated by a-interferon (Tiefenbrun et al., Mol Cell Biol 16:
3934-
3944 (1996)). Further, it has recently been shown that antisense
oligonucleotides
against Cdc25B inhibit the growth of a tumor cell line (Garnerhamrick et al.,
Int. ,l.
Cancer 76: 729-728 1998)._ These results support the idea that Cdc25
inhibitors may
block one or more pathways involved in cell transformation.
The x-ray crystal structure of human Cdc25A was reported by Saper et al. in
1998 (Saper et al., Cell 93: 617-625 (1998)). The structure does not provide
atomic-
level details of the catalytic loop or the amino acid residues at the carboxyl
terminus.
Further, the structure does not include a bound inhibitor of Cdc25A.
The Examples describe the preparation of polypeptides comprising the catalytic
domains of human Cdc25A, Cdc25B and Cdc25C and the crystallization of the
Cdc25A
and Cdc25B polypeptides. As used herein, the term "catalytic domain" refers to
any or all
of the following sites in Cdc25: the substrate binding site; the site where
the pentapeptide
inhibitor described below binds and the site where the cleavage of a substrate
occurs. For
Cdc25A, the catalytic domain is defined by amino acid residues from about
residue 336
to about residue 523 of SEQ >D NO: 1. For Cdc25B, the catalytic domain is
defined by
residues from about residue 378 to about residue 566 of SEQ ID NO: 2 (Xu et
al., J. Biol.
Chem., 271: 5118-5124 (1996)).
The polypeptides prepared are listed in Table 9, together with their N-
terminal
and C-terminal amino acid residues. The amino acid sequences of these
polypeptides
are presented in Figs. 4-14. The numbering of the residues in Table 9 refers
to the
appropriate residue in the amino acid sequence (SEQ >D NO: 1, 2 or 3) of the
corresponding native protein, as presented in Figs. 1, 2 and 3. The amino acid
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sequences of the native proteins (SEQ ID NOs: 1, 2 and 3) are taken as defined
in
SWISS-PROT (Bairoch et al. Nucleic Acid Res. 22:3578 (1994)). As described in
the
Examples, certain of these crystals were examined by x-ray crystallography and
atomic coordinates for the peptide were obtained. In certain cases, the
polypeptide
was unligated, that is, not complexed with a ligand. In other cases, the
polypeptide
was complexed with a ligand and the atomic coordinates of the ligand bound to
the
Cdc25 catalytic domain were also obtained.
The atomic coordinates for five crystals examined by x-ray crystallography are
presented in Figs. 15A-15PPP, 16A-16I, 17A-17EE, 18A-18X and 19A-19I. The
term "atomic coordinates" (or "structural coordinates") refers to mathematical
coordinates derived from mathematical equations related to the patterns
obtained on
diffraction of x-rays by atoms (scattering centers) of a crystalline
polypeptide
comprising a Cdc25 catalytic domain molecule. The diffraction data are used to
calculate an electron density map of the repeating unit of the crystal. The
electron
density maps are used to establish the positions of the individual atoms
within the unit
cell of the crystal. Atomic coordinates can be transformed as is known in the
art to
different coordinate systems without affecting the relative positions of the
atoms.
In particular, a high resolution crystal structure was obtained for the
polypeptide denoted Cdc25B (ONBB) in Table 9, complexed with the pentapeptide
inhibitor shown below, denoted "cdc1249" herein.
S03H
COOH COOH
w0 O w H~ H O
N N ~ NH
~ i 'H O ~ H O H O 2
COOH /
Polypeptide Cdc25B (~NBB) includes residues Leu 368 to Arg 562 of human
Cdc25B (SEQ ID NO: 2). The complex of this polypeptide and cdc1249
crystallized
in space group P43212, a = 70.29, c = 130.59. The term "space group" is a term
of art
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which refers to the collection of symmetry elements of the unit cell of a
crystal. The
results of the x-ray crystal structure determination for Cdc25B (ON1B)
indicated that
the unit cell includes eight polypeptide molecules. Atomic coordinates for the
non-
hydrogen atoms in the protein, the inhibitor in active site; a second molecule
of the
inhibitor (distal to the catalytic domain); water molecules; and sodium and
chloride
counter ions were determined and are provided in Fig. 15A-15PPP. For the
inhibitor
molecule in the active site, all heavy (non-hydrogen) atoms were observed
except for
the second Glu residue beyond CB and the C-terminal Glu-amide. For the second
inhibitor molecule, all non-hydrogen atoms were observed. For the present
purposes,
the carbon atoms in an amino acid side chain are designated CB, CG, CD, and so
forth, where CB is the carbon atom bonded to the a-carbon, CG is the side
chain
carbon atom bonded to CB and so forth. The letters designating the carbon
atoms are
ordered according to the corresponding Greek letters.
The structures determined for polypeptides comprising the Cdc25B catalytic
domain complexed with a ligand differ significantly from the structure
determined by
Saper et al. for Cdc25A. Most importantly, the Cdc25B protein structure has a
ligand
bound at the catalytic site, and all protein atoms in proximity to the ligand
are well
defined. In the Cdc25A structure of Saper et al. there is no bound ligand and
the
catalytic loop is very poorly resolved (the residues composing the catalytic
loop are
disordered). One particular residue of the catalytic loop, Arg 479, appears in
the
Cdc25A structure to be misplaced when compared to the structures determined
for
polypeptides comprising the Cdc25B catalytic domain and the structures of
other
known phosphatases. Consequently, no reliable information with regard to
ligand
binding can be directly obtained from the Cdc25A protein structure, and the
lack of
atomic resolution around the binding site means that molecular modeling
techniques
can not be reliably used to predict ligand binding modes or for ligand design.
Another
major difference between the Cdc25B catalytic domain structure described
herein and
the Cdc25A structure of Saper et al. is in the C-terminal region (residues 531-
547, .
Cdc25B numbering). This region of Cdc25B, which is well resolved in the
present
structures, contains an alpha-helix which is positioned against the bulk of
the protein,
and several residues of the helix, such as Met 531 and Arg 544, interact with
the
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bound ligand. In contrast, in the Cdc25A structure of Saper et al., this
region is
undefined beyond Asp 492 (Cdc25A numbering scheme), and the few residues that
are observed appear to be misplaced. For example, the sequence is directed
away
from the bulk of the protein and towards a symmetry related molecule in the
crystal.
The position of the C-terminus in the Cdc25A structure, thus, appears to be
determined by packing forces within the crystal.
The structure of the Cdc25B(~NBB)/cdc1249 complex shows that the phenyl
group of the ligand (H03SCH2)Phe residue is completely surrounded by
hydrophobic
groups including: Phe 475, Ser 477, and Glu 478 in the catalytic loop; by Met
531 on
the C-terminus; by the naphthyl group of the ligand; and by Pro 457 and Ile
458 of a
symmetry-related polypeptide molecule (crystal contacts). There is also a
hydrogen
bond from the ligand NH between PheCH2S03H and 2-OMe-naphth, to the carbonyl
oxygen atom of Pro 457. The naphthyl ring of the 2-OMe-naphth group also makes
van der Waals contact with the symmetry-related molecule at Pro 457 and with
with
the backbone at Lys 455 and Ser 456. The methyl group of the 2-O-Me-naphth
group
sits in a groove on the polypeptide molecule. This naphthyl group also
contacts Met
531 and Leu 540 on the C-terminus, and the Nal residue of the ligand. There is
an
important interaction, possibly a cation-pi, pi-pi or van der Waals,
interaction,
between the inhibitor Nal and Arg 544 (the sidechain of Arg 544 is hydrogen-
bonded
to Tyr 428 and it has one unfavorable torsion angle at CB-CG). This Nal
residue also
makes van der Waals contacts with the sidechains of Glu 478 and Arg 479 in the
catalytic loop, and Met 531 in the C-terminus.
The pentapeptide inhibitor adopts a helix-like conformation with a mixture of
3,~/a properties. The inhibitor exhibits two intramolecular H-bonds: amide O,
from
between PheCS03H and 2-OMe-Naphth, to backbone NH between the two Glu
residues, and to backbone NH between the second Glu and Nal. The hydrophobic
groups in the ligand are close to one another, a situation which can be
described as
hydrophobic collapse.
As mentioned above, no electron density was observed for the terminal
carboxylic acid in the ligand, a result which could indicate that there are a
number of
possible binding modes for this portion of the ligand.
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A second molecule of the inhibitor is observed in the crystal structure,
binding
to the protein at a site distal to the catalytic site. This molecule appears
to stabilize
the crystal by forming a number of favorable interactions at the interface of
two
symmetry related protein molecules. The conformation of the ligand molecule at
the
distal site is very similar to the conformation of the ligand at the binding
site. This
indicates that the ligand is in a low-energy conformation, one that is not
significantly
biased by interactions with the protein. This result has been confirmed by
molecular
modeling and conformational analysis.
The C-terminal region of the Cdc25B catalytic domain is helical and plays a
significant role in ligand binding. This region was not observed in the
structure of
Saper et al. This part of the protein may be highly flexible, with a geometry
dependent upon such factors as salt concentration, length of the construct,
protein-
protein interactions (CDK/cyclin), bound ligand, and pH, among others.
Analysis of the three dimensional structure of the Cdc25B catalytic domain
has indicated the presence of a number of subsites, each of which includes
molecular
functional groups capable of interacting with complementary moieties of an
inhibitor.
Subsites 1-16 of the Cdc25B catalytic domain are defined below. The catalytic
domain consists of the catalytic loop and surrounding area. Sixteen subsites
are
defined; subsites 1-9 correspond to pockets, clefts, grooves, etc., and the
remaining
seven are bumps, that is, the solvent exposed tops of amino acid side chains.
Figs. 20
(top view) and 21 (side view) illustrate the binding site region with the
subsites
labeled.
Subsites are characterized below according to the properties of chemical
moieties with which they are complementary, or with which they can interact.
Such
moieties can include hydrogen bond acceptors ("HA"), such as hydroxyl, amino,
and
carbonyl groups, halogen atoms, such as fluorine, chlorine, bromine and iodine
atoms;
and other groups including a heteroatom having at least one lone pair of
electrons,
such as groups containing trivalent phosphorous, di- and tetravalent sulfur,
oxygen
and nitrogen atoms; hydrogen bond donors ("HD"), such as hydroxyl, amino,
carboxylic acid groups and any of the groups listed under hydrogen atom
acceptors to
which a hydrogen atom is bonded; hydrophobic groups ("H"), such as linear,
branched or cyclic alkyl groups; linear, branched or cyclic alkenyl groups;
linear,
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branched or cyclic alkynyl groups; aryl groups, such as mon- and polycyclic
aromatic
hydrocarbyl groups and mono- and polycyclic heteroaryl groups; positively
charged
groups ("P"), such as primary, secondary, tertiary and quaternary ammonium
groups,
substituted and unsubstituted guanidinium groups, sulfonium groups and
phosphonium groups; and negatively charged groups ("N"), such as carboxylate,
sulfonamide, sulfamate, boronate, vanadate, sulfonate, sulfinate and
phosphonate
groups. A given chemical moiety can contain one or more of these groups.
Subsite 1: Catalytic loop; interacting chemical moieties: HA, H, N;
Residues involved: Cys 473; Glu 474; Phe 475; Ser 476; Ser 477; Glu 478; Arg
479;
Non-hydrogen atoms which interact with HA and N: Cys 473 S; Glu 474 N; Phe 475
N; Ser 476 N; Ser 477 N; Glu 478 N; Arg 479 N, NE, NHZ
Non-hydrogen atoms which interact with H: Glu 474 CA, CB, CG, CD; Phe 475 CB,
. CG, CD1, CD2, CE1, CE2, CZ; Ser 477 CB; Glu 478 CB, CG, CD
Subsite 2: Swimming pool
Interacting chemical moieties: HA, HD, H, N, P
Residues involved: Cys 426; Tyr 428; Pro 444; Leu 445; Glu 446; Glu 478; Arg
479;
Arg 482; Met 483; Arg 544; Thr 547; Arg 548
Non-hydrogen atoms which interact with HA and N: Tyr 428 OH; Arg 482 NHl or
NH2; Arg 544 NH 1
Non-hydrogen atoms which interact with HD and P: Cys 426 O; Tyr 428 OH; Pro
444
O; Glu 446 OE1, OE2; Thr 547 OG1.
Non-hydrogen atoms which interact with H: Leu 445 CA, CB, CDl; Glu 446 CA, CB,
CG, CD; Arg 479 CA, CB, CG, CD; Met 483 CA, CB, CG, SD, CE; Thr 547 CB,
CG2; Arg 548 CA, CB, CG, CD, CE
Subsite 3: Anion binding site
Interacting chemical moieties: HA, N
Residues involved: Arg 482; Arg 544
Non-hydrogen atoms which interact with HA and N: Arg 482 NH1, NH2; Arg 544
NHl, NH2
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Subsite 4: Groove between catalytic loop and swimming pool
Interacting chemical moieties: H
Residues involved: Glu 478; Arg 479; Met 531; Arg 544.
Non-hydrogen atoms which interact with H: Glu 478 CA, CB, CG, CD; Arg 479 CA,
CB, CG, CD, CZ; Met 531 CB, CG, SD, CE; Arg 544 CG, CD, CZ.
Subsite 5: Nal binding region
Interacting chemical moieties: HA, HD, H, N
Residues involved: Tyr 428; Glu 478; Arg 479; Met 531; Leu 540; Arg 544
Non-hydrogen atoms which interact with HA and HD : Tyr 428 OH
Non-hydrogen atoms which interact with H: Tyr 428 CD1,CE1; Glu 478 CA, CB,
CG, CD; Arg 479 CA, CB, CG, CD, CZ; Met 531 CG, SD, CE; Leu 540 CB, CG,
CD1, CD2; Arg 544 CG, CD, CZ
Non-hydrogen atoms which interact with N: Arg 544 NE, NH1, NH2
Subsite 6: 2-Me0-Nal binding region
Interacting chemical moieties: H
Residues involved: Phe 475; Met 531; Asn 532; Leu 540
Non-hydrogen atoms which interact with H: Phe 475 CG, CD1, CD2, CE1, CE2, CZ;
Met 531 CB, CG, SD, CE; Asn 532 CB, CG; Leu 540 CB, CG, CD1, CD2
Subsite 7: Interactions involving Ile 458 of the symmetry related polypeptide
Interacting chemical moieties: H
Residues involved: Phe 475; Ser 477
Non-hydrogen atoms which interact with H: Phe 475 C, CB, CG, CD1, CD2, CE1,
CE2, CZ; Ser 477 CB
Subsite 8: Interactions involving Pro 457 of the symmetry related polypeptide
Interacting chemical moieties: HA, HD, H
Residues involved: Glu 474; Phe 475; Met 531; Asn 532
Non-hydrogen atoms which interact with HA: Asn 532 ND2
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Non-hydrogen atoms which interact with HD: Glu 474 OE1, OE2; Asn 532 OD1
Non-hydrogen atoms which interact with H: Glu 474 CB, CG, CD; Phe 475 CG, CD1,
CD2, CE1, CE2, CZ; Met 531 C, CA, CB, CG, SD, CE; Asn 532 CA, CB, CG
Subsite 9: Region around Leu 540
Interacting chemical moieties: H
Residues involved: Tyr 428; Met 531; Lys 537; Leu 540; Lys 541; Arg 544
Non-hydrogen atoms which interact with H: Tyr 428 CD1, CE1; Met 531 CB, CG,
SD, CE; Lys 537 CA, CB, CG, CD, CE; Leu 540 CB, CG, CD1, CD2; Lys 541 CA,
CB, CG, CD, CE; Arg 544 CB, CG, CD
Subsite 10: Ser 477
Interacting chemical moieties: HA, HD
Residues involved: Ser 477
Non-hydrogen atoms which interact with HA and HD: Ser 477 OG
Subsite 11: Glu 478
Interacting chemical moieties: HD, P
Residues involved: Glu 478;
Non-hydrogen atoms which interact with HD and P: Glu 478 OE1, OE2
Subsite 12: Lys 394
Interacting chemical moieties: HA, N
Non-hydrogen atoms which interact with HA and N: Lys 394 NZ
Subsite 13: Arg 482
Interacting chemical moieties: HA, N
Non-hydrogen atoms which interact with HA and N: Arg 482 NE, NH 1 or NH2
Subsite 14: Arg 544
Interacting chemical moieties: HA, N
Non-hydrogen atoms which interact with HA and N: Arg 544 NE, NH2
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Subsite 15: Phe 475
Interacting chemical moieties: H
Non-hydrogen atoms which interact with H: Phe 475 CB, CG, CD1, CD2, CEI, CE2,
CZ
Subsite 16: Asn 532
Interacting chemical moieties: HA, HD, H
Non-hydrogen atoms which interact with HA: Asn 532 ND2
Non-hydrogen atoms which interact with HD: Asn 532 OD1
Non-hydrogen atoms which interact with H: Asn 532 CB, CG
Figs. 20-28 provide different views of the Cdc25 catalytic domain structure
and the interaction of cdc1249 with the polypeptide. For example, Fig. 20
provides a
view of the complex of Cdc25B and cdc1249 showing the protein secondary
structure, the ligand bound at the catalytic loop (thick bonds), and the
ligand bound at
the distal site (thin bonds). Fig. 21 is another view of this complex showing
two
symmetry related protein molecules interacting with the ligand bound at the
catalytic
site. Water molecules and ions are not shown. Fig. 22 shows a top view of the
molecular surface around the ligand binding area. The terminal atoms of Arg
482
have been removed so that the swimming pool can be clearly observed. Water
molecules and ions are not shown. The view of the complex presented in Fig. 23
is a
side view relative to the view in Fig. 22. Fig. 24 shows a top view of the
complex of
Cdc25B and cdc1249 with protein residues labeled. Water molecules and ions are
not
shown. Fig. 25 shows a side view of the complex relative to the view presented
in
Fig. 23. Fig. 26 presents a top view of the complex, showing the molecular
surface
around the ligand binding area, with each subsite labeled. The terminal atoms
of Arg
482 have been removed so that the swimming pool can be clearly observed. Water
molecules and ions are not shown. Fig. 27 shows a side view of the complex
relative
to the view in Fig. 26, and only subsites 1-6 are labeled. catalytic loop of
Cdc25A is
not shown and the well-defined catalytic loop of Cdc25B is shown in purple.
Figure
28 presents a side view of a potental tight-binding inhibitor complexed to
Cdc25B.
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The designed ligand binds in the catalytic loop and swimming pool, and spans
the
groove between the two.
In one embodiment, the present invention provides polypeptides comprising
the catalytic domain of Cdc25, crystalline forms of these polypeptides,
optionally
complexed with a ligand, and the three dimensional structure of the
polypeptides,
including the three dimensional structure of the Cdc25 catalytic domain. In
general,
these three dimensional structures are defined by atomic coordinates derived
from x-
ray crystallographic studies of the polypeptides. The polypeptides can include
the
catalytic domain of Cdc25 from any species, such as a yeast or other
unicellular
organism, an invertebrate or a vertebrate. Preferably, the polypeptide
includes the
binding domain of a mammalian Cdc25, such as a mammalian Cdc25A, Cdc25B or
Cdc25C. More preferably, the polypeptide includes the catalytic domain of
human
Cdc25A, Cdc25B or Cdc25C. In one embodiment, the polypeptide includes amino
acids Leu 336 to Thr 506 of SEQ 117 NO: 1, amino acids Leu 378 to Arg 548 of
SEQ
ID NO: 2 or amino acids Leu 282 to Val 453 of SEQ ID NO: 3. In particular
embodiments, the polypeptides can include amino acids Leu 336 to Leu 523; Gly
323
to Leu 523; Glu 326 to Arg 519; or Glu 326 to Thr 506 of SEQ ID NO: 1; Leu 378
to
Gln 566; Asp 365 to Gln 566; Glu 368 to Arg 562; Glu 368 to Ser 549 or Glu 368
to
Arg 548 of SEQ ID NO: 2; or amino acids Leu 282 to Pro 473 or Gly 280 to Val
453
of SEQ ID NO: 3.
The crystalline polypeptide, preferably, further includes a ligand bound to
the
Cdc25 catalytic domain. The ligand is, preferably, a small (less than about
1500
molecular weight) organic molecule, for example, a peptide, such as a
pentapeptide.
In one embodiment, the invention relates to a method of determining the three
dimensional structure of a first polypeptide comprising the catalytic domain
of a
CdC25 protein. The method includes the steps of (1) obtaining a crystal
comprising
the first polypeptide; (2) obtaining x-ray diffraction data for said crystal;
and (3) using
the x-ray diffraction data and the atomic coordinates of a second polypeptide
comprising the catalytic domain of a Cdc25 protein to solve the crystal
structure of
the first polypeptide, thereby determining the three dimensional structure of
the first
polypeptide. The second polypeptide can include the same Cdc25 catalytic
domain as
the first polypeptide, or a different Cdc25 catalytic domain. Either or both
of the first
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and second polypeptides can, optionally, be complexed with a ligand. That is,
the
crystal of the first polypeptide can comprise a complex of the first
polypeptide with a
ligand. The atomic coordinates of the second polypeptide can, optionally,
include the
atomic coordinates of a ligand molecule bound to the second polypeptide. The
atomic
S coordinates of the second polypeptide, generally, have been previously
obtained, for
example, by x-ray crystallographic analysis of a crystal comprising the second
polypeptide or a complex of the second polypeptide with a ligand. The atomic
coordinates of the second polypeptide can be used to solve the crystal
structure using
methods known in the art, for example, molecular replacement or isomorphous
replacement. Preferably, the second polypeptide comprises the catalytic domain
of a
mammalin Cdc25, more preferably a mammalian Cdc25B, and, most preferably,
human Cdc25B. For example the atomic coordinates which can be used include the
atomic coordinates presented herein, preferably the atomic coordinates
presented in
Fig. 1 SA to 1 SPPP.
1 S The invention also provides a method of identifying a compound which is a
potential inhibitor of Cdc25. The method comprises the steps of (1) obtaining
a
crystal of a polypeptide comprising the catalytic domain of Cdc25; (2)
obtaining the
atomic coordinates of the polypeptide by x-ray diffraction studies using said
crystal;
(3) using said atomic coordinates to define the catalytic domain of Cdc25; and
(4)
identifying a compound which fits the catalytic domain. The method can further
include the steps of obtaining, for example, from a compound library, or
synthesizing
the compound identified in step 4, and assessing the ability of the identified
compound to inhibit Cdc25 enzymatic activity.
The polypeptide preferably comprises the catalytic domain of a mammalian
Cdc25, such as a mammalian Cdc25A, Cdc25B or Cdc25C. More preferably the
polypeptide comprises the catalytic domain of human Cdc25A, Cdc25B or Cdc25C.
In a preferred embodiment, the polypeptide is a polypeptide of the present
invention,
as described above.
The polypeptide can be crystallized using methods known in the art, such as
the methods described in the Examples, to afford polypeptide crystals which
are
suitable for x-ray diffraction studies. A crystalline polypeptide/ligand
complex can be
produced by soaking the resulting crystalline polypeptide in a solution
including the
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ligand. Preferably, the ligand solution is in a solvent in which the
polypeptide is
insoluble.
The atomic coordinates of the polypeptide (and ligand) can be determined, for
example, by x-ray crystallography using.methods known in the art. The data
obtained
from the crystallography can be used to generate atomic coordinates, for
example, of
the atoms of the polypeptide and ligand, if present. As is known in the art,
solution
and refinement of the x-ray crystal structure can result in the determination
of
coordinates for some or all of the non-hydrogen atoms. The atomic coordinates
can
be used, as is known in the art, to generate a three-dimensional structure of
the Cdc25
catalytic domain. This structure can then be used to assess the ability of any
given
compound, preferably using computer-based methods, to fit into the catalytic
domain.
A compound fits into the catalytic domain if it is of a suitable size and
shape
to physically reside in the catalytic domain, thatis, if it has a shape which
is
complementary to the catalytic domain and can reside in the catalytic domain
without
1 S significant unfavorable steric or van der Waals interactions. Preferably,
the
compound includes one or more functional groups and/or moieties which interact
with
one or more subsites within the catalytic domain. Computational methods for
evaluating the ability of a compound to fit into the catalytic domain, as
defined by the
atomic coordinates of the polypeptide, are known in the art, and
representative
examples are provided below.
In another embodiment, the method of identifying a potential inhibitor of
Cdc25 comprises the step of determining the ability of one or more functional
groups
and/or moieties of the compound, when present in the Cdc25 catalytic domain,
to
interact with one or more subsites of the Cdc25 catalytic domain. Preferably,
the
Cdc25 catalytic domain is defined by the atomic coordinates of a polypeptide
comprising the Cdc25 catalytic domain. If the compound is able to interact
with a
preselected number or set of subsites, the compound is identified as a
potential
inhibitor of Cdc25.
A functional group or moiety of the compound is said to "interact" with a
subsite of the Cdc25 catalytic domain if it participates in an energetically
favorable, or
stabilizing, interaction with one or more complementary moieties within the
subsite.
Two chemical moieties are "complementary" if they are capable, when suitably
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positioned, of participating in an attractive, or stabilizing, interaction,
such as an
electrostatic or van der Waals interaction. Typically, the attractive
interaction is an
ion-ion (or salt bridge), ion-dipole, dipole-dipole, hydrogen bond, pi-pi or
hydrophobic interaction. For example, a negatively charged moiety and a
positively
charged moiety are complementary because, if suitably positioned, they can
form a
salt bridge. Likewise, a hydrogen bond donor and a hydrogen bond acceptor are
complementary if suitably positioned.
Typically, the assessment of interactions between the test compound and the
Cdc25 catalytic domain employs computer-based computational methods, such as
those known in the art, in which possible interactions of a compound with the
protein,
as defined by atomic coordinates, are evaluated with respect to interaction
strength by
calculating the interaction energy upon binding the compound to the protein.
Compounds which have calculated interaction energies within a preselected
range or
which otherwise, in the opinion of the computational chemist employing the
method,
have the greatest potential as Cdc25 inhibitors, can then be provided, for
example,
from a compound library or via synthesis, and assayed for the ability to
inhibit Cdc25.
The interaction energy for a given compound generally depends upon the ability
of
the compound to interact with one or more subsites within the protein
catalytic
domain.
In one embodiment, the atomic coordinates used in the method are the atomic
coordinates set forth in Figs. 15A to 15PPP, 16A to 16I, 17A to 17EE or 18A to
18X.
Preferably the atomic coordinates are the coordinates set forth in Fig. 15A to
15PPP.
It is to be understood that the coordinates set forth in Figs. 1 SA to 15PPP,
16A to 16I,
17A to 17EE and 18A to 18X can be transformed, for example, into a different
coordinate system, in ways known to those of skill in the art without
substantially
changing the three dimensional structure represented thereby.
In certain cases a moiety of the compound can interact with a subsite via two
or more individual interactions. A moiety of the compound and a subsite can
interact
if they have complementary properties and are positioned in sufficient
proximity and
in a suitable orientation for a stabilizing interaction to occur. The possible
range of
distances for the moiety of the compound and the subsite depends upon the
distance
dependence of the interaction, as is known in the art. For example, a hydrogen
bond
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typically occurs when a hydrogen bond donor atom, which bears a hydrogen atom,
and a hydrogen bond acceptor atom are separated by about 2.5 ~ and about 3.5
~.
Hydrogen bonds are well known in the art (Pimentel et al., The Hydrogen Bond,
San
Francisco: Freeman (1960)). Generally, the overall interaction, or binding,
between
the compound and the Cdc25 catalytic domain will depend upon the number and
strength of these individual interactions.
The ability of a test compound to interact with one or more subsites of the
catalytic domain of Cdc25 can be determined by computationally evaluating
interactions between functional groups, or moieties, of the test compound and
one or
more amino acid side chains in a particular protein subsite, such as subsites
1 to 16
above. Typically, a compound which is capable of participating in stabilizing
interactions with a preselected number of subsites, preferably without
simultaneously
participating in significant destabilizing interactions, is identified as a
potential
inhibitor of Cdc25. Such a compound will interact with one or more subsites,
preferably with two or more subsites and, more preferably, with three or more
subsites.
The invention further provides a method of designing a compound which is a
potential inhibitor of Cdc25. The method includes the steps of (1) identifying
one or
more functional groups capable of interacting with one or more subsites of the
Cdc25
catalytic domain; and (2) identifying a scaffold which presents the functional
group or
functional groups identified in step 1 in a suitable orientation for
interacting with one
or more subsites of the Cdc25 catalytic domain. The compound which results
from
attachment of the identified functional groups or moieties to the identified
scaffold is
a potential inhibitor of Cdc25. The Cdc25 catalytic domain is, generally,
defined by
the atomic coordinates of a polypeptide comprising the Cdc25 catalytic domain,
for
example, the atomic coordinates set forth in Figs. 15A-15PPP, 16A-16I, 17A-
17EE,
18A-18X or 19A-19I. Preferably, the Cdc25 catalytic domain is defined by the
atomic coordinates set forth in Fig. 15A-15PPP.
Suitable methods, as are known in the art, can be used to identify chemical
moieties, fragments or functional groups which are capable of interacting
favorably
with a particular subsite or set of subsites. These methods include, but are
not limited
to: interactive molecular graphics; molecular mechanics; conformational
analysis;
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energy evaluation; docking; database searching; pharmacophore modeling; de
novo
design and property estimation. These methods can also be employed to assemble
chemical moieties, fragments or functional groups into a single inhibitor
molecule.
These same methods can also be used to determine whether a given chemical
moiety,
fragment or functional group is able to interact favorably with a particular
subsite or
set of subsites.
In one embodiment, the design of potential human Cdc25 inhibitors begins from
the
general perspective of three-dimensional shape and electrostatic
complementarity for the
catalytic domain, encompassing subsites 1-16, and subsequently, interactive
molecular
modeling techniques can be applied by one skilled in the art to visually
inspect the
quality of the fit of a candidate inhibitor modeled into the binding site.
Suitable
visualization programs include INSIGHTII (Molecular Simulations Inc., San
Diego, CA),
QUANTA (Molecular Simulations Inc., San Diego, CA), SYBYL (Tripos Inc., St
Louis,
MO), RASMOL (Roger Sayle et al., Trends Biochem. Sci. 20: 374-376 (1995)),
GRASP
(Nicholls et al., Proteins 11: 281-289 (1991)), and MIDAS (Ferrin et al., J.
Mol.
Graphics 6:13-27 (1988)).
