Note: Descriptions are shown in the official language in which they were submitted.
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METHODS OF STRUCTURE-BASED DRUG DESIGN USING MS/NMR
Background of the Invention
A well established approach for drug discovery is the utilization of a
biological assay to screen a large database of proprietary compounds
(>100,000) to
identify initial leads that effect the activity of target proteins) in the
assay (for
reviews see - J. W. Armstrong, Am. Biotechnol. Lab. 17, 26, 28 ( 1999); J. E.
Gonzalez, P. A. Negulescu, Curr. Opin. Biotechnol. 9, 624-631 ( 1998); K. R.
Oldenburg, Annu. Rep. Med. Chem 33, 301-311 (1998); P. B. Fernandes, Curr.
Opin.
Chem. Biol. 2, 597-603 (1998); B. A. Kenny, M. Bushfield, D. J. Parry-Smith,
S.
Fogarty, J. M. Treherne, Prog. Drug Res 51, 245-269 ( 1998); L. Silverman, R.
Campbell, J. R. Broach, Curr. Opin. Chem. Biol. 2, 397-403 (1998). The
resulting
identification of lead chemical compounds from the high-throughput screening
(HTS)
effort initiates an iterative approach to optimizing the activity of the small
molecules
from feedback obtained from structural and biological activity data. A major
drawback of this method is the typical requirement that the biological assay
be
completely re-designed with the identification of each new protein target.
This
effectively requires a large commitment of resources and time before new drug
discovery projects can be initiated. Besides the difficulty associated with
the design
of a biological assay to properly screen the chemical library for the desired
activity,
there exists a number of other limitations that may hinder the analysis and
utility of
the assay. These are usually a result of the necessary complexity of the assay
to
reasonably mimic the cellular function of the target protein and to monitor
changes in
its activity. It is not uncommon for a biological assay to contain multiple
proteins, to
be a membrane based assay, or to even be a cell based assay. The consequence
of a
complex assay is the ambiguous nature of a positive hit since details of the
chemical
interaction between a target protein and small molecule is not readily
correlated to an
observed biological response. As a result, these assays greatly limit a
structure based
approach to drug optimization while making it extremely difficult to decipher
a
structure-activity relationship (SAR) from the initial chemical leads.
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NMR has been extensively used to evaluate ligand binding with an obvious
utility in structure based drug design (K. Wuthrich, NMR of Proteins and
Nucleic
Acids (John Wiley & Sons, Inc., New York, 1986); G. Otting, Curr. Opin.
Struct.
Biol. 3, 760-8 (1993); P. J. Whittle, T. L. Blundell, Annu. Rev. Biophys.
Biomol.
Struct 23, 349-75 (1994); T. L. Blundell, Nature 384, Suppl.), 23-26 (1996)).
The
"SAR by NMR" method previously described by Hajduk et al. illustrates this
utility
of NMR to screen small molecules for their ability to bind proteins from
observed
chemical shift perturbation in a 2D 'H-'SN-HSQC spectrum (P. J. Hajduk, et
al., J.
Med. Chem. 40, 3144-3150 (1997); P. J. Hajduk, et al., J. Am. Chem. Soc. 119,
5818-
5827 (1997); S. B. Shuker, P. J. Hajduk, R. P. Meadows, S. W. Fesik, Science
274,
1531-1534 (1996)). In addition to determining if the small molecule binds the
protein, the observed chemical shift perturbations also allow for the
identification of
the binding site of the protein. The concept of using NMR as a primary screen
has
some significant obstacles that may limit its use in a high-throughput format.
Mainly,
the relatively low sensitivity of NMR requires significant quantities of
isotope
enriched protein (> 0.2 mM) and data acquisition time (>10 minutes) per sample
which drastically impacts the number of compounds that can be screened (L. E.
Kay,
P. Keifer, T. Saarinen, J. Am. Chem. Soc. 114, 10663-5 (1992); J. Schleucher,
et al.,
J. Biomol. NMR 4, 301-6 ( 1994)). A response to these problems has been the
utilization of mixtures, but this then requires deconvolution of the positive
hits which
incurs a further commitment of sample supply and instrument resources.
Furthermore,
the utilization of mixtures may limit a compound's solubility below the
concentration
required by NMR while further complicating the necessity of maintaining
consistent
buffer conditions (pH, ionic strength) between samples. Additionally, the need
to
optimize the NMR data collection throughput usually results in a compromise
between data quality and acquisition time.
