Note: Descriptions are shown in the official language in which they were submitted.
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PEPTIDES AND PEPTIDOMIMETICS INHIBITING THE ONCOGENIC ACTION
OF P21 RAS
Field of the Invention
This invention relates to peptides effective in inhibiting
oncogenesis, particularly as related to inhibition of p21 ras and
adenocarcinomas of the colon, pancreatic carcinomas,
neuroblastomas, and other cancers which express the transformed
sequence of the ras gene product.
Backqround of the Invention
ras protooncogenes are activated by characteristic point
mutations in a wide variety of malignancies. The expressed p21
ras proteins are oncogenic by virtue of single substituted amino
acids, usually at position 12 or 61 of the 189-residue p21 ras
gene product. ras proteins act as membrane-associated molecular
switches that bind GTP and GDP and slowly hydrolyze GTP to GDP.
Mutations in ras are associated with the vast majority of
adenocarcinomas of the colon. Cancer of the colon is a highly
treatable and often curable disease when it remains localized to
the bowel. It is the second most frequently diagnosed malignancy
ln the United States as well as the second most common cause of
cancer death. Surgery is the primary treatment and results in
cure in approximately 50~ of patients. Adenocarcinoma is the
primary lesion in the majority of cases. Recurrence following
surgery is a major problem and often is the ultimate cause of
death. The prognosis for colon cancer patients is clearly
related to the degree of penetration of the tumor through the
bowel wall and the presence or absence of nodal involvement. For
locally advanced disease, the role of radiation therapy in colon
cancer is under clinical evaluation. There is no standard~ 30 therapy for advanced colon cancer and no evidence that
chemotherapy improves survival, although short-term palliation
may be achieved in approximately 10-20~ of patients.
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Pancreatic carcinoma has a high incidence of K-ras
mutations. Mutated K-ras sequences which can be identified by
polymerase chain reaction utilizing allele-specific primers can
even be found in the plasma or serum from patients with
pancreatic carcinoma. The c-Ki-ras oncogene is activated by
point mutations involving codon 12 in 72~-100~ of primary
pancreatic adenocarcinomas, but the gene is not activated in
nonneoplastic tissues. Cancer of the exocrine pancreas is rarely
curable. The highest cure rate (4~-12~) occurs if the tumor is
truly localized to the pancreas. Unfortunately, this stage of
disease accounts for fewer than 20~ of cases and, even with
surgical resection, results in little more than a 5~ 5-year
survival rate. For small cancers (less than 2 cm) in the head
of the pancreas with no lymph node metastases and no extension
beyond the "capsule" o~ the pancreas, the survival rate following
resection of the head of the pancreas approaches 20~. Overall
survival rate of all stages is less than 2~ at 5 years with most
patients dying within one year. Worldwide, very few patients
with cancers of the pancreatic tail or uncinate process have been
cured.
Lung cancers also frequently involve ras mutations. Point
mutations in codon 12 of the K-ras protooncogene occur more
frequently in lung adenocarcinomas from smokers (30~) than they
do in lung adenocarcinomas from nonsmokers (7~), suggesting that
smoking is an important factor in the induction of these
mutations. The ras oncogene may thus be a specific target of the
mutagenic activity of tobacco smoke, and suggest that DNA
alterations at this site can occur early and irreversibly during
the development of adenocarcinomas of the lung.
Mutations in the ras protooncogenes are the most frequently
observed molecular alteration in acute myeloid leukemia (AML).
Whether ras mutations occur as late or relatively early events
in the multistep process of myeloid transformation, remains an
open question. There is significant evidence that the ras
oncogene plays a role in experimental m~mm~y carcinogenesis; the
evidence in human breast cancer, however, is more limited.
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Similarly, there is significant evidence that the ras
oncogene plays a role in nitrosoamine-induced esophageal tumors
- in rats, but in human esophageal cancers ras gene mutations are
more rarely found. However, it is probable that there is a
~ 5 significant role of mutated ras genes in both cell proliferation
and malignant transformation of human esophageal cells.
Certain human neuroblastomas also show a high incidence of
oncogenic ras mutations. Indeed, one study suggested that
expressions of the oncogene N-myc and p21 together as detected
by immunohistochemical staining could be among the most reliable
prognostic indicators in neuroblastoma patients.
The ras proteins are key regulators of the growth of
eukaryotic cells. Some of the direct targets are unknown. These
target proteins include raf-1, qap, phosphatidylinositol-3-
hydroxykinase and, very recently, two nuclear proteins, C-JUN and
its kinase (JNK). The three-dimensional x-ray crystal structure
for a ras-related protein bound to a domain of raf-1 has been
elucidated. The ras-related protein (rak-1-a) binds to raf
directly, utilizing residues contained in a sequence involving
amino acids 35-37. All o~ the contact residues in the ras-
related protein are homologous to those in the corresponding
segment of ras-p-21. One of the inventors has shown that the p-
21 ras protein (35-47 segment) selectively inhibits the mitogenic
effects of oncogenic ras-p-21.
In addition to its role as an oncogene, the activation of
ras proteins is a key step in the signal transduction pathways
triggered by ligand-bound cell surface receptors, such as the
insulin receptor.
The classical target of the ras protein is the GTPase
activating protein GAP. This target protein is thought to play
an essential role in the regulation of ras activity by increasing
the GTPase activity of wild type, but not transformed ras. On
the other hand, there is a considerable super~amily of these GAP-
related proteins, which includes pl20-GAP. Other target proteins
besides mammalian gap itself include (1) IRA1 and IRA2, the
functional equivalents of GAP in yeast. They regulate the
ras-cyclic AMP pathway, controlling cell growth; (2) sarl, the
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fission yeast protein that regulates rasl in that organism; (3)
BUD2, a yeast protein that activates BUD1/RSR1 which participates
in the regulation of bud-site selection; (4) Human
neurofibromitosis (gene NF1). NFl is associated with type 1
neurofibromatosis, one of the most frequently inherited genetic
diseases characterized, in part, by multiple neural tumors. NF1
has been shown genetically and biochemically to interact with
and stimulate the GTPase activity of ras; (5) Drosophila Gapl,
which acts as a negative regulator of signalling by the Sevenless
(SOS) receptor tyrosine kinase involved in eye development.
Human SOS1 and SOS2 genes have also been recently identified
which encode proteins that control GDP--~GTP exchange on ras
proteins and are involved in signal transduction by tyrosine
kinase receptors. In si tu hybridization shows that SOS1 maps to
2p22-->pl6 and SOS2 to 14q21-->q22 in the human genome.
Another important target of ras is raf. The protein encoded
by the c-raf-1 protooncogene is thought to function downstream
of p21 ras because disruption of ra~ blocks signalling by ras in
a number of systems. A highly-conserved 81 residue region o~ the
N-terminus of raf protein has been to be shown to be critical as
the ras protein interaction region. Importantly, the raf gene
product interacts with both wild-type and activated ras protein.
In one study, approximately 50~ of the clones identified as
interacting with ras were encoded portions of the c-raf and A-raf
serine/threonine kinases. Thus, ras and the N-terminal region
of raf protein associate directly in vi tro and this interaction
is dependent on GTP bound to ras.
Within the superfamily of ras-related GTP-binding proteins,
only the ras protein itself has been shown to act as an oncogenic
protein. Many other proteins, however, have substantial amino
acid homology to ras. This ras superfamily of GTP-binding
proteins (, 50 members) regulates a diverse spectrum of
intracellular processes. These include cellular proliferation
and differentiation, intracellular vesicular tra~ficking,
cytoskeletal control, NADPH oxidase function, as well as others.
Some of these homologs may have biological activities which are
related to ras. For example, rhoA encodes a ras-related
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GTP-binding protein that was thought principally to play a role
in cytoskeletal organization. Recent evidence, however, has
- suggested both that rhoA could act either as a dominant oncogene,
since transfection of both normal and activated rho genes confer
a transformed phenotype on fibroblast cells in culture, or as a
recessive tumor suppressor gene, by virtue, in part, of its
chromosomal location at 3p21, a site deleted in many human
malignancies. Thus, it is important to consider these ras
homologs as potentially involved in cell growth and
transformation.
Azatyrosine strongly inhibits oncogenic ras-p-21. This
small molecule induces the EE~ gene, which encodes a proteinase
sequence showing 90~ amino acid sequence identity to lysyl
oxidase.
To acquire transforming potential, the precursor of the ras
oncoprotein must undergo farnesylation or similar modification
of the cysteine residue located in a carboxyl-terminal
tetrapeptide. These C-terminal lipid modifications are essential
for the interaction of ras-related proteins with membranes.
While all ras proteins are farnesylated and some palmitoylated,
the majority of other ras-related proteins are
geranylgeranylated. Thus selective peptide and peptidomimetic
inhibitors of ras lipidation have found potential utility as
anti-oncogenic agents.
In view of the foregoing, there is there a long~elt need in
the art for agents which inhibit the transforming ability of ras.
As described above, selective peptide and peptidomimetic
inhibitors or ras lipidation have found potential utility as
anti-oncogenic agents (Kohl et al. (1993) Science 260:1934-1937;
James et al. (1993) Science 260:1937-1942). Similarly, FR
patents 2694296 and 2690162 teach that peptides derived from the
GAP protein may serve to inhibit ras. However, neither '694296
nor '690162 describes peptides derived from the ras protein
itself. EP 203587 describes new ras oncogene polypeptides which
are used ~or producing antibodies for i~munogenic assays.
However, these sequences are derived from ras and its homologs
in the carboxyl terminal domain (residues 170 - 189 in SEQ ID
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NO:5) and are thus physically distant ~rom and completely
unrelated to any sequences claimed herein. Furthermore, these
sequences were claimed for the production o~ antibodies,
preferably by linking to an immunogenic carrier, and a claim for
direct therapeutic application was not made.
Thus, peptides constructed ~rom ~ and its homologs for
therapeutic application, namely by interfering with downstream
or upstream actions of ~ itsel~, are useful. Furthermore, the
method of identification o~ said peptides utilizing calculational
approaches is believed novel and has unexpectedly led us to these
cyclic peptides and peptidomimetics disclosed herein.
Sl~m~ry of the Inve~t;on
The present invention provides peptides, cyclized peptides
and peptidomimetics capable of inhibiting the oncogenic action
of p21 La~. The oncogenic ~-inhibiting cyclized peptides
correspond to domains of the oncogenic ~ protein which are most
flexible and important in ~interacting with target proteins
upstream and downstream from ~. The peptidomimetics are
obtained by molecular modeling, including the structural
minimization techniques o~ molecular dynamics.
The peptides are designated by the ~ormulas: Val-Val-Ile,
Lys-Arg-Val, Ile-Lys-Arg-Val-Lys-Asp (SEQ ID NO:1), Lys-Cys-Asp-
Leu-Ala (SEQ ID NO:2), Cys-Asp-Leu-Ala-Ala-Arg-Thr (SEQ ID NO:3),
Asp-Leu-Ala-Ala (SEQ ID NO:4) or physiologically acceptable salts
of the ~oregoing peptides.