A further embodiment of the present invention utilizes a database searching
program
which is capable of scanning a database of small molecules ofknown three-
dimensional
structure for candidates which fit into the target protein site. Suitable
software programs
include CATALYST (Molecular Simulations Inc., San Diego, CA), UNITY (Tripos
Inc.,
St Louis, MO), FLEXX (Rarey et al., J. Mol. Biol. 261: 470-489 (1996)), CHEM-
3DBS
(Oxford Molecular Group, Oxford, UK), DOCK (Kuntz et al., J. Mol. Biol 161:
269-288
(1982)), and MACCS-3D (MDL Information Systems Inc., San Leandro, CA). It is
not
expected that the molecules found in the search will necessarily be leads
themselves,
since a complete evaluation of all interactions will necessarily be made
during the initial
search. Rather, it is anticipated that such candidates might act as the
framework for
further design, providing molecular skeletons to which appropriate atomic
replacements
can be made. Of course, the chemical complimentary of these molecules can be
evaluated, but it is expected that the scaffold, functional groups, linkers
and/or monomers
may be changed to maximize the electrostatic, hydrogen bonding, and
hydrophobic
interactions with the enzyme. Goodford (Goodford JMed Chem 28:849-857 (1985))
has
produced a computer program, GRID, which seeks to determine regions of high
affinity
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for different chemical groups (termed probes) on the molecular surface of the
binding
site. GRID hence provides a tool for suggesting modifications to known ligands
that
might enhance binding.
A range of factors, including electrostatic interactions, hydrogen bonding,
hydrophobic interactions, desolvation effects, conformational strain, and
cooperative
motions of ligand and enzyme, all influence the binding effect and should be
taken into
account in attempts to design bioactive inhibitors.
Yet another embodiment of a computer-assisted molecular design method for
identifying inhibitors comprises searching for fragments which fit into a
binding region
subsite and link to a pre-defined scaffold. The scaffold itself may be
identified in such a
manner. Programs suitable for the searching of such functional groups and
monomers
include LUDI (Boehm, JComp. Aid. Mol. Des. 6:61-78 (1992)), CAVEAT (Bartlett
et al.
in "Molecular Recognition in Chemical and Biological Problems", special
publication of
The Royal Chem. Soc., 78:182-196 (1989)) and MCSS (Miranker et al. Proteins
11: 29-
34 ( 1991 )).
Yet another embodiment of a computer-assisted molecular design method for
identifying inhibitors of the subject phosphatase comprises the de novo
synthesis of
potential inhibitors by algorithmic connection of small molecular fragments
that will
exhibit the desired structural and electrostatic complementarity with the
active site of the
enzyme. The methodology employs a large template set of small molecules with
are
iteratively pieced together in a model of the Cdc25 active site. Programs
suitable for this
task include GROW (Moon et al. Proteins 11:314-328 (1991)) and SPROUT (Gillet
et al.
J Comp. Aid. Mol. Des. 7:127 (1993)).
In yet another embodiment, the suitability of inhibitor candidates can be
detemuned
using an empirical scoring function, which can rank the binding affinities for
a set of
inhibitors. For an example of such a method see Muegge et al. and references
therein
(Muegge et al., JMed. Chem. 42:791-804 (1999)).
Other modeling techniques can be used in accordance with this invention, for
example, those described by Cohen et al. (J. Med. Chem. 33: 883-894 (1994));
Navia et
al. (Current Opinions in Structural Biology 2: 202-210 (1992)); Baldwin et al.
(J Med.
Chem. 32: 2510-2513 (1989)); Appelt et al. (J Med. Chem. 34: 1925-1934
(1991)); and
Ealick et al. (Proc. Nat. Acad. Sci. USA 88: 11540-11544 (1991)).
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A compound which is identified by one of the foregoing methods as a potential
inhibitor of Cdc25 can then be obtained, for example, by synthesis or from a
compound
library, and assessed for the ability to inhibit Cdc25 in vitro. Such an in
vitro assay can
be performed as is known in the art, for example, by contacting Cdc25 in
solution with
the test compound in the presence of a substrate for Cdc25. The rate of
substrate
transformation can be determined in the presence of the test compound and
compared
with the rate in the absence of the test compound. Suitable assays for Cdc25
biological
activity are described in U.S. Patents No. 5,861,249; 5,695,950; 5,443,962;
and
5,294,538, the teachings of each of which are hereby incorporated by reference
herein in
their entirety.
An inhibitor identified or designed by a method of the present invention can
be a competitive inhibitor, an uncompetitive inhibitor or a noncompetitive
inhibitor.
A "competitive" inhibitor is one that inhibits Cdc25 activity by binding to
the same
kinetic form of Cdc25, as its substrate, thereby directly competing with the
substrate
for the active site of Cdc25. Competitive inhibition can be reversed
completely by
increasing the substrate concentration. An "uncompetitive" inhibitor inhibits
Cdc25
by binding to a different kinetic form of the enzyme than the substrate. Such
inhibitors bind to Cdc25 already bound with the substrate and not to the free
enzyme.
Uncompetitive inhibition cannot be reversed completely by increasing the
substrate
concentration. A "non-competitive" inhibitor is one that can bind to either
the free or
substrate bound form of Cdc25.
In another embodiment, the present invention provides Cdc25 inhibitors, and
methods of use thereof, which are capable of binding to the catalytic domain
of
Cdc25, for example, compounds which are identified as inhibitors of at least
one
biological activity of Cdc25 or which are designed by the methods described
above to
inhibit at least one biological activity of Cdc25. For example, the invention
includes
compounds which interact with one or more, preferably two or more, and more
preferably, three or more of Cdc25 subsites 1 to 16.
In one embodiment, the Cdc25 inhibitor of the invention comprises a moiety
or moieties positioned to interact with subsite 1, subsite 2 and at least one
other
subsite when present in the Cdc25 catalytic domain. For example, a functional
group
which can interact with subsite 1 can be a hydrogen bond acceptor, a
hydrophobic
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moiety or a negatively charged group. Preferably, the functional group
includes both
a negatively charged group and a hydrophobic group. A functional group which
can
interact with subsite 2 can be a hydrogen bond donor, a hydrogen bond
acceptor, a
hydrophobic moiety, a negatively charged group or a positively charged group.
In another embodiment, the Cdc25 inhibitor of the invention comprises
functional groups positioned to interact with subsites 1, 2 and 3, and,
optionally, one
or more additional subsites.
The Cdc25 inhibitors of the invention also include compounds having
functional groups positioned to interact with subsite l, subsite 3 and,
optionally, one
or more additional subsites. In another embodiment, the inhibitor has
functional
groups positioned to interact with subsite l, subsite 3, subsite 4, and,
optionally, one
or more additional subsites.
In other embodiments, the Cdc25 inhibitors of the invention include
compounds which have functional groups positioned to interact with the
following
1 S groups of subsites, each of which can, optionally, include one or more
additional
subsites:
subsites 1 and 5; subsites l, 4 and 5; subsites 1, 5 and 6; subsites 1, 7
and/or 8;
subsites 1, 2 and 9; subsites 1, 2, 4 and 9; subsites 1, 3 and 9; subsites 1,
3, 4 and 9.
A moiety of the inhibitor compound is "positioned to interact" with a given
subsite, if, when placed within the Cdc25 catalytic domain, as defined by the
atomic
coordinates presented in Fig. 1 SA to 1 SEE, the moiety is close enough to,
and
oriented properly relative to, the appropriate amino acid side chains within
the subsite.
As indicated in the description of the subsites above, several of subsites 1-
16
can potentially interact with two or more types of moieties. For each of the
subsites
listed below the preferred type of interacting moiety possessed by the
potential
inhibitor is indicated.
Subsite 1: negative charged (Arg 479) and hydrophobic moiety (Glu 474, Phe
475,
Ser 477, Glu 478)
Subsite 3: negative charged moiety (Arg 482; Arg 544)
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Subsite 5: hydrophobic, preferably aromatic, moiety (Tyr 428; Glu 478; Arg
479; Met
531; Leu 540; Arg 544)
Subsite 8: hydrophobic, preferably alkyl, moiety (Glu 474; Phe 475; Met 531;
Asn
532)
Subsite 11: positive charged moiety (Glu 478)
Subsite 12: negative charged moiety (Lys 394)
Subsite 13: negative charged moiety (Arg 482)
Subsite 14: negative charged moiety (Arg 544)
Subsite 16: hydrophobic and hydrogen donor/acceptor (Asn 532)
A preferred Cdc25 inhibitor of the invention inhibits Cdc25 enzymatic acitivty
with a Ki of at least about 1 mM, preferably at least about 100 p,M and more
preferably at least about 10 p.M.
In a preferred embodiment, the Cdc25 inhibitor of the invention comprises two
or more of the following when present at, or bound to, the Cdc25 catalytic
domain: (a)
a negatively charged functional group positioned to interact with Arg 479 of
human
Cdc25B; (b) a hydrogen bond donor or positively charged functional group
positioned
to interact with one or more of Cys 426, Tyr 428, Pro 444, Glu 446 and Thr 547
of
human Cdc25B; (c) a hydrogen bond acceptor or a negatively charged functional
group positioned to interact with one or more of Tyr 428, Arg 482 and Arg 544
of
human Cdc25B; (d) a hydrophobic moiety positioned to interact with one or more
of
Leu 445, Glu 446, Arg 479, Met 483, Thr 547 and Arg 548; (e) a negatively
charged
functional group positioned to interact with one or more of Arg 482 and Arg
544 of
human Cdc25B; (f) a hydrophobic moiety positioned to interact with one or more
of
Glu 478, Arg 479, Met 531 and Arg 544 of human Cdc25B; (g) a hydrophobic
moiety
positioned to interact with one or more of Tyr 428, Glu 478, Arg 479, Met 531,
Leu
540, and Arg 544 of human Cdc25B; (h) a hydrophobic moiety positioned to
interact
with one or more of Phe 475, Met 531, Asn 532 and Leu 540 of human Cdc25B; (i)
a
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hydrophobic moiety positioned to interact with one or more of Phe 475 and Ser
477 of
human Cdc25B; (j) a hydrophobic moiety positioned to interact with one or more
of
Glu 474, Phe 475, Met 531 and Asn 532 of human Cdc25B; (k) a hydrophobic
moiety
positioned to interact with one or more of Tyr 428, Met 531, Lys 537, Lys 541,
Leu
540 and Arg 544 of human Cdc25B; (1) a hydrogen bond donor or hydrogen bond
acceptor positioned to interact with Ser 477 of human Cdc25B; (m) a hydrogen
bond
donor or positively charged functional group positioned to interact with Glu
478 of
human Cdc25B; (n) a negatively charged functional group positioned to interact
with
Lys 394 of human Cdc25B; (o) a negatively charged functional group positioned
to
interact with Arg 482 of human Cdc25B; (p) a negatively charged functional
group
positioned to interact with Arg 544 of human Cdc25B; and (c~ a hydrophobic
moiety
and a hydrogen bond donor or hydrogen bond acceptor positioned to interact
with Asn
532 of human Cdc25B.
In preferred embodiments, the Cdc25 inhibitors of the invention comprise (a)
and (e); (a) and at least one of (b), (c) and (d); (a), (e) and at least one
of (b), (c) and
(d); (a), (e) and (f); (a) and (g); (a), (f) and (g); (a), (g) and (h); (a)
and at least one of
(i) and (j); (a), (k) and at least one of (b), (c) and (d).
Preferred Cdc25 inhibitors of the invention comprise a peptide, peptide
mimetic or other molecular scaffold or framework, to which the moieties and/or
functional groups which interact with the Cdc25 subsites are attached, either
directly
or via an intervening moiety. The scaffold can be, for example, a peptide or
peptide
mimetic backbone, a cyclic or polycyclic moiety, such as a monocyclic,
bicyclic or
tricyclic moiety, and can include one or more hydrocarbyl or heterocyclic
rings. The
molecular scaffold presents the attached interacting moieties in the proper
configuration or orientation for interaction with the appropriate residues of
Cdc25.
Information derived from the Cdc25B(ON8B)/cdc1249 crystal structure
described above has been successfully used to produce a potent inhibitor of
Cdc25A.
Compound cdc1671, shown below, inhibits Cdc25A with an ICSO of 5 pM.
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03H
OOH COOH
I
O ~ O
b~ a~ a
~ O ~ o ~ o
I~ ~ '
IrOOH
As discussed above, the cdc1249 molecule at the catalytic domain of Cdc25B
adopts a "turn" conformation, in which the peptide backbone has a helical
turn.
Further, the structure shows that the two internal glutamyl residues of
cdc1249 do not
interact significantly with residues of the catalytic domain. It was therefore
reasoned
that replacement of one glutamyl residue with a substituted or unsubstituted
prolyl or
dehydroprolyl residue, which would stabilize the "turn" conformation, would
result in
a more potent inhibitor. To test this idea, compound cdc1763, shown below, was
synthesized. This compound inhibits Cdc25A with an ICSO of 2.2 ~,M, a two-fold
increase in potency compared to cdc1671.
aH
/ O O~aw
I ~ O
N~
i~ O
O
COOH
Furthermore, the first glutamyl residue could be replaced by a neutral amino
acid such as the tert. butyl ester of glutamic acid itself or neutral amino
acids such as
norvaline or norleucine, thus changing the physicochemical properties of the
peptide.
The following pentapeptides, cdc 1719, cdc 1748 and cdc 1749 were prepared and
they
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have ICSO values comparable to or better than cdc1679 (1.1 ~.M, 2.5 pM, 1.5 pM
against Cdc25A respectively).
cdc1719 S03H COOH
O ~ O O~
b~ o
N~ ' s
to
I~ 1 ~ \
cootB~
cdc1748 S03H COOH
O
O 0
o . _
I b - O
N~ ' S
o ~ \
1
cdcl 749 S03H COOH
O / O O
I b~ N
__
I ~ O O
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In a preferred embodiment, the Cdc25 inhibitor of the invention is of Formula
I,
R~-A1-A2-A3-A4-Rz (I)
or a pharmaceutically acceptable salt or prodrug thereof, or a combination
thereof,
where Rl is R3-CO, R4RSN-CO, R6-SO2, R7R8NSOz, wherein R3, R4 , R5, R6, R7 and
Rg, are independently of each other, hydrogen, substituted or unsubstituted
alkyl,
substituted or unsubstituted aryl, substituted or unsubstituted cycloalkyl,
substituted
or unsubstituted heterocycloalkyl, E- or Z-aryl-CZ-C4-alkenyl or aryl-C2-Ca-
alkinyl.
Suitable alkyl substituents include hydrogen, hydroxy, C1_6alkoxy, phenoxy,
benzyloxy, halogen, amino, C~_balkylamino, di-C1_6alkylamino, C,_6alkyl-CO-NH,
substituted and unsubstituted cycloalkyl, substituted and unsubstituted
heterocycloalkyl, and substituted or unsubstituted aryl.
An aryl group can be, for example, a phenyl, naphthyl, anthracenyl,
phenanthrenyl, fluorenyl, pyridyl, pyridazinyl, pyridinonyl, furanyl, thienyl,
thiazolyl,
isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl, benzimidazolyl,
pyrazinyl,
pyrimidyl, quinolyl, isoquinolyl, benzofuranyl, benzodihydrofuranyl,
benzothienyl,
pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl or dibenzofuranyl group.
Substituted
aryl groups can be, for example, mono-, di- or trisubstituted and suitable
substituents
can be independently selected from C1_balkyl, halo, hydroxy, C~_6alkyl amino,
di-C~_
balkyl amino, CI_6alkoxy, CI_6alkylthio, C1_6alkylcarbonyl, phenylcarbonyl,
benzylcarbonyl, C1_6alkyl-sulfonyl, Cl_6alkyl-sulfonyl-amino, C1_6alkyl-
carbonyl-
amino, carboxyl, O- C1_6alkyl carboxyl, carboxylalkenyl, O- C1_6alkyl carboxyl
alkenyl, C1_6alkylcarbamoyl, cyano, nitro, trifluoromethyl and
oxytrifluoromethyl.
Suitable cycloalkyl groups include substituted and unsubstituted C3_8-
cycloalkyl, adamantyl, bicyclooct[3.3.0]-yl. Examples of suitable
heterocycloalkyl
groups include substituted and unsubstiuted pyrrolidinyl, piperazinyl,
tetrahydropyranyl, tetrahydrofuranyl, pyrrolidinonyl and morpholinyl. Suitable
substituents on the cycloalkyl or heterocycloalkyl group include one or more
of , for
example, C1_6alkyl, halo, hydroxy, CI_6alkyl amino, di-CI_balkyl amino,
C1_6alkoxy,
C1_balkylthio and Ci-6alkylcarbonyl.
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R4 and RS or R7 and R8 can also form together a four to seven-membered ring.
R3-CO can be further an amino acid residue of the formula R9-CO-G where R9 is
hydrogen, CI_6alkyl, phenyl, benzyl, naphthyl, benzyloxy or C1_6alkoxy and G
is an
Asp, Asn, Pro, Ala, Val, Lys , Gly , Arg, Ile, Ser, Thr, Leu, Trp, Cys, Tyr,
Met, Gln,
Glu , Phe or His residue.
A1 is an amino acid residue of the general formula II
X
Z
R10
N
R11
O
where Rlo is hydrogen or C1_6alkyl; Ri 1 is hydrogen or C1_balkyl; n is 0, 1
or 2; and X
is S03H, SOzNR1zR13, CHz-S03H, CFz-S03H, CHz-SOZNR1zR13, CFz-S02NR1zRi3,
where Rlz and R13 are independently hydrogen, C1_6alkyl or substituted or
unsubstituted phenyl, benzyl, furanyl, thiophenyl, thiazolyl, isothiazolyl,
pyrazolyl,
isoxazolyl or oxazolyl; or where Rlz is hydrogen, R13 can also be hydroxy,
C1_6-
alkoxy, CI_6-alkylcarbonyl or substituted or unsubstituted benzoyl; or X is
P03Hz,
CH2P03Hz, CFz-P03Hz, OCHZP03Hz, COOH, CHZCOOH, CF2COZH, OCHZC02H,
OCFZCOzH, OCH(COZH) z, O-CF(COZH) z, NH-SOz-R14, wherein Rl4 is C~_6alkyl,
benzyl or phenyl; or X is NH-CO-COO-Rls, wherein R15 is C,_6alkyl, benzyl or
phenyl. Preferably, X is at the 3 or 4 position of the phenyl ring. Z is
hydrogen, C1_
6alkyl, halo, hydroxy, C~_balkyl amino, di-C~_6alkylamino, C~_6alkoxy,
C1_6alkylthio,
CI_6alkylcarbonyl, halogen-substituted C1_6alkylcarbonyl, formyl,
phenylcarbonyl,
benzylcarbonyl, Cl_6alkyl-sulfonyl, CI_6alkyl-sulfonyl-amino , carboxyl, O-
Cl_6alkyl
carboxyl, carboxylalkenyl, O- C1_6alkyl carboxyl alkenyl, C1_balkylcarbamoyl,
cyano,
nitro, trifluoromethyl, oxytrifluoromethyl, or -(CHz)m NRI6R», wherein m is 0,
1 or 2
and R16 and R17 are independently of each other selected from hydrogen,
C1_6alkyl, C1_
6alkyl-carbonyl, amino-Cz_6alkyl, C1_6alkyl-amino-Cz_6alkyl, di-CI_6alkyl-
amino-Cz_
6alkyl, hydroxy-Cz_6alkyl, C,_6alkoxy-C1_6alkyl, aryl-Co_6alkyl,
C3_8cycloalkyl-Co_
6alkyl and heterocycloalkyl-Co_6alkyl. Aryl, cycloalkyl and heterocycloalkyl
are as
described above for R3, R4 and R5.
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A2 is an amino acid residue of the formula III
R18 R20
N
R19
O
where R,8 is hydrogen or C1_6alkyl; R,9 is hydrogen or C1_6alkyl; and RZO is
the side
chain of the amino acid Gly, Ala, Val, Leu, Ile, Nva, Nle, Asp, Glu, Lys, Asn,
Gln,
Phe, His, homoleucine, Glu(Cl_6alkyl), Asp(C1_6alkyl), Lys(Boc). RZO can also
be -
(CHZ)o-COORz, with o= 3-5 and R21 is hydrogen or C,_6alkyl. R19 and RZO can
also
form, together with the a-carbon, a three to seven-membered carbocyclic ring
system.
R18 and Rzo can also form, together with the nitrogen atom and a-carbon, a
four to
seven-membered heterocyclic ring system. A2 can, for example, be thioprolyl,
dehydroprolyl or substituted or unsubstituted prolyl, for example, mono- or
disubstituted prolyl, wherein the substituents are independently of each other
hydrogen, Ci_6alkyl, phenyl, hydroxy and C1_6alkoxy. R1$ and RZO can also form
a
bicyclic eight to twelve-membered nitrogen-containing ring system such as
isoindolinyl, octahydroindolyl or dihydroindolyl.
In preferred embodiments, A2 is aspartyl or an ester thereof; glutamyl or an
ester thereof; a-amino adipic acid or an ester thereof; valyl, norvalyl or
leucyl.
A3 is an amino acid of the general formula IV,
R22 R24
N
R23
O
where RZZ has the meaning stated above for R1g in Formula III, R23 has the
meaning
stated for R,9 in Formula III and R24 has the meaning stated above for RZO in
Formula
III.
In preferred embodiments, A3 is aspartyl or an ester thereof; glutamyl or an
ester thereof; a-amino adipic acid or an ester thereof; valyl, norvalyl or
leucyl; or R23
and R24 together form a three to seven-membered ring; or Rz2 and R24, together
with
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the nitrogen atom, form a substituted or unsubstituted heterocycle. For
example R3
can be prolyl or substituted prolyl, such as 2-methylprolyl, 3-methylprolyl, 5-
phenylprolyl, 3-hydroxyprolyl, 3-tert-butoxyprolyl, 3,3-dimethylprolyl;
dehydroprolyl, isoindolyl, octahydroindolyl or dihydroindolyl.
A4 is an amino acid of the general formula V
R25 R27
N
R26
O
where Rz5 is hydrogen or CI_6alkyl; Rz6 is hydrogen or C1_balkyl; R27 is -
(CHZ)p
(CH(RZ$))q-aryl; p is 0, 1 or 2; q is 0, 1 or 2 and RZ$ is hydrogen or methyl.
Suitable aryl groups include substituted and unsubstituted phenyl, naphthyl,
anthracenyl, phenanthrenyl, fluorenyl, pyridyl, pyridazinyl, pyridinonyl,
furanyl,
thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl,
benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuranyl,
tetrahydronaphthyl, benzodihydrofuranyl, quinazoline, benzothienyl, pyrazolyl,
indolyl, purinyl, isoxazolyl, oxazolyl, dibenzofuranyl.
Suitable substituents for the aryl groups include hydrogen, C1_6alkyl, halo,
hydroxy, C I _6alkyl amino, di-C 1 _6alkyl amino, C 1 _6alkoxy, C 1
_6alkylthio, C 1 _
6alkylcarbonyl, halogen-substituted C,_6alkylcarbonyl, formyl-,
phenylcarbonyl,
benzylcarbonyl, C1_6alkyl-sulfonyl, C,_6alkyl-sulfonyl-amino, carboxyl, O-
C1_6alkyl
carboxyl, carboxylalkenyl, O- C,_6alkyl carboxyl alkenyl, C1_6alkylcarbamoyl,
cyano,
nitro, trifluoromethyl, oxytrifluoromethyl, aryl, Y-(CHZ)S-C3_g-cycloalkyl, Y-
(CHZ)s-
aryl, where Y is O; S, NH and s is 0, 1, 2 or 3; Y-(CHZ)"-R29 where Y is O,
NH, or S,
a is 2 to 6 and R29 is OH, CHZ-OH, NHZ or NH(C=NH)NH2; Y-(CHZ)"-R3o where Y
is O, NH or S, v is 1- 6 and R3o is COC,_6alkyl, COOH or CONH2; Y -(CH=CH) -
R3u
where R3, is COC1_6alkyl, COOH, CONHZ or phenyl. Suitable aryl groups within
the
foregoing substituents include substituted and unsubstituted phenyl, pyridyl,
furanyl,
thienyl, thiazolyl, isothiazolyl, imidazolyl, pyrazinyl, pyrimidyl, pyrazolyl,
isoxazolyl
and oxazolyl, which can be independently substituted by one or more of
hydroxy,
amino, carboxyl, carboxamide, halo, hydroxy, C1_6alkyl amino, di-C,_6alkyl
amino,
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C i _6alkoxy, C i _balkylthio, C, _6alkylcarbonyl, Y-(CHZ)t-heterocycloalkyl,
where Y is
O, S or NH and t is 0, 1, 2 or 3, and the heterocycloalkyl group is selected
from the
group consisting of moipholinyl, pyrrolidinyl, piperazinyl, N-substituted
piperazinyl,
piperidinyl, tetrahydropyranyl, tetrahydrofuranyl, and pyrrolidinonyl. In a
preferred
embodiment, R27 is an aryl group selected from substtuted and unsubstituted
phenyl,
naphthyl, such as 1-naphthyl, and benzothienyl, such as 3-benzothienyl.
R2 is NR32R33, where R3z is hydrogen or C~_6alkyl; and R33 is (CH2)W W-
(CHZ)X-V, where W is a single bond, wherein the sum of w and x is 1 to 6, or,
where
w is 0, 1, 2 or 3 and x is 0, 1, 2 or 3, W can be aryl or aryl-T, where T is
O, S or NH.
Suitable aryl groups include substituted and unsubstituted phenyl, naphthyl,
pyridyl,
furanyl, thienyl and pyrimidyl. W can also be C3_8cycloalkyl, where w is 0, 1,
2 or 3
and x is 0, 1, 2 or 3. V is COOR34 where R34 is hydrogen or CI_6alkyl; or V is
COC1_
6alkyl, CONH2, S03H or NO2.
Rz can also be an amino acid AS of Formula VI
R35 R37
N U
R38 O
where R35 is hydrogen or C1_6alkyl; R36 is hydrogen or C1_6alkyl; R37 is the
side chain
of the amino acid Asp, Asn, Glu, Gln, Asp (CI_6alkyl), Glu(C1_6alkyl) or
(CHZ)y-
COOR42 where y is 3, 4 or 5; and R42 is hydrogen or C1_6alkyl; or R37 is
(CHZ)Z-
CONR4oR4~, where z is 1 to 5 and R4o and R41 are independently, hydrogen or C,-
6-
alkyl, or R4o, R4, and the nitrogen atom together form a 5- to 8-member
heterocycle;
or R37 is (CHZ)a-S03H, where a is 1, 2, 3, 4 or 5; or (CHZ)b-tetrazolyl where
b is l, 2,
3, 4 or 5; or R37 is (CHZ)d-phenyl-(CHz)e-COOR43 where d is 0 to 2, a is 0 to
2 and R43
is hydrogen or C1_6alkyl; or R3~ is (CHz)d-phenyl-(CHZ)e-CONR~R45 wherein d is
0
to 2 and a is 0 to 2 and R~ and R45 are independently hydrogen, C1_6alkyl or
R~ and
R45 and the nitrogen atom together form a S to 8-member heterocycle.
Preferably, R37
is the side chain of aspartic acid or glutamic acid; (CH2)y-COOR4z wherein y
is 3 to 5
and R4z is hydrogen or C1_balkyl; or -phenyl-(CHZ)e-COOR43, wherein a is 0, 1
or 2
and R43 is hydrogen or C1_6alkyl. U is hydroxy, C1_6alkoxy or NR38R39, where
R3$ and
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R39 are, independently of each other, hydrogen; substituted or unsubstituted
C1_lo-
alkyl, substituted or unsubstituted aryl or substituted or unsubstituted
cycloalkyl or
bicycloalkyl. Suitable alkyl substituents include hydrogen, hydroxy, halogen,
substituted and unsubstituted aryl and substituted and unsubstituted
cycloalkyl. The
aryl group can be selected from substituted and unsubstituted phenyl,
naphthyl,
anthracenyl, phenanthrenyl, fluorenyl, pyridyl, pyridazinyl, pyridinonyl,
furanyl,
thienyl, thiazolyl, isothiazolyl, imidazolyl, triazolyl, pyrrolyl, tetrazolyl,
benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuranyl,
benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl,
dibenzofuranyl.
Suitable aryl substituents are independently, C1_6alkyl, halo, hydroxy,
Cl_balkyl amino,
di-C1_6alkyl amino, C~_6alkoxy, C1_6alkylthio, C1_6alkylcarbonyl,
phenylcarbonyl,
benzylcarbonyl, C,_balkyl-sulfonyl, C1_6alkyl-sulfonyl-amino,Cl_6alkyl-
carbonyl-
amino, carboxyl, O- C1_6alkylcarboxyl, carboxylalkenyl, O- C1_6alkyl carboxyl
alkenyl, C~_balkylcarbamoyl, cyano, nitro, trifluoromethyl and
oxytrifluoromethyl.
Suitable cycloalkyl groups include C3_8-cycloalkyl, adamantyl and
bicyclooctyl.
Preferably, U is OH or NHR38 , wherein R38 is tert. butyl, isopropyl, 2,4-
dimethylpent-
3-yl, cyclopentyl, cyclohexyl, or bicyclooct[3.3.0]yl; or R38 and R39 ,
together with
the nitrogen atom, form a pyrrolidinyl or piperazinyl ring.
In one subset of compounds of Formula I, R2 is (CH2)w W-(CHZ)X COOR3a,
wherein W is a single bond, phenyl or C6-cycloalkyl.
By the terms "amino acid residue" and "peptide residue" is meant an amino
acid or peptide molecule without the -OH of its carboxyl group (C-terminally
linked)
or the proton of its amino group (N-terminally linked). In general the
abbreviations
used herein for designating the amino acids and the protective groups are
based on
, recommendations of the IUPAC-ILTB Commission on Biochemical Nomenclature
(see
Biochemistry (1972) 11:1726-1732). For instance Met, Ile, Leu, Ala and Gly
represent "residues" of methionine, isoleucine, leucine, alanine and glycine,
respectively. By the residue is meant a radical derived from the corresponding
a-amino acid by eliminating the OH portion of the carboxyl group and the H
portion
of the a-amino group. The term "amino acid side chain" is that part of an
amino acid
exclusive of the -CH(NH2)COOH portion, as defined by K. D. Kopple, "tides and
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Amino Acids", W. A. Benjamin Inc., New York and Amsterdam, 1966, pages 2 and
33; examples of such side chains of the common amino acids are -CH2CH2SCH3
(the
side chain of methionine), -CH(CH3)-CH2CH3 (the side chain of isoleucine), -
CH2CH(CH3)2 (the side chain of leucine) or H-(the side chain of glycine).
For the most part, the amino acids used in the application of this invention
are
those naturally occurnng amino acids found in proteins, or the naturally
occurring
anabolic or catabolic products of such amino acids which contain amino and
carboxyl
groups. Particularly suitable amino acid side chains include side chains
selected from
those of the following amino acids: glycine, alanine, valine, cysteine,
leucine,
isoleucine, serine, threonine, methionine, glutamic acid, aspartic acid,
glutamine,
asparagine, lysine, arginine, proline, histidine, phenylalanine, tyrosine, and
tryptophan.