Other attempts to minimize resource and sample requirements have focused
on the application of 1D NMR techniques, particularly diffusion-edited
measurements
and transfer NOES (M. J. Shapiro, J. R. Wareing, Curr. Opin. Drug Discovery
Dev. 2,
396-400 (1999); B. Meyer, T. Weimar, T. Peters, Eur. J. Biochem. 246, 705-709
(1997); M. Lin, M. J. Shapiro, J. R. Wareing, J. Am. Chem. Soc. 119, 5249-5250
(1997); M. Lin, M. J. Shapiro, J. R. Wareing, J. Org. Chem. 62, 8930-8931
(1997);
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P. J. Hajduk, E. T. Olejniczak, S. W. Fesik, J. Am. Chem. Soc. 119, 12257-
12261
( 1997)). The 1 D NMR experiments eliminate the need for labeled protein while
minimizing sample quantities and data acquisition time. Unfortunately, the 1D
NMR
experiments do not provide information on the location of the binding site.
They also
have a lower sensitivity to weak binders compared to the 2D 'H-'SN-HSQC
experiments while requiring a more complicated method for automated data
analysis.
Additionally, the utilization of mixtures is more difficult because of
spectral overlap.
Recently developed NMR cryoprobes and flow-through probes may provide some
solutions to these issues since they may provide a 3-4 fold increase in
sensitivity and
a method of increase throughput, respectively ( M. J. Shapiro, J. R. Wareing,
Curr.
Opin. Drug Discovery Dev. 2, 396-400 ( 1999)). Nevertheless, NMR may not be
ideal
for the initial stage of the screening process since typical NMR experiments
are time
consuming and resource intensive. Given the observation that most assays have
a hit
rate on the order of 0.1 to 1 % which means that >99% of the data collected is
negative
information, it appears to be a more logical approach to eliminate a majority
of the
compounds before the NMR analysis stage.
A new, rapid approach to drug design is provided by the present invention and
provides the details useful for structure based drug design, combined with the
capability to screen very small quantities of multiple compounds rapidly and
accurately.
Summary of the Invention
The present invention provides a method of screening a compound mixture to
identify compounds which bind to a target molecule by preparing a mixture of
compounds, each compound having a known molecular weight, and incubating the
mixture with target molecule to allow formation of bound compound-target
complex.
Mass spectral analysis is performed to determine the identity of bound
compound
based upon molecular weight. A complex of identified compound bound to target
molecule is prepared and the NMR chemical shift perturbation of the complex of
identified compound bound to target molecule is analyzed to identify the
location of
the binding site of compound on target molecule. Using the NMR data, a
molecular
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model can be prepared and computer assisted drug design can be used to design
high
affinity ligands for the target molecule.
The present invention also provides methods of designing a ligand having
improved affinity for a target molecule comprising preparing a mixture of
compounds
having known molecular weights and incubating the mixture with target molecule
to
allow formation of bound compound-target complex. The compound-target complex
is separated from unbound compound and mass spectral analysis is performed on
compound-target complex to determine the identity of bound compound based upon
molecular weight. A complex of identified compound bound to target molecule is
prepared and NMR is performed. The NMR shift perturbation of the complex of
identified compound bound to target molecule is analyzed to identify the
binding site
of the compound on the target molecule and a library of structural analogs
having
known molecular weights is designed based upon the chemical structure of the
identified compound and the identified binding site of the target molecule.
The library
of structural analogs is prepared and binding of the structural analogs to the
target
molecule is determined.
Further in accordance with the present invention is provided a method of
designing a high affinity ligand for a target molecule by preparing a mixture
of
compounds, each compound having a known molecular weight, and incubating the
mixture with target molecule to allow formation of bound compound-target
complex.
Mass spectral analysis is performed to identify bound compound. Complexes of
identified compounds bound to target molecule are prepared and the NMR shift
perturbation of complexes of identified compound bound to target molecule are
analyzed to identify at least two compounds having at least two different
binding sites
on the target molecule. The spatial orientation of the compounds on the target
molecule is determined and the structural information of at least two
identified
compounds are used to design a ligand which binds at the identified sites and
minimally affects the determined spatial orientation. Linking may be by
molecular
modeling or by chemical linkage.