Also provided in the present invention are cyclic analogues
of the above peptides and certain peptides and cyclic peptides:
cyclo [- R(1) R(2) Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val
Val Ile Asp R(3) R(4)-] (I);
cyclo [-R(1) R(2) Val Val Ile R(3) R(4)-] (II);
cyclo [-R(1) R(2) Tyr Arg Glu Gln Ile Lys Arg Val Lys Asp
Ser Asp Asp Val Pro R(3) R(4)-] (III);
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cyclo [-R(l) R(2) Lys Arg Val R(3) R(4)-] (IV);
~ cyclo [-R(1) R(2) Ile Lys Arg Val Lys Asp R(3) R(4)-] (V);
~ cyclo [-R(1) R(2) Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr
Val Glu R(3) R(4)-] (VI);
cyclo [-R(l) R(2) Lys Cys Asp Leu Ala R(3) R(4)-] (VII);
cyclo [-R(l) R(2) Cys Asp Leu Ala Ala Arg Thr R(3) R(4)]
(VIII);
cyclo [-R(1) R(2) Asp Leu Ala Ala R(3) R(4)-] (IX); and
NH2
O Asp
H2N-Thr-Ile-Glu-Asp-Ser-Tyr-Arg-Lys-Gln-NH-CH2-C-Val-NH-CH (X)
CH2 CH- 2
or physiologically acceptable salts thereof.
In cyclized peptide formulas (I)-(IX), R(1) R(2), R(3) and
R(4) represent, in the most general case, any amino acid which
can serve as an amino acid residue linker. Amino acid residue
linkers are usually at least one residue and can be most often
two to four residues, more often 1 to 10 residues, both ranges
being inclusive. Typical amino acid residues useful for linking
are tyrosine, cysteine, lysine, and glutamic and aspartic acid.
Most preferably [R(1), R(2)] and [R(3) , R(4)] are each
independently selected from either the group consisting of Glu,
Gln, Asp, Asn or from the group consisting of Lys, Arg, Orn.
The symbol - represents a bond between the carboxyl and
amino termini by which R(1) and R(4) can be interconnected to
each other via an lower alkenyl or lower alkynyl group, but most
preferably by a branched or unbranched methylene bridge of type
~ ~ (CH2) m ~ -or --(CH2) m~ -M- -(CH2)~--. In such an moiety, m and m'
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are integers ~rom 1 to 6, inclusive, and pre~erably ~rom 1 to 3,
inclusive; and M is NH, N[R(5)], O, S or CH-R(5), wherein R(5)
is lower alkyl, cycloalkyl or aryl and is preferably methyl,
ethyl, propyl, phenyl, X-phenyl, or heterocyclic, wherein X is
Cl-, CF3-, F-, substituted at the o-, m- , or p- positions on
the phenyl group M can contain a part of another diamino acid
within the same peptide, e.g., the omega amino group of the one
residue can be so linked to such an unnatural amino acid residue
in a terminal residue.
Furthermore, any amino acid in the sequences provided
hereinabove may be replaced with its D-analogue, with the proviso
that not more than 50~ of the total amino acids are so replaced.
Similarly, a homologous conservative substitution ~or any amino
acid is within the bounds of the present invention provided that
substitution does not eliminate the oncogenic ras p21-inhibiting
activity. Thus, depending on the applications for which the
peptides according to the invention are intended, it is also
possible to envisage intercalating between several amino acids,
or even between all the amino acids, o~ the peptides de~ined
above, dextrorotatory amino acids, and in particular
dextrorotatory phenylalanine or dextrorotatory tryptophan,
capable of preventing the action of the degradative enzymes in
the cell environment and thus of increasing their activity.
Another modification in this sense consists in replacing certain
amino acids, for example o~ the isoleucine type, by leucine.
In addition, a subject polypeptide can di~fer, unless
otherwise speci~ied, ~rom the natural sequences shown above by
the sequence being modified by terminal -NH2 acylation, e.g.,
acetylation, or by terminal-carboxylamidation, e.g., with
ammonia, alkylamines, and the like.
This invention ~urther relates to peptidomimetics which
model the critical semi-extended conformation of at least one
peptide of or cyclic peptide of the present invention,
exempli~ied by the compounds o~ Structure 1:
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x Y2 x x
Y ~ 3 1 711
X Y~l 1 1 l;-X
~2~ ~1 0/ ~8~ 1 5 y
Y~:, I 1, Y/\x
O \~
C = O R
~ OOC-( CH2) m
STRUCTURE 1
wherein the sidechain R attached at the carbon atom numbered 6
on the sterol nucleus can be NH-CH2-CH2NH3, alkylamino,
arylamino, or aralkylamino group, and wherein the sidechain
attached at the carbon number 3 can be replaced with -O-C(=O)-
-(CH2)~--COOH, where m is an integer ~rom 1 to 6, inclusive,
pre~erably ~rom 1 to 3, inclusive, and more pre~erably 2, and one
o~ X and Y at each position independently, can be one H, a small
alkyl group o~ C1 to C3, pre~erably C1; a halogen, preferably F,
or an amino group where the other o~ one o~ X and Y is H.
Pre~erably, each o~ X and Y is H.
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" ~ ~,3 17
1 1 l4
2 / '~o / \ 8 /
O / \ 4 / \ ~ /
C=O NH
OOC-CH2 iCH2
ICH2
+ NH3
STRUCTURE 2
An exemplary compound falling within Structure 1 is 3
malonoxy-6-(2-aminoethyl)aminocyclopentanoperhydrophenanthrene
(Structure 2).
Detailed Description of the Invention
The natural sequence of the human oncogenic ras p21 is given
in SEQ ID N0:5. The crystal X-ray structure has been determined
at high resolution for that portion of the human ras protein
corresponding to residues 1 to 166 of SEQ ID N0:5.
The regions o~ the p21 protein that are the most likely to
change their conformations upon activation o~ the protein, e.g.
by oncogenic amino acid substitutions have been computed using
two different methods. Both methods are based on the principle
that the linear sequence o~ amino acids in a protein determines
its unique three-dimensional structure. Given an amino acid
sequence of a polypeptide or protein, therefore, it should be
possible to predict its three-dimensional structure. This task
can be accomplished by using the principle that the observed
three-dimensional structure of a protein is the one of lowest
f-ree energy. There are a vast number of possible structures a
given polypeptide chain can adopt, but essentially only one of
these is observed. To allow folding to occur, therefore, the
interatomic interactions in the protein chain must greatly
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11
stabilize its final folded form, i.e., lower its conformational
energy substantially with respect to that of any other competing
structure Thus, to compute the lowest energy form of a protein,
it is necessary to be able first to compute the conformational
energy of a given conformation of the protein and then, second,
to generate its low energy con~ormations, or a representative
sampling of them. The structure of lowest conformational energy
so computed is then predicted to be the observed structure of the
protein. This structure may be the one determined by x-ray
crystallography or by 2- or 3-dimensional nuclear magnetic
resonance (NMR) techniques.
A set of potential energy functions, in the computer program
ECEPP (Empirical Conformational Energies of Peptides Program),
have been developed that accurately compute the conformational
energies of given conformations of proteins. The conformational
energy of a peptide can be expressed in Equation 1.
Etot = ~ DRl, + ~ ij Rij - 2 [ R j ] 6) + ~ ( 2 ) (licos(n~k))
where Etot is the total conformational energy of the protein, the
Q's are the charges on the ith and jth atoms; Rij is the distance
between the ith and jth atoms, D is the dielectrlc constant, ~ij
and Pij are the lowest non-bonded (Lennard-Jones) energy and the
distance at this lowest energy between atoms i and j in the
protein; Ak is the torsional barrier to rotation around specific
bonds; 9k iS the kth dihedral angle in the protein; n is a
degeneracy factor, i.e., 3 for single bonds and 2 for double
bonds; and the sign in the last summation term is positive for
single bonds and negative for double bonds such as occur in the
- peptide bond units.
This equation shows the total conformational energy as the
sum of three terms: the pairwise electrostatic interactions
between the individual atoms of a protein, each of which has a
partial charge, (first sum); a non-bonded energy term (second
term) that consists of an attractive term that varies as the
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12
inverse sixth power (tenth power for hydrogen-bonding atoms) of
the distance between the atoms (from an induced dipole-induced
dipole interaction term) and a repulsive term, from the overlap
of electron shells, that varies as the inverse twelfth power of
5the interatomic distance; and finally a torsional term (third
sum) that depends upon the bonds about which rotation takes
place. All of the constants in these terms have been determined
from experimental crystal packing data and reproduce the lattice
constants o~ all o~ the crystal structures of small molecules to
10which they have been applied and, where measured, the sublimation
energies of these crystals These potential functions have been
used to compute the low energy minima for single terminally
blocked amino acid residues, simple peptides, oligopeptides,
polypeptides, and proteins with excellent agreement between the
15lowest energy predicted structures and the structures determined
experimentally. These potentials have therefore been well-
tested, are based on experimental data, and have proved to be
reliable in prediction of structure from sequence.
These potential functions have been used to compute the
20average structure ~or the ras-p-21 protein in its normal and in
its oncogenic form using the perturbation method called the
electrostatically-driven Monte Carlo method (EDMC). Specific
regions of the oncogenic p21 protein undergo large conformational
changes compared with the structure of the normal, inactive
25protein. One o~ these regions has been found to be residues 35-
47. All of the segments that change conformation in the
oncogenic protein were found to be the most flexible in the
normal, inactive protein.
Of considerable significance has been the finding that a
30completely dif~erent method, viz. molecular dynamics, based upon
a completely different set of potential functions, i.e. the
program DISCOVER, yields identical results for the p21 protein.
Molecular dynamics is based on the principle that the
positions o~ the atoms of a molecule can be predicted as a
35function of time by solving Newton's equations of motion ~or the
molecule. The force on the molecule is the negative of the first
derivative of the potential function with respect to the
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13
coordinates of each of the atoms. Newton's equations of motion
are then integrated, using the Verlet algorithm, over a
~ trajectory such that the low energy region5 around the starting
structure are computed. The trajectories are computed over time
~ 5 intervals such that the total energy converges to a low, constant
value. The structures whose energies have converged are then
used to compute an average structure. Comparison of the
coordinates of the atoms of this average structure with those of
the starting structure reveals regions of the protein whose
conformations may change significantly. Furthermore, if the
variance of the coordinates of regions of the low energy
structures from the corresponding coordinates of the average
structure are high, these regions can be identified as being
flexible, i.e., are the ones most likely to be parts of effector
domains. Within this algorithm, for the p21 protein, up to 2000
water molecules have been generated around the protein in the
molecular dynamics simulations performed thus far.
Using these novel calchlational approaches, the present
inventors have identified important peptide regions of the
protein that are involved in the signal transduction process, and
these peptides can be used to design anti-cancer agents, as
taught herein. We have found that most particularly the 35-47,
96-110 and 115-126 peptides have strong and specific anti-
oncogenic p21 activity. Even more particularly, we found that
these domains contain unique extended structures and/or short
beta-bend structures which are hypothesized to account in large
part for their biological uni~ueness. This suggested that
cyclization of the peptide structures to force the beta-bend
conformation in place would serve to enhance therapeutic
activity.