However, the term amino acid residue further includes analogs, derivatives
and congeners of any specific amino acid referred to herein. For example, the
present
invention contemplates the use of amino acid analogs wherein a side chain is
lengthened or shortened while still providing a carboxyl, amino or other
reactive
precursor functional group for cyclization, as well as amino acid analogs
having
variant side chains with appropriate functional groups). For instance, the
subject
peptidomimetic can include an amino acid analog as for example, a-
cyanoalanine,
canavanine, djenkolic acid, norleucine, 3-phosphoserine, homoserine,
dihydroxyphenylalanine, 5-hydroxytryptophan, 1-methylhistidine, or 3-
methylhistidine. Other naturally occurring amino acid metabolites or
precursors
having side chains which are suitable herein will be recognized by those
skilled in the
art and are included in the scope of the present invention.
Also included are the D and L stereoisomers of such amino acids when the
structure of the amino acid admits of stereoisomeric forms. The configuration
of the
amino acids and amino acid residues herein are designated by the appropriate
symbols
D, L or DL, furthermore when the configuration is not designated the amino
acid or
residue can have the configuration D, L or DL. It will be noted that the
structure of
some of the compounds of this invention includes asymmetric carbon atoms. It
is to
be understood accordingly that the isomers arising from such asymmetry are
included
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within the scope of this invention. Such isomers are obtained in substantially
pure
form by classical separation techniques and by sterically controlled synthesis
and
have arbitrarily been named, for example, as isomers #1 or #2. For the
purposes of
this application, unless expressly noted to the contrary, a named amino acid
shall be
construed to include both the D or L stereoisomers, preferably the L
stereoisomer.
The phrase "protecting group" as used herein means temporary substituents
which protect a potentially reactive functional group from undesired chemical
transformations. Examples of such protecting groups include esters of
carboxylic
acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and
ketones,
respectively. The field of protecting group chemistry has been reviewed
(Greene,
T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 3"d ed.; Wiley: New
York, 1999; and Kocienski, P.J. Protecting Groups, Georg Thieme Verlag: New
York, 1994).
The phrase "N-terminal protecting group" or "amino-protecting group" as used
herein refers to various amino-protecting groups which can be employed to
protect the
N-terminus of an amino acid or peptide against undesirable reactions during
synthetic
procedures. Examples of suitable groups include acyl protecting groups such
as, to
illustrate, formyl, dansyl, acetyl, benzoyl, trifluoroacetyl, succinyl and
methoxysuccinyl; aromatic urethane protecting groups as, for example,
carbonylbenzyloxy (Cbz); and aliphatic urethane protecting groups such as t-
butyloxycarbonyl (Boc) or 9-Fluorenylmethoxycarbonyl (FMOC). Peptidomimetics
of the present invention which have sidechain or azepine ring substituents
which
include amino groups -such as where R3 is a lysine or arginine, or where Rg,
R1, R2
or Y comprise a free amino group, can optionally comprise suitable N-terminal
protecting groups attached to the sidechains.
The phrase "C-terminal protecting group" or "carboxyl-protecting group" as
used herein refers to those groups intended to protect a carboxylic acid
group, such as
the C-terminus of an amino acid or peptide. Benzyl or other suitable esters or
ethers
are illustrative of C-terminal protecting groups known in the art.
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In addition to a variety of sidechain replacements which can be carried out to
generate the subject peptidomimetics, the present invention specifically
contemplates
the use of conformationally restrained mimics of peptide secondary structure
or
mimetics, which are not-cleavable by hydrolytic enzymes. Numerous surrogates
have
been developed for the amide bond of peptides. Frequently exploited surrogates
for
the amide bond include the following groups (i) trans-olefins, (ii)
fluoroalkene, (iii)
methylene-oxy, (iv) methylene-amino, (v) methylene-thio, (vi)
dihydroxyethylene,
(vii) phosphonamides, (viii) sulfonamides and (ix) ketomethylene.
0
amide bond
F
~~pi~
trans-olefine fluoro-olefine methylene-oxy
OH
nHi nSi
H
methylene-amino methylene-thio dihydroxyethylene
I O
OH
phosphoramidone sulfonamide ketomethylene
Additionally, peptidomimietics based on more substantial modifications of the
backbone of the peptide can be used. Peptidomimetics which fall in this
category
include (i) retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called
peptoids).
Furthermore, the methods of combinatorial chemistry are being brought to bear,
e.g., by G.L. Verdine at Harvard University, on the development of new
peptidomimetics. For example, one embodiment of a so-called "peptide morphing"
strategy focuses on the random generation of a library of peptide analogs that
comprise a
wide range of peptide bond substitutes.
In an exemplary embodiment, the peptidomimetic can be derived as a retro-
inverso analog of the peptide. Such retro-inverso analogs can be made
according to
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the methods known in the art, such as that described by the Sisto et al. U.S.
Patent
4,522,752.
Retro-enantio analogs such as this can be synthesized from commercially
available D-amino acids (or analogs thereof) and standard solid- or solution-
phase
peptide-synthesis techniques. For example, in a preferred solid-phase
synthesis
method, a suitably amino-protected (fluorenyl-methoxycarbonyl, Fmoc) D-;~,
residue
(or analog thereof) is covalently bound to a solid support such as chlortrityl
chloride
resin. The resin is washed with diemthylformamide, dichloromethane (DCM) and
methanol, and the Fmoc protecting group removed by treatment with piperidine
in
DCM. The resin is washed and neutralized, and the next Fmoc-protected D-amino
acid (D-A2) is introduced by coupling with diisopropylcarbodiimide. The resin
is
again washed, and the cycle repeated for each of the remaining amino acids in
turn
(D-A3, D-A4, etc). When synthesis of the protected retro-enantio peptide is
complete, the protecting groups are removed and the peptide cleaved from the
solid
support by treatment with trifluoroacetic acid. The final product is purified
by HPLC
to yield the pure retro-enantio analog.
In still another illustrative embodiment, trans-olefin derivatives can be made
for the subject polypeptide. For example, an exemplary olefin analog is
derived for
the illustrative pentapeptide:
2-Et0-Naphthyl-CO-Smp-NH-CH(CH3)-CH=CH-CH(CH3)-CO-Phe-Glu-NHtBu
The trans olefin analog of the pentapeptide can be synthesized according to
the
method of Y.K. Shue et al. (Tetrahedron Letters, 28: 3225 (1987)). Referring
to the
illustrated example, Boc-amino L-Ala is converted to the corresponding a-amino
aldehyde, which is treated with a vinylcuprate to yield a diastereomeric
mixture of
alcohols, which are carned on together. The allylic alcohol is acetylated with
acetic
anhydride in pyridine, and the olefin is cleaved with osmium tetroxide/sodium
periodate to yield the aldehyde, which is condensed with the Wittig reagent
derived
from a protected alanine precursor, to yield the allylic acetate. The allylic
acetate is
selectively hydrolyzed with sodium carbonate in methanol, and the allylic
alcohol is
treated with triphenylphosphine and carbon tetrabromide to yield the allylic
bromide.
This compound is reduced with zinc in acetic acid to give the transposed traps
olefin
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as a mixture of diastereomers at the newly formed center. The diastereomers
are
separated and the pseudodipeptide is obtained by selective transfer
hydrogenolysis to
unveil the free carboxylic acid. Other synthetic approaches to traps olefin
building
block are described by J.S. Wai et al., (Tetrahedron Letters 36: 3461 (1995)),
T.
Ikuba et al. (J. Org. Chem. 56: 4370 (1991)) and J.A. McKinney (Tetrahedron
Letters
35: 5985 ( 1994)).
The pseudodipeptide in its Fmoc-portected form is then coupled instead of A2
and A3 in the sequence. Other pseudodipeptides can be made by the method set
forth
above merely by substitution of the appropriate starting Boc amino acid and
Wittig
reagent. Variations in the procedure may be necessary according to the nature
of the
reagents used, but any such variations will be purely routine and will be
obvious to
one of skill in the art.
It is further possible to couple the pseudodipeptides synthesized by the above
method to other pseudodipeptides, to make peptide analogs with several
olefinic
functionalities in place of amide functionalities. For example,
pseudodipeptides
corresponding to Fmoc-protected Glu-Ala or Tyr-Glu, etc. could be made and
then
coupled together by standard techniques to yield an analog of the pentapeptide
which
has alternating olefinic bonds between residues.
Still another class of peptidomimetic derivatives includes the phosphonate
derivatives. The synthesis of such phosphonate derivatives can be adapted from
known synthesis schemes. See, for example, Loots et al. in Peptides: Chemistry
and
Biology, (Escom Science Publishers, Leiden, 1988, p. 118); Petrillo et al. in
Peptides:
Structure and Function (Proceedings of the 9th American Peptide Symposium,
Pierce
Chemical Co. Rockland, IL, 1985).
Other peptidomimetic structures are known in the art and can be readily
adapted for use in the subject peptidomimetics. They would replace two
adjacent
amino acids in the general formula, preferrably the amino acid sequence A2-A3.
The
synthetic procedures to incorporate this peptidomimetics are similar to the
usual
peptide synthesis. The Fmoc-protected form of the peptidomimetic is used
instead of
the corresponding two amino acid in the build-up of the sequence from the C-
terminus. Peptidomimetics PM-1 to PM-18, shown below, can be coupled under the
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usual peptide coupling conditions providing structures such as R1-A1-(PM-x)-A4-
R2
with x is 1 to 18
In one embodiment, the invention provides compounds of Formula I in
which A2 and A3 together form a peptidomimetic residue selected from (a) 6-
amino-
5-oxoperhydropyrido[2,1-b][1,3]thiazole-3-carboxylic acid, preferably (R,S,S)-
6-
amino-5-oxoperhydropyrido[2,1-b][1,3]thiazole-3-carboxylic acid (PM-1); (b) 6-
amino-5-oxoperhydro-3-indolizinecarboxylic acid, preferably (S,S,S)- 6-amino-5-
oxoperhydro-3-indolizinecarboxylic acid (PM-2); (c) (S, R)- 6-amino-5-
oxoperhydro-
8a-indolizinecarboxylic acid or (R,R)-6-amino-5-oxoperhydro-8a-
indolizinecarboxylic acid (PM-3); (d) (R, S)- 6-amino-5-oxoperhydro-8a-
indolizinecarboxylic acid or (S,S)-6-amino-5-oxoperhydro-8a-
indolizinecarboxylic
acid (PM-4); (e) 2-(3-amino-2-oxo-1,2-dihydro-1-pyridinyl)acetic acid (PM-5);
(f) 2-
(3-amino-2-oxo-6-phenyl-1,2-dihydro-1-pyridinyl)acetic acid (PM-6); (g) 3-
amino
benzoic acid (PM-7); (h) 4-aminobenzoic acid (PM-8); (i) 3-aminomethyl benzoic
acid (PM-9-1); (j) (S)-3-(1-aminoethyl)benzoic acid or (R)-3-(1-
aminoethyl)benzoic
acid (PM-9-2); (k) (S)-3-(1-aminopropyl) benzoic acid or (R)- 3-(1-
aminopropyl)
benzoic acid PM-9-3); (1) (S)- 3-(1-aminobutyl) benzoic acid or (R)-3-(1-
aminobutyl)
benzoic acid (PM-9-4); (m) 2-(3-amino-2-oxo-1-azepanyl)acetic acid, preferably
(S)-
2-(3-amino-2-oxo-1-azepanyl)acetic acid (PM-10); (n) 2-[8-(aminomethyl)-3,6-
dimethyl-9,10,10-trioxo-9,10-dihydro-1076-thioxanthen-1-yl]acetic acid (PM-
11); (o)
2-(2-oxopiperazino)acetic acid (PM-12); (p) 2-[8-(aminomethyl)-2-oxo-5-phenyl-
2,3-
dihydro-1H 1,4-benzodiazepin-1-yl]acetic acid (PM-13-1); (q) 2-[8-
(aminomethyl)-2-
oxo-5-methyl-2,3-dihydro-1H 1,4-benzodiazepin-1-yl]acetic acid (PM-13-2); (r)
3-
aminopropanoic acid (PM-14); (s)4-aminobutanoic acid (PM-15); (t) 5-
aminopentanoic acid (PM-16); (u) 2-[2-(2-aminoethoxy)ethoxy]acetic acid (PM-
17);
and (v) 2-(3-amino-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-1-yl)acetic acid,
preferably (S)- 2-(3-amino-2-oxo-2,3,4,5-tetrahydro-1H 1-benzazepin-1-
yl)acetic
acid.
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0
NH,,
O NH
NH,, ' NH,, N N
N~ N
S/ J
S H H O O
P M-1 P M-2 P M-3 P M-4
NH O O NH ~ R O
N~ NH \ I I NH \
/ R p ~ / I
R =H PM-5
R= P h P M-6 P M-7 P M-8 P M-9
NH I
NH' /,O ~ R"
~N \ O \ N N
/ R'~O O
o'S'~o
PM-10 PM-11 PM-12
O
NH
ZO N O NH n I
R",
/ ~N
n = 0 PM-14
n =1 PM-15
PM-13 n = 2 PM-16
2$ NH~
~O
NH~O~O J N
PM-17 PM-18
30 The peptidomimetic residues are shown here in similar way as the "amino
acid
residue" or "peptide residue" has been defined before. The term
"peptidomimetic
residue" means without the -OH of its carboxyl group (C-terminally linked) or
the
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proton of its amino group (N-terminally linked). Some of the peptidomimetics
are
commercially available in their acid form with or without protection of the
amino
group such as PM-1, PM-7, PM-8, PM-10, PM-14, 15 and 16. Synthesis of the
different peptidomimetics are described for PM 5 and 6 by P.D. Edwards et al.
(J.
S Med. Chem. 1996, 39, 1112) and by F.J. Brown et al. (J. Med. Chem. 1994, 37,
1259), for PM 3 and 4 by J. D. Gramberg et al. (Tetrahedron Letters 1994, 35,
861),
for PM2 by H.-G. Lombart et al. (J.Org. Chem. 61: 9437 (1996)), for PM-12 by
A.
Pohlmann et al. (J.Org. Chem. 62: 1016 (1997)), for PM-9 by L. Chen et al.
(Tetrahedron Letters 36: 8715 (1995)), for PM-17 by A.M.P. Koskinen (Biorg. &
Med. Chem. Lett. 5: 573 (1995)), for PM-13 by W.C. Ripka et al. (Tetrahedron
49:
3593 (1993)), for PM-11 by M. Sato et al. (Biochem. Biophys. Res. Commun. 187:
199 (1992)) and for PM-1 by U. Nagai (Tetrahedron Letters 26: 647 (1985)). The
peptidomimetic may incorporate the 1-azabicyclo[4.3.0]nonane surrogate (see
Kim et
al., J. Org. Chem. 62: 2847 (1997)), or an N acyl piperazic acid (see Xi et
al., J. Am.
Chem. Soc. 120: 80 (1998)), or a 2-substituted piperazine moiety as a
constrained
amino acid analogue (see Williams et al., J. Med. Chem. 39: 1345-1348 (1996)).
In
still other embodiments, certain amino acid residues can be replaced with aryl
and bi-
aryl moieties, e.g., monocyclic or bicyclic aromatic or heteroaromatic
nucleus, or a
biaromatic, aromatic-heteroaromatic, or biheteroaromatic nucleus.
The subject peptidomimetics can be optimized by, e.g., combinatorial
synthesis techniques combined with such high throughput screening as described
herein.
Moreover, other examples of mimetopes include, but are not limited to,
protein-based compounds, carbohydrate-based compounds, lipid-based compounds,
nucleic acid-based compounds, natural organic compounds, synthetically derived
organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or
fragments
thereof.
Numerous efficient methods for the synthesis of racemic unnatural amino
acids have been described in literature. The Strecker synthesis involves the
reaction
of an aldehdye with ammonia or with a substituted primary amine and hydrogen
cyanide to form an alpha-amino nitrite which is hydrolyzed to the
corresponding
amino acid (Houben-Weyl, Methoden der organischen Chemise Vol. 11/2, p. 305
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(1958)). The condensation of an amine R1-NH2, an aldehyde RzCHO, an acid R3-
COOH and an isocyanide R4-NC (the Ugi reaction), is a general method of
prepraring
,racemic amino acids of the type (R3-CO)NR1-CHRZ-CO-NHR3 (Ugi et al., Liebigs
Ann. Chem. 1967, 709, 1; I. Ugi et al. Angew,. Chem. Int. ed. 1962, 1, 8).
Solid phase
S versions of the Ugi reaction have been described (Strocker et al.,
Tetrahedron Letters
37: 1149 (1996); Zhang et al., Tetrahedron Letters 37: 751 (1996); Tempest et
al.,
Angew. Chem. Int. Ed. 35: 640 (1996); Sutherlin et al., J. Org. Chem. 61: 8350
(1996); Short et al., Tetrahedron Letters 37: 7489 (1996)). Furthermore,
racemic
amino acids can be prepared by deprotonation of (N-diphenylmethylene)glycine
derivatives with strong bases such as sodium hydride or lithium
diisopropylamide and
reaction of the anion with corresponding alkylating agents, for example
substituted
bromomethylphenyl-derivatives or bromomethylnaphthyl-derivatives. Protection
(Fmoc, Cbz, Boc, Alloc) or acylation of the amino moiety and subsequent
hydrolysis
affords the corresponding amino acid derivatives. An illustrative example to
the
1 S building blocks (R1-CO-(2-Br)-Smp-OH and Rl-CO-(2-CHO)-Smp-OH) is shown in
scheme I:
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Scheme I
Br 1. BH3 Br Br
COOH 2. NBS Me2S SO nPn
3. NazS03 I ~ S03Na 1. Me2N~=CHCI d- ~ ~ a
$ ~ ~ then NEt3,nPnOH
2. NBS Br
LDA, THF, DMPU
PhZC=N~HZ-COZEt
Br Br
1~ ~ S03nPn 1. MeONHZ, HCI ~ Sp3nPn
2. R~-CO-d,
0 ~ PY~dne /
Ph
R1 p COZEt ~ ~ N COZEt
15 Pd(PPh3),,
Vinyltributylstannane
HO
1. AQmix-beta
S03nPn 2. Na104 ~ S03Na
20 I 3. NaOH
O
O
R1 H COZEt R1 ~ H COOH
Another route to amino acids consists of reacting the anion of (N-
25 diphenylmethylene)-glycine derivatives with aldehydes, hydrogenation of
thus
obtained dehydroamino acids yields the racemic amino acids. Furthermore,
hydroxy
napthyl alanine derivatives have been prepared by Vela et al. (J. Org. Chem.
55: 2913
( 1990)).
The Wittig reaction or its Horner-Emmons-modification of an alpha-
30 phosphoryl-glycine derivatives with aldehydes can be used to synthesize the
corresponding dehydroamino acids as described by Ciattini (Ciattini et al.,
Synthesis
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2: 140 (1988)) and Shin (Shin et al., Tetrahedron Lett. 28: 3827 (1987); Shin,
et al.,
Chem. Pharm. Bull. 38: 2020 (1990)).
Hydrogenation of the dehydro amino acid derivatives with metal catalysts
such as palladium on carbon, phosphine or amine complexes of rhodium,
ruthenium
or palladium gives the racemic amino acid derivatives. By using chiral
compounds as
metal ligands, amino acids can be obtained in high enantiomeric excesses as
described
in I. Ojima, "Catalytic Asymmetric Synthesis", Yerlag Chemie, 1993, chapter 1,
p.6
and R. Noyori, "Asymmetric Catalysis in Organic Synthesis", John Wiley, 1994,
chapter 2, p.16. For example, the synthesis of D- and L-alpha aminoadipate,
pimelate
and suberate have been described by T. Pham et al. (J. Org. Chem. 59: 3676
(1994)).
Furthermore, methods for preparation of optically active alpha-amino acids
can be found in R.M. Williams, "Synthesis of active alpha-amino acids"
Pergamon
Press, 1989, and in L.M. O'Donnell "alpha-amino acid synthesis", Tetrahedron
symposia-in-print, 44: 5253 (1988). Thus, syntheses of chiral amino acids can
be
achieved by using chiral glycine derivatives in a similar way as described
above. The
final chiral amino acid is obtained after removal or cleavage of the chiral
auxiliaries.
Depending on the chiral auxiliary, either the D or the L-amino acid can be
obtained in
high enantiomeric excess. Preferred methods are the method described by U.
Schoellkopf (Schoellkopf et al., Angew, Chem. Int. Ed. 18: 863 (1979);
Schoellkopf et
al., Angew, Chem. Int. Ed. 20: 798 (1981); Schoellkopf et al., Synthesis, 969
(1981);
Schoellkopf et al., Synthesis, 866 (1982); Schoellkopf et al., Synthesis, 861
(1982);
Schoellkopf et al., Synthesis, 37 (1983); Schoellkopf et al., Synthesis, 271
(1984)) the
methods described by R. M. Williams using the 5,6-diphenyl-2,3,5,6-tetrahydro-
4H-
1,4-oxazin-2-one template (Williams et al.,
J. Am. Chem. Soc. 113: 9276 (1991); Williams et al., Tetrahedron Letters 29:
6075
(1988); Solas et al., J. Org. Chem. 61: 1537 (1996); Williams et al., J. Am.
Chem.
Soc. 110: 1547 ( i 988); Williams et al., J. Am. Chem. Soc. 110: 482 (1988);
Williams
et al., J. Am. Chem. Soc. 108: 1103 (1986)), the methods described by D.
Seebach
(Fitzi et al., Tetrahedron 44: 5277 (1988), Seebach et al., Liebigs Ann.
Chem., 1215
(1989); Schickli et al., Liebigs Ann. Chem., 1323 (1991); Seebach et al.,
Liebigs Ann.
Chem., 1145 (1992); Mueller et al., Helv. Chim. Acta, 75: 855 (1992); Seebach
et al.,
Angew. Chem. 105: 1780 (1993)); and the method described by A. Myers (Myers et
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al., J. Am. Chem. Soc. 117: 8488 (1995); Myers et al., J. Am. Chem. Soc. 119:
656
( 1997)).
An illustrative example, the syntheses of Fmoc-(6-OH)-Nal-OH or Fmoc-(7-
OH)Nal-OH, is shown in scheme II.
Scheme II:
Br
O 1. MeMgBr, then HZSOQ HO Me 1. TBS-CI,
Me0 2. DDQ imidazole TBSO
\ 3. BBr3 \ \ 2. NBS \ \
/ / ~ / /
LiN(TMS)Z, DMPU
Cbz'
' BOON
Ph'
Ph
TBSO
/
TBSO I 1. Hz. PdCl2 \
\ 2. Fmoc-OSuccinimid
Cbz\ O
\ N
.~O
frrloc~ Ph ~~
COOH Ph
Another route is the electrophilic amination of chiral enolates and subsequent
hydrolysis of the chiral auxiliaries as exemplified by D. Evans (D.Evans et
al., J. Am.
Chem. Soc. 112: 4011 (1990); D. Evans et al., Tetrahedron 44: 5525 (1988)).
Alpha, alpha-disubstituted amino acid derivatives can be obtained by methods
described by D. Seebach (Seebach et al., Liebigs Ann. Chem., 217 (1995);
Seebach et
al., Tetrahedron Letters, 25: 2545 (1984)).
1 S Chiral amino acids are also obtained by resolution of racemic amino acid
esters
by enzyme-catalyzed acylation as described by Stuermer et al. (BASF AG) Ger.
Offen., DE 19727517.
The amino acids obtained by the above methods can be further modified or
transformed using standard organic reactions, such as reductive amination,
alkylation,
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esterification, etherification, Mitsunobu reaction, performed with the amino
acid in
suitable protected form (for example with Boc, Fmoc, Alloc, Cbz) or in a
peptidyl
environment such as the penta- and tetrapetidyl intermediate, obtained in
routes 1 to 4
as described below. The modification or transformation reaction can be carried
out in
S solution or with the amino acid or the peptide derivative bound to a
suitable solid
phase. For example, the synthesis of Fmoc-N-methyl amino acids has been
described
by Yang et al., Tetrahedron Letters 42: 7307 (1997), using a solid phase
methodology.
An illustrative example for the modification of the pentapeptide is the
reductive amination of the 2-formyl-smp-derivative as shown in scheme III:
2-CI-trityl-resin
S03H
OHC
'O
OJ O ' ~ N ~~N b
H II H
\ H ~~O O / S O
O _
\ I
1. 2-THF-CHZ-NHZ (5 equiv.),
NaBH(OAc}~ (5 equiv. )
dichloroethane, rt , 20 h
2. AczO (20 equi.v),
diisopropylethylamine, rt, 2 h
3. 95% TFA/H20 (3 mL, rt. 1 h,
then ether preapitation
O N S03H
COOH
of o ' ~ N a~ a
\ N ~ O O / S O
H
/ I a ~ -
\
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Another illustrative example for modification of a peptidyl intermediate is
the
Mitsunobu reaction of a Nal-derivative on solid phase as shown in scheme IV:
i I COOtBu COOtBu
O ~ O O H
R1 ~~ ~ ~ ~ N-Resin
O ~ ~ O
COOtBu I /
TBSO
1. Bu,NF (5 equiv.), THF, 15 min., rt
2. Ph-CHzOH, PBu3 (10 equiv.),
MCP (10 equiv.), NEt3 (25 equiv. )
THF:dichloromethane, 1:1, rt , 20 h
3. 95% TFA/HZO, (3 mL, rt. 1 h,
then ether precipitation
25
COOH COOH
O ~ O O
R1~~ ~ ~~N NH2
O ~ .. _ H O
COOH
The novel compounds of the general formula I can be prepared by known
methods of peptide chemistry. Thus, the peptidyl derivatives can be assembled
sequentially from amino acids or by linking suitable small peptide fragments.
In the
sequential assemblage, starting at the C terminus the peptide chain is
extended
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stepwise by one amino acid each time. In fragment coupling it is possible to
link
fragments of different lengths, and the fragments in turn can be obtained by
sequential
assemblage from amino acids or themselves by fragment coupling. Both in the
sequential assemblage and in the fragment coupling it is necessary to link the
units by
forming an amide linkage. Enzymatic and chemical methods are suitable for
this.
Chemical methods for forming the amide linkage are described in detail by
Miiller,
Methoden der organischen Chemie Vol. XV/2, pp 1 to 364, Thieme Verlag,
Stuttgart,
1974; Stewart, Young, Solid Phase Peptide Synthesis, pp 31 to 34, 71 to 82,
Pierce
Chemical Company, Rockford, 1984; Bodanszky, Klausner, Ondetti, Peptide
Synthesis, pp 85 to 128, John Wiley & Sons, New York, 1976 and other standard
works on peptide chemistry. Particular preference is given to the azide
method, the
symmetric and mixed anhydride method, in situ generated or preformed active
esters,
the use of urethane protected N-carboxy anhydrides of amino acids and the
formation
of the amide linkage using coupling reagents (activators, especially
dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-
ethoxycarbonyl-
2-ethoxy-1,2-dihydroquinoline (EEDQ), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide hydrochloride (EDCI), n-propane-phosphonic anhydride (PPA), N,N-
bis(2-oxo-3-oxazolidinyl)imido-phosphoryl chloride (BOP-Cl), bromo-tris-
pyrrolidinophosphonium hexafluorophosphate (PyBrop), diphenyl-phosphoryl azide
(DPPA), Castro's reagent (BOP, PyBop), O-benzotriazolyl-N,N,N',N'-
tetramethyluronium salts (HBTU), diethylphosphoryl cyanide (DEPCN), 2,5-
diphenyl-2,3-dihydro-3-oxo-4-hydroxy-thiophene dioxide (Steglich's reagent;
HOTDO), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
(TBTU), O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU) and 1,1'-carbonyl-diimidazole (CDI). The coupling
reagents can be employed alone or in combination with additives such as N,N-
dimethyl-4-aminopyridine (DMAP), N-hydroxy-benzotriazole (HOBt), 1-hydroxy-7-
azabenzotriazole (HOAt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide
(HOSu) or 2-hydroxypyridine.
Whereas it is normally possible to dispense with protective groups in
enzymatic peptide synthesis, reversible protection of reactive groups not
involved in
formation of the amide linkage is necessary for both reactants in chemical
synthesis.
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Four conventional protective group techniques are preferred for the chemical
peptide
synthesis: the benzyloxycarbonyl (Cbz), the t-butoxycarbonyl (Boc), the
allyloxycarbonyl (Alloc) and the 9-fluorenylmethoxycarbonyl (Fmoc) techniques.
Identified in each case is the protective group on the a-amino group of the
chain-
s extending unit. A detailed review of amino-acid protective groups is given
by Miiller,
Methoden der organischen Chemie Vol. XV/1, pp 20 to 906, Thieme Verlag,
Stuttgart, 1974. The units employed for assembling the peptide chain can be
reacted
in solution, in suspension or by a method similar to that described by
Merrifield in J.
Amer. Chem. Soc. 85: 2149 (1963). Particularly preferred methods are those in
which
peptides are assembled sequentially or by fragment coupling using the Cbz,
Boc,
Alloc or Fmoc protective group technique, with one of the reactants in the
said
Merrifield technique being bonded to an insoluble polymeric support (also
called
resin hereinafter). This typically entails the peptide being assembled
sequentially on
the polymeric support using the Boc, Alloc or Fmoc protective group technique,
the
growing peptide chain being covalently bonded at the C terminus to the
insoluble
resin particles. This procedure makes it possible to remove reagents and
byproducts
by filtration, and thus recrystallization of intermediates is unnecessary. The
protected
amino acids can be linked to any suitable polymers, which merely have to be
insoluble in the solvents used and to have a stable physical form which makes
filtration easy. The polymer must contain a fimctional group to which the
first
protected amino acid can be firmly attached by a covalent bond. Suitable for
this
purpose are a wide variety of polymers, eg. cellulose, polyvinyl alcohol,
polymethacrylate, sulfonated polystyrene, chloromethylated
styrene/divinylbenzene
copolymer (Merrifield resin), 4-methylbenz-hydrylamine resin (MBHA-resin),
phenylacetamidomethyl-resin (Pam-resin), Rink amide resin, chlorotrityl
chloride
resin, p-benzyloxy-benzyl-alcohol-resin, benzhydryl-amine-resin (BHA-resin), 4-
(hydroxymethyl-)-benzoyl-oxymethyl-resin, the resin of Breipohl et al.
(Tetrahedron
Letters 28 (1987) 565; supplied by BACHEM), 4-(2,4-
dimethoxyphenylaminomethyl)phenoxy-resin (supplied by Novabiochem) or o-
chlorotrityl-resin (supplied by Biohellas).
Suitable for peptide synthesis in solution are all solvents which are inert
under
the reaction conditions, especially water, N,N-dimethylformamide (DMF),
dimethyl
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sulfoxide (DMSO), acetonitrile, dichloromethane (DCM), 1,4-dioxane,
tetrahydrofuran (THF), N-methyl-2-pyrrolidinone (NMP) and mixtures of the said
solvents.
Peptide synthesis on the polymeric support can be carried out in all inert
organic solvents in which the amino-acid derivatives used are soluble;
however,
preferred solvents additionally have resin-swelling properties, such as DMF,
DCM,
NMP, acetonitrile and DMSO, and mixtures of these solvents. After synthesis is
complete, the peptide is cleaved off the polymeric support. The conditions
under
which cleavage of the various resin types is possible are disclosed in the
literature.