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Brief Description of the Figures
Figure 1 is a ESI mass spectral analysis of filtrate after passing MMP-1
inhibitors through Sephedex G-25 columns in the presence and absence of MMP-1.
(A) 45 ~M compound 1 (MW 393) and 45 E.tM MMP-1, (B) 45 ~M compound 1
alone, (C) 250 ~,M compound 2 (MW 457) and SO ~M MMP-1, (D) 250 ~.M
compound 2 alone, (E) 8 mM compound 3 (MW 394) and 0.4 mM MMP-1, (F)
8 mM compound 3 alone.
Figure 2 is an ESI (positive ionization) mass spectral analysis of the
filtrate
from the gel-filtration titration of compound 2 (MW 457) with MMP-1 (A) MMP-1
alone at 50 ~.M; (B-E) increasing amount of MMP-1 (B) 20 ~t.M, (C) 30 ~.M, (D)
40 ~.M and (E) 50 ~M and increasing amount of compound 2 from (B) 100 ~,M, (C)
150 ~M, (D) 200 ~,M and (E) 250 ~,M; and (F) 250 ~.M compound 2 alone .
Figure 3 is an ESI (negative ionization) mass spectral analysis of the
filtrate
from the gel-filtration analysis of a mixture containing 1 mM each of ten
known
MMP-1 inhibitors (TOP) with 0.1 mM MMP-1 and (BOTTOM) without MMP-1.
The mass ions for the ten compounds are highlighted on the spectrum. The
mixture is
composed of compounds 4-13 listed in Table 1.
Figures 4A-C are 2D 'H-'SN HSQC spectra of free MMP-1 (multiple contours)
overlayed with MMP-1 complexed with (A) compound 1, (B) compound 2 and (C)
compound 3 (1-2 contours) identified as binders from the gel-filtration/mass
spectral
analysis (Figure 1).
Figure 5 (A) A GRASP(32) surface of the NMR solution structure of MMP-1
where residues that incurred a perturbation in the 'H-'SN HSQC spectrum in the
MMP-l:compound 1 complex are colored black, indicating the location of the
ligand
interaction with the protein.
Figure 5(B) NMR structure of the MMP-l:compound 1 complex. Compound
1 is shown with thicker bonds.
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Detailed Description of the Invention
The present invention provides a method of screening compounds to identify
compounds which specifically bind to a target molecule and to identify the
site of
binding. This invention also provides a fast and efficient method of designing
ligands
for a given target molecule.
In accordance with methods of the present invention, mixtures of ligands or
compounds such as small molecules are prepared. The ligands may be for
example,
from commercial sources, from preexisting chemical libraries, or prepared
according
to need, such as based upon previous structure activity relationship
information. Each
mixture is comprised of a group of ligands, each having a known molecular
weight. In
some preferred embodiments of the present invention each ligand has a unique
molecular weight which preferably differs from other ligands of the mixture by
more
than 3Da to allow for clear identification of each component. In some aspects
of the
invention, the molecular weight of each ligand is preferably less than about
2000, and
where linkage of one or more compounds is anticipated, the molecular weight
may be
more preferably less than about 350. In addition to molecular weight, ligands
may be
chosen based upon, for example, acidity, reactivity, shape and functional
groups of
the compounds. Diversity of libraries is generally preferred. Ligand
concentration
will vary depending upon the number of ligands forming the mixture. In general
the
compound mixture comprises at least about 0.1 nM of each compound to be
screened,
and more preferably at least about 1 nM of each compound.
The compound mixture is incubated with a target molecule (such as a protein,
nucleic acid, etc.). Target molecule may be obtained from commercial sources,
may
be purified from natural sources or may be prepared recombinantly. In general,
the
incubation mixture contains at least about 10 ~,M of target molecule and
preferably
from about 50 ~,M to about 200 ~M and most preferably about 100 ~,M.
Complexes of bound compound-target molecule are separated from unbound
compounds by running the mixture through a size exclusion column such as by
suction filtration or centrifugation. Such chromatography techniques such as
gel
permeation chromatography (GPC) spin column are described in J. Mass. Spect.
33:
264-273 (1998) which is incorporated by reference herein. Size exclusion
chromatography is based upon the premise that low molecular weight compounds
are
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retained in the column, and high molecular weight compounds are passed through
the
column. Thus, compounds which elute from the column should have bound to the
target molecule and are thus highly likely to be active in a biological assay
involving
the target.