The results of these studies indicate that a domain of
particular interest is the domain from residues 35 through 47 of
SEQ ID NO:5, i.e., Thr-Ile-Glu-Asp-Ser-Tyr-Arg-Lys-Gln-Val-Val-
Ile-Asp (SEQ ID NO:6), of even more particular interest the
peptide corresponding to residues 44 to 46 in SEQ ID NO:5, i.e.,
Val-Val-Ile, of still more interest is the se~uence from residues
96 to 110 o~ SEQ ID NO:5, i.e., Tyr-Arg-Glu-Gln-Ile-Lys-Arg-Val-
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14
Lys-Asp-Ser-Asp-Asp-Val-Pro (SEQ ID NO:7), of even more
particular interest is the sequence from residues 101-103 in SEQ
ID NO:5, i.e., Lys-Arg-Val; and the sequence corresponding to
residues 100 to 105 in SEQ ID NO:5, i.e., Ile-Lys-Arg-Val-Lys-Asp
(SEQ ID NO:1); the sequence corresponding to residues 115 to 126
of SEQ ID NO:5, i.e., Gly-Asn-Lys-Cys-Asp-Leu-Ala-Ala-Arg-Thr-
Val-Glu (SEQ ID NO:8); and most particularly the sequence
corresponding to residues 117 to 121 of SEQ ID NO:5, i.e, Lys-
Cys-Asp-Leu-Ala (SEQ ID NO:2) and the sequence corresponding to
residues 118 to 124 of SEQ ID NO:5, i.e., Cys-Asp-Leu-Ala-Ala-
Arg-Thr (SEQ ID NO:9); and the sequence corresponding to residues
119 to 122 of SEQ ID NO:5, i.e., Asp-Leu-Ala-Ala (SEQ ID NO:4).
Additional sequences homologous to the various preferred
sequences recited hereinabove can be derived by one skilled in
the art from the sequences of closely related ras proteins. Such
sequences may possess enhanced therapeutic activity. Nonlimiting
examples of such proteins closely related to the ras gene product
which represent the parent sequences having identical or nearly
identical three dimensional structures and from which homologs
of the sequences given in the preceding paragraph can be derived
by one normally skilled in the art are:
ras-related protein Ara-3 [Arabidopsis thaliana (mouse ear
cress)] (SEQ ID NO:10);
ras-related protein Ara-2 [A. thaliana] SEQ ID NO:11;
ras-related protein Ara-1 [A. thaliana] SEQ ID NO:12;
ras-related protein OraB-1 [Disco~Yae ommata (electric
ray)] SEQ ID NO:13;
ras-related protein Rab-lA [Lymnea staqnalis (great pond
snail)] SEQ ID NO:14;
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ras-related protein Rab-2 [Homo sa~iens (human)] SEQ ID
NO:15;
ras-related protein Rab-2 [L. staqnalis] SEQ ID NO:16;
ras-related protein Rab-2 [OrYctolaaus cuniculus (rabbit)]
SEQ ID NO:17;
ras-related protein Rab-2 [Rattus norveqicus (rat)] SEQ ID
NO:18;
ras-related protein Rab-3 [Drosophila melanoqaster
(fruit~ly)] SEQ ID NO:19;
ras-related protein Rab-4 [R. norveqicus] SEQ ID NO:20;
ras-related protein Rab-6 [Caenorhabditis eleaans] SEQ ID
NO:21;
~-related protein Rab-6 [H. sa~iens] SEQ ID NO:22;
ras-related protein Rab-7 [Canis ~amiliaris (dog)] SEQ ID
NO:23;
ras-related protein Rab-7 [Dictyostelium discoideum (slime
mold)] SEQ ID NO:24;
ras-related protein Rab-8 [C. ~amiliaris] SEQ ID NO:25;
ras-related protein RabC [D. discoideum] SEQ ID NO:26;
ras-related protein Rac-1 [C. eleqans] SEQ ID NO:27;
ras-related protein Rac-lA [D. discoideum] SEQ ID NO:28;
ras-related protein RacB [D. discoideuml SEQ ID NO:29;
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16
ras-related protein RacC [D. discoideum] SEQ ID NO: 30;
ras-related protein Ral-A [H. sapiensl SEQ ID NO:31;
ras-related protein Ral-B [H. saPiens] SEQ ID NO:32;
ras-related protein O-Ral [D. ommata] SEQ ID NO:33;
ras-related protein Ora-1 [D. ommata] SEQ ID NO:34;
ras-related protein Ora-2 [D. ommatal SEQ ID NO:35;
ras-related protein Ora-3 [D. ommata] SEQ ID NO:36;
ras-related protein Rap-1 [D. discoideuml SEQ ID NO:37;
ras-related protein Rap-2A [H. sapiens] SEQ ID NO:38;
ras-related protein Rap-2B [H. saPiens] SEQ ID NO:39;
ras-related protein O-KREV [D. ommata] SEQ ID NO:40;
ras-related protein Rap-lA [H. sapiens] SEQ ID NO:41;
ras-related protein Rap-lB [H. saPienS] SEQ ID NO:42;
ras-like protein GNROR3 [D. melanoqaster] SEQ ID NO:43;
ras-like protein rasA [D. discoideum] SEQ ID NO:44;
ras-like protein rasB [D. discoideum] SEQ ID NO:45;
ras-like protein rasC [D. discoideum] SEQ ID NO:46;
ras-like protein rasG [D. discoideum] SEQ ID NO:47;
ras-like protein F54C8.5 [C. eleqans] SEQ ID NO:48;
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ras-like protein CC-ras [Coprinus cinereus (inky cap
fungus)]
SEQ ID NO:49;
ras-like protein [Geodia cvdonium (sponge)] SEQ ID NO:50;
ras-related protein Rab-10 [C. familiaris] SEQ ID NO:51;
ras-related protein Rab-11 [H. sa~iens] SEQ ID NO:52.
In addition, as described hereinabove, the therapeutic
activity of these sequences is enhanced by cyclization.
Cyclization forces and maintains the conformations of these
peptides in unique structures like beta-bends. The following are
representative, nonlimiting examples of cyclized peptides useful
for inhibiting the oncogenic activity of the ras protein, said
peptides having formulas as given below:
cyclo [- R(1) R(2) THR ILE GLU ASP SER TYR ARG LYS GLN VAL VAL
ILE ASP R(3) R(4)-] (I)
cyclo [-R(1) R(2) VAL VAL ILE R(3) R(4)-] (II)
cyclo [-R(1) R(2) TYR ARG GLU GLN ILE LYS ARG VAL LYS ASP SER ASP
ASP VAL PRO R(3) R(4)-] (III)
cyclo [-R(1) R(2) LYS ARG VAL R(3) R(4)-] (IV)
cyclo [-R(1) R(2) ILE LYS ARG VAL LYS ASP R(3) R(4)-] (V)
cyclo [-R(1) R(2) GLY ASN LYS CYS ASP LEU ALA ALA ARG THR VAL GLU
R(3) R(4)-] (VI)
cyclo [-R(1) R(2) LYS CYS ASP LEU ALA R(3) R(4)-] (VII)
cyclo [-R(1) R(2) CYS ASP LEU ALA ALA ARG THR R(3) R(4)-] (VIII)
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cyclo [-R(1) R(2) ASP LEU ALA ALA R(3) R(4)-] (IX); and
NH2
I
O Asp
H2N-Thr-Ile-Glu-Asp-Ser-Tyr-Arg-Ly9-Gln-NH-CH2-C-Val-NH-CH (X)
CH2 CH2
or a physiologically acceptable salt thereof.
In the aforementioned cyclized peptide formulas I-IX, R(1)
R(2), R(3) and R(4) represent, in the most general case, any
amino acid, such that they serve as amino acid residue linkers.
Amino acid residue linkers are usually at least one residue and
can be most often two to four residues, more often 1 to 10
residues. Typical amino acid residues used for linking are
tyrosine, cysteine, lysine, glutamic and aspartic acid. Most
preferably [R(1), R(2)] and [R(3) , R(4)] independently are
selected from either the groups [Glu, Gln, Asp, Asn] or tLys,
Arg, Orn].
The term - represents a bond between the carboxyl and amino
termini by which R(1) and R(4) can be interconnected to each
other via an lower alkyl, alkenyl or lower alkynyl group, but
most preferably by a branched or unbranched methylene bridge of
type --(CHz)m --or --(CH2)m--M--(CH2)m--. In such an moiety, m
and m' are integers from 1 to 6, inclusive, and preferably from
1 to 3, inclusive; and M is NH, N[R(5)], O, S or CH-R(5), wherein
R(5) is lower alkyl, cycloalkyl or aryl and is preferably methyl,
ethyl, propyl, phenyl, X-phenyl, or heterocyclic, wherein X is
Cl-, CF3, F-, substituted at the o-, m- , or p- positions on the
phenyl group. M can contain a part of another diamino acid
within the same peptide, e.g., the omega amino group of the one -I
residue can be so linked to such an unnatural amino acid residue
in a terminal residue.
Furthermore, any amino acid in the cyclized peptide
sequences (I)-(X) recited herein may be replaced with its D-
analogue, insofar as not more than 50~ of the total amino acids
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19
are so replaced. Similarly, a homologous conservative
substitution for any amino acid is within the bounds of the
- present invention. Conservative substitutions include Glu for
Asp, Gln for Asn and Val for Ile, among others, as well-known to
the art. Depending on the applications for which the peptides
according to the invention are intended, it is also possible to
intercalate between several amino acids, or even between all the
amino acids, of the peptides defined above, dextrorotatory amino
acids, and in particular dextrorotatory phenylalanine or
dextrorotatory tryptophan, capable of preventing the action of
the degradative enzymes in the cell environment and thus of
increasing their activity. Another modification in this sense
consists ln replacing certain amino acids, for example of the
proline type, by D-tryptophan.
In addition, a subject polypeptide can differ, unless
otherwise specified, from any of the natural sequences shown
herein above by the sequence being modified by terminal -NH2
acylation, e.g., acetylation,~ or by terminal-carboxylamidation,
e.g., with ammonia, alkylamines, and the like.
The placement of hydrophobic amino acid residues is highly
dependent on the peptide sequence. For example, for the 35-47
peptide sequence, corresponding to amino acids 35-47 of SEQ ID
NO:5, there is a distinct hydrophobic region for the amino acid
residues corresponding to amino acids 44-46 of SEQ ID NO:5. The
bridge in Compound (X) occurs at what corresponds in structure
to amino acids 44-46 in SEQ ID NO:5. It is possible to extend
this hydrophobic segment without sacrificing activity. For
example, the carboxyl terminal Asp residue can be replaced with
one or more hydrophobic residues such as Val or Ile, and the
result is greater efficiency in crossing cell membranes.