The cleavage reactions most commonly used are acid- and palladium-catalyzed,
especially cleavage in liquid anhydrous hydrogen fluoride, in anhydrous
trifluoromethanesulfonic acid, in dilute or concentrated trifluoroacetic acid,
palladium-catalyzed cleavage in THF or THF-DCM-mixtures in the presence of a
weak base such as morpholine or cleavage in acetic
acid/dichloromethane/trifluoro-
ethanol mixtures. Depending on the chosen protective groups, these may be
retained
or likewise cleaved off under the cleavage conditions.
More specifically, the following routes can be used to prepare the novel
compounds of general formula I.
a) Route 1: Synthesis of RI- Al-A2-A3-A4-AS-U.
The peptide sequence is built up stepwise from the C-terminus by coupling the
corresponding Fmoc-amino acid (Fmoc-AS-OH) to the free amino group on a resin
such as Rink amide AM resin, then removal of the Fmoc-protecting group to
liberate
the amino group for the next coupling, for example with piperidine. The
coupling and
deprotection is repeated for each of the amino acids in the order Fmoc-A4-OH,
Fmo-
A3-OH, Fmoc-A2-OH and Fmoc-A1-OH to yield the intermediate NHZ-A1-A2-A3-
A4-AS-NH-resin.
The final capping is done by coupling the corresponding acid R3-COOH to the
above resin intermediate or by reacting the corresponding acid chloride R3-
COCI, acid
fluoride R3-COF or anhydride (R3-CO)ZO in presence of a base such as tertiary
amines or alcoholates or inorganic bases with the resin intermediate. In case
of the
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areas the resin intermediate is treated with the corresponding isocyanate R4-
NCO or
with R4RSNCOC1, in the case of the sulfonamides with the corresponding
sulfonylchloride R6-SOZCI and in the case of the sulfonylurea with R7R8SOzC1
in the
presence of a base.
The final cleavage of the compound from the resin is achieved by treating the
resin with acids such as hydrochloric acid or trifluoroacetic acid. Depending
on the
strength of the acid used and the reaction time, simultaneous deprotection of
the side
chains could be achieved. The resulting compounds could be further purified by
standard techniques such as column chromatography.
b) Route 2: Synthesis of R1- Al-A2-A3-A4-AS- NR38R39.
In the case that the final compound has an amino side chain containing an acid
residue such as Glu, Asp or Aad, the corresponding amino acid Fmoc-Xaa(tBu)-OH
was coupled in solution with the corresponding amine HNR3gR39 using standard
peptide coupling techniques to yield Fmoc-Xaa(tBu)-NR38R39 which is then
deprotected with 95% trifluoroacetic acid to yield Fmoc-Xaa- NR38R39 with a
free
carboxylic acid moiety in the side chain. This amino acid is coupled to a
resin such as
the chlor(tritylchloride resin) to yield the resin-bound ester. The Fmoc group
can now
be deprotected with bases such as piperidine to yield the resin-bound amine.
The
coupling and deprotection is repeated for each of the amino acids in the order
Fmoc-
A4-OH, Fmo-A3-OH, Fmoc-A2-OH and Fmoc-A1-OH to yield the intermediate
NHz-A1-A2-A3-A4-AS(resin)- NR3gR39. The final capping is done by coupling the
corresponding acid R3-COOH to the above resin intermediate or by reacting the
corresponding acid chloride R3-COCI, acid fluoride R3-COF or anhydride (R3-
CO)20
in presence of a base such as tertiary amines or alcoholates or inorganic
bases with the
resin intermediate. In case of the areas the resin intermediate is treated
with the
corresponding isocyanate R4-NCO or with RaRSNCOC1, in the case of the
sulfonamides with the corresponding sulfonylchloride R6-S02C1 and in the case
of the
sulfonylurea with R7R8SOzCl in the presence of a base.
The final cleavage of the compound from the resin is achieved by treating the
resin with acids such as hydrochloric acid or trifluoroacetic acid. Depending
on the
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strength of the acid used and the reaction time, simultaneous deprotection of
the side
chains could be achieved. The resulting compounds could be further purified by
standard techniques such as column chromatography.
c) Route 3: R1- A1-A2-A3-A4- OH and R1- A1-A2-A3-A4- NR3zR33
The tetrapeptide acid sequence is built up stepwise from the C-terminus as
described previously by coupling the corresponding Fmoc-amino acid A1 to the
chlorotrityl chloride resin, then removal of the Fmoc-protecting group to
liberate the
amino group for the next coupling. The coupling and deprotection is repeated
for
each of the amino acids in the order Fmoc-A3-OH, Fmo-A2-OH and Fmoc-A1-OH
and Fmoc-A1-OH to yield the intermediate NHZ-A1-A2-A3-A4-resin.
The final capping is done by coupling the corresponding acid R3-COOH to the
above resin intermediate or by reacting the corresponding acid chloride R3-
COCI, acid
fluoride R3-COF or anhydride (R3-CO)z0 in presence of a base such as tertiary
amines or alcoholates or inorganic bases with the resin intermediate. In case
of the
areas the resin intermediate is treated with the corresponding isocyanate R4-
NCO or
with R4RSNCOCI, in the case of the sulfonamides with the corresponding
sulfonylchloride R6-SOZCI and in the case of the sulfonylurea with R7RgSOzCI
in the
presence of a base.
The final cleavage of the compound from the resin is achieved by treating the
resin with acids such as hydrochloric acid or trifluoroacetic acid. Depending
on the
strength of the acid used and the reaction time, simultaneous deprotection of
the side
chains could be achieved. The resulting final compounds Rl-A1-A2-A3-A4-OH
could be further purified by standard techniques such as column
chromatography.
These compounds are also used as intermediate to couple the corresponding
amine NR32R33 in solution to yield the final amides Rl-A1-A2-A3-A4-NR32Rs3
which
can be further purified by standard techniques such as column chromatography.
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d) Route 4: R,-A1-A2-A3-A4-A5-OH and R~- A1-A2-A3-A4-AS- NR38R39
The above described route to the tetrapeptides can also be used to prepare the
corresponding pentapeptides by coupling with the amino acid A5 to the resin
such as
chlorotrityl chloride resin and then proceeded in similar fashion of repeated
coupling
of the next amino acids and deprotection, final capping and cleavage form the
resin to
yield the final compounds Rl-A1-A2-A3-A4-A5-OH. These compounds are also
used as intermediate to couple the corresponding amine HNR38R39 in solution to
yield
the final amides R~-A1-A2-A3-A4-NR3gR39 which can be further purified by
standard
techniques such as column chromatography.
The amino acids used are either commercially available or their syntheses are
described in the literature. The amino acid A can be considered a pTyr-
mimetic.
Examples of non-hydrolizable phosphor-containing pTyr mimetics have been
described in literature such as phosphonomethyl phenylalanine (Pmp, I.
Marseigne et
al., J. Org. Chem. 53: 3621-3624 (1988)) and phosphonodifluoromethyl
phenylalanine (FZPmp, T.R. Burke Jr. et al., J. Org. Chem. 58: 1336-1340
(1993)).
Examples for non-phophorous containing pTyr mimetics include O-
malonyltyrosine (Tyr(Mal), K.H. Kole et al., Biochem. Biophys. Res. Commun.
209:
817-822 (1995); B. Ye et al., J. Med. Chem. 38: 4270 -4275 (1995)), fluoro-O-
malonyl-tyrosine (Tyr(Fmal), T.R. Burke Jr., J. Med. Chem. 39: 1021-1027
(1996)),
O-carboxymethyl-tyrosine (T.R. Burke Jr. et al., Tetrahedron 54: 9981-9994
(1998)),
3-carboxy-4-(O-carboxymethyl)-tyrosine (T.R. Burke Jr. et al., Tetrahedron
54:9981-
9994 (1998)), 3,4-Di(O-carboxymethyl)-tyrosine (T.R. Burke Jr. et al.,
Tetrahedron
54: 9981-9994 (1998)), O-(carboxydifluoromethyl)-tyrosine (H. Fretz,
Tetrahedron
54: 4849-4858 (1998)) and hydroxysulfonylmethyl phenylalanine (I. Marseigne et
al.,
J. Med. Chem. 32: 445-449 (1989)).
To improve cellular penetration prodrugs can be used for the different acid
functionalities of the compounds with the general structure of formula I.
Typical
prodrug forms for carboxylic acid residues are described in R.B. Silverman,
The
Organic Chemistry of Drug Design and Drug Action, Academic Press, 1992,
chapter
8. For the phosphonate function in amino acid A1 suitable prodrugs are simple
and
substituted alkyl and aryl ester, acyloxyalkyl esters as described in the
review by J.P.
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Krise et al. (Advanced Durg Delivery Reviews, 19: 287-310 (1996)), S-
acylthioethyl
esters as described by X. Li et al. (Bioorg. Med. Chem. Lett. 8: 57-62 (1998))
or
pivaloylmethyl esters as described by C.J. Stankovic et al. (Bioorg. Med.
Chem. Lett.
7: 1909 ( 1997)).
In one embodiment, the present invention relates to a method of treating a
Cdc25-mediated condition in a patient. The method comprises the step of
administering to the patient a therapeutically effective amount of a Cdc25
inhibitor as
described above. The patient can be any animal, and is, preferably, a mammal
and,
more preferably, a human.
A "Cdc25-mediated condition" is a disease or medical condition in which the
catalytic activity of one or more Cdc25 homologues plays a role, for example,
in the
development of the disease or condition. For example, in one embodiment, the
condition is characterized by excessive cellular proliferation.
In one embodiment, the Cdc25-mediated condition is cancer, such as a tumor.
For example the condition to be treated can include lymphoma, such as
Hodgkin's
disease and non-Hodgkin's lymphoma, and tumors of the head, neck, breast,
lung,
such as non-small cell lung carcinoma, and stomach.
The Cdc25 mediated condition can also be a condition in which
hyperproliferation of non-cancer cells plays an important role, such as
restinosis and
reocclusion of the coronary arteries following angioplasty, both of which
result from
abnormal proliferation of smooth muscle cells.
In another embodiment, the Cdc25-mediated condition is an inflammatory
disease which is characterized by abnormal cell proliferation, such as
rheumatoid
arthritis, Reiter's disease, systemic lupus
A therapeutically effective amount, as this term is used herein, is an amount
which results in partial or complete inhibition of disease progression or
symptoms.
Such an amount will depend, for example, on the size and gender of the
patient, the
condition to be treated, the severity of the symptoms and the result sought,
and can be
determined by one skilled in the art.
The compound of the invention can, optionally, be administered in
combination with one or more additional drugs which, for example, are known
for
treating andlor alleviating symptoms of the condition mediated by Cdc25. The
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additional drug can be administered simultaneously with the compound of the
invention, or sequentially. For example, the Cdc25 inhibitor can be
administered in
combination with another anticancer agent, as is known in the art.
The invention further provides pharmaceutical compositions comprising one
or more of the Cdc25 inhibitors described above. Such compositions comprise a
therapeutically (or prophylactically) effective amount of one or more Cdc25
binding
inhibitors, as described above, and a pharmaceutically acceptable carrier or
excipient.
Suitable pharmaceutically acceptable carriers include, but are not limited to,
saline,
buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
The
Garner and composition can be sterile. The formulation should suit the mode of
administration.
Suitable pharmaceutically acceptable carriers include but are not limited to
water, salt solutions (e.g., NaCI), alcohols, gum arabic, vegetable oils,
benzyl
alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,
amylose or
starch, cyclodextrin, magnesium stearate, talc, silicic acid, viscous
paraffin, perfume
oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidone, etc. The
pharmaceutical preparations can be sterilized and if desired, mixed with
auxiliary
agents, e.g., lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for
influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic
substances
and the like which do not deleteriously react with the active compounds.
The composition, if desired, can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. The composition can be a liquid
solution,
suspension, emulsion, tablet, pill, capsule, sustained release formulation, or
powder.
The composition can be formulated as a suppository, with traditional binders
and
carriers such as triglycerides. Oral formulation can include standard carriers
such as
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,
polyvinyl
pyrollidinone, sodium saccharine, cellulose, magnesium carbonate, etc.
The composition can be formulated in accordance with the routine procedures
as a pharmaceutical composition adapted for intravenous administration to
human
beings. Typically, compositions for intravenous administration are solutions
in sterile
isotonic aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic to ease pain at the site of the
injection.
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Generally, the ingredients are supplied either separately or mixed together in
unit
dosage form, for example, as a dry lyophilized powder or water free
concentrate in a
hermetically sealed container such as an ampoule or sachet indicating the
quantity of
active agent. Where the composition is to be administered by infusion, it can
be
S dispensed with an infusion bottle containing sterile pharmaceutical grade
water, saline
or dextrose/water. Where the composition is administered by injection, an
ampoule of
sterile water for injection or saline can be provided so that the ingredients
may be
mixed prior to administration.
The pharmaceutical compositions of the invention can also include an agent
which controls release of the Cdc25 inhibitor compound, thereby providing a
timed or
sustained release composition.
The Cdc25 inhibitor can be administered subcutaneously, intravenously,
parenterally, intraperitoneally, intradermally, intramuscularly, topically,
enteral (e.g.,
orally), rectally, nasally, buccally, sublingually, vaginally, by inhalation
spray, by
drug pump or via an implanted reservoir in dosage formulations containing
conventional non-toxic, physiologically acceptable Garners or vehicles. The
preferred
method of administration is by oral delivery. The form in which it is
administered
(e.g., syrup, elixir, capsule, tablet, solution, foams, emulsion, gel, sol)
will depend in
part on the route by which it is administered. For example, for mucosal (e.g.,
oral
mucosa, rectal, intestinal mucosa, bronchial mucosa) administration, nose
drops,
aerosols, inhalants, nebulizers, eye drops or suppositories can be used. The
compounds and agents of this invention can be administered together with other
biologically active agents, such as analgesics, anti-inflammatory agents,
anesthetics
and other agents which can control one or more symptoms or causes of a Cdc25-
mediated condition.
In a specific embodiment, it may be desirable to administer the agents of the
invention locally to a localized area in need of treatment; this may be
achieved by, for
example, and not by way of limitation, local infusion during surgery, topical
application, transdermal patches, by injection, by means of a catheter, by
means of a
suppository, or by means of an implant, said implant being of a porous, non-
porous,
or gelatinous material, including membranes, such as sialastic membranes or
fibers.
For example, the agent can be injected into the joints.
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EXAMPLES
Example 1 - Synthesis of Peptidic Cdc25 inhibitors
General materials and methods
The following amino acid abbreviations are used herein, for the natural amino
acids the three letter code:
Asp = aspartic acid; Asn = asparagine; Pro = proline; Ala = alanine; Val =
valine; Lys =
Lysine; Gly = glycine; Arg = arginine; Ile = isoleucine; Ser = serine; Thr =
threonine;
Leu = leucine; Trp = tryptophan; Cys = cysteine; Tyr = tyrosine; Met =
methionine; Gln
= glutamine; Glu = glutamic acid; Phe = phenylalanine; His = histidine,
for other amino acids: Nal = 2-amino-3-(naphth-1-yl)-propanoic acid (or
napthyl
alanine); Bta - 2-amino-3-benzo[bJthiophen-3-yl-propanoic acid (or 3
benzothienylalanine); Aad = alpha-aminoadipic acid; Smp = Phe(4-CHZ-S03H); Pmp
=
Phe(4-CHZ-PO(OH)z); FZPmp = Phe(4-CF2-PO(OH)2); Asu = alpha aminosuberic acid;
Hyp = 4-hydroxyproline; Nle = norleucine; Nva = norvaline; hLeu = homoleucine;
Pip =
pipecolinic acid; 3-MePro = 3-methyl-proline; 2-MePro = 2-methyl-proline; Isc
= 1-
isoindolinecarboxylic acid; Oic = octahydroindolyl-2-carboxylic acid; Ac6 =1-
amino-1-
cyclohexanecarboxylic acid; Ac5 = 1-amino-1-cyclopentanecarboxylic acid; Ac3 =
1-
amino-1-cyclopropanecarboxylic acid; Thiopro=L-thiazolidine-4-carboxylic acid;
Iva=
2-amino-2-methylbutanoic acid; Tyr(mal) = Tyr(O-(CH(COOH)Z); Tyr(Fmal) = Tyr(O
(CF(COOH)Z); 2-Oxn = (8-hydroxy-quinolin-2-yl )methyl glycine; dehydrPro = 3,4
dehydroproline; dehydroVal = dehydrovaline; Aib = alpha-aminoisobutyric acid;
Pra =
propargylic glycine; Phg = phenyl glycine ; SmPhg = Phg(4-CHZ-S03H)
Furthermore the following abbreviations have the meaning of Xaa= amino
acid, hXaa = homo amino acid, (NMe)Xaa = amino acid methylated at the amino
group , MeXaa = amino acid methylated at the alpha carbon or the position
indicated,
Xaa(R) = amino acid with a group R functionalized side chain, and D,L-Xaa =
mixture of D- and L-isomer (50/50 or the ratio indicated below in
parenthesis).
In the synthetic procedures the amino group of the amino acid is usually
protected with the Fmoc-group, in a few cases with the Alloc- or Boc-group.
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Carboxylic acid moieties in the side chain of the amino acids are protected as
tent.
butyl esters, when the carboxylic acid moiety is desired in the final product.
The tert.
butyl esters are hydrolysed under the certain conditions used for the cleavage
of the
peptidyl derivatives from polymers or resin.
All natural amino acids and their corresponding D-isomers can be purchased
from
Novabiochem or Bachem.
The other amino acids were purchased from:
Fmoc-Nal(1)-OH (Synthetech Inc.), Fmoc-Bta-OH (Peptech Corp.), Fmoc-Aad(tBu)
OH (Bachem), Fmoc-Smp-OH (RSP Amino Acid Analogues), Fmoc-(Pmp(Et)Z}-OH
(Neosystem), Fmoc-Asu(tBu)-OH (Peninsula Labs), Fmoc-Hyp(tBu)-OH
(Novabiochem), Fmoc-Nle-OH (Novabiochem), Fmoc-Nva-OH (Novabiochem),
Fmoc-hLeu-OH (Neosystem), Fmoc-Pip-OH (Bachem), Fmoc-(2-Me)Pro-OH
(Bachem), Fmoc-Isc-OH (Neosystem), Fmoc-Oic-OH (Bachem), Fmoc-Ac6 -OH
(Neosystem), Fmoc-Ac5-OH (Neosystem), Fmoc-Ac3-OH (Advanced Chem Tech),
Fmoc-Thiapro-OH (Neosystem), Fmoc-Iva-OH (Acros), Fmoc(Tyr(mal(tBu)Z)-OH
(Bachem), Fmoc-DehydrPro-OH (Bachem), Fmoc-DehydroVal-OH (Advanced Chem
Tech), Fmoc-Aib-OH (Senn Chemicals), Fmoc-Pra-OH (Advanced Chemtech),
Fmoc-Phg-OH (Novabiochem), SmPhg (RSP Amino Acid Analogues), Fmoc-(3-Cl-
Phe)-OH (Peptech), Fmoc-(2-Cl- Phe)-OH (Peptech), Fmoc-(4-Cl- Phe)-OH
(Bachem), Fmoc-(4-CONHZ- Phe)-OH (RSP Amino Acid Analogues), Fmoc-(4-NHz-
Phe)-OH (Bachem), Fmoc-(3-NHZ- Phe)-OH (RSP Amino Acid Analogues), ), Fmoc-
(4-NHAc-Phe)-OH (RSP Amino Acid Analogues), Fmoc-(4-COOtBu-Phe)-OH
(Bachem), Fmoc-(4-CF3- Phe)-OH (Apollo), Fmoc-(3-NOZ- Phe)-OH (Peptech),
Fmoc-(4-Ph-Phe)-OH (Bachem), Fmoc-3-NH-pyridinon-1-yl-CHZ-COOH
(Neosystem), Fmoc-3-NH-caprolactam-1-CHZ-COON (Neosystem), Fmoc-6-NH-5-
oxo-perhydropyrido[2,1-b]-(1,3)-thiazol-3-COOH (Neo-system), Fmoc-azetidine-
carboxylic acid (Neosystem).
The following amino acids were prepared according to the following
references:
Synthesis of Fmoc-(FZPmp(Et)2)-OH, ref. T.R. Burke Jr. et al., J. Org. Chem.
1993,
58, 1336 -1340; synthesis of Fmoc-(Tyr(Fmal(tBu)2)-OH), ref. T.R. Burke Jr. et
al.,
J. Med. Chem. 1996, 39, 1021-1027; synthesis of Fmoc-(O-carboxymethyl)-Tyr-OH
,
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ref. T.R. Burke Jr. et al., Tetrahedron 1998, 54,9981-9994; synthesis of Fmoc-
(2-
Oxn)-OH, ref. G.K. Walkup et al., J. Org. Chem. 1998, 63, 6727 - 6731 .
Furthermore, the following abbreviations are used herein:
EDCI = 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
HATU = O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HBTU = 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HOAt = 1-hydroxy-7-azabenzotriazole
TBTU = 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate
Fmoc = fluorenylmethoxycarbonyl
Rink amide AM = 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-
phenoxyacetamido-norleucyl aminomethyl.
The Rink amide AM is commercially available from Novabiochem.
The 2-Chlorotritylchloride resin was purchased from Novabiochem.
Analytical HPLC conditions
Method A-E:
Column: Vydac 300 Angstrom, C18, flow rate: 0.8 mL/min (~, = 214 nm)
Solvents: solvent A = 0.1 % trifluoroacetic acid/water, solvent B = 0.1
trifluoroacetic acid/acetonitrile
Gradients:
Method A: 30 to 90% Solvent B in 30 min.
Method B: 5 to 45% Solvent B in 40 min.
Method C: 5 to 65% Solvent B in 60 min.
Method D: 15 to 60% Solvent B in 30 min.
Method E: 40 to 90% Solvent B in 25 min.
Methods F-Q
Column: C18 (4 mm x 300 mm), flow rate: 0.5 mL/min with CH3CN/H20 (0.1
TFA)
Solvents: solvent A = 0.1 % trifluoroacetic acid/water, solvent B = 0.1
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trifluoroacetic acid/acetonitrile
Gradients:
Method F: 10% Solvent B for 5 min., from 10% to 90% in 20 min.
Method G: 50% Solvent B for 5 min., from 50% Solvent B to 90% in 20 min.
Method H: 10% Solvent B for 5 min., from 10% Solvent B to 100% in 12.5 min
Method I: 20% Solvent B for 5 min., from 20% Solvent B to 70% in 20 min.
Method J: 25% Solvent B for 5 min., from 25% Solvent B to 65% in 13 min,
from 65% to 100% in 4 min.
Method K: 15% Solvent B for S min., from 15% Solvent B to 80% in 20 min
Method L: 40% Solvent B to 100% in 20 min.
Method M: 10% Solvent B for 5 min., from 10% Solvent B to 90% in 25 min.
Method N: 30% Solvent B for 5 min., from 30% Solvent to 70% in 12.5 min.,
from 70% to 100% in 5 min.
Method O: 50% Solvent B to 90% in 20 min
Method P: 25% Solvent B for 7 min., from 25% Solvent B to 100% in 20 min.
Method Q: 25% Solvent B for 5 min., from 25% Solvent B to 100% in 15 min.
Method R: Column: Vydac (2.1 mm x 150 mm) 300 Angstrom C18, flow rate: 0.2
mL/min (~, = 214 nm)
Solvent: solvent A = 0.02% trifluoroacetic acid/0.08% formic acid/ water,
solvent B =
0.02% trifluoroacetic acid/0.08% formic acid/ acetonitrile
Method R: 5% Solvent B to 98% in 10 min.
Synthesis of 2-CH30-Naphtyl-1-CO-Smp-Glu-Glu-Nal-Glu-NH2, cdc1249
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03H
COOH COOH
O / O O
\ ~ ~~ NHz
\ \ O
OOH
/ /
/
The peptide sequence is built up stepwise from the C-terminus by coupling the
corresponding Fmoc-amino acid to the free amino group on the resin, then
removal of
the Fmoc-protecting group to liberate the amino group for the next coupling.
The final
capping is done with 2-methoxy-naphthyl-carboxylic acid.
Rink amide AM resin (223 mg resin, 0.1 mmol) was washed with
dimethylformamide (2 x 25 mL), then removal of the Fmoc protection group was
achieved with 20% piperidine in dimethylformamide (2 x 25 mL). All amino acids
(0.15 mmol, 1.5 eq) were coupled using the coupling reagent TBTU (48 mg, 0.15
mmol, 1.5 eq) and the base N,N-diisopropylethylamine (38 mg, 0.3 mmol, 3 eq)
in
dimethylformamide except Fmoc- Phe(4-CHZS03H)-OH and 2-methoxy-naphth-1-yl-
carboxylic acid which were coupled with HATU (57 mg, 0.15 mmol, 1.5 eq and 76
mg, 0.2 mmol, 2.0 eq respectively) and N,N-diisopropylethylamine (39 mg, 0.3
mmol, 3.0 eq, and 65 mg, 0.5 mmol, 5.0 eq respectively) in dimethylformamide
(25
mL) . Fmoc deprotections were done with 20% piperidine in dimethylformamide (2
x
25 mL). The completion of coupling and deprotection reactions was assessed by
Kaiser test (ninhydrin). After each step, the resin was washed with
dimethylformamide (2 x 25 mL), dichloromethane (3 x 25 mL) and methanol (2 x
25
mL) then dried in vacuo. Cleavage of the final peptide from the resin and
simultaneous deprotection of the side chains was achieved with trifluoroacetic
acid/water (95:5) at room temperature for two hours. Trifluoroacetic acid was
removed in vacuo, then the remaining residue was triturated with diethylether
(30
mL). The resulting solid was dried in vacuo.
HPLC purification was done on Waters Deltapack C1g reverse phase silica gel
using a 40 mm x 200 mm 300 Angstrom column.
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HPLC conditions: flow rate: 10 mL/min
Gradient: 10%-30% B in 57min then 30%-90% B in 200 min
A= 0.1 % trifluoroacetic acid/water
B= 0.1 % trifluoroacetic acid/acetonitrile
Yield: 25 mg (0.024 mmol) 2-CH30-1-naphthyl-1-CO-Smp-Glu-Glu-Nal-Glu-NHz
Analytical data
Rt 32.1 min (Method B)
MS (ESI): MH+ 1027
H1 NMR (d6-DMSO, 400MHz): 8 4.83 (m, 1H), 4.69 (m, 1H), 4.43 (m, 1H), 4.26
(m, 1 H), 4.19 (m, 1 H) (characteristic alpha-protons).
Synthesis of 2-Et0-1-naphthyl -1-CO-Smp-Glu-Glu-Bta-Glu-NH2, cdc 1659
sH
I ~ COOH COOH
O ~ O p
~~N ~~ NH2
~ ~f H ~
O
I / COOH
Rink amide AM resin (379 mg, 0.25 mmol) was washed with dimethylformamide (2 x
mL) then Fmoc deprotected with 20% piperidine in dimethylformamide (2 x 25
mL). The first four amino acids (1.0 mmol) from the C-terminus were coupled in
1-
methyl-2-pyrrolidinone using 0.45 M HBTU in dimethylformamide (2 g , 0.9 mmol)
20 and 2 M N,N-diisopropylethylamine in 1-methyl-2-pyrrolidinone ( 1.0 mL).
Fmoc-
Phe(4-CHZS03H)-OH (180 mg, 0.375 mmol) was then coupled with TBTU (120 mg,
0.375 mmol) and N,N-diisopropylethylamine (113 mg, 0.875 mmol) in
dimethylformamide (25 mL). The resin was split at this point and 0.1 mmol was
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coupled with 2-ethoxy-1-naphthoic acid (43 mg, 0.2 mmol) using HATU (76 mg,
0.2
mmol) and N,N-diisopropylethylamine (58 mg, 0.45 mmol) in dimethylformamide
(25 mL). Fmoc deprotections of each amino acid were done with 20% piperidine
in
dimethylformamide (2 x 25 mL). The completion of coupling and deprotection
reactions was assessed by Kaiser test (ninhydrin). The resin was washed with
dimethylformamide (2 x 25 mL), dichloromethane (3 x 25 mL) and methanol (2 x
25
mL) then dried in vacuo. The peptide was cleaved from the resin and the side
chains
were deprotected with trifluoroacetic acid/water (95:5) at room temperature
for
approx. 2 hours. The trifluoroacetic acid was removed in vacuo then the
remaining
residue was triturated with Et20 (30 mL). The resulting solid was dried in
vacuo.
HPLC purification was done on Waters Deltapack C18 reverse phase silica gel
using a
40 mm x 200 mm 300 Angstrom column.
Yield: 22 mg (0.021 mmol) 2-Et0-1-naphthyl-1-CO-Smp-Glu-Glu-Bta-Glu-NHZ
Analytical data
R~ 34.3 min (Method B)
MS (ESI): MH+ 1047
H' NMR (db-DMSO, 400MHz): b 4.87 (m, 1H), 4.70 (m, 1H), 4.41 (m, 1H), 4.28
(m, 1 H), 4.19 (m, 1 H), (characteristic alpha-protons), 4.11 (q, 2H, CHZ),
1.19 (t, 3H,
CH3 ).
Synthesis of 2-CH30-1-naphthyl-CO-Smp-Glu-Glu-Bta-Aad-NHtBu, cdc1671
00
\
H
I\ ~ O w
/ COOH
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A. 2-chlorotritylchloride resin loading.
Fmoc-Aad(tBu)-OH (377 mg, 0.847 mmol) was dissolved in dichloromethane (15
mL) under a nitrogen atmosphere. 2-Chlorotritylchloride resin (1.5 g, 1.43
mmol) was
added to this solution, followed by N,N-diisopropylethylamine (442 mg, 3.43
mmol).
The suspension was stirred under nitrogen at room temperature for 6 hours. The
reaction completion was checked by thin layer chromatography, monitoring the
consumption of amino acid. The resin was filtered and washed with a mixture of
dichloromethane / methanol / N,N-diisopropylethylamine (17:2:1, 3 x 25 mL),
dichloromethane (3 x 25 mL), and dimethylformamide (2 x 25 mL). The Fmoc
protecting group was removed with 20% piperidine in dimethylformamide (2 x 25
mL, monitored by Kaiser test). The resin was washed with dimethylformamide (2
x
25 mL), dichloromethane (3 x 25 mL) and methanol (2 x 25 mL), then dried in
vacuo.
Loading: approximately 0.6 mmol/g.