A compound from the mixture may be easily identified once bound to a target
molecule, on the basis of its molecular weight as determined by mass
spectrometry
which is performed on the filtrate in the molecular weight range for the
compounds in
the mixture. Since the molecular weights are known for each compound in the
mixture, the observation of an ion peak in the mass spectrometer
simultaneously
identifies the presence of a hit and the compound identity. In preferred
embodiments
of the present invention, each of the compounds of the mixture has a unique
molecular weight. A target-specific assay to identify candidates from a
mixture is
avoided allowing for easy automation. In addition, deconvolution is generally
avoided. Where deconvolution is necessary such as when the molecular weight of
a
hit corresponds to more than one compound of the mixture or fragment thereof,
it is
generally of limited scope and can be rapidly carried out.
In some preferred embodiments of the present invention, the size exclusion
column can be prepared with any size-exclusion resin such as Sephadex G25
resin
(Pharmacia) that allows large molecular weight compounds to pass through the
column while retaining smaller molecular weight compounds (such as those less
than
2000 MW). The resin can be packed into individual columns prepared with, for
instance, disposable syringes or, more preferably a 96-well filtration plate
containing
a low-protein-binding filter such as hydrophilic durapore filter or silanized
glasswool.
The small column length of the 96-well plates minimizes sample requirements
and
because of the high-sensitivity of MS only picomoles of the target protein are
required for each sample. The protein-compound mixture can be loaded onto the
size-
exclusion column under a number of conditions, where the buffer conditions,
number
of compounds in the mixture and the protein-compound molar ratios may be
varied.
The filtrate from the column is collected in a standard 96-well plate by
either
centrifugation or suction filtration of the resin-filled 96-well filtration
plate. The
technique is sensitive to weak protein-drug interactions.
Mass spectral analysis may be performed on the mixture without separating
bound and unbound compound. Mass spectral analysis is performed such as with
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_g_
electrospray ionization (ESI) MS methods in both positive and negative
ionization
modes. Background noise is differentiated from unique molecular ion peaks and
the
molecular weight leading to the identity of bound compounds, is determined
based
upon the difference between the weight of the target molecule and the weight
of any
complex which correlates to a peak corresponding to a unique chemical entity.
Alternatively, matrix assisted laser desorption/ionization MALDI/MS can be
used.
These steps can be easily automated using robotics. For instance, a Gilson
215 liquid handler may be used to transfer the filtrate from the 96-well
plates to the
mass spectrometer.
Once the identity of a compound (ligand) which binds to a target is known, the
specific binding site may be determined using NMR spectroscopy, for instance,
by
mapping NMR chemical shift perturbations onto the structure of the target. The
three
dimensional structure of the target may be obtained from standard X-ray, NMR
or
homology modeling techniques and the NMR resonance assignments from standard
NMR protocols. The chemical shift perturbations may be obtained by comparing
the
NMR spectra of the free target with the NMR spectra of the target complexed
with
the identified ligand, where the NMR spectra may correspond to standard 2D 'H-
'SN
HSQC, 2D 'H-'3C HSQC, 2D 'H-'SN HMQC or 2D 'H-'3C HMQC experiments using
either 'SN-enriched or "C/'SN-enriched proteins or targets. The observed NMR
resonances for the target that exhibit a chemical shift perturbations in the
presence of
the ligand are assigned to a residue in the target by utilizing the NMR
resonance
assignments for the free target. The residues in the target that experience
chemical
shift perturbations in the presence of the ligand are then mapped onto the
structure of
the target to define the binding site of the ligand on the target. Any
enriched target
molecule may be used, and preferably polypeptides serve as the target. The
target
molecule can be labeled with '3C or '5N using methods known in the art. In
preferred
embodiments the target molecule is prepared in recombinant form using
transformed
host cells. The "SAR by NMR" procedure utilizing NMR-chemical shift
perturbation
and linking of molecular fragments for drug design has been disclosed in
international
patent application publication Number WO 97/18469 and WO 97/18471; and
published in Science 274:1531-1534 (1996); JACS 119:5818-5827 (1997) and J.
Med. Chem.40:3144-3150 ( 1997).