Short half-lives of peptides, a major problem, can be at
least partially extended by the addition of D-amino acids to
either or both of the amino and carboxyl terminal ends of the
peptide. These D-amino acid residues block the action of exo-
proteases that degrade peptides ~rom their amino or carboxyl
ends. In addition, the cyclization of the peptide further
renders the peptide less susceptible to proteolysis.
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Recent advances in the field o:E peptides have been directed
towards the stabilization o~ these peptides against enzymatic or
hydrolytic degradation. It would be extremely valuable to
stabilize these peptides :Erom degradation by proteolytic enzymes
in order to improve their pharmacokinetic properties. Enhanced
resistance to enzymatic degradation would increase the use~ulness
o:E these peptides as therapeutic agents. However, since they
only exhibit short hal~ lives in vivo, large amounts o:E such
peptides must typically be administered to a subject in order to
achieve the desired e~Eect. Alternatively, smaller quantities
may be prescribed to an individual, but more ~requent dosages
would be required to achieve the same level o~ potency.
It is ~urther well-known to those normally skilled in the
art that it is possible to replace peptides with peptidomimetics.
Peptidomimetics are generally pre:Eerable as therapeutic agents
to peptides owing to their enhanced bioavailability and relative
lack o~ attack from proteolytic enzymes. The present inventors
have used the techniques o:E molecular modeling supra to design
a peptidomimetic which mimics the critical beta-bend aspects o~
the peptide corresponding in sequence to amino acids 96-llO o:E
SEQ ID NO:5 (p21 ;~). The bend structure occurs at amino acids
102-103 in the p21 ~ protein. These residues have been
implicated in the binding o~ ;~ p21 to SOS.
Peptidomimetric compounds which inhibit the oncogenic or
transEorming activity o~ the p21 ras protein are provided by the
compounds oi~ Structure I:
Y x x
Y ~ 1 3 1 7/~
X \ ~ l 1 1 G--X
~2/ ~1 0/ ~8~ -- 1 5 y
Y~l I 1, Y \x
~~ ~4~ ~6/
C=O R
OOC- ( CH2) m
STRUCTURE I
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wherein the sidechain R attached at the carbon atom numbered 6
on the sterol nucleus can be NH-CH2-CH2NH3+, alkyl amino,
arylamino, or aralkylamino group, and wherein the sidechain
attached at the carbon number 3 can be replaced with -O-C(=O)-
-(CH2)m--COOH, where m is an integer from 1 to 6, inclusive,
preferably from 1 to 3, inclusive, and more preferably 2, and one
of x and y at each position independently, can be one H, a small
alkyl group of C1 to C3, preferably Cl; a halogen, preferably F,
or an amino group where the other of one of x and y is H.
Preferably, each of x and y is H.
Without wishing to be bound by any particular theory, the
structure believed to be the optimally designed L~-inhibiting
peptidomimetic is illustrated below in Structure II:
1 1 ~ ~1 3 1 7
-1 L 14 /
2/ ~1 0/ \8/ 5
li 1~
~~ ~4/ \6/
C=O NH
OOC- CH2 ICH2
CH2
NH3
STRUCTURE 2
The instant invention comprises novel peptides of medicinal
importance most particularly for the treatment of adenocarcinomas
of the colon, pancreatic carcinomas, neuroblastomas, and other
cancers of undefined germ cell origin which express the
transformed sequence of the La~ protein. These peptide sequences
were unexpectedly obtained by the use of molecular dynamic
simulations on ~ p21 to define which domains of the protein
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22
were most flexible and were thus most important in interacting
with target proteins upstream and downstream from ras. These
peptides are identified by the following amino acid sequences:
Thr-Ile-Glu-Asp-Ser-Tyr-Arg-Lys-Gln-Val-Val-Ile-Asp (SEQ ID
NO:6),Val-Val-Ile,Tyr-Arg-Glu-Gln-Ile-Lys-Arg-Val-Lys-Asp-Ser-
Asp-Asp-Val-Pro (SEQ ID NO:7), Lys-Arg-Val, Ile-Lys-Arg-Val-Lys-
Asp (SEQ ID NO:1), Gly-Asn-Lys-Cys-Asp-Leu-Ala-Ala-Arg-Thr-Val-
Glu (SEQ ID NO:8), Lys-Cys-Asp-Leu-Ala (SEQ ID NO:2), Cys-Asp-
Leu-Ala-Ala-Arg-Thr (SEQ ID NO:9), and Asp-Leu-Ala-Ala (SEQ ID
NO:4).
Including the cyclic analogues of the above peptides,
namely:
cyclo [- R(1) R(2) Thr-Ile-Glu-Asp-Ser-Tyr-Arg-Lys-Gln-Val-Val-
Ile-Asp-R(3) R(4)-] (I);
cyclo [-R(1) R(2) Val-Val-Ile-R(3) R(4)-] (II);
cyclo [-R(1) R(2) Tyr-Arg-Glu-Gln-Ile-Lys-Arg-Val-Lys-Asp-Ser-
Asp-Asp-Val-Pro-R(3) R(4)-] (III);
cyclo [-R(1) R(2) Lys-Arg-Val R(3) R(4)-] (IV);
~ cyclo [-R(1) R(2) Ile-Lys-Arg-Val-Lys-Asp R(3) R(4)-] (V);
cyclo [-R(1) R(2) Gly-Asn-Lys-Cys-Asp-Leu-Ala-Ala-Arg-Thr-Val-Glu
R(3) R(4)-] (VI);
cyclo [-R(1) R(2) Lys-Cys-Asp-Leu-Ala R(3) R(4)-] (VII);
cyclo [-R(1) R(2) Cys-Asp-Leu-Ala-Ala-Arg-Thr R(3) R(4)-] (VIII);
cyclo [-R(1) R(2) Asp-Leu-Ala-Ala R(3) R(4)-] Z (IX); and
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NIH2
O Asp
H2N-Thr-Ile-Glu-Asp-Ser-Tyr-Arg-Lys-Gln-NH-CH2-C-Val-NH-CH (X)
CH2 CH2
or a physiologically acceptable salt thereof.
Wherein for cyclized peptide formulas designated by (I)-(IX)
hereinabove, R(l) R(2), R(3) and R(4) represent, in the most
general case, any amino acid, such that they serve as amino acid
residue linkers. Amino acid residue linkers are usually at least
one residue and can be most often two to four residues, more
often 1 to 10 residues. Typical amino acid residues used for
linking are tyrosine, cysteine, lysine, glutamic and aspartic
acid, or the like. Most preferably [R(l)~ R(2)] and [R(3),
R(4)] independently are selected from either the groups [Glu,
Gln, Asp, Asn] or [Lys, Arg, Orn].
The symbol - represents a bond between the carboxyl and
amino termini by which R(l) and R(4) can be interconnected to
each other via an lower alkenyl or lower alkynyl group, but most
preferably by a branched or unbranched methylene bridge of type
--(CH2)m --or --(CH2) m~ -M--(CH2) m~~~ In such an moiety, m and m'
are integers from 1 to 6 and preferably from 1 to 3; and M is NH,
N [R(5)]~ O~ S CH-R(5) or does not exist, wherein R(5) is lower
alkyl, cycloalkyl or aryl and is preferably methyl, ethyl,
propyl, phenyl, X-phenyl, or heterocyclic, wherein X is Cl-, CF3-
, F-, substituted at the o-, m- , or p- positions on the phenyl.
M can contain a part of another diamino acid within the same
peptide, e.g., the omega amino group of the one residue can be
so linked to such an unnatural amino acid residue in a terminal
residue.
Furthermore, any amino acid in the se~uences provided may
be replaced with its D-analogue, insofar as not more than 50~ of
the total amino acids are so replaced. Conservative
substitutions include Glu for Asp, Gln for Asn and Val for Ile,
among others, as is well known to those of ordinary skill in the
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24
art. Similarly, a homologous conservative substitution for any
amino acid is within the bounds of the present invention.
Depending on the applications for which the peptides according
to the invention are intended, it is also possible to envisage
intercalating between several amino acids, or even between all
the amino acids, of the peptides defined above, dextrorotatory
amino acids, and in particular dextrorotatory phenylalanine or
dextrorotatory tryptophan, capable of preventing the action of
the degradative enzymes in the cell environment and thus of
increasing their activity. Another modification in this sense
consists in replacing certain amino acids, for example of the
proline type, by D-tryptophan.
In addition, a subject polypeptide can differ, unless
otherwise specified, from the natural sequences shown above by
the sequence being modified by terminal -NH2 acylation, e.g.,
acetylation, or by terminal-carboxylamidation, e.g., with
ammonia, alkylamines, and the like.
The instant invention also comprises a method of use of the
peptides supra for the treatment of adenocarcinomas of the colon,
pancreatic carcinomas, neuroblastomas, and other cancers of
undefined germ cell origin which express the transformed sequence
of the ras protein.
It is also an object of the present invention to provide
peptides and cyclized peptide homologs from the sequences listed
in SEQ ID NOS:10-52.
The amino acid residues described herein are preferred to
be in the "L" isomeric form. However, residues in the "D"
isomeric form can be substituted for any L-amino acid residue,
as long as the desired functional property is retained by the
polypeptide. NH2 refers to the free amino group present at the
amino terminus of a polypeptide. COOH refers to the free
carboxyl group present at the carboxy terminus of a polypeptide.
In keeping with standard polypeptide nomenclature described in
J. Biol. Chem. 243:3552-3559 (1969) and adopted at 37 C.F.R.
1.822 (b) (2)), The list of variable amino acids, capable oE
participating in the composition of this peptide is as follows:
Y, Tyr, tyrosine; G, Gly, glycine; F, Phe, phenylalanine; M, Met,
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methionine; A, Ala, alanine; S, Ser, serine; I, Ile, isoleucine;
L, Leu, leucine; T, Thr, threonine; V, Val, valine; P, Pro,
proline; K, Lys, lysine; H, His, histidine; Q, Gln, glutamine;
E, Glu, glutamic acid; W, Trp, tryptophan; R, Arg, arginine;
- 5 D, Asp, aspartic acid; N, Asn, asparagine; C, Cys, cysteine.
Amino acid residue sequences are presented herein in the
conventional left-to-right direction of amino-terminus to
carboxy-terminus. In addition, the phrase "amino acid residue"
is broadly defined to include the amino acids listed hereinabove,
and modified and unusual amino acids, such as those listed in 37
C.F.R. 1. 822 (b)(4), incorporated herein by reference.
Furthermore, it should be noted that a dash at the beginning or
end of an amino acid residue sequence indicates either a peptide
bond to a further sequence of one or more amino acid residues or
a covalent bond to an amino or hydroxyl end group.
Polypeptide and peptide are terms used interchangeably
herein to designate a linear series of amino acid residues
connected one to the other by peptide bonds between the
alpha-amino and carboxy groups of adjacent residues.
Protein is a term used herein to designate a linear series
of greater than about 20 amino acid residues connected one to the
other as in a polypeptide.