1 S B. Amino acid couplings
The Aad(tBu) loaded trityl resin (417 mg, 0.25 mmol) was suspended in 1-methyl-
2-
pyrrolidinone (4 mL). The amino acids Fmoc-Bta-OH, Fmoc-Glu(tBu)-OH and,
Fmoc-Glu(tBu)-OH (1.0 mmol each) were coupled using 0.45 M HBTU in
dimethylformamide (2 g , 0.9 mmol) and 2 M N,N-diisopropylethylamine in 1-
methyl-2-pyrrolidinone (1.0 mL). The resin was split at this point and 176 mg
of the
resin (0.083 mmol) was used for the coupling of Fmoc- Phe(4-CHZS03H)-OH (60
mg,
0.125 mmol) with TBTU (40 mg, 0.125 mmol) and N,N-diisopropylethylamine (43
mg, 0.332 mmol) as coupling reagents. The final capping of the peptide was
done
with 2-methoxy- 1-naphthoic acid (34 mg, 0.166 mmol) using HATU (63 mg, 0.166
mmol) and N,N-diisopropylethylamine (48 mg, 0.374 mmol). Fmoc deprotections
after each coupling step were done with 20% piperidine in dimethylformamide (2
x
25 mL). The completion of coupling and deprotection reactions was assessed by
Kaiser test (ninhydrin). The resin was washed with dimethylformamide (2x 25
mL),
dichloromethane (3x 25 mL), and methanol (2x 25 mL). Cleavage of the peptide
from the resin was achieved with dichloromethane/trifluoroacetic acid /acetic
acid
(8:1:1, 10 mL) at room temperature for one hour. The side-chain protected
pentapeptide acid was collected as a white solid (52 mg).
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C. C-terminal amide synthesis
The side chain protected pentapeptide acid 2-Et0-1-naphthoyl-Phe(4-CHZS03H)-
Glu(tBu)-Glu(tBu)-Bta-Glu-OH (52 mg, 0.042 mmol) was dissolved in
dichloromethane (3 mL) , then tert-butylamine (6.2 mg, 0.084 mmol), HOAt (5.7
mg,
0.042 mmol), EDCI (16 mg, 0.084 mmol), and N,N-diisopropylethylamine (22 mg,
0.168 mmol) were added. The reaction mixture was stirred at room temperature
for 18
hours. The reaction mixture was diluted with dichloromethane (150 mL) and
washed
with 1:1 water/brine (75 mL). The organic layer was dried over sodium sulfate,
concentrated in vacuo and the remaining solid was dried in vacuo. The tert.-
butyl
esters were deprotected using trifluoroacetic acid/ water (95:5) at room
temperature
for 1 hour. The reaction mixture was concentrated in vacuo and the remaining
residue
was triturated with diethylether (30 mL). The resulting solid was dried in
vacuo.
HPLC purification was done on Waters Deltapack C1g reverse phase silica gel
using a
40 mm x 200 mm 300 Angstrom column.
HPLC conditions: flow rate: l OmL/min
Gradient: 20%-40%B in 57min then 40%-100%B in 257min
A= 0.1 % trifluoroacetic acid/water
B= 0.1 % trifluoroacetic acid/acetonitrile
Yield: 13 mg (0.012 mmol) 2-CH30-1-naphthyl-CO-Smp-Glu-Glu-Bta-Aad-NHtBu
Analytical data
Rt 39 min (Method B)
MS(ESI): MH+ 1103
H1 NMR (d6-DMSO, 400MHz): b 4.82 (m, 1H), 4.71 (m, 1H), 4.45 (m, 1H), 4.30
(m, 1 H), 4.17 (m, 1 H) (characteristic alpha-protons), 1.24 (s, 9H, tBu).
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Synthesis of 2-Et0-1-naphthyl-CO-Smp-Nva-(3-Me)Pro-Bta-Aad-NHtBu, cdc 1747
S03H COOH
H O H
/ O ~ N
\ O O ~~N
~ - H O
N~N ~ S
\ 'N
H O
O
a) Synthesis of 6-(tert-butylamino)-5-(((9H 9-fluorenylmethoxy)carbonyl)amino)-
6-
oxohexanoate
Fmoc-L-a-aminoadipic acid-8-tert.-butylester (2 g, 4.55 mmol) was dissolved in
dichloromethane (80 ml), then tert.-butylamine (0.665 g, 9.1 mmol), 1-hydroxy-
7-
azabenzotriazole (0.619 g, 4.55 mmol), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (1.74 g, 9.1 mmol), and diisopropylethylamine
(2.05
g, 15.92 mmol) were added. The yellow solution was stirred at room temperature
for
21 hours. The reaction mixture was diluted with dichloromethane, then washed
with
saturated aqueous sodium bicarbonate, 5% aqueous citric acid and with a one to
one
mixture of water and brine. The organic layer was dried over sodium sulfate
and
concentrated under reduced pressure to give 2.07 g (4.19 mmol) tert-butyl 6-
(tert-
butylamino)-5- f [9H 9-fluorenylmethoxy)carbonyl]amino}-6-oxohexanoate.
MS(ESI): MH+= 495
Rt= 23.5 (Method E)
The crude tert-butyl 6-(tert-butylamino)-5- f [9H 9-fluorenylmethoxy)carbonyl]
amino}-6-oxohexanoate (2.07 g, 4.19 mmol) was dissolved in minimal
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dichloromethane, then diluted with 95% trifluoroacetic acid/ water (30 ml).
The
reaction mixture was stirred at room temperature for 1.5 hours then
concentrated
under reduced pressure. The remaining oil was triturated with diethylether and
filtered. The diethylether filtrate was concentrated under reduced pressure to
give
2.48 g of 6-(tert-butylamino)-5-(((9H 9-fluorenylmethoxy)carbonyl)amino)-6-
oxohexanoate.
MS(ESI): MH+ = 439
Rt = 14.6 (Method E)
b) Loading of 5-amino-6-(tert-butylamino)-6-oxohexanoic acid on chlorotrityl
chloride resin
6-(tert-butylamino)-5-(((9H 9-fluorenylmethoxy)carbonyl)amino)-6-
oxohexanoate (2.48 g, 5.66 mmol) was dissolved in dichloromethane (80 ml)
under a
nitrogen atmosphere then added chlortritylchloride resin (4.85 g, 7.23 mmol)
and
diisopropylethylamine (2.92 g, 22.64 mmol). The dark purple reaction mixture
was
stirred for 5 hours under a nitrogen atmosphere. The suspension was filtered,
then
washed the resin three times with a mixture of dichloromethane/ methanol/
diisopropylethylamine (17:2:1); three times with dichloromethane and twice
with
dimethylformamide. The resin was suspended in dimethylformamide (25 ml), then
treated with 20% piperidine in dimethylformamide the first time for 5 min, a
second
time for 20 min, followed by washing it five times with dimethylformamide.
Finally,
the resin was washed with three times with dichloromethane , twice with
methanol
and dried in vacuo to give 5.21 g of loaded resin.
c) 2-Et0-1-naphthyl-CO-Smp-Nva-(3-Me)Pro-Bta-Aad-NHtBu,
The peptide sequence was built up stepwise from the C-terminus by coupling
the corresponding Fmoc-amino acid to the free amino group on the resin, then
removal of the Fmoc-protecting group to liberate the amino group for the next
coupling. The final capping is done with 2-ethoxy-naphthyl-carboxylic acid.
The previously loaded trityl resin (187 mg resin, 0.15 mmol) was suspended in
dimethylformamide (25 ml). The four amino acids (in the order Fmoc-Bta-OH,
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Fmoc-(3-Me)Pro-OH, Fmoc-Nva-OH, Fmoc-Smp-OH) (0.225 mmol, 1.5 eq) were
coupled using the coupling reagent TBTU (72 mg, 0.225 mmol, 1.5 eq) and the
base
N,N-diisopropylethylamine (77 mg, 0.6 mmol, 4 eq) in dimethylformamide,
whereas
the final coupling with 2-ethoxy-naphth-1-yl-carboxylic acid was done with
HATU
(114 mg, 0.3 mmol, 2 eq) and N,N-diisopropylethylamine (87 mg, 0.675 mmol, 4.5
eq) in dimethylformamide (25 mL). Fmoc deprotections were done with 20%
piperidine in dimethylformamide (2 x 25 mL). The completion of coupling and
deprotection reactions were assessed by Kaiser test (ninhydrin). After each
step, the
resin was washed with dimethylformamide (2 x 25 mL), dichloromethane (3 x 25
mL)
and methanol (2 x 25 mL) then dried in vacuo. Cleavage of the final peptide
from the
resin was achieved with dichloromethane/trifluoroethanol/ acetic acid (8:1:1,
10 ml)
at room temperature for 45 min. The suspension was filtered, washing with
dichloromethane. The filtrate was concentrated under reduced pressure and the
remaining residue was triturated with diethylether. The resulting solid was
dried in
vacuo to give 2-Et0-naphth-1-yl-CO-Smp-Nva-(3-Me)Pro-Bta-Aad-NHtBu as a pale
yellow solid; ( 1 OS mg, 66%).
MS(ESI): MH+ = 1069
Rt = 15.8 (Method A)
The compounds presented in Tables 1-6 were obtained in a similar fashion as
the four pentapeptides described above. The column Cdc# gives the reference
number for the compounds. The columns Rl, A1, A2, A3, A4, A5, U, RZ, these are
the resiudes of the peptidyl compounds as defined for Formula I, above. The
tables
also provide mass spec data (MH+ ) and analytical data for the compounds
(retention
time and the HPLC method used).
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Table 1:
Cdc# R, A1 A2 A3 A4 A5 U MH+ HPLC-Meth.
Ret.
R~
(
1788 Ac Smp Nva Pro Bta GluNHtBu 885 9.3 A
1789 9-Anthracenyl-COSmp Nva Pro Bta GluNHtBu 104715.9 A
1790 Mesityl-502 Smp Nva Pro Bta GluNHtBu 102527.5 D
1797 2-Me-naphthyl-COSmp Nva Pro Bta GluNHtBu 101114.6 A
1814 2,6-diMe-Bzl-COSmp Nva Pro Bta GluNHtBu 975 13.4 A
1815 2,6-di(Me0)-Bzl-COSmp Nva Pro Bta GluNHtBu 100712.3 A
1799 Ac-Aib Smp Nva Pro Bta GluNHtBu 970 10.5 A
1823 9-Anthracenyl-COSmp Glut2-MeProBta AadNHtBu 116118.6 A
Bu)
1949 2,4-Di-OMe-5-Me-Ph-Smp Nva 2-MeProBta AadNHtBu 104913.8 A
CO
1948 2-Naphthyl-NH-COSmp Nva 2-MeProBta AadNHtBu 104016.6 A
1947 1-Naphthyl-NH-COSmp Nva 2-MeProBta AadNHtBu 104015.6 A
1946 Ph-CHZ-NH-CO- Smp Nva 2-MeProBta AadNHtBu 100414.1 A
1945 Ph-NH-CO Smp Nva 2-MeProBta AadNHtBu 990 14.4 A
1944 Pivaloyl Smp Nva 2-MeProBta AadNHtBu 955 13.6 A
1943 Adamantyl-CO Smp Nva 2-MeProBta AadNHtBu 103316.9 A
1939 1-Dibenzofuranyl-COSmp Nva 2-MeProBta AadNHtBu 106516.7 A
1938 Di-Phenyl-1-CO Smp Nva 2-MeProBta AadNHtBu 105115.7 A
1937 Naphthyl-1-CO Smp Nva 2-MeProBta AadNHtBu 102515 A
1936 4-Fluorenyl-CO Smp Nva 2-MeProBta AadNHtBu 106316.1 A
1935 1-Fluorenyl-CO Smp Nva 2-MeProBta AadNHtBu 106317.3 A
1934 3-Indolyl-CO Smp Nva 2-MeProBta AadNHtBu 101413.7 A
1933 1-Et0-Naphthyl-2-COSmp Nva 2-MeProBta AadNHtBu 105516.3 A
1907 2,3-Di-OMe-Ph-COSmp Nva 2-MeProBta AadNHtBu 103514.1 A
1906 4-Phenanthrenyl-CO-Smp Nva 2-MeProBta AadNHtBu 107516.4 A
2060 9-Fluorenyl-CO-Smp Nva 2-MeProBta AadNHtBu 106316.5 A
1972 5-NMez-Naphthyl-1-SOZSmp Nva 2-MeProBta AadNHtBu 110410.8 A
2284 3,5-diCl-Ph-CO Smp Nva Pro Nal AadNHtBu 102320 Q
2285 2,5-diCl-Ph-CO Smp Nva Pro Nal AadNHtBu 102318 Q
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Cdc# R, A A2 A3 A4 A5 U MH+ HPLC-Meth.
1 Ret.
R
2286 2-OH-5-Cl-Ph-COSmp Nva Pro Nal Aad NHtBu 100518 Q
2287 2-OH-3,5-diCl-Ph-COSmp Nva Pro Nal Aad NHtBu 103921 Q
2288 2,3-diCl-Ph-CO Smp Nva Pro Nal Aad NHtBu 102318 Q
2289 4-OH-3-Cl-Ph-COSmp Nva Pro Nal Aad NHtBu 100517 Q
2290 2,6-diOMe-5-Cl-Ph-COSmp Nva Pro Nal Aad NHtBu 104918 Q
2291 2,3,5-triCl-Ph-COSmp Nva Pro Nal Aad NHtBu 105918 Q
2292 2,3,6-triCl-Ph-COSmp Nva Pro Nal Aad NHtBu 105918 Q
2293 2-F-5-Cl-Ph-CO Smp Nva Pro Nal Aad NHtBu 100718 Q
2294 3-F-5-Cl-Ph-CO Smp Nva Pro Nal Aad NHtBu 100718 Q
2295 2-Br-5-Cl-Ph-COSmp Nva Pro Nal Aad NHtBu 106918 Q
2296 Ac Smp Nva Pro Nal Aad NHtBu 893 18 Q
2297 2-Furanyl-CO Smp Nva Pro Nal Aad NHtBu 945 17 Q
2298 2-Thiophenyl-COSmp Nva Pro Nal Aad NHtBu 961 16 Q
2299 N Me-pyrrolyl-2-COSmp Nva Pro Nal Aad NHtBu 958 17 Q
2300 3-pyridyl-CO Smp Nva Pro Nal Aad NHtBu 956 17 Q
2301 Ph-C C-CO Smp Nva Pro Nal Aad NHtBu 979 18 Q
2302 4-F-3-(E~- Smp Nva Pro Nal Aad NHtBu 109920 Q
(CHCHCOZtBu)-Ph-CO
2303 2-OMe-3-(E~- Smp Nva Pro Nal Aad NHtBu
(CHCHCOZtBu)-Ph-CO
2304 4-Me-3-(E~- Smp Nva Pro Nal Aad NHtBu 109520 Q
(CHCHCOZtBu)-Ph-CO
2305 5-OMe-3-(~- Smp Nva Pro Nal Aad NHtBu 111120 Q
(CHCHCOztBu)-Ph-CO
2306 2-Me-5-OMe-3-((E~-Smp Nva Pro Nal Aad NHtBu 112520 Q
(CHCHCOZtBu)-Ph-CO
2307 5-(E~-(CHCHCOztBu)-Smp Nva Pro Nal Aad NHtBu 112320 Q
2,3-dihydrobenzofuranyl-
7-CO
2308 3-Cyano-Ph-CO Smp Nva Pro Nal Aad NHtBu 980 17 Q
2309 3,5-di-tBu-Ph-COSmp Nva Pro Nal Aad NHtBu 106720 Q
2310 Cyclopropy-CO Smp Nva Pro Nal Aad NHtBu 919 17 Q
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Cdc# R, A A2 A3 A4 A5 U MH+ HPLC-Meth.
1 Ret.
R
2311 Cyclobutyl-CO Smp Nva Pro Nal Aad NHtBu 933 17 Q
2312 Benzocyclobutyl-COSmp Nva Pro Nal Aad NHtBu 981 18 Q
2313 Cyclopentyl-CO Smp Nva Pro Nal Aad NHtBu 947 17 Q
2314 Cyclohexyl-CO Smp Nva Pro Nal Aad NHtBu 961 18 Q
2315 Cycloheptyl-CO Smp Nva Pro Nal Aad NHtBu 975 18 Q
2316 2-Ethyl-butanoylSmp Nva Pro Nal Aad NHtBu 949 18 Q
2317 Pivaloyl Smp Nva Pro Nal Aad NHtBu 935 17 Q
2318 2-Tetrahydrofuranyl-COSmp Nva Pro Nal Aad NHtBu 949 16 Q
2319 4-N-Me-piperazin-1-yl-Smp Nva Pro Nal Aad NHtBu 976 17 Q
CO
2320 MeCOCHZCHzCO Smp Nva Pro Nal Aad NHtBu 949 16 Q
2321 HCONHCHZCO Smp Nva Pro Nal Aad NHtBu 936 16 Q
2322 MeCONHCH2C0 Smp Nva Pro Nal Aad NHtBu 950 16 Q
2323 MeZNCHzCO Smp Nva Pro Nal Aad NHtBu 936 17 Q
2324 MeOCHZCO Smp Nva Pro Nal Aad NHtBu 923 17 Q
2325 2-Pyrrolidinone-5-COSmp Nva Pro Nal Aad NHtBu 962 16 Q
2326 Bz10CH2C0 Smp Nva Pro Nal Aad NHtBu 999 18 Q
2327 (E~-Ph-CH=CH-COSmp Nva Pro Nal Aad NHtBu 981 18 Q
2328 (CycIoNHCONHCHZCH)Smp Nva Pro Nal Aad NHtBu 962 16 Q
-CO
2329 3-Furanyl-CO Smp Nva Pro Nal Aad NHtBu 945 17 Q
2330 3-Thiophenyl-COSmp Nva Pro Nal Aad NHtBu 961 17
2342 Ph-CHzOCO-Gly Smp Nva Pro Nal Aad NHtBu 101219 Q
2344 Ph-CHZOCO-Ala Smp Nva Pro Nal Aad NHtBu 102617
2346 Ph-CHZOCO-Val Smp Nva Pro Nal Aad NHtBu 105418 Q
2348 Ph-CHZOCO-Leu Smp Nva Pro Nal Aad NHtBu 106818 Q
2350 Ph-CHzOCO-Phg Smp Nva Pro Nal Aad NHtBu 108818
2351 Ph-CHZOCO-Tyr Smp Nva Pro Nal Aad NHtBu 111818 Q
2353 Ph-CHZOCO-Pro Smp Nva Pro Nal Aad NHtBu 105219 Q
2355 Ph-CHZOCO-D-AlaSmp Nva Pro Nal Aad NHtBu 102617 Q
2036 Piperonyloyl Smp Nva Pro Nal Aad NHtBu 999 5.2 R
2037 4-(~-(CH=CH-COZtBu)-Smp Nva Pro Nal Aad NHtBu 10815.8 R
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Cdc#R, A1 A2 A3 A4 A5 U MH+HPLC-Meth.
Ret.
R~
(
Ph-CO
2038(3-NOZ-Ph)-CO Smp Nva Pro Nal Aad NHtBu 10005.3 R
20393-Cl-Ph-CO Smp Nva Pro Nal Aad NHtBu 9895.3 R
20403-MeS02-Ph-CO Smp Nva Pro Nal Aad NHtBu 10335.1 R
20413-AcNH-Ph-CO Smp Nva Pro Nal Aad NHtBu 10125 R
20423-Me0-Ph-CO Smp Nva Pro Nal Aad NHtBu 9855.2 R
2043(3-CF30-Ph)-CO Smp Nva Pro Nal Aad NHtBu 10395.5 R
20443-NHCOEtPh Smp Nva Pro Nal Aad NHtBu 10265.1 R
20453,4-diCl-Ph-CO Smp Nva Pro Nal Aad NHtBu 10255.5 R
20463-(Ph-CO-)Ph-COSmp Nva Pro Nal Aad NHtBu 10595.4 R
20473,5-diBr-anisoylSmp Nva Pro Nal Aad NHtBu 11435.7 R
20483-(E)-(CH=CH-C02tBu)-Smp Nva Pro Nal Aad NHtBu 10815.7 R
Ph-CO
21785-Me-2-OH-Ph-COSmp Nva Pro Nal Aad NHtBu 98523 F
21795-I-2-OH-Ph-CO Smp Nva Pro Nal Aad NHtBu 109723 F
22004-(E~-(CH=CH-COZH)-Smp Nva Pro Nal Aad NHtBu 102521 F
Ph-CO
22013-(E~-(CH=CH-COZH)-Smp Nva Pro Nal Aad NHtBu 102522 F
Ph-CO
23884-F-3-(E~-(CH=CH-Smp Nva Pro Nal Aad NHtBu 10435.5 R
COZH)-Ph-CO
23892-OMe-3-(E~-(CH=CH-Smp Nva Pro Nal Aad NHtBu 10555.5 R
COZH)-Ph-CO
23904-Me-3-(E~-(CH=CH-Smp Nva Pro Nal Aad NHtBu 10395.5 R
COZH)-Ph-CO
23915-OMe-3-(~-(CH=CH-Smp Nva Pro Nal Aad NHtBu 10555.5 R
COZH)-Ph-CO
23925-OMe-2-Me-3-(E~-Smp Nva Pro Nal Aad NHtBu 10695.5 R
(CH=CH-COzH)-Ph-CO
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Table 2:
Cdc R1 A1 A2 A3 A4 AS U MH+ HPLC-Meth.
#
Ret.
R~
(min.)
17312-Et0- Smp Nva Pip Bta Aad NHtBu 1069 16.6 A
naphthyl-1-
CO
17472-Et0- Smp Nva 3-MeProBta Aad NHtBu 1069 15.8 A
naphthyl-1-
CO
17492-Et0- Smp Nva MePro Bta Aad NHtBu 1069 15.7 A
naphthyl-1-
CO
17612-Et0- Smp Nva Pro (NMe)Aad NHtBu 1069 15.7 A
naphthyl-1- -Bta
CO
17652-Et0- Smp Nva D,L-IscBta Aad NHtBu 1103 16.8 A
naphthyl-1-
CO
17722-Et0- Smp Nva Hyp(tBu)Bta Aad NHtBu 1127 17.7 A
naphthyl-1-
CO
17872-Et0- Smp Nva Hyp Bta Aad NHtBu 1071 13.4 A
naphthyl-1-
CO
17942-Et0- Smp Nva Oic Bta Aad NHtBu 1109 18.2 A
naphthyl-1-
CO
18082-Et0- Smp Nva 5-phenyl-Bta Aad NHtBu 1131 19.1 A
naphthyl-1- Pro
CO
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18162-Et0- Smp Nva Ac6 Bta Aad NHtBu 1083 19.5 A
naphthyl-1-
CO
18172-Et0- Smp Nva (N Bta Aad NHtBu 1043 15.2 A
naphthyl-1- Me)Ala
CO
17712-Et0- Smp Nva Pro Bta Aad NHtBu 1069 15.6 A
naphthyl-1-
CO
17462-Et0- Smp Nva ThioproBta Aad NHtBu 1073 15.6 A
naphthyl-1-
CO
17962-Et0- Smp Nva Ac5 Bta Glu NHtBu 1055 18.1 A
naphthyl-1-
CO
18072-Et0- Smp Nva Aib Bta Glu NHtBu 1029 16.1 A
naphthyl-1-
CO
18102-Et0- Smp Nva ThioproBta Glu NHtBu 1059 15.6 A
naphthyl-1-
CO
20592-Et0- Smp Nva Pro Bta MeGlu OH 999 12.6 A
naphthyl-1-
CO
19702-Et0- Smp Nva Pro Bta Aad NH- 1095 16.4 A
naphthyl-1- Cyclohexyl
CO
19412-Et0- Smp Nva Pro Bta Aad(tBu)NH- 1151 20.7 A
naphthyl-1- Cyclohexyl
CO
17212-Et0- Smp Nva Pro Bta Glu NHtBu 1041 15.1 A
naphthyl-1-
CO
19042-Et0- Smp Nva 2-MeProBta Aad NH-(2,4- 1111 18 A
naphthyl-1- diMe-pent-3-
CO y1)
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19031-EtO- SmpNva 2-MeProBta Aad NH-3a- 1121 17.9 A
naphthyl-2- (bicyclooct-
CO [3.3.0]-yl)
19012-Et0- SmpNva 2-MeProBta Aad NH-(2-Me-1083 16.6 A
naphthyl-1- but-2-yl)
CO
18982-Et0- SmpNva 2-MeProBta Aad N 1067 14.3 A
naphthyl-1- Pyrrolidinyl
CO
18942-Et0- SmpNva 2-MeProBta Aad(tBu)OH 1070 17.9 A
naphthyl-1-
CO
18122-Et0- SmpNva 2-MeProBta Aad(N NHtBu 1122 16.9 A
naphthyl-1- pyrrolidinyl)
CO
19052-Et0- SmpNva Ac5 Bta Aad(NH- NHtBu 1126 19.2 A
naphthyl-1- OtBu)
CO
19402-Et0- SmpNva Ac5 Bta Aad(NHOH)NHtBu 1070 17.2 A
naphthyl-1-
CO
2275PhthalimidSmpNva 2-MeProBta Aad NHtBu 1001 13.7 A
1243Ac SmpGlu Glu Nal Glu NHS 885 23.7 B
1517Indolyl-2-SmpGlu Glu Bta Glu NHz 992 32.6 B
CO-
1660BenzothiophSmpGlu Glu Bta Glu NHz 1009 35.5 B
enyl-2-CO
16613,5-Di-tBu-SmpGlu Glu Bta Glu NHZ 1065 46 C
Benzoyl
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16702-Me- Smp Glu Glu Bta Glu NHZ 1017 35.1 B
naphthyl-1-
CO
17632-Et0- Smp Glu Pro Bta Aad NHtBu 1085 12.2 A
naphthyl-1-
CO
13032-Me0- Smp Glu Asp Nal Glu NHZ 1013 31.8 B
naphthyl-1-
CO
13732-Me0- Smp Glu Gln Nal Glu NHz 1026 30.6 B
naphthyl-1-
CO
13762-Me0- Smp Glu Glu Nal Glu NHz 1026 31.6 B
naphthyl-1-
CO
14162-Me0- Smp Glu Glu Nal. Gln NHz 1026 31 B
naphthyl-1-
CO
14192-Me0- Smp Glu Glu Bta Glu NHZ 1033 31.7 B
naphthyl-1-
CO
15162-Me0- Smp Glu Glu Bta Glu OH 1034 32.3 B
naphthyl-1-
CO
15212-Me0- Smp Glu Glu Bta Glu NHtBu 1089 37.5 B
naphthyl-1-
CO
15222-Me0- Smp Glu Glu Bta Glu NH-5- 1149 43.1 C
naphthyl-1- Indanyl
CO
15652-Me0- Smp Glu Glu Bta Glu NH- 1167 44 C
naphthyl-1- Adamantyl
CO
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16592-Et0- Smp Glu Glu Bta Glu NH2 1047 34.3 B
naphthyl-1-
CO
17202-Et0- Smp Glu D-Glu Bta Glu NHtBu 1103 37.7 B
naphthyl-1-
CO
18992-Et0- Smp Glu 2-MeProBta Asu NH-tBu 1183 18.2 A
naphthyl-1-
CO
16792-Et0- Smp Glu(tBPro Bta Glu NHtBu 1127 18.7 A
naphthyl-1- u)
CO
17192-Et0- Smp Glu(tBPro Bta Aad NHtBu 1141 17.2 A
naphthyl-1- u)
CO
18202-Et0- Smp Glu(tBAc5 Bta Aad NHtBu 1155 20 A
naphthyl-1- u)
CO
18222-Et0- Smp Glu(tB2-MeProBta Aad NHtBu 1155 17.6 A
naphthyl-1- u)
CO
19022-Et0- Smp Glu(tB4,4- Bta Aad NHtBu 1169 18.7 A
naphthyl-1- u) diMePro
CO
18932-Et0- Smp Glu(tBIva Bta Aad NHtBu 1143 19.1 A
naphthyl-1- u)
CO
18922-Et0- Smp Glu(tBIva Bta Aad NHtBu 1143 18.9 A
naphthyl-1- u)
CO
18912-Et0- Smp Glu(tBAib Bta Aad NHtBu 1129 18 A
naphthyl-1- u)
CO -
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18372-Et0- SmpGlu(tBAc3 Bta Aad NHtBu 1127 16.5 A
naphthyl-1- u)
CO
18362-Et0- SmpGlu(tBDehydro-Bta Aad NHtBu 1141 17.8 A
naphthyl-1- u) val
CO
22372-Et0- SmpGlu(tB2-MeProAla Aad NH-3a- 1075 15 A
naphthyl-1- u) (bicyclooct-
CO [3.3.0]-yl)
22062-Et0- SmpGlu(tB2-MeProNal Aad NH-3a- 1201 20.1 A
naphthyl-1- u) (bicyclooct-
'
CO [3.3.0]-yl)
21672-Et0- SmpGlu(tB2-MeProBta Aad NH-3a- 1207 19.8 A
naphthyl-1- u) (bicyclooct-
CO [3.3.0]-yl)
18192-Et0- SmpMeGluPro Bta Glu NHtBu 1141 17.3 A
naphthyl-1- (tBu)
CO
1897Ac (N Glu(tBAib Bta Aad NHtBu 987 13.2 A
Me)Su)
mp
18962-Et0- MeSGlu(tBAib Bta Aad NHtBu 1143 17.4,17.A
naphthyl-1-mp u) 7
CO
18952-Et0- MeSGlu(tBMeAib Bta Aad NHtBu 1143 17.3 A
naphthyl-1-mp u)
CO
21681-Fluorenyl-SmpGlu(tB2-MeProBta Aad NH-3a- 1201 20.2 A
CO u) (bicyclooct-
[3.3.0]-yl)
22191-Fluorenyl-SmpGlu(tB2-MeProBta Aad(Me) NH-3a- 1215 21.9 A
CO u) (bicyclooct-
[3.3.0]-yl)
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15642-Me0- Smp Glu(tBGlu(tBu)Bta Glu(tBu) OH 1202 14.g E
naphthyl-1- u)
CO
16722-Et0- Smp Glu(tBGlu(tBu)Bta Glu OtBu 1216 17.6 E
naphthyl-1- u)
CO
16732-Et0- Smp Glu(tBGlu(tBu)Bta Glu NHtBu 1215 16.8 E
naphthyl-1- u)
CO
17302-Et0- Smp Val Pro Bta Aad NHtBu 1055 15 A
naphthyl-1-
CO
17432-Et0- Smp Leu Pro Bta Aad NHtBu 1069 16.1 A
naphthyl-1-
CO
17482-Et0- Smp Nle Pro Bta Aad NHtBu 1069 16 A
naphthyl-1-
CO
17592-Et0- Smp Pro Pro Bta Aad NHtBu 1053 13.3 A
naphthyl-1-
CO
17642-Et0- Smp D-ProPro Bta Aad NHtBu 1053 14.7 A
naphthyl-1-
CO
17622-Et0- Smp Pro D-Pro Bta Aad NHtBu 1053 19.9 A
naphthyl-1-
CO
17232-Et0- Smp Phe Pro Bta Glu NHtBu 1089 16.3 A
naphthyl-1-
CO
17922-Et0- Smp Ac6 Pro Bta Glu NHtBu 1067 14.4 A
naphthyl-1-
CO
22182-Et0- Smp Lys 2-MeProNal Aad NH-3a- 1144 16.6 A
naphthyl-1- (bicyclooct-
CO [3.3.0]-yl)
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22082-Et0- SmpLys(B2-MeProNal Aad NH-3a- 1245 19.4 A
naphthyl-1- oc) (bicyclooct-
CO [3.3.0]-yl)
18132-Et0- SmpAib Aib Bta Aad NHtBu 1029 14.2 A
naphthyl-1-
CO
18112-Et0- SmpAib Nva Bta Aad NHtBu 1043 15.7 A
naphthyl-1-
CO
16782-Et0- SmpAla Ala Bta Glu NHtBu 987 26.4 D
naphthyl-1-
CO
17602-Et0- Smp2- Nva Bta Aad NHtBu 1069 14.8 A
naphthyl-1- MePro
CO
23622-Et0- SmpNva Pro Bta Aad NH-(2,4- 1097 19 L
naphthyl-1- diMe-pent-3-
Co y1)
18432-Me0- SmpPro Gly Nal Glu NHz 923 A
naphthyl-1-
CO
18442-Me0- SmpPro Ala Nal Glu NHZ 937 A
naphthyl-1-
CO
18452-Me0- SmpAla Pro Nal Glu NHZ 937 A
naphthyl-1-
CO
18462-Et0- SmpAsp(tPro Nal Aad NHtBu 1121 17 A
naphthyl-1- Bu)
CO
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18472-Et0- Smp Aad(tPro Nal Aad NHtBu 1149 16 A
naphthyl-1- Bu)
CO
18482-Et0- Smp Glu(cyPro Nal Aad NHtBu 1161 19 A
naphthyl-1- clohex
CO y1)
18492-Et0- Smp Cys(MPro Nal Aad NHtBu 1067 15 A
naphthyl-1- e)
CO
18502-Et0- Smp Met Pro Nal Aad NHtBu 1081 15 A
naphthyl-1-
CO
18512-Et0- Smp Pra Pro Nal Aad NHtBu 1045 14 A
naphthyl-1-
CO
18522-Et0- Smp 3- Pro Nal Aad NHtBu 1035 14 A
naphthyl-1- MeAla
CO
18532-Et0- Smp Ser(MPro Nal Aad NHtBu 1051 14 A
naphthyl-1- e)
CO
18542-Et0- Smp hLeuPro Nal Aad NHtBu 1077 17 A
naphthyl-1-
CO
18552-Et0- Smp Nva Pro Nal Aad NHtBu 1049 15 A
naphthyl-1-
CO
18562-Et0- Smp Nva azetane-Nal Aad NHtBu 1035 15 A
naphthyl-1- CO
CO
18572-Et0- Smp Glu(tBazetane-Nal Aad NHtBu 1121 17 A
naphthyl-1- u) CO
CO
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18582-Et0- Smp Aad(tazetane-Nal Aad NHtBu 1135 18 A
naphthyl-1- Bu) CO
CO
18592-Et0- Smp Nva 3,4- Nal Aad NHtBu 1047 A
naphthyl-1- dehydro-
CO Pro
18602-Et0- Smp Glu(tB3,4- Nal Aad NHtBu 1133 17 A
naphthyl-I- u) dehydro-
CO Pro
18612-Et0- Smp Aad(t3,4- Nal Aad NHtBu 1147 17 A
naphthyl-1- Bu) dehydro-
CO Pro
17332-Me0- Smp Gly Pro Nal Glu NHZ 923 10.6 R
naphthyl-1-
CO
17342-Me0- Smp Glu Pro Nal Glu NHz 995 10.1 R
naphthyl-1-
CO
17352-Me0- Smp D-GluPro Nal Glu NHZ 995 10.6 R
naphthyl-1-
CO
18852-Et0- Smp Glu Glu 1-NalGlu OH 1042 23 F
naphthyl-1-
CO
18882-Et0- Smp Nva Pro Bta Aad NHz 999
naphthyl-
I -
CO
1890Ac Smp Nva Pro Bta Aad NHZ 843
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Table 3:
CdcR, A 1 A2 A3 A4 AS U MH+ HPLC
Meth.