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A preferred means of preparing adequate quantities of uniformly labeled
polypeptides is to transform a host cell with an expression vector that
contains a
polynucleotide that encodes the polypeptide and culture the transformed cells
in a
medium that contains assimilable sources of radiolabel. Such sources are well
known
in the art. For instance, 'SNH4Cl, '3C Glucose or ('SNHQ)ZS04 may be used.
Means for preparing expression vectors that contain polynucleotides encoding
specific polypeptides are well known in the art, as are means for transforming
host
cells with vectors and culturing those transformed cells so that the
polypeptide is
expressed.
Given the protein and compound structure and the general location of the
compound binding site from the NMR chemical shift perturbations, standard
modeling techniques are applied to define a computer model of the complex. The
resulting computer model of the complex may be verified by consistency between
predicted short (< 5 ~) hydrogen pair distances and NOEs observed in NMR
spectra
of the complex and/or X-ray structures of the complex.
The affinity of the compound for the protein (Kd and/or IC50) can be
determined from a variety of accepted techniques which may include Kd
measurements from NMR diffusion coefficient changes or chemical shift
perturbations and/or IC50 determination from a specific biological assay for
the
protein target to determine biological relevance of the hit.
If more than one ligand having a unique binding site is identified, the three
dimensional structure and spatial orientation of the ligands in relation to
the target, as
well as in relation to each other may be determined. Spatial orientation of
each ligand
to the target molecule allows for identification of portions of the ligand
which are in
close proximity to the atoms in the target, as well as portions which are
distal from
atoms in the binding site and which may be involved in interactions with other
molecules in situ.
Once the specific binding site has been identified, three dimensional models
may be generated using any one of a number of methods known in the art, and
include, but are not limited to, homology modeling as well as computer
analysis of
raw structural coordinate data generated using crystallographic or
spectroscopy
techniques. Computer programs used to generate such three dimensional models
and/or perform the necessary fitting analysis include, but are not limited to:
GRID
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(Oxford University, Oxford, UK), MCSS (Molecular Simulations, San Diego, CA),
AUTODOCK (Scripps Research Institute, La Jolla, CA), DOCK (University of
California, San Franscisco, CA), F1o99 (Thistlesoft, Morris Township, NJ),
Ludi
(Molecular Simulations, San Diego, CA), QUANTA (Molecular Simulations, San
Diego, CA), Insight (Molecular Simulations, San Diego, CA), SYBYL (TRIPOS,
Inc., St. Louis, MO), and LEAPFROG (TRIPS, Inc., St. Louis, MO).
These and other computer programs will be well known to those of ordinary
skill in the art. Once the relevant data has been analyzed by such programs,
candidate
ligands can be identified, prepared and tested for their ability to bind to a
target and
for its biological activity.
Identified ligands which bind to the target molecule may then be tested in
biological systems to confirm that biological activity correlates with the
observed
binding. In traditional systems, IC50 values are obtained for each ligand from
the
biological assay that provides an initial ranking of the effectiveness of the
chemical
leads. As a follow up Kd values might be obtained from NMR titration data or a
variety of other analytical techniques. The present invention inverts these
typical
steps, thereby eliminating the need to convert a standard biological assay to
a high
throughput format. Rather, the number of leads is reduced so that the standard
assay
need not be converted.
Following verification of biological activity, a refined structure of the
protein-
ligand complex may be elucidated by NMR, X-ray and/or modeling.
Further, a library of structural analogs may be prepared based upon the
initial
lead or leads, and tested for binding in accordance with the present
invention, thereby
further optimizing the affinity and activity of the ligand. For instance, a
lead
compound may be derivatized at one or more positions in the molecule based
upon
points of interaction at the binding site in accordance with known chemical
principals
to provide structural analogs. Combinatorial syntheses may be particularly
useful for
these purposes. In addition, where more than one ligand having unique binding
sites
is identified, the spatial orientation of the ligands with the binding site
can be used to
design new high affinity ligands. New ligands can be designed by modeling
techniques or by chemical linkage of two compounds. In this way two or more
compounds having a given affinity for a target may be linked resulting in a
compound
with improved affinity for a target. The design of a linker is based on the
distances
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and angular orientation needed to maintain each of the ligands in proper
orientation to
the target. Suitable linkers are well known and can easily be identified by
those
skilled in the art. J. of Computer Aided Molecular Design 6:61-78 (1992),
Perspectives in Drug Discovery and Design 3:21-33 ( 1995), J. Med. Chem.
27(5),
557-563 (1984), Science 263:380-384 (1994).