The term synthetic peptide refers to a chemically produced
chain of amino acid residues linked together by peptide bonds
that is free of naturally occurring proteins and fragments
thereof. The term peptide encompasses linear and cyclic
peptides.
(D,L), (D), or (L) preceding the amino acid designation
means that this amino acids exists in that specific isomeric
form, i.e. (D,L) Phe means that the amino acid phenylalanine
exists as a racemic mixture; (D) Phe or D-Phe means that the
amino acid phenylalanine exists as the D-stereoisomer or implied
R configuration; (L) Phe means that the amino acid phenylalanine
exists as the L stereoisomer or implied S configuration.
Alkyl as used herein means methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl,
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26
neopentyl, 2 -methylbutyl, 1-methylbutyl, 1-ethylpropyl,
1,1-dimethylpropyl, n-hexyl, 1-methylpentyl, 2-methylpentyl,
3-methylpentyl, 4-methylpentyl, 3,3-dimethylbutyl,
2,2-dimethylbutyl, 1,1-dimethylbutyl, 2 -ethylbutyl, 1-ethylbutyl,
1~3-dimethylbutyl~ n-heptyl, 5-methylhexyl, 4-methylhexyl,
3-methylhexyl, 2-methylhexyl, 1-methylhexyl, 3-ethylpentyl,
2-ethylpentyl, 1-ethylpentyl, 4,4-dimethylpentyl,
3,3-dimethylpentyl, 2,2-dimethylpentyl, 1,1-dimethylpentyl,
n-octyl, 6-methylheptyl, 5-methylheptyl, 4-methylheptyl,
3-methylheptyl, 2-methylheptyl, 1-methylheptyl, 1-ethylhexyl,
1-propylpentyl~ 3-ethylhexyl, 5,5-dimethylhexyl,
4,4-dimethylhexyl, 2,2-diethylbutyl, 3,3-diethylbutyl,
1-methyl-1-propylbutyl.
Cycloalkyl re~ers to a hydrocarbon ring having ~rom 3 to 7
carbon atoms, inclusive. Examples o~ cycloalkyl groups are
cyclopropyl, cyclopentyl, cycloheptyl, cyclooctyl, cyclononyl,
and the like.
The term aryl re~ers to aromatic groups which have at least
one ring having a conjugated pi electron system and includes
carbocyclic aryl, heterocyclic aryl aralkyl, and biaryl groups,
all of which may be optionally substituted.
Heterocyclic groups means groups having ~rom 1 to 3
heteroatoms as ring atoms in the aromatic ring and the remainder
o~ the ring atoms carbon atoms. Suitable heteroatoms include
oxygen, sul~ur, and nitrogen, and their heterocyclic compounds
can include ~uranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl
pyrrolo, pyrimidyl, pyrazinyl, imidazolyl, and the like, all
optionally substituted.
Substituted heterocyclic re~ers to any heterocyclic aryl
group substituted by a alkyl, aryl, cycloalkyl, halo, sul~onate,
or tri~luoromethyl group.
The term alkyl amino re~ers to the groups --NRR' wherein
respectively, (a) R is alkyl and R' is hydrogen or alkyl; (b) R
is aryl and R' is hydrogen or aryl, (c) R is cycloalkyl and R'
is hydrogen or alkyl, (d) R is hydrogen and R' is itsel~ linear
aminoalkyl, (e) R is alkyl and R' is itsel~ linear aminoalkyl.
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27
The term aminoalkyl re~ers to the groups -(CH2)m-NRR',
wherein m is an integer ~rom 1 to 6, inclusive and -NRR' is alkyl
- amino, as defined supra .
Halo encompasses fluoro, chloro, bromo and iodo.
- 5 The phrase protecting group, as used herein, means
substituents which protect the reactive functional group from
undesirable chemical reactions. Examples of such protecting
groups include esters of carboxylic acids, ethers of alcohols and
acetals and ketals of aldehydes and ketones.
The phrase N-protecting group or N-protected as used herein
means those groups intended to protect the N-terminus o~ an amino
acid or peptide, to protect an amino group against undesirable
reactions during synthetic procedures and includes, but is not
limited to, sulfonyl, acetyl, pivaloyl, t-butyloxycarbonyl (Boc),
carbonylbenzyloxy (Cbz), benzoyl and an L- or D-aminoacyl
residue, which may itself be N-protected similarly. The
deprotection conditions for the above protecting groups
necessarily vary with the choice of protecting group. Thus, for
example, an acyl group such as an alkanoyl, alkoxycarbonyl or an
aroyl group may be removed for example, by hydrolysis with a
suitable base such as an alkali metal hydroxide, for example
lithium or sodium hydroxide. Alternatively an acyl group such
as a t-butoxycarbonyl group may be removed, for example, by
treatment with a suitable acid such as hydrochloric, sulfuric or
phosphoric acid or tri~luoroacetic acid and an
arylmethoxycarbonyl group such as a benzyloxycarbonyl group may
be removed, for example, by hydrogenation over a catalyst such
as palladium-on-charcoal.
The phrase COOH-protecting group or carboxyl-protecting
group is, an esterifying group, for example an alkyl group
(especially methyl or ethyl) or an arylmethyl group (especially
benzyl). The deprotection conditions for the above protectin~
groups necessarily vary with the choice o~ protecting group.
Thus, for example, an esteri~ying group such as an alkyl or
arylmethyl group may be removed, :Eor example, by hydrolysis with
a suitable base such as an alkali metal hydroxide, ~or example
lithium or sodium hydroxide. Alternatively an esteri~ying group
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28
such as an arylmethyl group may be removed, for example, by
hydrogenation over a catalyst such as palladium-on-charcoal using
either hydrogen or ammonium ~ormate as a hydrogen source by
methods well-known to those skilled in the art.
Electrolyte means a solution that has suf~icient acid
strength to render a basic starting material essentially
protonated.
Chemical derivative re~ers to a subject polypeptide having
one or more residues chemically derivatized by reaction o~ a
functional side group. Such derivatized molecules include ~or
example, those molecules in which ~ree amino groups have been
derivatized to ~orm amine hydrochlorides, p-toluene sul~onyl
groups, carbobenzoxy groups, t-butyloxycarbonyl groups,
chloroacetyl groups or ~ormyl groups. Free carboxyl groups may
be derivatized to form salts, methyl and ethyl esters or other
types o~ esters or hydrazides. Free hydroxyl groups may be
derivatized to ~orm O-acyl or O-alkyl derivatives. The imidazole
nitrogen o~ histidine may be derivatized to ~orm N-imidazolyl-
benzylhistidine. Also included as chemical derivatives are those
peptides which contain one or more naturally occurring amino acid
derivatives of the twenty standard amino acids. For examples,
4-hydroxyproline may be substituted ~or proline; 5-hydroxylysine
may be substituted ~or lysine; 3-methylhistidine may be
substituted ~or histidine; homoserine may be substituted ~or
serine; and ornithine may be substituted ~or lysine.
Polypeptides o~ the present invention also include any
polypeptide having one or more additions and/or deletions or
residues relative to the sequence o~ a polypeptide whose sequence
is shown herein, so long as the requisite activity is maintained.
As used herein, ~ragment means any subject peptide or
polypeptide having an amino acid residue sequence shorter than
that o~ a peptide or polypeptide whose ~ull length amino acid
residue sequence is shown herein.
A pharmaceutically acceptable salt is one which is prepared
by contacting a compound o~ ~ormulas (I) - (X) according to the
speci~ications therein with an acid whose anion is generally
considered suitable for human consumption. Examples o~
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29
pharmacologically acceptable acid addition salts include the
hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate,
- acetate, propionate, lactate, maleate, malate, succinate, and
tartrate salts. All of these salts may be prepared by
~ 5 conventional means by reacting, for example, the appropriate acid
with the corresponding compound of structure of Formulas (I) -
(X) .
Unless otherwise indicated, the preparation methods
disclosed herein result in product distributions which include
all possible structural isomers. It is understood that
physiological response may vary according to stereochemical
structure. The isomers may be separated by conventional means
such as fractional crystallization or High Pressure Liquid
Chromatography (HPLC). Briefly, the absolute configuration of
a compound relates to how its substituents are oriented in space
about a central atom. This notion becomes significant when
coupled with the rigors of chirality. Chirality involves the
identity of the substituents~about that central atom. Thus, in
general, a compound is said to be chiral when four distinctly
different groups are bound to a central carbon atom. These
groups may be spatially aligned in more than one manner without
repeating their individual orientations. That is, a chiral
compound may exhibit a mirror image which is also chiral. These
mirror images are termed meso configurations, and are each
absolute configurations of a chiral compound.
Pharmaceutical compositions according to the present
invention comprise one or more peptides and/or peptidomimetics
of the invention in association with a pharmaceutically
acceptable carrier or excipient, adapted for use in human or
veterinary medicine. The compositions may contain from 0.001-99~
of the active material. Such compositions may be presented ~or
use in conventional manner in admixture with one or more
physiologically acceptable carriers of excipients. The
pharmaceutical compositions according to the invention may also
contain other active ingredients such as antimicrobial agents,
or preservatives. The compositions may optionally further
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contain one or more other therapeutic agents which may, if
desired, be a chemotherapeutic antiviral agent.
Pharmaceutically acceptable salts of the peptides of this
invention may be ~ormed conventionally by reaction with an
appropriate acid. The addition salts so formed from addition by
acid may be identi~ied by hydrochloric, hydrobromic, phosphoric,
acetic, fumaric, salicylic, citric, lactic, mandelic, tartaric,
oxalic, methanesul~onic, and the like.
Thus, the peptides and peptidomimetics according to the
present invention may be ~ormulated for oral, buccal, parenteral,
topical or rectal administration. In particular, these peptides
and peptidomimetics may be formulated for injection or for
infusion and may be presented in unit dose form in ampoules or
in multidose containers with an added preservative. The
compositions may take such forms as suspensions, solutions, or
emulsions in oily or aqueous vehicles, and may contain
~ormulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the active ingredient may be
in powder form for constitution with a suitable vehicle, e.g.
sterile, pyrogen-free water, be~ore use.
The present invention ~urther provides a process for
preparing a pharmaceutical composition which comprises bringing
a peptide and/or peptidomimetic o~ the invention into association
with a pharmaceutically acceptable excipient or carrier.
For administration by injection or in~usion, the daily
dosage as employed for treatment o~ an adult human o~
approximately 70 kg body weight will range ~rom 0.01 mg to 10 mg
of each active ingredient, preferably 0.1 to 5 mg, which may be
administered in 1 to 4 doses, for example, depending on the route
of administration and the condition of the patient. The dosage
of the peptide used in the treatment will vary, depending on the
seriousness of the disorder, the weight of the patient, the
relative ef~icacy of the peptide and the judgment o~ the treating
physician. However, suitable unit dosages in humans may be
between about 0.05 mg to about 100 mg. For example, a unit
dosage may be ~rom between about 0.2 mg to about 50 mg. Such a
unit dosage, described herelnabove, may be administered more than
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once a day, e g., two or three times a day. Thus, the total
daily dosage is in the range of about 0.01 mg to 10 mg/kg. Such
~ therapy may extend ~or several weeks, in an intermittent or
uninterrupted manner, until the patient's symptoms are
~ 5 eliminated.