# -Ret.
R,
(min.)
21722-Et0- Pmp(Et)Z Nva 2-MeProBta Aad NHtBu 112522.5A
naphthyl-
1-CO
19002-Me0- D,L-SmphgGlu Glu Bta Glu NHz 103331.9,B
naphthyl- 31.2
1-CO
10352-Me0- Tyr(mal) Glu Glu Phe Glu NHZ 100131.1B
'
naphthyl-
1-CO
1040Ac Tyr(mal) Glu Glu Nal Glu NHZ 909 26.5B
1041Ac Tyr(Fmal)Glu Glu Nal Glu NHZ 927 25.7B
13152-Me0- Tyr(O- Glu Glu Nal Glu NHZ 104333.3B
naphthyl-CHZP03H2)
1-CO
13742-Me0- FzPmp Glu Glu Nal Glu NHZ 106333.1B
naphthyl-
1-CO
13752-Me0- D-FZPmp Glu Glu Nal Glu NHz 106334 B
naphthyl-
1-CO
14202-Me0- 4- Glu Glu Nal Glu NHz 102638 B
naphthyl-(NHSOZMe)-
1-CO Phe
1025Ac Tyr(mal) Glu Glu Phe Glu NHZ 859 20.7B
1244Ac 4-SO3H-PheGlu Glu Nal Glu NHZ 871 22.7B
12452-Me0- 4-S03H-PheGlu Glu Phe Glu NHZ 101330.5B
naphthyl-
1-CO
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17982-Et0- D,L-SmphgNva Pro Nal Glu NHtBu 102714.7,A
naphthyl- 15.0
1-CO
18212-Et0- D,L-SmphgNva Ac5 Bta Aad NHtBu 105517.7.A
naphthyl- 18.5
1-CO
23942-Et0- Pmp Nva Pro Bta Aad 3-NH-2,4-109514 L
Naphthyl- diMe-
1-CO pentyl
23952-Et0- 4-(P03H2)-PheNva Pro Bta Aad 3-NH-2,4-1083
Naphthyl- diMe-
1-CO pentyl
23962-Et0- Tyr(Fmal)Nva Pro Bta Aad 3-NH-2,4-114016 L
Naphthyl- diMe-
1-CO pentyl
23972-Et0- 4-S03H-PheNva Pro Bta Aad 3-NH-2,4-108112 L
Naphthyl- diMe-
1-CO pentyl
24162-Et0- Tyr(O- Nva Pro Bta Aad 3-NH-2,4-1113
Naphthyl-CHZP03HZ) diMe-
1-CO pentyl
24202-Et0- 4- Nva Pro Bta Aad 3-NH-2,4-1075
Naphthyl-(CHZCOzMe)- diMe-
1-CO Phe pentyl
24392-Et0- 4-(CHzCO2H)-Nva Pro Bta Aad 3-NH-2,4-1061
Naphthyl-Phe diMe-
1-CO pentyl
23582-Et0- Pmp(Et)2 Nva Pro Bta Aad 3-NH-2,4-115319 L
Naphthyl- diMe-
1-CO pentyl
23592-Et0- Tyr(mal) Nva Pro Bta Aad 3-NH-2,4-112116 L
Naphthyl- diMe-
1-CO pentyl
23602-Et0- 4-COOH-PheNva Pro Bta Aad 3-NH-2,4-104717 L
Naphthyl- diMe-
1-CO pentyl
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23612-Et0- 4- Nva Pro Bta Aad3-NH-2,4-111819 L
Naphthyl-(NHCOCOZEt diMe-
1-CO )-Phe pentyl
23632-Et0- 4-(PO(OEt)Z)-Nva Pro Bta Aad3-NH-2,4-113919 L
Naphthyl-Phe diMe-
1-CO pentyl
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Table 4:
Cdc#R, A1 A2 A3 A4 AS U MH+HPLC-Meth.
Ret.
(min.)
24352-Et0- 3-(CHZ-NH-CHZ-2-Nva Pro Bta Aad 3-NH-2,4-12107.3 R
Naphthyl-tetrahydrofuranyl)- diMe-pentyl
1-CO Smp
24362-Et0- 3-(CHZ-N(Ac)-CHZ-Nva Pro Bta Aad 3-NH-2,4-12526.9 R
Naphthyl-2-tetrahydrofuranyl)- diMe-pentyl
1-CO Smp
24372-Et0- 3-(CHZ-NH-CHz-Nva Pro Bta Aad 3-NH-2,4-11976.8 R
Naphthyl-CHZ-NMe2)-Smp diMe-pentyl
1-CO
24382-Et0- 3-(CH2-N(Ac)-CHz-Nva Pro Bta Aad 3-NH-2,4-12396.8 R
Naphthyl-CHz-NMe2)-Smp diMe-pentyl
1-CO
24522-Et0- 2-(CHZ-N(Ac)-CHZ-Nva Pro Bta Aad 3-NH-2,4-1272
Naphthyl-CHZ-Ph)-Smp diMe-pentyl
1-CO
24532-Et0- 3-(CHZ-NH- Nva Pro Bta Aad 3-NH-2,4-11847 R
Naphthyl-(CHZ)30H)-Smp diMe-pentyl
1-CO
24542-Et0- 3-(CHZ-N(Ac)-Nva Pro Bta Aad 3-NH-2,4-12266.7 R
Naphthyl-(CHZ)30H)-Smp diMe-pentyl
1-CO
24552-Et0- 3-(CHZ-NH-(CHz)3-Nva Pro Bta Aad 3-NH-2,4-12517.1 R
Naphthyl-N pyrrolidinonyl)- diMe-pentyl
1-CO Smp
24562-Et0- 3-(CHz-N(Ac)-Nva Pro Bta Aad 3-NH-2,4-12936.7 R
Naphthyl-(CHz)3-N- diMe-pentyl
1-CO pyrrolidinonyl)-Smp
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24792-Et0- 2-(E~-(CH=CH-Nva Pro Bta Aad 3-NH-2,4-1167 20 N
Naphthyl-COOH)-Smp diMe-pentyl
1-CO
24802-Et0- 2-Br-Smp Nva Pro Bta Aad 3-NH-2,4-1177 24 N
Naphthyl- diMe-pentyl
1-CO
24082-Et0- 3-CHO-Smp Nva Pro Bta Aad 3-NH-2,4-1125
Naphthyl- diMe-pentyl
1-CO
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Table 5:
CdcR, A A2 A3 A4 AS U MH+ HPLC- Meth.
1
# Ret.
R~
(min.)
22802-Et0-Naphthyl-Smp Glu Glu Phe Glu OH 992 16 H
1-CO
22432-Et0-Naphthyl-Smp Glu Glu 3-Cl-Phe Glu OH 102617 H
1-CO
22442-Et0-Naphthyl-Smp Glu Glu 2-Cl-Phe Glu OH 102617 H
1-CO
22452-Et0-Naphthyl-Smp Glu Glu 4-CI-Phe Glu OH 102617 H
1-CO
22462-Et0-Naphthyl-Smp Glu Glu 4-COOH-PheGlu OH 103616 H
1-CO
22472-Et0-Naphthyl-Smp Glu Glu 4-CONHZ-PheGlu OH 103516 H
1-CO
22482-Et0-Naphthyl-Smp Glu Glu 4-NHCOMe-Glu OH 104916 H
1-CO Phe
22492-Et0-Naphthyl-Smp Glu Glu 4-NHz-PheGlu OH 100715 H
1-CO
24342-Et0-Naphthyl-Smp Glu Glu 3-NHZ-PheGlu OH 10075.4 R
1-CO
22632-Et0-Naphthyl-Smp Glu Glu 4-NHCOzEt-Glu OH 108016 H
1-CO Phe
22642-Et0-Naphthyl-Smp Glu Glu 4-NHCOPh--Glu OH 111117 H
1-CO Phe
22702-Et0-Naphthyl-Smp Glu Glu 3-NHAc-PheGlu OH No 16 H
1-CO MS
22712-Et0-Naphthyl-Smp Glu Glu 3-NHCOPh--Glu OH 111017 H
1-CO Phe
22812-Et0-Naphthyl-Smp Glu Glu 4-CF3-PheGlu OH 106017 H
1-CO
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22822-Et0-Naphthyl-Smp Glu Glu 4-Ph-Phe Glu OH 106817 H
1-CO
22832-Et0-Naphthyl-Smp Glu Glu 3-NOZ-PheGlu OH 103716 H
1-CO
23352-Et0-Naphthyl-Smp Nva Pro 4-CF3-PheAad 3-NH-2,4-110923 O
1-CO diMepentyl
23362-Et0-Naphthyl-Smp Nva Pro 4-NHAc-PheAad 3-NH-2,4-109830 P
1-CO diMepentyl
23372-Et0-Naphthyl-Smp Nva Pro 4-COOH-PheAad 3-NH-2,4-108529 P
1-CO diMepentyl
23382-Et0-Naphthyl-Smp Nva Pro 4-Ph-Phe Aad 3-NH-2,4-111714 O
1-CO diMepentyl
23642-Et0-Naphthyl-Smp Glu Glu 3-NHCOzEt-Glu OH 10795.1 R
1-CO Phe
23682-Et0-Naphthyl-Smp Nva Pro 4-NHCOPh-Aad 3-NH-2,4-116027 P
1-CO Phe diMepentyl
23692-Et0-Naphthyl-Smp Nva Pro 2-Cl-Phe Aad 3-NH-2,4-107528 P
1-CO diMepentyl
23702-Et0-Naphthyl-Smp Nva Pro 3-Cl-Phe Aad 3-NH-2,4-107528 P
1-CO diMepentyl
23712-Et0-Naphthyl-Smp Nva Pro 4-CONHz-PheAad 3-NH-2,4-108426 P
1-CO diMepentyl
23722-Et0-Naphthyl-Smp Nva Pro Phe Aad 3-NH-2,4-104120 L
1-CO diMepentyl
23732-Et0-Naphthyl-Smp Nva Pro 4-NHCOzEt-Aad 3-NH-2,4-112816 L
1-CO Phe diMepentyl
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Table 6:
CdcR, A A2 A3 A4 A5 U MH+ HPLC-
1 Meth.
Ret.
~ (gin.)
19502-Et0- Smp Nva Pro(7-O)-Nal Aad NHtBu 106513.7 A
naphthyl-1-CO
18752-Et0- Smp Glu Glu7-(OiBu)-1-NalGlu NHZ 111419 J
Naphthyl-1-CO
18762-Et0- Smp Glu Glu7-(OCHzCH2-NGlu NHZ 11696.6 R
Naphthyl-1-CO piperazinyl)-1-Nal
18782-Et0- Smp Glu Glu7-(OBzI)-1-NalGlu NHZ 1147
Naphthyl-1-CO
19232-Et0- Smp Glu Glu7-Me0-1-Nal Glu OH 107224.5 F
Naphthyl-1-CO
19242-Et0- Smp Glu Glu7-(CHZ-C6H")-1-Glu OH 115427 F
Naphthyl-1-CO Nal
19252-Et0- Smp Glu Glu7-(OCHZCH(OH)-Glu OH 113223 F
Naphthyl-1-CO CHzOH)-1-Nal
19262-Et0- Smp Glu Glu7-OPr-1-Nal Glu OH 110029 F
Naphthyl-1-CO
19272-Et0- Smp Glu Glu7-(OCHZCHZ-1-Glu OH 116924 I
Naphthyl-1-CO piperazinyl)-1-Nal
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19582-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z-NHz)-1-NalGlu OH 110122 F
1-CO
19592-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)3-NHz)-1-NalGlu OH 111523 F
1-CO
19602-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z-C6H")-1-NalGlu OH 116827 F
1-CO
19612-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)3-C6H")-1-NalGlu OH 118227 F
1-CO
19622-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)4-C6H")-1-NalGlu OH 119628 F
1-CO
19632-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)zOH)-1-NalGlu OH 110223 K
1-CO
19992-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z-CH(OH)-Glu OH 114724 F
1-CO CHZOH)-1-Nal
20002-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)zN- Glu OH 117124 F
1-CO morpholinyl)-1-Nal
20012-Et0-Naphthyl-Smp Glu Glu 7-(O-CSH9)-1-NalGlu OH 112626 F
1-CO
20022-Et0-Naphthyl-Smp Glu Glu 7-OiPr-1-Nal Glu OH 110026 F
1-CO
20092-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z-4-pyridyl)-1-Glu OH 116323 F
1-CO Nal
20102-Et0-Naphthyl-Smp Glu Glu 7-(OCHz-COOH)-1-NalGlu OH 111723 F
1-CO
20112-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)3COOH)-1-NalGlu OH 114524 F
1-CO
20122-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z-(4-HO-)Ph)-Glu OH 117824 F
1-CO 1-Nal
20132-Et0-Naphthyl-Smp Glu Glu 7-O(CHz)z-(4-NHz)Ph)-1-Glu OH 117825 F
1-CO Nal
20142-Et0-Naphthyl-Smp Glu Glu 7-OH-1-Nal Glu OH 115919 F
1-CO
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20262-Et0-Naphthyl-Smp Glu Glu 6-OH-1-Nal Glu OH 105616 H
1-CO
20272-Et0-Naphthyl-Smp Glu Glu 7-(O(CHZ)Z-2-thienyl)-Glu OH 116627 F
1-CO 1-Nal
20282-Et0-Naphthyl-Smp Glu Glu 7-(O(CHZ)3COMe)-1-Glu OH 114024 F
1-CO Nal
20492-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)30H)-1-NalGlu OH 111420 F
1-CO
21592-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z-(2-Me-Glu OH 118126 F
1-CO thiazolyl))-1-Nal
21602-Et0-Naphthyl-Smp Glu Glu 7-(O(CHZ)2-2-pyridyl)-Glu OH 116320 M
1-CO 1-Nal
21822-Et0-Naphthyl-Smp Glu Glu 6-(O(CHZ)3-C6H")-1-Glu OH 118220 H
1-CO Nal
21862-Et0-Naphthyl-Smp Glu Glu 7-O(CHZ)3- Glu OH 115721 M
1-CO NHC(NH)NHZ)-1-Nal
21872-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z- Glu OH 114321 M
1-CO NHC(NH)NHz)-1-Nal
21882-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z-4- Glu OH 117023 M
1-CO tetrahydropyranyl)-1-
Nal
21892-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)z-2- Glu OH 115623 M
1-CO tetrahydofuranyl)-1-
Nal
21902-Et0-Naphthyl-Smp Glu Glu 7-(O(CHZ)Z-(4-Glu OH 120623 M
1-CO COOH)Ph)-1-Nal
21912-Et0-Naphthyl-Smp Glu Glu 7-(O(CHz)Z-4- Glu OH 116921 M
1-CO piperazinyl)-1-Nal
21922-Et0-Naphthyl-Smp Glu Glu 7-(OCH(Me)COOH)-Glu OH 113021 M
1-CO 1-Nal
21932-Et0-Naphthyl-Smp Glu Glu 7-(O(CHZ)2-N Glu OH 116921 M
1-CO pyrrolidinyl)-1-Nal
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21942-Et0- Smp Glu Glu 6-(O(CHz)z-NGlu OH 116917 H
Naphthyl-1-CO pyrrolidinonyl)-
1-Nal
21952-Et0- Smp Glu Glu 6- Glu OH 111516 H
Naphthyl-1-CO (O(CHz)3NHz)-
1-Nal
21972-Et0- Smp Glu Glu 6-(O(CHz)z-4-Glu OH 116316 H
Naphthyl-1-CO pyridyl)-1-Nal
21982-Et0- Smp Glu Glu 6-(O(CHz)z-4-Glu OH 117018 H
Naphthyl-1-CO tetrahydropyrany
1)-1-Nal
21992-Et0- Smp Glu Glu 6- Glu OH 114616 H
Naphthyl-1-CO (O(CHz)zCH(O
H)CHzOH)-1-
Nal
22252-Et0- Smp Glu Glu 7-(CHCH-Ph)-1-Glu OH 1144
Naphthyl-1-CO Nal
23392-Et0- Smp Glu Glu 7-(CHz)z- Glu OH 11143.6R
Naphthyl-1-CO COOH)-1-Nal
23402-Et0- Smp Nva Pro 2-Oxn Aad 3-NH-2,4-diMe-11086.4R
Naphthyl-1-CO pentyl
22042-Et0- Smp Glu(tB2- (alpha- Aad NH-3a- 120521.8A
naphthyl-1-CO u) MeProMe(tetrahydro- (bicyclooct[3.3.0]-
Nal)) y1)
21742-Et0- Smp Glu(tB2- (beta-Me)NalAad NH-3a- 121521.2A
naphthyl-1-CO u) MePro (bicyclooct[3.3.0]-
yl)
21692-Et0- Smp Glu(tB2- (tetrahydro-Nal)Aad NH-3a- 120521.7A
naphthyl-1-CO u) MePro (bicyclooct[3.3.0]-
yl)
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Table 7:
cdc R, A1 A2 A4 AS U M'=HPLC-Ret.Meth.
#
17932-Et0-naphthyl-Smp 3-NH-pyridinon-1-yl-BtaGlu NHtBu 99513.75 A
1-CO CHZ-CO
17852-Et0-naphthyl-Smp 3-NH-caprolactam-1-BtaGlu NHtBu 101313.4 A
1-CO CHZ-CO
17862-Et0-naphthyl-Smp 6-NH-5-oxo- BtaGlu NHtBu 104315.23 A
1-CO perhydropyrido[2,1-b]-
( 1,3)-thiazolyl-3-CO
17952-Et0-naphthyl-Smp 3-NH-Ph-CO BtaAad NHtBu 97814.92 A
1-CO
16802-Et0-naphthyl-Smp NH-(CHZ)4-CO BtaGlu NHtBu 94415.92 A
1-CO
Synthesis of 2-Et0-1-naphthyl-CO-Smp-Nva-Pro-Bta-NH-CH2-(3-HOOC-CHZ)-Ph,
cdc2276
S03H
_I O ~ O
I \ O \ O
\ N ~N ~ ~ ~ COOH
H ~
/ O ~ S i
The tetrapeptide acid sequence is built up stepwise from the C-terminus as
described
previously (see example 3) by coupling the corresponding Fmoc-amino acid (Fmoc-
Bta-OH) to the chlortrityl chloride resin, then removal of the Fmoc-protecting
group
to liberate the amino group for the next coupling. The final capping is done
with 2-
ethoxy-naphthyl-carboxylic acid. The tetrapeptide is cleaved from the resin in
a
similar fashion as described in example 3.
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a) 2-Et0-1-naphthyl-CO-Smp-Nva-Pro-Bta-NH-CHZ-(3-Me00C-CHZ)-Ph
The tetrapeptide acid (50 mg, 0.058 mmol) was dissolved in dichloromethane (5
ml) then methyl 2-[3-(aminomethyl)phenyl]acetate hydrochloride (23 mg, 0.116
mmol), 1-hydroxy-7-azabenzotriazole (8 mg, 0.058 mmol), 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (22 mg, 0.116 mmol),
and
diisopropylethylamine (49 mg, 0.377 mmol) were added. The reaction mixture was
stirred at room temperature for 18 hours. The reaction mixture was diluted
with
dichloromethane and washed with 5% aqueous citric acid (lx). The organic layer
was
dried over sodium sulfate and concentrated under reduced pressure. The
remaining
residue was redissolved in dichloromethane. Heptane was then added until a
precipitate formed. The mixture was concentrated in vacuo and the remaining
solid
dried in vacuo to give the product as a white solid 60 mg of 2-Et0-1-naphthyl-
CO-
Smp-Nva-Pro-Bta-NH-CHz-(3-Me00C-CHZ)-Ph).
MS (ESI): MH+= 1018.0
Rt = 16.49 (Method A)
a) 2-Et0-1-naphthyl-CO-Smp-Nva-Pro-Bta-NH-CHZ-(3-HOOC-CHz)-Ph
The crude tetrapeptide ester (60 mg, 0.06 mmol) was dissolved in
tetrahydrofuran
(5 ml) and water (1 ml), then 1N aqueous lithium hydroxide (150 p,1, 0.15
mmol) was
added. The reaction mixture was stirred at room temperature for 2 days. The
reaction
was incomplete based on HPLC, therefore more 1N aqueous lithium hydroxide (60
p.1, 0.06 mmol) was added at room temperature and the reaction mixture was
then
heated to 40 °C for 3 hours. The reaction mixture was cooled to 0
°C and quenched
with aqueous 1N hydrochloric acid (240 ~l, 4 eq). The mixture was concentrated
under reduced pressure. The remaining residue was redissolved in minimal
acetonitrile/water and then lypholized to give 50 mg 2-Et0-1-naphthyl-CO-Smp-
Nva-Pro-Bta-NH-CHZ-(3-HOOC-CHZ)-Ph as a mixture of diastereomers (ratio 85:6)
MS(ESI): MH+= 1004
R~ = 14.51, 14.98 (Method A)
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The following tetrapeptides were prepared to the method described above:
Table 8
cdc#R, A1 A2 A3 A4 Rz M'= HPLC- Meth.
Ret.~
(ratio) R~
(min)
23782-Et0- Smp Nva Pro L,D-BtaNH-CHZ-(3-NOZ-Ph)991 15.2, A
naphthyl-1-CO
(75:15) 15.6
23772-Et0- Smp Nva Pro L,D-BtaNH-CHZ-(2-(COOH-101816.6, A
naphthyl-1-CO (CHZ)z)-Ph)
(76:13) 17
22772-Et0- Smp Nva Pro L,D-BtaNH-CHz-(4-(HOOC-100414.1, A
naphthyl-1-CO CHZ)-Ph)
(76:13) 14.5
22762-Et0- Smp Nva Pro L,D-BtaNH-CHZ-(3-(HOOC-100414.5, A
.
naphthyl-1-CO CHz)-Ph)
(85:6) 15
22742-Et0- Smp Nva Pro Bta NH-CHz-CHZ-CHz-942 12.7 A
naphthyl-1-CO COOH
22732-Et0- Smp Nva Pro L,D-BtaNH-CHZ-(4-COOH-Ph)990 13.7, A
naphthyl-1-CO
(65:13) 14.2
22722-Et0- Smp Nva Pro L,D-BtaNH-CHz-CHZ-(4-100414.1, A
naphthyl-1-CO COOH-Ph)
(78:18) 14.5
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22542-Et0- Smp Nva Pro L,D-BtaNH-CHz-(4-(HOOC-1018 14.6, A
naphthyl-1-CO (CHZ)Z)-Ph)
(71:12) 15
22532-Et0- Smp Nva Pro L,D-BtaNH-CHZ-(3-(HOOC-1018 15.1, A
naphthyl-1-CO (CHZ)2)-Ph)
(67:15) 15.5
22522-Et0- Smp Nva Pro L,D-BtaNH-(3-(HOOC-CHZ-1006 15, A
naphthyl-1-CO O)-Ph)
(63:13) 15.6
22512-Et0- Smp Nva Pro L,D-BtaNH-CHz-CHZ-(3-1004 17.1, A
naphthyl-1-CO COOH-Ph)
(95:5) 17.8
22362-Et0- Smp Nva 2- Bta NH-(4-(HOOC-CHZ)-1004 15.5 A
naphthyl-1-CO MePro Ph
22352-Et0- Smp Nva 2- Bta NH-(3-HOOC-CHz)-1004 16.2 A
naphthyl-1-CO MePro Ph
22072-Et0- Smp Nva 2- Bta NH-(CHz)4-COOH970 13.7 A
naphthyl-1-CO MePro
22052-Et0- Smp Nva 2- Bta NH-CHZ-t-(4-HOOC)-1010 14.2 A
naphthyl-1-CO MePro cycloC6H,o
21712-Et0- Smp Nva 2- Bta NH-(3-COOH)-Ph990 16.4 A
naphthyl-1-CO MePro
21702-Et0- Smp Nva 2- Bta NH-CHZ-(3-COON-1004 14.9 A
naphthyl-1-CO MePro Ph)
20582-Et0- Smp Nva Pro Bta NH-CHZ-CHz-Ph960 17.2 A
naphthyl-1-CO
20562-Et0- Smp Nva Pro Bta NH-(CHz)4-CH3940 18.7 A
naphthyl-i-CO
22092-Et0- Smp Glu Glu Bta NH-CHz-(3-COOH-1052 10.1 A
naphthyl-1-CO Ph)
22172-Et0- Smp Glu(tBuGlu(tBBta NH-CHz-(3-COOH-1164 18.7 A
naphthyl-1-CO ) u) Ph)
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14282-Me0- Smp Glu Glu Nal NHZ 898 32.5 B
naphthyl-1-CO
1 2-Me0- Smp Glu Glu Bta NH-(CHZ)5-COOH101834.4 B
S
18
naphthyl-1-CO
15192-Me0- Smp Glu Glu Bta NH-(CHz)4-COOH100433.4 B
naphthyl-1-CO
15202-Me0- Smp Glu Glu Bta NH-(CHz)3-COOH990 33.1 B
naphthyl-1-CO
15612-Me0- Smp Glu(tBuGlu(tBBta NH-(CHZ)3-COOH110250.8 C
naphthyl-1-CO ) u)
15662-Me0- Smp Glu(MeGlu Bta NH-(CHZ)3-COOH100421.4 D
naphthyl-1-CO )
15672-Me0- Smp Glu Glu(MBta NH-(CHZ)3-COOH100421.7 D
naphthyl-1-CO e)
15702-Me0- Smp Glu Glu Bta NH-(CHZ)3-COOCH3100422.1 D
naphthyl-1-CO
15712-Me0- Smp Glu(MeGlu(MBta NH-(CHZ)3-COOH101823.5 D
naphthyl-1-CO ) e)
15722-Me0- Smp Glu(MeGlu Bta NH-(CHz)3-COOCH3101823.7 D
naphthyl-1-CO )
15732-Me0- Smp Glu(MeGlu(MBta NH-(CHZ)3-COOCH3103225.3 D
naphthyl-1-CO ) e)
Example 2 Protein Purification
GST-tagged Proteins (ON1A, ~N1B, ON1C):
Expression cloning of the open reading frames for the GST-tagged expression
of the catalytic domains of Cdc25A, Cdc25B, and Cdc25C was performed by
cloning
the appropriate fragment into the PGEX-2T or PGEX-KT (Pharmacia) vector
utilizing
the EcoRI and HinDIII sites (see Table 9). The resulting plasmids were
transformed
into BL-21 (DE3) and the proteins were overproduced by induction of mid-log
cells
with 0.5 mM IPTG for 3 h at 25°C.
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Purification of Cdc25 catalytic domain from GST-Cdc25.
Cell pellets from an approximately 4 liter E. coli high density fermentation
were
thawed. The cells were resuspended in 600 ml phosphate-buffered saline (PBS),
10
mM dithiothreitol w/ a protease inhibitor cocktail (complete tablets
Boehringer
Mannheim cat#1697498) supplemented w/ 1/1000 pepstatin A (10 mg/ml; 10 ug/ml
final). The cells were lysed via 2 passes through a microfluidizer at 15,000
psi. The
resulting suspension was centrifuged in a GSA rotor (Beckman) at 12,000 rpm
for 1
hour.