The following examples are meant to illustrate the effectiveness of methods of
the present invention by employing compounds previously tested against a given
target, MMP-1. The examples are not meant to be limiting of the present
invention.
EXAMPLES
Example 1
Compounds having known affinities for MMP-1 were chosen.
The compounds are provided in Table 1.
Table 1 Inhibitors of MMP-1.
CompoundChemical Name IC50
Number (nM
1 N2-(4-Methoxy-benzenesulfonyl)-N2-[(pyridin-3-yl)-9 393
meth 1]-N-h droX -D-valinamide
2 N-Hydroxy-2-[(4-methoxy-benzenesulfonyl)-pyridin-3-9900 457
lmeth 1-amino]-iso hthalamic acid
3 [(2-Hydroxycarbamoyl-6-methylphenyl)-(4-methoxy-8900 394
benzenesulfon 1)-amino -acetic acid 0
4 2-[Benzyl-(4-methoxy-benzenesulfonyl)-amino]-N-408 426
h drox -5-meth 1-benzamide
5 8-Methoxy-4-[(4-methoxy-benzenesulfonyl)-pyridin-3-46 494
ylmethyl-amino]-quinoline-3-carboxylic
acid
h drox amide
6 N-Hydroxy-2-(4-methoxy-benzenesulfonyl)-2-methyl-139 399
3-na hthalen-2- 1- ro ionamide
7 2-(4-Methoxy-benzenesulfonyl)-5-methyl-2-pyridin-3-760 407
lmeth 1-hexanoic acid h drox amide
8 4-(Methyl-[4-(pyridin-4-yloxy)benzenesulfonyl]-1012 450
amino)- uinoline-3-carbox lic acid h
drox amide
9 1-(Furan-2-carbonyl)-4-(4-methoxybenzenesulfonyl)-17 471
2,3,4,5-tetrahydro-1H-[ 1,4]-benzodiazepine-3-
carbox lic acid h drox amide
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CompoundChemical Name IC50 Mw
Number (nM
4-(4-Butoxy-benzenesulfonyl)-1-methyl-piperidine-4-3417 370
carbox lic acid h drox amide
11 5-Bromo-N-hydroxy-3-methyl-2-[methyl(naphthalene-1095 449
2-sulfon 1 -amino]-benzamide
12 4-(4-Butoxy-benzenesulfonyl)-1-ethyl-piperdine-4-7062 384
carbox lic acid h drox amide
13 3-[4-(2-Azepan-1-yl-ethoxy)-phenyl]-N-hydroxy-2-(4-540 491
methox -benzenesulfon 1)-2- ro ionamide
Example 2
Protein-single compound incubation
5 1 mM each of compounds 1, 2 and 3 (Table 1) were dissolved in DMSO and each
incubated alone or in the presence of MMP-1 at a 0.1 mM in a buffer consisting
of 20
mM Tris, 100 mM NaCI, 5 mM CaCl2, 0.1 mM ZnClz, 2 mM NaN3 and 3.5 mM
DTT at pH 6.5 at room temperature for 30 minutes. The final concentration of
DMSO
in the MMP-l:compound mixture was 5%. A total volume of 25 ~1 of each sample
10 was loaded on a Sephedex G25 column in a Millipore multiscreen filtration
system
composed of a 0.65 ~m hydrophilic durapore filter. The samples were eluted
using
centrifugation (15,OOOxg for 3 minutes). Samples were collected and analyzed
by
mass spectroscopy using automated ESI/MS methods in both positive and negative
ionization modes with a Micromass LCT quadrapole time of flight mass
spectrometer
and Quattro I triple-quadrapole mass spectrometer each equipped with a Gilson
215
liquid handler. Results in Figure 1 show that unbound compound is retained
(Figures
1B (Compound 1), 1D (Compound 2) and 1F (Compound 3)), while compound bound
to MMP-1 is eluted (Figures 1A (Compound 1+MMP-1), 1C (Compound 2+MMP-1)
and 1E (Compound 3+MMP-1)).
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Example 3
Titration
Increasing amounts of Compound 2 were incubated alone or with increasing
amounts of MMP-1 as described in Example 2. Figure 2A-E provide ESI (positive
ionization) mass spectral analysis of these filtrates.