The present invention also provides pharmaceutical
compositions which comprise a pharmaceutically e~ective amount
o~ the one or more peptides and/or peptidomimetics o~ this
invention, or pharmaceutically acceptable salts thereo~, and,
pre~erably, a pharmaceutically acceptable carrier or adjuvant.
Therapeutic methods of this invention comprise the step of
treating patients in a pharmaceutically acceptable manner with
those peptides or compositions. Such compositions may be in the
~orm o~ tablets, capsules, caplets, powders, granules, lozenges,
suppositories, reconstitutable powders, or liquid preparations,
such as oral or sterile parenteral solutions or suspensions.
In order to obtain consistency of administration, it is
pre~erred that a composition o~ the invention is in the ~orm o~
a unit dose. The unit dose presentation forms ~or oral
administration may be tablets and capsules and may contain
conventional expedients. For example binding agents, such as
acacia, gelatin, sorbitol, or polyvinylpyrrolidone; ~illers, such
as lactose, sugar, maize-starch, calcium phosphate, sorbitol or
glycine; tabletting lubricants such as magnesium stearate;
disintegrants, such as starch, polyvinylpyrrolidone, sodium
starch glycolate or microcrystalline cellulose; or
pharmaceutically acceptable wetting agents such as sodium lauryl
sul~ate.
The solid oral compositions may be prepared by conventional
methods oi~ blending, i~illing, tabletting, or the like. Repeated
blending operations may be used to distribute the active agent
throughout those compositions employing large ~uantities o~
~illers. Such operations are, o~ course, conventional in the
art. The tablets may be coated according to methods well-known
in normal pharmaceutical practice, in particular with an enteric
coating.
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Oral liquid preparations may be in the ~orm o~ emulsions,
syrups, or elixirs, or may be presented as a dry product ~or
reconstitution with water or other suitable vehicle be~ore use.
Such liquid preparations may or may not contain conventional
additives. For example suspending agents, such as sorbitol,
syrup, methyl cellulose, gelatin, hydroxyethylcellulose,
carboxymethylcellulose, aluminum stearate gel, or hydrogenated
edible ~ats; emulsi~ying agents, such as sorbitan monooleate or
acacia; non-aqueous vehicles (which may include edible oils),
such as almond oil, ~ractionated coconut oil, oily esters
selected from the group consisting o~ glycerine, propylene
glycol, ethylene glycol, and ethyl alcohol; preservatives, ~or
instancemethylpara-hydroxybenzoate, ethylpara-hydroxybenzoate,
n-propyl parahydroxybenzoate, or n-butyl parahydroxybenzoate or
sorbic acidi and, i~ desired, conventional ~lavoring or coloring
agents.
For parenteral administration, ~luid unit dosage ~orms may
be prepared by utilizing the peptide and a sterile vehicle, and,
depending on the concentration employed, may be either suspended
or dissolved in the vehicle. In preparing solutions, the
peptides o~ this invention may be dissolved in water, whereas
opiates used hereto~ore showed only marginal solubility in
aqueous media or physiological ~luids. Once in solution, the
peptide may be injected and filter sterilized be~ore ~illing a
suitable vial or ampoule and subsequently sealing the carrier or
storage package. Adjuvants, such as a local anaesthetic, a
preservative or a bu~fering agent, may be dissolved in the
vehicle prior to use. Stability o~ the pharmaceutical
composition may be enhanced by ~reezing the composition a~ter
~illing the vial and removing the water under vacuum, e.g.,
~reeze drying the composition. Parenteral suspensions may be
prepared in substantially the same manner, except that the
peptide should be suspended in the vehicle rather than being
dissolved. A sur~actant or wetting solution may be
advantageously included in the composition to ~acilitate uni~orm
distribution o~ the peptide.
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The stability of the peptides and cyclized peptides of the
present invention exceeds that of naturally occurring peptides
- if substitution is made with D-amino acids in at least 20~, but
not more than 50~, of those residues which are naturally present
~ 5 in the (L) configuration. Without being bound by theory, we
believe that the increased resistance to enzymatic degradation
over of the peptides of the present invention as compared to
natural peptides is due to the presence of D-amino acids in the
peptides. This switch from L to D amino acids neutralizes the
digestion capabilities of many of the ubiquitous peptidases found
in the digestive tract. Alternatively, the ~nh~n~ed stability
of the peptides of this invention may also be the result of the
introduction of modifications of traditional peptide linkages.
For example, the introduction of a cyclic ring within the peptide
backbone may confer enhanced stability in order to circumvent the
effect of many proteolytic enzymes known to digest small peptides
in the stomach or other digestive organs and in serum.
The compounds of the~present invention are initially
synthesized by either solution or by solid phase techniques.
Specific exemplary syntheses are described in the examples
hereinbelow. The peptides of this invention may be prepared by
initially reacting a first appropriately protected amino acid
with a second appropriately protected amino acid in an organic
solvent inert to the reactants, in the presence of a suitable
peptide coupling agent according to the following scheme:
Z(l)NH-AA(1)-COOH + coupling agent + NH2-AA(2)-COOZ( 2)
Z(1)-~nH-~(l)-C0 ~ -~(2)-COOZ(2)
wherein Z(1) is a suitable nitrogen protecting group and Z(2) is
a suitable carboxyl protecting group and AA represents any
natural or unnatural amino acid residue. The desired peptides
may be prepared by utilizing the appropriate amino acids and
repeating this reaction sequence as required until a peptide with
three to ten amino acid residues has been prepared. A suitable
deprotection method is then employed to remove specified or all
of the remaining protecting groups or the peptide from the resin.
The first appropriately protected amino acid and, for
instance, an appropriately protected tyrosine may be reacted
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together in the presence o~ a suitable peptide coupling agent in
a suitably inert organic solvent with stirring, shaking, or
agitation to ~orm a protected tyrosine containing dipeptide.
Introducing this dipeptide to appropriate protecting group
removal conditions af~ords a selectively deprotected dipeptide
which is well-suited for continued peptide synthesis. Contacting
this mono-deprotected tyrosine containing dipeptide with an
appropriately protected amino acid having a side chain
represented as above, in the presence o~ a suitable peptide
coupling agent in a suitably inert organic solvent with stirring,
shaking, or agitation ~orms a protected tyrosine containing
tripeptide. This method may be repeated as many times as
necessary to achieve the desired peptide.
The method o~ preparation ~or peptide synthesis requires
speci:Eic i~unctional groups to react with other substituents to
link amino acid residues in a desired manner to ~orm a peptide
possessing a known and desired sequence of amino acid residues.
Since amino acids possess ~at least two reactive ~unctional
groups, suitable protection, blocking, or masking o~ these groups
is required to ensure that reaction will occur only at
speci~ically desired sites.
These protecting groups should be introduced to the moiety
ef~icaciously while their removal should be per~ormed under
conditions which do not a~ect other portions o~ the molecule.
In this manner, certain reactions and modiEications may be
per~ormed on the amino acid, peptide, or other compound, with
assurance that the protected ~unctionality will not inter~ere
with the desired reaction. Further, by choosing a protecting
group that is sensitive and labile to certain reactive
conditions, a reaction scheme may be outlined to advantageously
utilize these characteristics to e~ectively remove the
protecting group once the synthesis is complete.
Both N-protecting groups and COOH-protecting groups (see
de~initions) may be used within the scope o~ this invention. A
variety o:E protecting groups known in the ~ield o~ peptide
synthesis and recognized by conventional abbreviations therein,
may be ~ound in T. Greene, Protective Gxou~s In Orqanic
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Synthesis, Academic Press (1981). Among the preferred protecting
groups that may be utilized for suitable protection of reactive
nucleophilic substituents include, for example, benzyl (Bz),
carbobenzyloxy (Cbz), t-butoxycarbonyl (Boc), or
~ 5 9-fluorenylmethyloxy-carbonyl (Fmoc).
Coupling o~ amino acids, which may be the same or di~ferent
as those described above, to yield small peptides in route to
peptides comprised o~ greater numbers of amino acid residues may
be accomplished by employing established techniques in the field
of peptide chemistry. A broad range of suitable reactions are
described in E. Gross and J Meinhofer, The Peptides: Analysis,
SYnthesis, Bioloqv; Modern Techniques of PePtide and Amino Acid
Analysis, John Wiley & Sons, (1981) and M. Bodanszky, Principles
Of PePtide SYnthesis, Springer-Verlag (1984). The peptide
coupling agents which may be used to assist condensation o~ amino
a n d c a rb o xy l i c a ci d m o i e t i e s i n c l u de
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-carbonyl diimidazole
(CDI), l-hydroxy benzotriazole (HOBt), ethyl chloroformate,
b e n z y 1 c h 1 o r o f o r m a t e
1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ),
benzotriazoyl-oxy-tris-(dimethyl)amino-phosphonium hexafluoro
phosphate (BOP) and the like. A pre~erred technique uses DCC as
the coupling reagent. The DCC method may be used with or without
catalytic additives such as 4-dimethylaminopyridine (DMAP),
copper (II) chloride or HOBt to hasten the reaction and suppress
the racemization of the desired compound.
The DCC reaction is often performed at room temperature but
may be carried out ~rom about -78 ~C to gentle re~lux in a
variety of solvents that are inert with respect to the reactants.
The solvents are normally organic solvents which are polar and
aprotic. Preferred solvents include, ~or example,
dichloromethane, chloroform, diethyl ether, tetrahydro~uran
(THF), N,N'-dimethylformamide (DMF), and the like. Particularly
pre~erred solvents are dichloromethane and DMF. In general, the
coupling reaction may be carried out at atmospheric pressure a
temperature o~ -78 oc to reflux for a period o~ between 1 and 48
hours. Pre~erably, the reaction is carried out at about -10~ C
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36
to 25~ C with stirring, shaking or agitation, over a period of
between 4 and 6 hours.
Alternatively, synthesis may be achieved prepared using
solid phase synthesis, such as that described by Merrifield, J
Am. Chem. Soc., 85, p 2149 (1964), although other equivalent
chemical syntheses known in the art can also be used.
Solid-phase synthesis is commenced from the C-terminus of the
peptide by coupling a protected alpha-amino acid to a suitable
resin as generally set forth in U.S. Pat. No. 4,244,946.
As an example, Ile protected by BOC is coupled to the a BHA
resin using methylene chloride and dimethylformamide. Following
the coupling of BOC-Ile to the resin support, the alpha-amino
protecting group is removed, as by using trifluoroacetic acid
(TFA) in methylene chloride, TFA alone or with HCl in dioxane.
Preferably 50 volume ~ TFA in methylene chloride is used with 0-5
weight ~ 1, 2 ethanedithiol. The deprotection is carried out at
a temperature between about 0~ C and room temperature. Other
standard cleaving reagents and conditions for removal of specific
alpha-amino protecting groups may be used as described in
Schroder & Lubke, The Peptides, pp 72-75 (Academic Press 1965).