The supernatant was batch bound to 250 ml GSH sepharose 4-B (Amersham
Pharmacia Biotech) for 1 hour at 4 degrees with gentle rocking. The
supernatant was
decanted and resuspended with GSH sepharose 4-B and packed into xk50 column at
ml/min. The column was washed with 5-10 column volumes of 50 mM Tris pH
8.0, 0.500 mM NaCI, 1 mM EDTA,ImM DTT. The column was then washed at a
rate of 5 ml/min. with 5 column volumes of 50 mM tris, pH 8.0, 1 mM EDTA, 1 mM
15 DTT. The column was then eluted at a rate of 1.5 ml/min.with 50 mM tris pH
8.0, 25
mM reduced GSH, 1mM EDTA, 1 mM DTT. The eluate was collected in 4 mL
fractions.
The fractions were then subjected to size exclusion chromatography using a
5300
Sephacryl xk 50/100 column eluted at 4 ml/min, equilibrated with 50 mM tris pH
8.0,
20 150 mM NaCI, 1 mM EDTA, 5mM DTT. The eluate was collected as 10 mL
fractions. GST-CDC25 eluted both as an aggregate in the column void volume and
as
a dimeric peak. Fractions corresponding to the dimer peak were pooled.
The dimeric GST-CDC25 was bound to fresh GSH sepharose beads at 4 mg
fusion protein per 1 ml GSH beads. The beads were washed with 10 column
volumes
of 25 mM tris pH 8.0, 150 mM NaCI, 2.5 mM CaCl2, 1 mM DTT, 100 uM EDTA.
The beads were resuspended in two volumes of buffer and then digested with 5
units
thrombin (Calbiochem cat# 604980 sp. activity 1900 units/mg) per mg fusion
protein
for 90 minutes at room temperature with gentle rocking.
The beads were filtered using a 0.45 ~,m cellulose acetate bottle top
filtration
system (Corning) to remove supernatant, and the beads were washed with 1
volume
buffer. The wash was added to the pool. The thrombin was removed using ATIII
agarose beads (Sigma cat# A-8293) at a ratio of 1 mL beads per 100 ug thrombin
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added. The solution was incubated for 1 hour at 4°C with gentle rocking
and then
filtered using a0.45 p,m cellulose acetate bottle top filtration system
(Corning) to
remove the beads. 10 mM EDTA and 0.5 mM AEBSF (Calbiochem cat#101500)
were then added to inactivate any remaining thrombin. The solution was then
concentrated to 6 mL using a centriprep 10,000 dalton molecular weight cutoff
device
(Millipore) and filtered through 0.22 p,m filter.
The concentrated, filtered solution was injected onto a Superdex 75 xk26/100
column equilibrated in 50 mM NaPi pH 6.75,100 mM NaCI, l mM DTT,1mM EDTA at
2 ml/min, and the elute was collected as 2.5 mL fractions. Fractions
containing the
Cdc25 catalytic domain were pooled and concentrated to 20 mg/ml vs BSA. EDTA
was
added to SmM , DTT to 10 mM, AEBSF to 0.5 mM, and Na azide to 0.02% final
concentrations.
Native Proteins (ONSA, ONBA, ONBA-c17, ONSB, ONBB, ON8B-c17, ON8B-c18,
~N9C):
Expression cloning of the open reading frames for the native expression of the
catalytic domains of Cdc25A, Cdc25B, and Cdc25C was performed by cloning the
appropriate fragment into the pET-3d vector (Novagen) by incorporation of a
NcoI
site at the start codon and a HinDIII site following the stop codon (see Table
9). The
resulting plasmids were transformed into BL-21 (DE3) and the proteins were
overproduced by induction of mid-log cells with 0.5 mM IPTG for 3 h at
25°C. All
steps in the purification were performed at 4°C and phosphatase
activity was followed
by assays using pNPP as a substrate. In a typical preparation, 33 g of frozen
cell
pellets were thawed in 150 mL of buffer A (3 mM potassium phosphate (pH 7.4),
75
mM NaCI, 1 mM EDTA, 1 mM DTT, and a cocktail of protease inhibitors (0.001
mg/ml Aprotinin, 0.001 mg/ml Leupeptin, 0.01 mg/ml Soybean Trypsin Inhibitor,
0.01 mg/ml
L-1-Chloro-3-(4-tosylamido)-7-amino-2-heptanone hydrochloride and 0.01 mg/ml
L-1-Chloro-3-(4-tosylamido)-4-phenyl-2-butanone). Following centrifugation at
18K for 30 min the cleared lysate was bound to 15 mL of SP-Sepharose
equilibrated
in buffer A. The Cdc25 protein was eluted with buffer A containing 150 mM
NaCI,
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or by a gradient in buffer A up to 250 mM NaCI, and fiuther purified by S-200
chromatography in phosphatase reaction buffer (50 mM Tris-HCl (pH 8.0), 50 mM
NaCI, 1 mM EDTA, 1 mM DTT). Protein yields varied from 1 - 25 mg per liter of
cell culture.
Table 9 Polypeptides comprising Cdc25 catalytic domain
Construct derived fromSource N-terminus C-terminus
name
ON1A Cdc25A GST-tagged (GS)-Leu336 Leu523
(2T)
ON1B Cdc25B GST-tagged (GS)-Leu378 Gin566
(2T)
ON1C Cdc25C GST-tagged (GS)-Leu282 Pro473
(KT)
~NSA Cdc25A Native G1y323 Leu523
~N8A Cdc25A Native G1u326 Arg519
NBA-c17 Cdc25A Native G1u326 Thr506
ONSB Cdc25B Native (M)-Asp365 G1n566
ONBB Cdc25B Native (M)-G1u368 Arg562
~NBB-c17 Cdc25B Native (M)-G1u368 Ser549
ON8B-c18 Cdc25B Native (M)-G1u368 Arg548
ON9C Cdc25C Native (M)-G1y280 Va1453
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Example 3 Crystallization of polypeptides and crystal structure determination
Crystallization of Cdc25A(ON1A)
Frozen Cdc25A (~N1A construct; 25 mg/ml in 25 mM Tris.HCl, pH 7.5, 100
mM NaCI, 10 mM DTT, 5 mM EDTA, 0.5 mM AEBSF; 25 pL) was thawed and
mixed with 1 pL DTT (100 mM), 1 pL Na2W04 (100 mM), and 23 pL HzO. This
protein solution (1 p,1) was mixed with 1 p,L of a reservoir solution
consisting of 15%
(w/v) polyethylene glycol (PEG) 4000, 100 mM sodium citrate, pH 5.6, and
suspended over the reservoir on the underside of a siliconized glass cover
slip at 4°C.
Long, pyramidal crystals appeared in one day. Crystals also grew under these
conditions in the presence of varying amounts of PEG 4000, in the presence of
0-400
mM ammonium acetate, and at pH values from 4.8 to 5.6.
Cryoprotection of a Cdc25A(ON1A) Crystal
A Cdc25A(ON1A) crystal (crystal 1) grown as described above was
transferred into a series of cryoprotective buffers containing 21-24% (w/v)
PEG 4000,
100 mM sodium citrate, pH 5.6, 2 mM Na2W04, and 0, 5, 10, 15, and 20% (v/v)
glycerol. The crystal was first soaked in the 0% glycerol buffer for two min,
and then
allowed to soak sequentially in the 5, 10, 15, and 20% glycerol buffers for 5
min each.
The crystal was picked up with a fiber loop and flash-cooled by plunging into
liquid
nitrogen. The crystal was stored in a liquid nitrogen refrigerator.
X-ray Diffraction Data Collection from a Cdc25A(DIV1A) Crystal Grown from PEG
X-ray diffraction data were collected from crystal 1 on a Siemens SRA
rotating anode generator (50 kV, 108 mA, 40% bias, graphite-monochromated Cu
Ku,
radiation) equipped with a MAR Research image plate detector using the
rotation
method. The Cdc25A(~N1A) crystal was maintained at a temperature of 100 K with
an Oxford Cryosystems Cryostream cooler during data collection. For each frame
of
data (225 total) the crystal was rotated by 0.4°. The crystal was then
re-oriented
(approximately 60° rotation around the x-ray beam) and 225 additional
data frames
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were collected. The data were processed with the CCP4 Suite of programs
(Collaborative Computational Project, Number 4, 1994). After determining the
crystal orientations with REFIX (Kabsch, 1993) and IDXREF (Collaborative
Computational Project, Number 4, 1994), the data were integrated (in space
group P4~
or P43, a = 43.79 ~, c = 117.37 ~) with MOSFLM (Leslie, 1992), scaled and
merged
with SCALA (Evans, 1997), and placed on an absolute scale and reduced to
structure
factor amplitudes with TRUNCATE (French & Wilson, 1978). Five percent of the
unique reflections were assigned, in a random fashion, to the "free" set, for
calculation of the free R-factor (Rfree); the remaining 95% of the reflections
constituted the "working" set, for calculation of the R-factor (R). These data
are
summarized in Table 10.
Comparison of the Cdc25A(ON1A) Structure in Crystals Grown from Ammonium
Sulfate or PEG
The diffraction data from crystal 1 described above were indexed in a
tetragonal unit cell, space group P41 (or P43). In space groups P41 and P43,
the
direction of the polar c axis cannot be determined without reference to a
molecular
model for (part of) the unit cell contents. Accordingly; the data as initially
indexed,
"UP", were reindexed, using the transformation h' _ -k, k' _ -h, I' _ -1, to
provide the
"DOWN" indexing. Structure factors were calculated (Collaborative
Computational
Project, Number 4, 1994) in space group P4~ using the partially-refined
structural
coordinates derived from crystals of Cdc25A(O1V1A) (Fig. 16A to 16I) grown
from
ammonium sulfate as described below, which form in a similarly-sized
tetragonal unit
cell. These calculated structure factors were scaled against the "UP"-indexed
data
and the "DOWN'-indexed data. The "DOWN"-indexed data scaled substantially
better than the "UP"-indexed data (Rfree 39.1 vs. 52.4%, respectively),
confirming that
the correct indexing of the data was "DOWN". Examination of SigmaA-weighted
(Collaborative Computational Project, Number 4, 1994) 2F°-F~ and
F°-F~ electron-
density maps showed that the partially-refined structural coordinates for the
Cdc25A(ON1A) molecule in the crystals grown from ammonium sulfate accounted
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for most of the structure of Cdc25A(ON1A) in the crystals grown from
polyethylene
glycol ('PEG").
Growth of Cdc25A( NlA) crystals from ammonium sulfate
Cdc25A( N1A) was crystallized from 1.9-2.3 M (NH4)ZSO4, SOmM sodium
phosphate pH 6.5-7.0, 2mM sodium tungstate at 4°C. Long square-based
pyramidal
crystals, with a slight tapering along their length, and often coming to a
sharp point at
the apex, grew over two weeks time. The crystals had unit cell dimensions
a=b=44.17
Angstrom, c=118.65 Angstrom, and belonged to the tetragonal space group P4(1).
There was one molecule of the protein in the crystallographic asymmetric unit.
The
phases required to obtain an interpretable electron density map were derived
with 3
heavy atom derivatives of these crystals prepared by contacting the crystals
with ( 1 )
KAu(CN)2; (2) KzPtCl4; and (3) Thiomersal. Heavy atom phases were improved by
solvent flattening. The atomic structure was refined using diffraction data to
2.1
Angstrom resolution (X-Plor). The R-factor was 26%, with an R(free) of 31 %.
The
resulting atomic coordinates are presented in Figs. 16A to 16I.
Preliminary Refinement of the PEG Cdc25A(ON1A) Crystal Structure (Crystal 1)
The partially-refined structural coordinates derived from crystals of
Cdc25A(ONlA) grown from ammonium sulfate were refined against the "DOWN"-
indexed data of the crystal described above using the program x-PLOR (Brunger,
1992). Rigid-body, Powell minimization, slowcool simulated annealing molecular
dynamics, and temperature factor refinement resulted in an R of 31.7% (Rfree
36.3%)
for all reflections with /F/ > 2.O6F between 20 and 1.80 ~ resolution.
Examination of
SigmaA-weighted 2F° F~ and F° F~ electron-density maps revealed
that the active site
loop (residues 431-434) was substantially disordered, and that no ligand (i.e.
tungstate) was bound in the active site. Also, residues 493-523, at the C-
terminus of
the. protein, were not located in the electron-density maps and were not
included in the
structural coordinates. These data are summarized in Table 10.
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Soaking of a Cdc25A(ON1A) Crystal with cdc1316
A Cdc25A(ONlA) crystal grown as described above was transferred to 100
pL of a solution of 24% PEG 4000, S mM sodium citrate, pH 5.6 at 4°C.
After
soaking for 2 min, the crystal was transferred to a fresh solution containing
in addition
2 mM cdc1316. After 23 hrs, the crystal was transferred through a series of
cryoprotective buffers containing 24% (w/v) PEG 4000, 10 mM sodium citrate, pH
5.6, 2 mM cdc 1316, and 5, 10, and 20% (v/v) glycerol (5 min each). After an
additional 5 min soak in the 20% glycerol buffer, the crystal was picked up
with a
fiber loop and flash-cooled by plunging into liquid nitrogen.
X-ray Diffraction Data Collection from a Cdc25A(ON1A) Crystal Soaked with
cdc1316
A total of 235 data frames (0.4° each) were collected from a
Cdc25A(ON1A)
crystal soaked with cdc1316 as described above (crystal 2). The crystal was
maintained at a temperature of 100 K during data collection. The diffraction
data
were processed and reduced to structure factor amplitudes, as described for
crystal 1,
in space group P41, a = 43.69 t~, c = 117.27 ~. The unique reflections were
assigned
to the same "free" and "working" sets as used for crystal 1. These data are
summarized in Table 10. Additional data were collected from this crystal at
the
National Synchrotron Light Source (NSLS; beamline X25, ~, =1.100 ~, Brandeis
B4
CCD detector). The crystal was maintained at a temperature of 100 K with an
Oxford
Cryosystems Cryostream cooler during data collection. The diffraction data
were
processed and reduced to structure factor amplitudes, as described above. Only
a
small fraction of the unique data were collected due to instrumental
limitations.
These data show, however, that the crystals diffract x-rays to 1.35 ~
resolution.
These additional data are also summarized in Table 10.
Further Refinement of the PEG Cdc25A(DIV1A) Crystal Structure
The partially-refined structural coordinates for crystal 1 were further
refined
against the diffraction data collected from the Cdc25A(ON1A) crystal 2 using X-
PLOR.
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Refinement alternated with manual rebuilding of the structural coordinates
(the
"model") using the molecular graphics program o (Jones et al., 1991). Rigid-
body,
Powell minimization, slowcool simulated annealing molecular dynamics, and
individual temperature factor refinement resulted in an R of 29.9% (R~e 32.6%)
for
all reflections with /F /> 1.SaF between 20 and 1.80 ~ resolution. The model
was
rebuilt aided by inspection of simulated annealing omit maps. Several more
rounds of
rebuilding and refinement brought the R to 27.0% (R~e 28.9%; /F/> I.SaF, 20-
1.80
t~). Additional refinement with ttEF~c (Murshudov et al., 1997) brought the R
to
23.0% (Renee 24.3%; /F/ > 0.0 6F, 20-1.80 ~). This model ("Refmacl") includes
Cdc25A residues 335-413, 419-431, and 435-492 and 74 water molecules. No
interpretable electron-density was present for either the rest of the active
site loop
(residues 432-434) or the ligands tungstate or cdc1316. Also, residues 493-
523, at the
C-terminus of the protein, were not located in the electron-density maps and
were not
included in the structural coordinates. Weak, but not readily interpretable
density was
present for residues 414-418. These data are summarized in Table 10.
Crystallization of Cdc25A(ON8A)
Frozen Cde25A (ON8A construct; 17 mg/ml in 50 mM Tris.HCl, pH 7.5, 50
mM NaCI, 1 mM DTT, 1 mM EDTA; 150 ~L) was thawed and mixed with 1.5 ~L
DTT (1 M), 0.3 ~L NaN3 (1.5 M), and 1.5 ~L NazW04 (100 mM). This protein
solution (1 ~L) was mixed with 1 ~.L of a reservoir solution consisting of 20%
(w/v)
PEG 3000, 600 mM Li2S04, 100 mM sodium citrate, pH 5.6, and suspended over the
reservoir on the underside of a siliconized glass cover slip at 4°C.
Long, pyramidal
crystals appeared in about 2 weeks. Crystals also grew under these conditions
in the
presence of varying amounts of PEG 3000 or Li2S04, in the presence of other
salts
instead of Li2S04 (e.g. ammonium acetate or ammonium sulfate), in the absence
of
PEG 3000 entirely, and at pH values from 5.6 to 5.8.
Cryoprotection of a Cdc25A(ON8A) Crystal
A Cdc25A(ON8A) crystal (crystal 3) grown as described above was
transferred into a series of cryoprotective buffers containing 20% (w/v) PEG
3000,
100 mM sodium citrate, pH 5.6, and 0 and 5% (v/v) glycerol, and same
containing
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10% glycerol and 25% or 30% PEG 3000. The crystal was soaked sequentially in
the
0% glycerol, 5% glycerol, 25% PEG 3000/10% glycerol, and 30% PEG 3000/10%
glycerol buffers for five sec each. The crystal was picked up with a fiber
loop and
flash-cooled by plunging into liquid nitrogen. The crystal was stored in a
liquid
nitrogen refrigerator.
X-ray Diffraction Data Collection from a Cdc25A(ON8A) Crystal
A total of 225 data frames (0.4° each) were collected from crystal
3 as
described above. The crystal was maintained at a temperature of 100 K during
data
collection. The diffraction data were processed and reduced to structure
factor
amplitudes, as described above in space group P4,, a = 43.94 ~, c = 117.39 ~.
The
unique reflections were assigned to the same "free" and "working" sets as used
for
crystal 1.
1 S Annealing of a Cdc25A(~NBA) Crystal
Crystal 3 was thawed after x-ray data collection in a cryoprotective buffer
containing 30% (w/v) PEG 3000, 100 mM sodium citrate, pH 5.6, and 10% (v/v)
glycerol at 4°C. After 1 S min, the crystal was picked up with a fiber
loop and flash-
cooled again by plunging into liquid nitrogen.
X-ray Diffraction Data Collection from the Annealed Cdc25A(ONBA) Crystal
A total of 180 data frames (0.5° each) were collected from the annealed
crystal
3 using the method described above. The crystal was maintained at a
temperature of
100 K during data collection. The diffraction data were processed, merged with
the
data collected as described above, and reduced to structure factor amplitudes
in space
group P4,, a = 43.92 ~, c = 117.38 t~. These annealed data extended to higher
resolution than the pre-annealed data (2.15 vs. 2.40 t~). The annealed crystal
also had
a lower mosaic spread (0.3° vs. 0.6°). The unique reflections
were assigned to the
same "free" and "working" sets as used for crystal 1. These final crystal 3
data are
summarized in Table 10.
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Refinement of the Cdc25A(ONBA) Crystal Structure
The refinement of the Cdc25A(ONBA) crystal structure (crystal 3) began with
the intermediate "Powe118" structural coordinates for Cdc25A(ON1A), described
above, from which water molecules had been removed. Rigid-body, Powell
minimization, and overall and individual temperature factor refinement
resulted in an
R of 27.1 % (Rfree 29.2%) for all reflections with /F/ > 2.OaF between 20 and
2.15 ~
resolution. Examination of SigmaA-weighted 2F° F~ and F°-F~
electron-density maps
revealed that the active site loop (residues 431-434) was partially ordered,
in a
conformation different from that observed in other phosphatases. Further
refinement
with ~FNt~,C (Murshudov et al., 1997) brought the R to 22.8% (Rfree 27.1%; /F/
>
O.OaF, 20-2.15 ~). This model ("Refinacl") includes Cdc25A residues 335-413
and
419-492, and 67 water molecules. No interpretable electron-density was present
for
the ligand tungstate. Weak, but not readily interpretable density was present
for
residues 414-418. Active site loop residues 431-433 were built as alanines.
Tungstate, residues preceding 335, and residues 493-523 were not located in
the
electron-density maps and were not included in the structural coordinates.
These data
are summarized in Table 10.
Soaking of a Cdc25A(ON8A) with Na2W04 and X-ray Diffraction Data Collection
Crystal 3 was annealed for three days at 4°C in a buffer supplemented
with 10
mM NaZW04. The crystal was flash-cooled in liquid nitrogen. A total of 40 data
frames (1.0° each) were collected from the crystal (now crystal 4) as
described above.
The crystal was maintained at a temperature of 100 K during data collection.
The
diffraction data were processed and reduced to structure factor amplitudes, as
described above, in space group P41, a = 43.98 ~, c = 117.72 ~. These soaked
data
were much weaker than those of crystal 3 (3.00 vs. 2.15 A). The unique
reflections
were assigned to the same "free" and "working sets" as used for crystal 1.
These data
are summarized in Table 10.
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Refinement of the Na2W04-Soaked Cdc25A(~NBA) Crystal Structure
Refinement of the NazW04-soaked Cdc25A(ONBA) crystal structure (crystal
4) began with the "Powell8" structural coordinates for crystal 1 in which
water
molecules had been removed. Rigid-body, Powell minimization, and temperature
factor refinement resulted in an R of 22.7% (Rfree 25.7%) for all reflections
with /F/ >
2.OaF between 20 and 3.00 ~ resolution. Examination of SigmaA-weighted
2F°-F~
and F° F~ electron-density maps showed that tungstate was not present
in the active
site. The model was not further refined. These data are summarized in Table
10.
Crystallization of the Cdc25B(DIV1B).cdc1249 Inhibitor Complex
Frozen Cdc25B (ON1B construct; 17.5 mg/ml in 10 mM sodium phosphate,
pH 6.7, 50 mM NaCI, 10 mM DTT, 5 mM EDTA; 212 ~L) was thawed and mixed
with 0.4 p,L NaN3 (1.5 M) and 7.4 p,L cdc1249 (30 mM in 25 mM sodium HEPES,
pH 7.5). This protein solution was aged for 4 days at 1°C. A slight
precipitate that
formed was removed by centrifugation. The supernatant (1 ~L) was mixed with 1
pL
of a reservoir solution consisting of 4.4 M NaCI, 50 mM sodium HEPES, pH 7.0,
and
suspended over the reservoir on the underside of a siliconized glass cover
slip at 4°C.
Block-like crystals appeared within 2-7 days. Crystals also grew under these
conditions in the presence of varying amounts of NaCI, and at pH values from
5.75 to
8Ø
Crystallization of the Cdc25B(~NBB).cdc1249 Inhibitor Complex.
Frozen Cdc25B (ONBB construct; 27.5 mg/ml in SO mM Tris.HCl, pH 7.5, 50
mM NaCI, 1 mM DTT, 1 mM EDTA; 25 pL) was thawed and mixed with 0.25 pL
NaN3 (0.3 M), 0.2 pL DTT (1 M), and 0.85 pL cdc1249 (30 mM in 25 mM sodium
HEPES, pH 7.5). This protein solution (2 p.L) was mixed with 2 ~,L of a
reservoir
solution consisting of 82.5% saturated NaCI (~4.5 M), 50 mM sodium MES, pH
6.5,
and suspended over the reservoir on the underside of a siliconized glass cover
slip at
4°C. Block-like crystals appeared in one day.
The Cdc25B(ONBB-c17).cdc1249, Cdc25B(ON8B-cl8).cdc1249,
Cdc25B(ON1B).cdc1671, Cdc25B(ON1B).cdc1885, Cdc25B(~NBB).cdc1659, and
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Cdc25B(ON8B).cdc1671 complexes were also crystallized using this general
procedure.
Cryoprotection of Cdc25B(~N1B), Cdc25B(ON8B), Cdc25B(ONBB-cl7), and
Cdc25B(ONBB-c18) Inhibitor Complex Crystals
A Cdc25B inhibitor complex crystal grown as described above was transferred
into a series of cryoprotective buffers containing 4.5 M NaCI, 50 mM sodium
MES,
pH 6.5 or 50 mM sodium HEPES, pH 7.0, and 0, 5, 10, and 16.5% (v/v) glycerol.
The crystal was soaked sequentially in the 0 and 5% glycerol buffers, and then
the 10
and 16.5% glycerol buffers (each of which also contained 0.5-2.0 mM of the
appropriate inhibitor), for 5 min each. The crystal was picked up with a fiber
loop
and flash-cooled by plunging into liquid nitrogen. The crystal was stored in a
liquid
nitrogen refrigerator.
X-ray Diffraction Data Collection from a Cdc25B(ON1B).cdc1249 Inhibitor
Complex
Crystal
A total of 359 data frames (0.25° each) were collected from a
Cdc25B(ON1B).cdc1249 inhibitor complex crystal (crystal 5) using the equipment
described above. The crystal was maintained at a temperature of 100 K during
data
collection. The diffraction data were processed and reduced to structure
factor
amplitudes, as described above, in space group P41212 or P43212, a = 70.15 ~,
c =
130.35 ~. Five percent of the unique reflections were assigned, in a random
fashion,
to the "free" set, for calculation of the free R-factor (RfTee); the remaining
95% of the
reflections constituted the "working set", for calculation of the R-factor
(R). These
data are summarized in Table 10.
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Molecular Replacement Solution of the Cdc25B(ON1B).cdc1249 Inhibitor Complex
Crystal Structure
A cross-rotation function was calculated with the Cdc25B(ON1B).cdc1249
inhibitor complex crystal data described above, using the program AMO~
(Navaza,
1994). The search model was the partially-refined structural coordinates of
Cdc25A(ON1A) (crystal 1). The cross-rotation function had one obvious
solution, at
Eulerian angles [27.66, 63.02, 94.53], which was 11.3 standard deviations
above the
mean level of the cross-rotation function; the next highest peak was 6.9
standard
deviations above the mean. The translation function was calculated (AMOK) in
space
groups P4122, P4322, P41212, and P43212. One solution was obvious, in space
group
P432~2, with an R-factor of 49.3%, and a correlation coefficient of 31.4% (15-
3.01
resolution).
Synchrotron X-ray Diffraction Data Collection from the Cdc25B(ON1B).cdc1249
Inhibitor Complex Crystal
A total of 35 data frames (1.0° each) were collected from crystal S
at the
National Synchrotron Light Source (NSLS; beamline X25, ~. = 1.100 ~, Brandeis
B4
CCD detector). The crystal was maintained at a temperature of 100 K with an
Oxford
Cryosystems Cryostream cooler during data collection. The diffraction data
were
processed and reduced to structure factor amplitudes, as described above, in
space
group P43212, a = 70.15 ~, c = 130.35 ~. The unique reflections were assigned
to the
same "free" and "working" sets as used for the laboratory data for crystal S.
These
data are summarized in Table 10.
Refinement of the Cdc25B(ON1B).cdc1249 Inhibitor Complex Crystal Structure
The partially-refined structural coordinates of Cdc25A(~NlA) (crystal 1) were
refined against the Cdc25B(ON1B).cdc1249 inhibitor complex crystal data using
x-
PLOR. Refinement alternated with manual rebuilding of the model using the
molecular
graphics program o. The molecular replacement model was modified by changing
many of the amino acid side chains that differed between Cdc25A and Cdc25B to
the
Cdc25B amino acids. These side chains were located in clear electron-density.
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Unclear amino acids were truncated as alanine. Bulk solvent correction (~60%
solvent
by volume in these crystals), Powell minimization, overall and then group
temperature factor refinement, and manual rebuilding reduced the R to 37.7%
(Rfree
43.9%) for all reflections with /F/ > 1.56F between 20 and 2.40 ~ resolution.
Torsion
angle molecular dynamics improved the electron-density maps such that two
missing
loops (residues 456-464 and 474-477, the active site loop) could be built into
very
clear density. Clear density was also present at this stage of the refinement
for much
of cdc 1249 bound in the active site. Continued refinement (including
individual
temperature factors) and rebuilding allowed the addition of residues 533-548
(the C-
terminal a-helix), the first 5 (of 6 total) residues for cdc 1249, and several
water
molecules (R 27.0%, R~.ee 31.2%). Further refinement including slowcool
simulated
annealing molecular dynamics revealed the presence of another molecule of
cdc1249
bound, not at the active site, but between two Cdc25B(OlVlB) molecules related
by
crystallographic symmetry. Addition of this second molecule of cdc1249,
several
water molecules, a Na+ ion, and several Cl- ions followed by more refinement
reduced
the R to 22.8% (Rfree 26.1 %). The model was rebuilt aided by inspection of
simulated
annealing omit maps. Several rounds of refinement brought the R to 20.3%
(Rfree
22.9%; /F/> O.SaF, 20-2.30 ~). These structural coordinates ("Powel114") for
the
Cdc25B(ON1B).cdc1249 inhibitor complex consist of Cdc25B residues 377-548, two
cdc1249 molecules, 129 water molecules, one Na+ ion, and five Cl- ions. At
this point
the synchrotron data described above became available. Further refinement with
~FMac, which included the addition of several side chains in alternate
conformations, resulted in an R of 21.8% (Rfree 24.4%; 1F1> O.OaF, 20-1.95 ~).
These
final structural coordinates ("Refinac 1 ") for the Cdc25B(ON1B).cdc 1249
inhibitor
complex consist of Cdc25B residues 377-548, two cdc1249 molecules, 158 water
molecules, one Na+ ion, and five Cl- ions. These data are summarized in Table
10.
X-ray Diffraction Data Collection from a Cdc25B(ON1B).cdc1249 Inhibitor
Complex
crystal Soaked with cdc1316
A Cdc25B(ON1B).cdc1249 inhibitor complex crystal prepared as described
above was transferred to 50 pL of a buffer containing 4.5 M NaCI, 50 mM sodium
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HEPES, pH 7.0, and 2 mM cdc1316. After 70 min, the crystal was cryoprotected
and
flash-cooled (buffers contained 2 mM cdc1316). A total of 180 data frames
(0.5°
each) were collected from this soaked crystal (crystal 6). The crystal was
maintained
at a temperature of 100 K during data collection. The diffraction data were
processed
and reduced to structure factor amplitudes, as described previously, in space
group
P43212, a = 70.21 ~, c = 130.09 t~. The unique reflections were assigned to
the same
"free" and "working" sets as used for crystal 5. These data are summarized in
Table
10.
Refinement of the Structure of the Cdc25B(ON1B).cdc1249 Inhibitor Complex
Crystal Soaked with cdc 1316
Refinement against the data collected with crystal 6 began with an
intermediate model ("Powelll l") for the Cdc25B(~N1B).cdc1249 inhibitor
complex
from which both molecules of cdc1249 had been deleted. Slowcool simulated
1 S annealing molecular dynamics, Powell minimization, and individual
temperature
factor refinement lowered R to 27.4% (Rcree 32.9%; /F/ > 1.OaF, 20-2.50 ~).
Examination of electron-density maps showed strong, clear electron-density for
cdc1249 in the active site. There was no evidence for replacement of cdc1249
by
cdc1316 at the active site. The positive F° F~ electron-density was not
as strong at the
crystallographic symmetry site, however, suggesting that partial replacement
of
cdc 1249 by cdc 1316 may have occurred at this site. These data are summarized
in
Table 11.