Fi Com ound MMP-1
ure
2A 0 50
2B 100uM 20uM
2C 150uM 30uM
2D 200uM 40uM
2E 250uM SOuM
2F 250uM
I
Figure 2 shows that the relative intensity of the [M+H]'+ (m/z) 457.9 ion
correlates
with the increase in MMP-1 concentration.
Example 4
Protein-mixture incubation
A mixture of ten compounds described in Example 1 are provided at an
approximate concentration of 1 mM each was dissolved in DMSO. The ligand
mixture was incubated alone or with MMP-1 at a concentration of 0.1 mM in a
buffer
consisting of 20 mM Tris, 100 mM NaCI, 5 mM CaCl2, 0.1 mM ZnCl2, 2 mM NaN3
and 3.5 mM DTT at pH 6.5 at room temperature for 30 minutes. The final
concentration of DMSO in the MMP-l:compound mixture was 5%.
Example 5
Gel Filtration/Mass Spectroscopy collection of samples
A total volume of 25 ~1 of the MMP-1-compound mixture is loaded on a
Sephedex G25 column in a Millipore multiscreen filtration system composed of a
0.65
pm hydrophilic durapore filter. The samples were eluted using centrifugation
(15,OOOxg for 3 minutes). Samples were collected and analyzed by mass
spectroscopy
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using automated ESI/MS methods in both positive and negative ionization modes
with
a Micromass LCT quadrapole time of flight mass spectrometer and Quattro I
triple
quadrupole mass spectrometer each equipped with a Gilson 215 liquid handler.
Results are shown in Figures 3A (with MMP-1) and B (without MMP-1). Mass ions
for the ten compounds are highlighted on the spectra.
Example 6
NMR Analysis of MS Hits
MMP-1 was labeled as described in Moy, J. Biomol. NMR, Vol. 10: 9-19
(1997). Compounds 1, 2 and 3 were selected from Example 5. The gradient
enhanced
2D 'H-'SN HSQC spectra were collected on a 0.2 mM 'SN-MMP-1 in a buffer
consisting of 20 mM Tris, 100 mM NaCI, 5 mM CaCl2, 0.1 mM ZnCl2, 2 mM NaN3
and 3.5 mM DTT in 90% H20 and 10 % DZO at pH 6.5 and 35°C with
compounds
titrated to achieve concentrations of compound 1, 2 and 3 ranged from 0.2 -4.0
mM.
The 2D'H-'SN HSQC spectra were recorded with 256 complex points in t1, 2048
real
points in t2, and 192 scans per increment. Spectra windows for t1 and t2 were
1723.7
and 8064.5 Hz, respectively, with the carrier at 4.75 and 115.2 ppm,
respectively.
Data were processed and analyzed using NMRPipe, NMRWish [F. Delaglio, S.
Grzesiek, G. W. Vuister, G. Zhu, J. Pfeifer, and A. Bax J. Biomol. NMR 6, 277
(1995). ] and PIPP [ D. S. Garrett, R. Powers, A. M. Gronenborn, and G. M.
CloreJ.
Magn. Reson. 95, 214-20 (1991).] on a Sun Ultra 10 workstation. Figure 4
provides
the spectra of free MMP-1 (multiple contours) overlayed with MMP-1 complexed
with Compound 1 (Figure 4A), Compound 2 (Figure 4B) and Compound 3 (Figure
4C) ( 1-2 contours). All three compounds induce chemical shift perturbations
for
residues in the vicinity of the catalytic Zn and S 1' pocket in the MMP-1
active site.
Particularly, residues 80-83, 114-119 and 136-142 exhibited the largest
chemical shift
changes in the presence of the inhibitors. The extent of the chemical shift
perturbations and the number of residues exhibiting the chemical shift change
is
directly related to the observed IC50 for each of the compounds. (Figure 4A,
4B, 4C),
i.e. stronger binding contributes to greater perturbations and weaker binding
to less
perturbations.
CA 02401014 2002-08-20
WO 01/62688 PCT/USO1/05495
-15-
Example 7
Modeling
Using computer modeling, a GRASP surface of the NMR solution structure of
MMP-1 was designed (Figure SA) where residues that incurred a perturbation in
the
spectra from Example 7 in the MMP-l:Compound 1 complex are colored blue,
indicating the location of the ligand interaction with the protein. An NMR
structure is
designed of the MMP-l:Compound 1 complex. (Figure SB). Compound 1 is shown
with thicker bonds.