After removal of the alpha-amino protecting group of Ile,
the rem~;n;ng alpha-amino- and side chain-protected amino acids
are coupled step-wise in the deslred order to obtain the
intermediate compound defined hereinbefore. As an alternative
to adding each amino acid separately in the synthesis, some of
them may be coupled to one another prior to addition to the solid
phase reactor. The selection o~ an appropriate coupling reagent
is within the skill of the art. Particularly suitable as
coupling reagents are N,N'-dicyclohexyl carbodiimide (DCC) and
N,N'-diisopropyl carbodiimide (DICI), or
N-ethyl-N'-(3-dimethylaminopropyl) carbodiimide. Other
activating reagents and their use in peptide coupling are
described by Schroder & Lubke, supra, in Chapter III and by
Kapoor (1970) J. Phar. Sci. 59:127.
Each protected amino acid or amino acid sequence is
introduced into the solid phase reactor in about a fourfold
excess, and the coupling is carried out in a medium of
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37
dimethyl~ormamide: dichloromethane (1:1) or in DMF or
dichloromethane alone. In instances where the coupling is
- carried out manually, the success o~ the coupling reaction at
each stage o~ the synthesis is monitored by the ninhydrin
reaction, as described by E. Kaiser et al. (1970) Anal. Biochem.
34:595. In cases where incomplete coupling occurs, the coupling
procedure is repeated be~ore removal o~ the alpha-amino
protecting group prior to the coupling o~ the next amino acid.
The coupling reactions can be per~ormed automatically, as on a
Applied Biosystems automatic synthesizer.
After the desired amino acid sequence has been completed,
the intermediate peptide is removed ~rom the resin support by
treatment with a reagent, such as liquid hydrogen ~luoride, which
not only cleaves the peptide ~rom the resin but also cleaves all
remaining side chain protecting groups and the alpha-amino
protecting group (unless it is an acyl group which is intended
to be present in the ~inal peptide) to obtain the peptide. When
using hydrogen ~luoride ~or cleaving, anisole or cresol and
methylethyl sul~ide are included in the reaction vessel as
scavengers. When Met is present in the sequence, the BOC
protecting group may be cleaved with tri~luoroacetic acid
(TFA)/ethanedithiol prior to cleaving the peptide ~rom the resin
to eliminate potential S-alkylation.
All patents and publications re~erred to in the examples,
and throughout the specii~ication, are incorporated herein by
re~erence, without admission that such is prior art.
The ~ollowing nonlimiting examples are provided to
illustrate the invention. The skilled artisan will recognize
that there may be substitutions and variations o~ the exempli~ied
methods and compositions which are apparent and can be practiced
without departing ~rom the essence o~ the invention.
EXAMPLES
Exam~le 1. Pe~tide Synthesis
The synthesis of the peptide o~ SEQ ID NO:1 (Thr-Ile-Glu-
Asp-Ser-Tyr-Arg-Lys-Gln-Val-Val-Ile-Asp) is conducted in a
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stepwise manner on a MBHA hydrochloride resin, such as available
from Bachem, Inc. (Torrance, CA) having a substitution range of
about 0.1 to 0.5 mmoles/gm. resin.
All equipment employed in the examples is commercially
available. Unless otherwise indicated, all starting materials
employed in the examples are commercially available. Sources ~or
these materials include Sigma Chemical Co. (St. Louis, MO),
Aldrich Chemical Co. (Milwaukee, WI), Lancaster Synthesis
(W;n~lh~m, NH), Fisher Scientific (Pittsburgh, PA), Boehringer
Mannheim Biochemicals (Indianapolis, IN), Fluka Chemical Corp.
(Ronkonkoma, NY) and Chemical Dynamics Corp. (South Plain~ield,
NJ). Most o~ the starting materials were obtained ~rom Aldrich
Chemical Co. (Milwaukee, WI).
All solvents used in the peptide preparations described
herein, e.g. methylene chloride dichloromethane, 2-propanol,
dimethyl~ormamide (DMF), and methanol, were Burdick and Jackson
"distilled in glass" grade and used without additional
distillation. Tri~1uoroacetic acid (TFA), diisopropylethylamine
(DIPEA), piperidine (PIP), dicyclohexylcarbodiimide (DCC),
1-hydroxybenzotriazole (HOBt), and [benzotriazole-1-yl-
oxy-tris(dimethyl) phosphonium hexa~luorophosphate] (BOP) were
purchased ~rom Chemical Dynamics Corp. and were "sequenal" grade
purity. 1~2-ethanedithiol (EDT) was purchased ~rom Sigma
Chemical Co. and used without ~urther puri~ication. All
protected amino acids were o~ the L-con~iguration unless
otherwise indicated and were obtained ~rom Bachem (Torrance, CA).
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39
The synthesis is performed on an Applied Biosystems peptlde
synthesizer (Foster City, CA) using a suitable program,
~ preferably as follows:
STEP REAGENTS AND OPERATIONS MIX TIMES
1 Dichloromethane-80 ml. 2
2 Methanol(MeOH) wash-30 ml. 2
3 Dichloromethane-80 ml. 3
4 50 percent TFA plus 5 percent 1,2-ethane- dithiol in 2
dichloromethane-70 ml.
Isopropanol wash-80 ml. 2
6 TEA 12.5 percent in dichloromethane -70 ml. 2
7 MeOH wash-40 ml. 2
8 Dichloromethane wash-80 ml. 3
9 Boc-amino acid (10 mmoles) in 30 ml. of either DMF or
dichloromethane, depending upon the solubility of the
particular protected amino acid, (1 time) plus DCC (10
mmoles) in dichloromethane (reaction time 20-200 min)
Note: All wash and mix times three minutes except where noted.
Coupling of BOC-ASP(OBz) results in the substitution of
about 0 35 mmol ASP per gram of resin. All solvents that are
used are carefully degassed, preferably by sparging with an inert
gas, e.g., helium or nitrogen, to insure the absence of oxygen.
After deprotection and neutralization, the peptide chain is
built stepwise on the resin. Generally, one to two mmol. of
BOC-protected amino acid in methylene chloride is used per gram
of resin, plus one equivalent of 2 M DCC in methylene chloride,
for two hours. When BOC-Arg(Tos) is being coupled, a mixture of
50~ DMF and methylene chloride is used. Bzl is used as the
hydroxyl side-chain protecting group for Ser and Thr
p-nitrophenyl ester(ONp) can be used to activate the carboxyl end
of Asn or Gln; for example, BOC-Asn(ONp) can be coupled overnight
using one equivalent o~ HOBt in a 50~ mixture of DMF and
methylene chloride. The amido group of Asn or Gln is protected
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by Xan when DCC coupling i5 used instead of the active ester
method. 2-Cl-CBZ is used as the protecting group for the Lys
side chain. Tos is used to protect the guanidine group of Arg
and the imidazole group of His, and the side-chain carboxyl group
of Glu or Asp is protected by OBzl.
To cleave and deprotect the resulting protected
peptide-resin, it is treated with 1.5 ml anisole, 0.5 ml of
methylethylsulfide and 15 ml liquid hydrogen fluoride (HF) per
gram of peptide-resin, first at -20 ~C for 20 min and then at 0
~C for 30 min. This reaction must be performed with great care
owing to the highly toxic and corrosive nature of hydrogen
fluoride. Thls reaction is performed in a commercially available
teflon apparatus (Peninsula Research, Inc., Richmond, CA). After
complete elimination of HF under high vacuum using a KOH trap,
the resin-peptide is washed alternately with dry diethyl ether
and chloroform, and the peptides are then extracted with degassed
2 N aqueous acetic acid and separated from the resin by
filtration on a Hirsch funne~.
The peptide is purified by gel permeation followed by
preparative HPLC as described in Marki et al.(1981) J. Am. Chem.
Soc. 103:3178i Rivier, et al. (1984) J. Chromatography
288:303-328; and Hoeger, et al. (1987) BioChromatography
2:134-142. The chromatographic fractions are carefully monitored
by HPLC (see below), and only the fractions showing substantial
purity are pooled.
To confirm that the desired sequence is achieved, the
peptide is hydrolyzed in sealed evacuated tubes containing
constant boiling HCl, 3 ~l of thioglycol/ml and 1 nmol of Nle (as
an internal standard) for 9 hours at 140 ~C. Amino acid analysis
of the hydrolysates using a Beckman 121 amino acid analyzer to
determine amino acid ratios allows confirmation that the desired
peptide structure has been obtained.
Exam~le 2. Cyclization of Active Pe~tides .
Cyclization "traps" the bioactive conformation o~ the
peptide by making the active conformation part of a ring system
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41
that allows it much less conformational flexibility. In this
procedure, aspartate or glutamate residues are introduced into
~ the sequence either in place of non-essential amino acid residues
or as added residues in the chain. The new peptide is then
~ 5 subjected to electro-oxidation in which the two residues are
decarboxylated, in an intramolecular Kolbe electro-oxidative
coupling reaction, resulting in the joining of their respective -
CH2 groups, forming a ring as shown in Fig. 1. This method has
been used to make a cyclized ~-bend of the dipeptide, Pro-Gly,
by placing a glutamic acid residue on the amino and carboxyl ends
of this dipeptide and then performing the Kolbe electro-oxidation
to form the tetra-(CH2)-bridge. The Pro-Gly peptide, which has
a variety of conformations in solution, when cyclized, was found
to adopt the ~-bend structure uniquely (Joran, A.,
"Conformationally restricted biologically active peptides,
methods for their production and uses thereof," U.S. Patent No.
5,364,851.) This method has been used quite recently to
synthesize cyclized forms of the peptide vasopressin; these forms
have been tested in an in vitro adenylate cyclase system and have
been found to have prolonged half-lives and greater activity than
the native peptide. Therefore, this cyclization procedure may
result in enhanced peptide inhibition and in increased half-life.
Introduction of the cyclizing rigidifying agent reduces the
flexibility of the peptide and concurrently introduces non-polar
aliphatic groups into the peptide (such as the tetra-methylene
bridge shown in Scheme I) that help promote transport of the
peptide through the cell membrane.
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42
Scheme I
H H ~ H H O
I _ 1 11 1 1 11
~rv~_~~ NC C------~rv~~~~v~v~ N C C ~
l I
CH CH
2 I Z
Cl 2 Cl 2
COO-- COO G1U
GI~L
Kolbe
electro Iytic
decarboxylation
1~ H ~ H H O
~~A N--C C ~\N C - C ~~
~CH2
2 c~2 'H2
Di-arninosuberic acid ring
S~ )TE SHEET (RULE 26
_
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43
Scheme I illustrates an exemplary result o~ using
~ electrolytic decarboxylation to cyclize peptides to trap them in
their active con~ormations. Either two gultamate, two aspartate,
or one glutamate and one aspartate residues are introduced in the
chain represented by the wavy line. Under electro-oxidation
shown here ~or two glutamate residues, a tetramethylene bridge
forms cyclizing the region o~ the peptide that is to be held
~ixed in its bioactive con~ormation. The two connected residues
shown constitute the diamino suberic acid moiety.