X-ray Diffraction Data Collection from Cdc25B(ONBB).cdc1249 Inhibitor Complex
Crystals
A total of 201 data frames (0.5° each) were collected from two
Cdc25B(ONBB).cdc1249 inhibitor complex crystals (crystals 7 and 8). The
crystals
were maintained at a temperature of 100 K during data collection. The
diffraction
data were processed and reduced to structure factor amplitudes, as described
previously in space group P43212, a = 70.28 ~, c = 130.97 E~. The unique
reflections
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were assigned to the same "free"and "working" sets as used for crystal 5.
These data
are summarized in Table 10.
Refinement of the Cdc25B(ONBB).cdc1249 Inhibitor Complex Crystal Structure.
Refinement of the Cdc25B(~NBB).cdc1249 inhibitor complex crystal data
began with the "Powe1114" structural coordinates for the Cdc25B(~1V1B).cdc1249
inhibitor complex. Rigid body, Powell minimization, and individual temperature
factor refinement lowered R to 23.7% (Rfree 25.5%; /F/> 2.OaF, 20-2.00 ~).
Electron-
density maps revealed the presence of additional N terminal amino acid
residues, as
expected for the Cdc25B(ON8B) construct compared to the Cdc25B(ON1B)
construct.
Both molecules of cdc 1249 were in their expected locations at the active site
and at
the crystallographic symmetry site. Rebuilding followed by additional
refinement
lowered R to 22.6% (Rfree 24.3%; /F/ > l.SaF, 20-2.00 ~). These structural
coordinates ("Powell2") for the Cdc25B(DIV8B).cdc1249 inhibitor complex
consist of
Cdc25B residues 370-548, two cdc1249 molecules, 128 water molecules, one Na+
ion,
and five Cl- ions. These data are summarized in Table 11.
Soaking of Cdc25B(~NBB).cdc1249 Inhibitor Complex Crystals with Low Molecular
Weight Organic Compounds
Eight crystals of the Cdc25B(ONBB).cdc1249 inhibitor complex were
transferrred sequentially through solutions containing 4.5 M NaCI, 50 mM
sodium
HEPES, pH 7.0, 0.5-1.5 mM cdc1249, and increasing concentrations (0, 50, 100,
250,
and 1000 mM) of t-BuNH2, imidazole, (R)-3-hydroxypyrrolidine, or 2-methyl-1-
propanol 0500 mM maximum, saturated solution) at 4°C. Crystals were
soaked for
10-1 S min in each solution, and were then left in the final solution for 3 or
23 hrs.
Crystals were then cryoprotected by the addition of glycerol (7.5% (v/v), 5
min;
17.5% (v/v), 5 min) and then flash-cooled by plunging into liquid nitrogen.
Test x-
ray diffraction images of all eight crystals showed that the crystalline order
of each
had been substantially unaffected by the soaking procedure, as the crystals
diffracted
x-rays to a maximum resolution of 2.2-2.6 t~. The crystals were stored in a
liquid
nitrogen refrigerator.
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X-ray Diffraction Data Collection from Cdc25B(ONBB).cdc1249 Inhibitor Complex
Crystals Soaked with t-BuNHz, Imidazole, or 2-Methyl-1-propanol
A total of 100 data frames (0.5° each) were collected from the
Cdc25B(ONBB).cdc1249 inhibitor complex crystal (crystal 9) that had been
soaked
for 3 hrs with ~0.5 M 2-methyl-1-propanol using the equipment described above.
The
crystal was maintained at a temperature of 100 K during data collection. The
diffraction data were processed and reduced to structure factor amplitudes in
space
group P432,2, a = 70.01 ~, c = 129.92 t~. Similarly, a total of 225 data
frames (0.4°
each) were collected from the Cdc25B(ON8B).cdc1249 inhibitor complex crystal
(crystal 10) that had been soaked for 23 hrs with 1 M t-BuNH2. The crystal was
maintained at a temperature of 100 K during data collection. The diffraction
data
were processed and reduced to structure factor amplitudes in space group
P43212, a =
70.38 ~, c = 131.15 ~. Similarly, a total of 112 data frames (0.5°
each) were
collected from the Cdc25B(ONBB).cdc1249 inhibitor complex crystal (crystal 11)
that
had been soaked for 23 hrs with 1 M imidazole. The crystal was maintained at a
temperature of 100 K during data collection. The diffraction data were
processed and
reduced to structure factor amplitudes in space group P43212, a = 70.46 ~, c =
131.36
~. The unique reflections for all three data sets were assigned to the same
"free" and
"working" sets as used for crystal S. These three data sets are summarized in
Table
10.
X-ray Diffraction Data Collection from Cdc25B(~NBB).cdc1249 Inhibitor Complex
Crystals Soaked with t-BuNH2 or (R)-3-hydroxypyrrolidine
X-ray diffraction data were collected at NSLS as described above at a
temperature of 100 K. A total of 60 data frames (1.0° each) were
collected from the
Cdc25B(ON8B).cdc 1249 inhibitor complex crystal (crystal 12) that had been
soaked
for 3 hrs with 1 M t-BuNH2. The diffraction data were processed and reduced to
structure factor amplitudes in space group P43212, a = 70.24 ~, c = 131.57 ~.
Similarly, a total of 67 data frames (1.0° each) were collected
from the
Cdc25B(ONBB).cdc1249 inhibitor complex crystal (crystal 13) that had been
soaked
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for 3 hrs with 1 M (R)-3-hydroxypyrrolidine. The diffraction data were
processed and
reduced to structure factor amplitudes in space group P43212, a = 70.04 ~, c =
130.34
t~. And, a total of 99 data frames (0.5° each) were collected from the
Cdc25B(~1V8B).cdc1249 inhibitor complex crystal (crystal 14) that had been
soaked
for 23 hrs with 1 M (R)-3-hydroxypyrrolidine. The diffraction data were
processed
and reduced to structure factor amplitudes in space group P43212, a = 70.46 ~,
c =
131.00 ~. The unique reflections for these three data sets were assigned to
the same
"free" and "working" sets as used for crystal 5. These three data sets are
summarized
in Table 10.
Structural Refinement of the Cdc25B(~NBB).cdc1249 Inhibitor Complex Crystals
Soaked with Low Molecular Weight Organic Compounds
Refinement of the soaked Cdc25B(ONBB).cdc1249 inhibitor complex crystal
data began with the "Powe112" structural coordinates for the
Cdc25B(~NBB).cdc1249
inhibitor complex from which water molecules and a Cf ion in the "Swimming
Pool"
region of the active site, adjacent to the Nal residue of cdc1249, had been
removed.
For crystal 12, rigid body, Powell minimization, individual temperature factor
refinement and slowcool simulated annealing molecular dynamics lowered R to
24.2% (Rcree 27.7%; /F/ > l.OaF, 20-1.60 ~). Electron-density maps showed that
t-
BuNH2 was not present in the "Swimming Pool". The model for crystal 13 was
refined similarly, resulting in an R of 24.7% (Rfree 27.8%; /F/ > I.OaF, 20-
1.60 ~).
Electron-density maps showed that (R)-3-hydroxypyrrolidine was not present in
the
"Swimming Pool". The model for crystal 9 was refined as above, but without
slowcool simulated annealing molecular dynamics, resulting in an R of 21.3%
(Rfree
25.1%; /Fl > 2.OaF, 30-2.45 ~). Electron-density maps showed that 2-methyl-1-
propanol was not present in the "Swimming Pool". The model for crystal 10 was
refined similarly, resulting in an R of 22.3% (Rfree 25.6%; /Fl > 2.OaF, 20-
2.15 ~).
Electron-density maps showed that t-BuNH2 was not present in the "Swimming
Pool". The model for crystal 14 was refined as for crystal 10 resulting an R
of 23.2%
(RfTee 25.2%; /Fl > 2.OaF, 30-2.30 A). Electron-density maps showed that
imidazole
was not present in the "Swimming Pool". Finally, the model for crystal 14 was
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refined as for crystals 12 and 13 (with an additional cycle of Powell
minimization and
individual temperature factor refinement) resulting in an R of 24.6% (Rfree
27.9%; ~F~
> O.O~F, 20-1.60 ~). Electron-density maps showed that (R)-3-
hydroxypyrrolidine
was not present in the "Swimming Pool". These data are summarized in Table 11.
X-ray Diffraction Data Collection from Crystals of the Cdc25B(~N1B).cdc1671,
Cdc25B(ONBB).cdc1659, and Cdc25B(ON8B).cdc1671 Inhibitor Complexes
A total of 195 data frames (0.4° each) were collected from the
Cdc25B(ON1B).cdc1671 inhibitor complex crystal (crystal 15). The crystals were
maintained at a temperature of 100 K during data collection. The diffraction
data
were processed and reduced to structure factor amplitudes in space group
P43212, a =
70.03 A, c = 130.07 ~. A total of 120 data frames (0.5° each) were
collected similarly
from a Cdc25B(ONBB).cdc1659 inhibitor complex crystal (crystal 16). The data
were
processed and reduced to structure factor amplitudes in space group P43212, a
= 70.10
~, c = 129.47 t~. Test data were also collected from a Cdc25B(ON8B).cdc1671
inhibitor complex crystal (crystal 17) space group P43212, a = 70.10 ~, c =
130.13 ~),
but were judged not worthy of full data collection due to weak diffraction
(maximum
resolution, 3.00 ~) compared to the Cdc25B(ON1B).cdc1671 inhibitor complex
crystal. The unique reflections for crystals 15 and 16 were assigned to the
same
"free" and "working" sets as used for crystal 5. These two data sets are
summarized
in Table 10.
X-ray Diffraction Data Collection from a Crystal of the Cdc25B(ON1B).cdc1885
Inhibitor Complex
A total of 100 data frames (0.5° each) were collected from the
Cdc25B(ON1B).cdc1885 inhibitor complex crystal (crystal 18). Additional data
(32
frames, 1.0° each) were collected at NSLS as described previously. The
crystals were
maintained at a temperature of 100 K during data collection. The laboratory
and
NSLS diffraction data were processed, merged, and reduced to structure factor
amplitudes in space group P43212, a = 70.10 ~, c = 130.13 ~. The unique
reflections
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were assigned to the same "free" and "working" sets as used for crystal 5.
These data
are summarized in Table 10.
Refinement of the Cdc25B(~N1B).cdc1671 Inhibitor Complex Crystal Structure
Refinement of the Cdc25B(ON1B).cdc1671 inhibitor complex crystal data
(crystal 15) began with the "Powe1115" structural coordinates for the
Cdc25B(ON1B).cdc1249 inhibitor complex. The cdc1249 molecules, water
molecules, and ions were removed from the model, which was then subjected to
rigid
body, Powell minimization, and individual temperature factor refinement.
Electron-
density maps showed that both molecules of cdc1671 were in their expected
locations
(comparable to cdc1249) at the active site and at the crystallographic
symmetry site.
These inhibitors were added to the model. Additional refinement alternating
with
rebuilding lowered R to 20.2% (Rfree 22.8%; /F/ > 1.06F, 20-3.00 t~). These
structural
coordinates ("Powell6") for the Cdc25B(~N1B).cdc1671 inhibitor complex consist
of
Cdc25B residues 377-548, two cdc1671 molecules, 32 water molecules, one Na+
ion,
and five Cf ions. These data are summarized in Table 11.
Refinement of the Cdc25B(ONBB).cdc1659 Inhibitor Complex Crystal Structure
Refinement of the Cdc25B(ONBB).cdc1659 inhibitor complex crystal data
(crystal 16) began with the "Powe112" structural coordinates for the
Cdc25B(~NBB).cdc1249 inhibitor complex. The cdc1249 molecules were removed
from the model, which was then subjected to rigid body, Powell minimization,
and
individual temperature factor refinement. Electron-density maps showed that
both
molecules of cdc1659 were in their expected locations (comparable to cdc1249)
at the
active site and at the crystallographic symmetry site. These inhibitors were
added to
the model. Additional refinement alternating with rebuilding lowered R to
22.2%
(Rfree 25.3%; /F/ > I.OaF, 20-2.20 ~). These structural coordinates
("Powe113") for
the Cdc25B(ONBB).cdc1659 inhibitor complex consist of Cdc25B residues 370-548,
two cdc1659 molecules, 128 water molecules, one Na+ ion, and five Cl- ions.
These
data are summarized in Table 11.
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Refinement of the Cdc25B(ON1B).cdc1885 Inhibitor Complex Crystal Structure
Refinement of the Cdc25B(DIV1B).cdc1885 inhibitor complex crystal data
(crystal 18) began with the "Powe1114" structural coordinates for the
Cdc25B(ON1B).cdc1249 inhibitor complex. The cdc1249 molecules were removed
from the model, which was then subj ected to rigid body, Powell minimization,
and
individual temperature factor refinement. Electron-density maps showed that
both
molecules of cdc1885 were in their expected locations (comparable to cdc1249)
at the
active site and at the crystallographic symmetry site. These inhibitors were
added to
the model. Additional refinement alternating with rebuilding lowered R to
22.8%
(Rfree 25.1%; /F/ > l.OaF, 30-1.70 ~). These structural coordinates
("Powe112") for
the Cdc25B(ON1B).cdc1885 inhibitor complex consist of Cdc25B residues 377-548,
two cdc1885 molecules, 128 water molecules, one Na+ ion, and five Cl- ions.
These
data are summarized in Table 11.
Synchrotron X-ray Diffraction Data Collection from a Cdc25B(ONBB).cdc1249
Inhibitor Complex Crystal Grown in the Presence of cdc1900
A total of 121 data frames (0.5° each) were collected from a
Cdc25B(~NBB).cdc1249 inhibitor complex crystal grown in the presence of
cdc1900
(crystal 19) at NSLS as described previously. The crystals were maintained at
a
temperature of 100 K during data collection. The diffraction data were
processed and
reduced to structure factor amplitudes in space group P43212, a = 70.29 ~, c =
130.59
~. The unique reflections were assigned to the same "free" and "working" sets
as
used for crystal 5. These data are summarized in Table 10.
High-Resolution Refinement of the Cdc25B(ON8B).cdc1249 Inhibitor Complex
Crystal Structure
Refinement of the Cdc25B(ONBB).cdc1249 inhibitor complex crystal structure
against the synchrotron data collected from crystal 19 began with the
"Powe112"
structural coordinates for the Cdc25B(ON8B).cdc1249 inhibitor complex. Rigid
body, Powell minimization, and individual temperature factor refinement with x-
PLOR, followed by the addition of several side chains in alternate
conformations and
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refinement with ~F~c resulted in an R of 22.4% (Rfree 24.1 %; /F/ > O.OaF, 15-
1.45
~). Further refinement with individual anisotropic temperature factors
resulted in an
R of 21.1 % (Rfree 23.1 %; /F/ > O.OaF, 15-1.45 t~). These final structural
coordinates
("Refinac3") for the Cdc25B(ON8B).cdc1249 inhibitor complex consist of cdc25B
residues 369-548, two cdc1249 molecules, 235 water molecules, one Na+ ion, and
six
Cl' ions. The inhibitor cdc1900 was not located, even though it had been
included in
the crystallization mixture. These data are summarized in Table 11.
X-ray Diffraction Data Collection from a Crystal of the Cdc25B(ONBB-
c17).cdc1249
Inhibitor Complex
A total of 113 data frames (0.5° each) were collected from a
Cdc25B(~NBB-
c17).cdc1249 inhibitor complex crystal (crystal 20) using the equipment
described
above. The crystals were maintained at a temperature of 100 K during data
collection.
The diffraction data were processed and reduced to structure factor
amplitudes, as
described in 3, in space group P43212, a = 70.05 ~, c = 131.44 t~. The unique
reflections were assigned to the same "free" and "working" sets as used for
crystal 5.
These data are summarized in Table 10.
Refinement of the Cdc25B(ONBB-c17).cdc1249 Inhibitor Complex Crystal Structure
Refinement of the Cdc25B(~NBB-c17).cdc1249 inhibitor complex structure
against the data collected from crystal 20 began with the "Powe112" structural
coordinates for the Cdc25B(ONBB).cdc1249 inhibitor complex. The cdc1249
molecules were removed from the model, which was then subjected to rigid body,
Powell minimization, and individual temperature factor refinement with X-PLOR.
Electron-density maps showed that both molecules of cdc1249 were in their
expected
locations at the active site and at the crystallographic symmetry site. These
inhibitors
were added to the model. Additional refinement alternating with rebuilding
lowered R
to 22.6% (RfTee 27.4%; /F/ > 1.OaF, 30-2.70 t~). These final structural
coordinates
("Powell2") for the Cdc25B(ONBB-c17).cdc1249 inhibitor complex consist of
Cdc25B residues 370-548, two cdc1249 molecules, 128 water molecules, one Na+
ion,
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and five Cf ions. The structure was extremely similar to that of the
Cdc25B(ON8B).cdc1249 complex. These data are summarized in Table 11.
X-ray Diffraction Data Collection from a Crystal of the Cdc25B(ONBB).cdc1249
Inhibitor Complex Grown in the Presence of cdc1973
A total of 140 data frames (0.5° each) were collected from a
Cdc25B(ONBB).cdc1249 inhibitor complex crystal grown in the presence of
cdc1973
(crystal 21). The crystals were maintained at a temperature of 100 K during
data
collection. The diffraction data were processed and reduced to structure
factor
amplitudes in space group P43212, a = 70.22 ~, c = 131.29 ~. The unique
reflections
were assigned to the same "free"and "working" sets as used for crystal 5.
These data
are summarized in Table 10.
Refinement of the Cdc25B(ONBB).cdc1249 Inhibitor Complex Crystal Structure,
Crystal Grown in the Presence of cdc 1973
Refinement of the Cdc25B(ONBB).cdc1249 inhibitor complex structure
against the data collected from crystal 21 began with the "Powe112" structural
coordinates for the Cdc25B(ON8B).cdc1249 inhibitor complex. The cdc1249
molecules were removed from the model, which was then subjected to rigid body,
Powell minimization, and individual temperature factor refinement with X-PLOR.
Electron-density maps showed that both molecules of cdc1249 were in their
expected
locations at the active site and at the crystallographic symmetry site. These
inhibitors
were added to the model. Additional refinement alternating with rebuilding
lowered R
to 20.5% (Rfree 24.4%; /F/ > l.OaF, 30-2.52 ~). These final structural
coordinates
("Powell2") for the Cdc25B(ON8B).cdc1249 inhibitor complex consist of Cdc25B
residues 369-548, two cdc1249 molecules, 128 water molecules, one Na+ ion, and
five
Cl' ions. There was no evidence for the replacement of cdc1249 by cdc1973 at
either
site. These data are summarized in Table 11.
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Soaking of Cdc25B(DIV8B).cdc1249 Inhibitor Complex Crystals with Other
Inhibitors
Four crystals of the Cdc25B(ONBB).cdc1249 inhibitor complex prepared as
described above were soaked in a buffer containing 4.5 M NaCI, 50 mM sodium
HEPES, pH 7.0, 4°C. The buffer also contained 2 mM cdc1659, cdc1671,
cdc1748,
or cdc1973. After one day, the crystal in the cdc1748 soak had dissolved; the
other
crystals were intact. Similar soaks were set up at room temperature, using
cdc1659,
cdc 1671, or cdc 1973. After four additional days, the crystals were
cryoprotected as
described previously. Additional crystals were soaked in a similar fashion (1
mM
inhibitor) for one day, and then cryoprotected as described previously.
X-ray Diffraction Data Collection from Crystals of the Cdc25B(DIV8B).cdc1249
Inhibitor Complex Soaked in the Presence of cdc1659, cdc1671, or cdc1973
The x-ray diffraction characteristics of the soaked crystals described above
(five days soak time) were examined. The crystals were maintained at a
temperature
1 S of 100 K during data collection. Only the crystal that had been soaked
with 2 mM
cdc 1973 at 4°C diffracted x-rays. A total of 88 data frames
(0.5° each) were collected
from this crystal, crystal 22. The diffraction data were processed and reduced
to
structure factor amplitudes in space group P43212, a = 70.03 ~, c = 131.30 ~.
The
unique reflections were assigned to the same "free" and "working" sets as used
for
crystal S. These data are summarized in Table 10.
Refinement of the Cdc25B(ONBB).cdc1249 Inhibitor Complex Crystal Structure,
Crystal Soaked in the Presence of cdc 1973
Refinement of the Cdc25B(ONBB).cdc1249 inhibitor complex structure
against the data collected from crystal 22 began with the "Powell2" structural
coordinates for the Cdc25B(ONBB).cdc1249 inhibitor complex. The cdc1249 and
water molecules were removed from the model, which was then subjected to rigid
body, Powell minimization, and individual temperature factor refinement with X-
PLOR. Electron-density maps showed that both molecules of cdc1249 were in
their
expected locations at the active site and at the crystallographic symmetry
site. These
inhibitors were added to the model. Additional refinement alternating with
rebuilding
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lowered R to 21.6% (R~e 25.3%; /F/ > 2.06F, 30-3.50 ~). These final structural
coordinates ("Powe113") for the Cdc25B(DIV8B).cdc1249 inhibitor complex
(soaked
with cdc1973) consist of Cdc25B residues 370-548, two cdc1249 molecules, one
Na+
ion, and five Cl- ions. A SigmaA-weighted F° F~ electron-density map
showed no
interpretable difference density for the cdc1249 molecule in the active site.
At the
crystallographic symmetry lattice site, however, a strong negative peak (>3a)
was
centered over the sulfonate moiety of cdc1249, suggesting that some
substitution of
cdc 1973 for cdc 1249 had occurred at this site only. These data are
summarized in
Table 11.
X-ray Diffraction Data Collection from Crystals of the Cdc25B(ONBB).cdc1249
Inhibitor Complex Soaked in the Presence of cdc1659 or cdc1671 at Room
Temperature
The x-ray diffraction characteristics of the soaked crystals described above
(one day soak time at room temperature) were examined using the equipment
described previously. The crystals were maintained at a temperature of 100 K
during
data collection. Only the crystal that had been soaked with 2 mM cdc1659
diffracted
x-rays. A total of 59 data frames (0.75° each) were collected from this
crystal, crystal
23. The diffraction data were processed and reduced to structure factor
amplitudes in
space group P432~2, a = 70.05 ~, c = 129.95 ~. The unique reflections were
assigned
to the same "free" and "working" sets as used for crystal 5. These data are
summarized in Table 10.
Refinement of the Cdc25B(ONBB).cdc1249 Inhibitor Complex Crystal Structure,
Crystal Soaked in the Presence of cdc1659 at Room Temperature
Refinement of the Cdc25B(ONBB).cdc1249 inhibitor complex structure
against the data collected from crystal 24 began with the "Powe112" structural
coordinates for the Cdc25B(ONBB).cdc1249 inhibitor complex. The cdc1249 and
water molecules were removed from the model, which was then subjected to rigid
body and, Powell minimization, and overall temperature factor refinement with
x-
PLOR. Electron-density maps showed that both molecules of cdc 1249 were in
their
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expected locations at the active site and at the crystallographic symmetry
site. These
inhibitors were added to the model. Additional refinement alternating with
rebuilding
lowered R to 24.3% (Rfree 26.3%; /F/ > I.OaF, 30-2.70 ~). These final
structural
coordinates ("Powell2") for the Cdc25B(~1V8B).cdc1249 inhibitor complex
(soaked
with cdc1659) consist of Cdc25B residues 370-548, two cdc1249 molecules, one
Na+
ion, and five Cl- ions. A SigmaA-weighted F° F~ electron-density map
showed no
interpretable difference density for either cdc1249 molecule, suggesting that
no
substitution of cdc1659 for cdc1249 had occurred. These data are summarized in
Table 10.
Table 10 Summary of x-ray diffraction data collection
Crystal Poly-Inhibitor ResolutionUnique CoverageMultipli<1/a,>R5~"
PeptideOr Soak ~. Reflections% * city
1 ON1A NaZW04 1.79 20,146 96.6 5.8 19.4 3.6
(65.2) (3.2)
(3.0)(37.4)
2 ON1A 2 mM cdc1316,1.79 19,644 95.5 3.5 15.0 3.7
(54.5) (1.8)
23 hrs
(2.3)(44.4)
I
2 ~N1A 2 mM cdc1316,1.35 14,015 29.3 1.1 5.8 8.7
(34.1) (1.1)
(NSLS) 23 hrs ( (43.9)
1.0)
3 ONBA NaZW04 2.1 12,117 100 4.8 10.7 8.4
S ( 100) (3.5)
(2.7)(46.5)
I
4 ON8A BASF-32 3.00 3,968 88.5 1.8 5.5 13.0
thawed, (94.6) (1.7)
soaked in (2.2)(30.7)
10 mM
NaZW04,
re-
frozen
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~N1B cdc1249 2.30 15,06999.5 6.2 13.2 9.1
(96.4) (5.6)
(2.8)(55.6)
5 ON1B cdc1249 1.95 23,61697.2 2.6 7.9 7.8
(97.0) (2.6)
(NSLS) (1.9)(46.7)
6 ON1B Cdc1249; 2.50 11,87899.9 6.6 12.5 11.0
70 min (100) (6.7)
soak, 2 (3.4)(51.5)
mM
cdc1316
7 & 8 ~NBB cdc1249 2.00 22,65898.8 6.6 12.7 7.9
(95.4) (4.1)
(2.7)(44.0)
12 ~NBB Cdc1249; 1.53 48,28896.0 3.8 9.9 5.9
3 hr (81.7) (1.8)
(NSLS) soak, 1 (1.3)(49.0)
M
t-BuNHz
13 ONBB Cdc1249; 1.56 45,97798.2 4.4 10.4 6.0
3 hr (85.2) (2.6)
(NSLS) soak, 1 (1.6)(51.6)
M (R)-3-
HO-pyrrolidine
9 ~NBB Cdc1249; 2.45 12,45999.7 3.4 6.3 14.3
3 hr (98.9) (3.8)
soak, ~0.5 (2.3)(44.6)
M 2-
Methyl-1-
propanol
16 ~NBB cdc 1249; 2.16 18,22899.2 6.5 11.1 11.1
3 hr (95.8) (5.8)
soak, 1 (2.9)(52.2)
M
t-BuNHz
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14 ONBB cdc1249; 1.60 38,903 88.2 3.1 8.2 7.9
23 hr (77.8) (1.9)
(NSLS) soak, 1 (2.0)(37.8)
M (R)-3-
HO-pyrrolidine
15 ON1B cdc1671 2.90 7,651 99.5 5.6 8.0 17.9
(98.5) (5.9)
(3.3)(45.6)
11 ~1V8Bcdc1249; 2.30 14,698 95.7 3.7 7.3 12.7
23 hr (89.6) (2.4)
soak, 1 (2.0)(40.8)
M
Imidazole
16 ONBB cdc1659 2.20 17,098 99.9(99.6)4.4(4.5)8.9(2.4)10.0
(52.5)
18 ON1B cdc1885 1.70 21,718 60.0 2.6 8.1 8.6
(14.2) (1.3)
(1.4)(43.3)
19 ONBB cdc 1249 1.45 48,439 82.8 3.7 11.6 6.2
+ (45.2) (
1.4)
cdc1900 (1.7)(35.1)
20 ONBB-cdc1249 2.70 9,521 99.7 4.3 10.7 9.9
(99.9) (4.4)
c17 (3.7)(36.9)
21 ONBB cdc1249 2.52 11,507 98.5 3.6 8.4 10.9
+ (98.6) (3.4)
cdc1973 (3.1)(33.9)
22 ONBB cdc 1249;
soaked
5 days at 3.50 3,963 90.1 3.5 4.7 21.4
4 C w/ (93.1) (3.5)
2 mM cdc1973 (3.6)(28.2)
23 ON8B cdc1249;soaked
1 day at 2.70 8,650 93.3 3.5 8.4 11.5
22 C (98.2) (3.4)
w/ 2 mM (2.8)(38.9)
cdc1659
*Highest resolution shell in parentheses.
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Table 11. Summary of Crystallographic Refinement Statistics
CrystalProtein Inhibitor ResolutionR Rfre
No. Constructor Soak .~ ~/a
1 ON1A Na2W04 1.8 31.7 36.3
2 ~N1A 2 mM cdc1316,1.80 23.0 24.3
23
hrs
3 ON8A NaZW04 2.15 22.8 27.1
4 ON8A Crystal 3 3.00 22.7 25.7
thawed,
soaked in
10 mM
NaZW04, re-frozen
ON 1 Cdc 1249 1.95 21.8 24.4
B
6 ON1B Cdc1249; 2.50 27.4 32.9
70 min
soak, 2 mM
cdc1316
7+8 ON8B cdc 1249 2.00 22.6 24.3
12 DIV8B Cdc 1249; 1.60 24.2 27.7
3 hr soak,
1 M t-BuNHz
13 ON8B cdc1249; 1.56 24.7 27.8
3 hr soak,
1 M (R)-3-HO-
pyrrolidine
9 ON8B cdc1249; 2.45 21.3 25.1
3 hr soak,
~0.5 M 2-Methyl-1-
propanol
~N8B cdc1249; 2.16 22.3 25.6
23 hr
soak, 1 M
t-BuNHz
14 ON8B cdc 1249; 1.60 24.6 27.9
23 hr
soak, 1 M
(R)-3-
HO-pyrrolidine
ON 1 cdc 1671 3.00 20.2 22.8
B
11 ~NBB cdc1249; 2.30 23.2 25.2
23 hr soak,
1 M Imidazole
16 ~NBB cdc 1659 2.20 22.2 25.3
18 ON1B cdc1885 1.70 22.8 25.1
19 ~N8B cdc1249 + 1.45 21.1 23.1
cdc1900
O1V8B-c cdc 1249 2.70 22.6 27.4
17
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21 ~NBB cdc 1249 2.52 20.5 24.4
+ cdc 1973
22 ONBB cdc1249; 3.50 21.6 25.3
soaked 5
days at 4C
w/ 2 mM
cdc1973
23 ON8B cdc 1249; 2.70 24.3 26.3
soaked 1
day at 22Cw/
2 mM
cdc1659
References:
Briinger, A. T. ( 1992) X PLOR Version 3. l, A System for Crystallography and
NMR (Yale
University Press, New Haven, CT).
Collaborative Computational Project, Number 4. (1994). Acta Cryst. D50, 760-
763.
Evans, P.R. (1997). Joint CCP4 and ESF EACBMNewsletter 33, 22-24.
French, S. & Wilson, K. (1978). Acta Cryst. A34, 517-525.
Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjelgaard, M. (1991). Acta Cryst. A47,
110-119.
Kabsch, W. (1993). J. Appl. Cryst. 24,795-800.
Leslie, A.G.W. (1992). CCP4 and ESF EACMB Newsletter on Protein
Crystallography
No. 26.
Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997) Acta Crystallogr. D53,
240 255.
Navaza, J. (1994). Acta Cryst. A50, 157-163.
While this invention has been particularly shown and described with
references to preferred embodiments thereof, it will be understood by those
skilled in
the art that various changes in form and details may be made therein without
departing from the scope of the invention encompassed by the appended claims.