It should be noted that, in the synthesis o~ this new
peptide, there are possibly other aspartate and glutamate amino
acid residues that can undergo the oxidative decarboxylation.
To prevent these reactions ~rom occurring, these Asp and Glu
residues are protected as esters during the solid phase synthesis
o~ the peptide. The i~ree Glu residues at positions 44 and 46 are
then allowed to undergo the cyclization reaction, a~ter which the
protected acid groups are then deprotected.
This cyclization procedure can be per~ormed on other regions
o~ this peptide and on the other two active peptides.
The electrooxidative coupling reaction used to prepare the
cyclic peptides o~ the invention can be performed in a divided
or an undivided cell such as a standard glass H-cell, as
described in Orqanic Electrochemistrv (2nd Ed.), M. Baizer and
H. Lund, eds., Marcel Dekker, New York, 1983, Chap. 5, p 168
For large scale runs, the reaction can be carried out in a plate
and ~rame ~low cell as described in Technique o~ Electroorqanic
Synthesis, Part III, N. Weinberg and B. Tilak, ed., John Wiley
& Sons, New York, 19 8 2, Chap. III, p 179.
Cathode materials useful ~or the preparation o~ the
compounds o~ the invention include, but are not limited to, high
hydrogen overvoltage materials such as mercury, lead or cadmium.
Anode materials include, but are not limited to, materials such
as mercury, lead, graphite, or graphite paste, which are stable
under electrolysis conditions.
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44
The electrooxidative coupling can occur in aqueous, or
aqueous organic electrolytes, comprising solutions of Bronsted
acids, such as sulfuric, fluoroboric, and tri~luoroacetic acids.
Any electrolyte may be selected that has sufficient acid strength
to render a basic starting material protonated. A dilute
solution of trifluoroacetic acid is most preferred.
Although the preferred method of electrolysis to obtain the
compounds of this invention takes place under constant current
conditions, the oxidative coupling could also be performed using
controlled potential electrolysis, as understood by those skilled
in the art. Typical current densities are between 1 and 5000
milliamps(mA)/cm2, preferably between 10 and 100 mA/cm2. The
reaction is preferably carried out at a temperature in the range
of about 0 ~C to 37 ~C, more preferably about 10 ~C.
A standard glass H-cell (200 ml volume, glass frit
separator) was equipped with a mercury pool cathode 12 cm2 area),
a magnetic stirrer, and a platinum foil anode. The cell
reservoir was filled with 40 ~M trifluoroacetic acid (110 ml) and
placed in a water bath maintained at 10 ~C. The catholyte was
purged with nitrogen. The starting peptide (20 mg) was added to
the catholyte and constant current electrolysis was initiated at
0.1 A. The reaction progress was followed by HPLC and after
passage of 1,060 coulombs, all the substrate had been consumed
and the electrolysis was terminated. The catholyte was recovered
and adjusted to pH 8 with NaOH. The pH-adjusted catholyte was
extracted with chloroform (2 times 70 ml). The extract was
freeze dried and the resultant powdery material extracted with
acetonitrile (HPLC grade). This was filtered through a sintered-
glass filter (fine porosity) and was reduced in volume on a
rotary evaporator using a mechanical vacuum pump to a volume of
2 ml. This material was puri~ied by reversed-phase high pressure
liquid chromatography using a Waters HPLC system with a 0.46 x
0.25 cm column packed with 5 ~m C1~ silica, 300 A pore size. I
Buffer A is an aqueous 0.1~ (vol/vol) trifluoroacetic acid .i
solution (1.0 ml of TFA per 1000 Ml solution); Buffer B is 100~
acetonitrile. The determination is run at room temperature with
a gradient from 15.5~ Buffer B to 75~ Buffer B over a 30 min.
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The flow rate is 2.2 ml per minute, and the retention time is
25.0 min.
~ The structure was confirmed by 300 MHz lH NMR, 13C NMR, and
electrospray mass spectroscopy.
The amounts of the reactants and the conditions required to
facilitate reaction and encourage efficient completion of the
aforementioned Examples may vary widely. However, in general,
the amounts of material employed to induce reaction in the
processes discussed above will be substantially stoichiometric,
unless otherwise specified. In the following examples, reaction
concentrations are generally held at 0.1 M for the reactants,
unless a higher concentration or dilution would be particularly
useful for influencing the direction of a specific reaction. In
practice, the amounts used will depend upon variations in
reaction conditions and the nature of the reactants as readily
apparent to one of ordinary skill in the art.
In any of the methods described hereinabove, the desired
products may be isolated- from the reaction mixture by
crystallization. Alternatively, chromatographic techniques
including, but not limited to, normal phase, reverse phase,
ion-exchange, affinity, or gel permeation, may be employed, as
well as electrophoresis or extraction or other means.
Example 3. Oocvte Maturation AssaY
Using the method described in Chung et al. (1991) Anticancer
Res. 11:1373-1378, test peptides, cyclized peptides and/or
peptidomimetics are injected into immature oocytes at various
doses. The oocytes are co-injected with recombinant transforming
ras p21 obtained from the National Cancer Institute of Japan.
Alternatively, the oncogenic ras p21 can be prepared by the
ordinary skilled artisan without the expense of undue
experimentation as described in Chung et al. (1991) supra and in
Chung et al. (1992) Exp. Cell. Res. 203:329-335 The maturation
of the oocytes is evaluated microscopically at low power (20 X),
using a Nikon Diaphot microscope, for example. Percent
inhibition is calculated based on comparisons with oocytes which
are injected with 0. 05 mg/ml oncogenic ras p21.
CA 02232750 1998-03-23
WO 97/10836 PCT~US96/15098
46
The ~ollowing results were obtained using a dose o~ each
peptide equivalent to an internal oocyte concentration o~ 50 nM:
Peptide ORSequence ID Percent Inhibition of
Numberras-lnduced Maturation
6 28
Val-Val-lle 34
7 56
Lys-Arg-Val 22
76
8 92
2 38
9 65
4 22
The peptidomimetics and cyclic peptides o~ the present
invention will be similarly e~ective in inhibiting oocyte
maturation in response to the oncogenic ras p21 protein, and in
inhibiting oncogenesis.
CA 02232750 1998-03-23
W O 97/10836 PCTrUS96/15098
47
Example 4. SYnthesis oi~ PePtidomimetic p21 ras Inhibitor
A representative peptidomimetic o~ the present invention is
synthesized according to Scheme II, as ~urther described
hereinbelow.
Scheme II
f ~ H2NOH~CI
H J ~, NaOAc,ESOH ~ Na. nPrO~
HO ~ ~ux,~h,72~~ ~O ~ ~flu~.79Y~
~OH
R z c~3C~(CH~3C~(C~)2
~ BnOCOCI. THF ~ t8uO~OH
HO ; K2CC~ ooC--rt HO ~ cr~ DMAP. CH2CIz
NH2 NH(CBZ) 92Y~
3 4
R R
~ ~z,~ P~C ~;~
E~H.~ g3Y~ R'5
NlttCE3Z) NH2
FI' _ tBI~OzC C i 12C O
'
Steroid 1 was commercially available, and used without ~urther
puri~ication. Mono-t-butylmalonate was prepared by literature
methods [Brunwin, D.M.; et al. (1971) J. ChelZI. Soc. C. 3756].
THF was distilled ~rom sodium/benzophenone under Ar. Methylene
chloride and ethanol (absolute) were distilled from CaH2 under
Ar. Benzylchloro~ormate and n-propyl alcohol are commercially
CA 022327~0 1998-03-23
WO 97/10836 PCT~US96/15098
48
available, and used without further purification or drying. All
reactions performed under an atmosphere of Ar unless otherwise
noted
5-~-3-~-hydroxy-6-o~;m;nochole~tane (2): Cholestanone 1 (1.00
g. 2.5 mmol), NaOAc (352 mg, 4.3 mmol) and hydroxylamine
hydrochloride (197 mg, 2.8 mmol) were heated at reflux in
absolute ethanol (15 ml). The reaction was efficiently stirred
under Ar for llh at reflux. The reaction was cooled, and the
solvent removed on the rotary evaporator. The resulting white
solid was dissolved in CHC13, and washed once with brine (80 ml).
The organic layer was dried over Na2SO4, filtered, and
concentrated in vacuo. The yield of crude material was 1.068 g
of a lightly colored solid. The material was recrystallized from
absolute ethanol (15.5 mL) to give 752 mg (72~) of white needles,
mp = 202-204~C (dec). 1H NMR(CDCl3): ~3.60 (m, lH), 3.33 (dd,
= 4.37, 13.6 Hz, lH), 2.10-1.75 (broad m, 6H), 1.70-1.45 (broad
m, 5H), 1.42-1.28 (broad m, 8H), 1.14 (m, 9H), 0.92 (d, J = 6.87
Hz, 3H), 0.88 (d, ~ = 6.54 Hz, 6H), 0.77 (s, 3H), 0.67 (s, 3H).
l3C NMR (CDCl3): ~159.55, 70.77, 56.39, 55.91, 54.04, 49.26,
42.60, 39.42, 39.20, 38.57, 35.92, 35.84, 35.63, 35.43, 31.24,
30.40, 29.39, 27.87, 27.70, 23.80, 23.53, 22.51, 22.26, 21.17,
18.36, 12.34, 11.79, IR (neat film): 3354, 2941, 1667, 1467,
1065, 978 cm~1.
5-~-6-~-amino-3-~-hydroxycholestane (3): Oxime 2 (752 mg, 1.8
mmol) was dissolved with good stirring in boiling n-propyl
alcohol (32 mL). The flask was removed from the oil bath, and
small pieces of freshly prepared sodium wire (2.898 g, 126 mmol)
were added at a rate sufficient to maintain the reflux. After
the addition of the sodium was complete, the flask was lowered
into the bath, and stirred at reflux for 2h (a thick, white crust
forms). The reaction was cooled to room temperature, and
carefully quenched by slow, dropwise addition of water under an
inert atmosphere. The quenched reaction was extracted twice with
CHCl3 (50 mL), and the extracts were washed with brine, dried .I
over Na2SO4, filtered, and concentrated. The yield of a vanilla
colored solid was 723 mg. The material was puri~ied by flash
chromatrography on silica in CHCl3/MeOX (3:2) to give 577 mg
CA 022327~0 l99X-03-23
WO 97/10836 PCTrUS96/15098
49
(79~) of a vanilla colored solid. Alternatively, the product can
be recrystallized from EtOAc. lH NMR (CDCl3): ~3.59 (m, lH),
2.60 (dd, ~ = 2.87, 10.35, 20.73Hz, lH), 2.09 (d, J = 11.79Hz,
lH), 1.99 (d, ~ = 12.94Hz, lH), 1.85 (m, 3H), 1.78 - 1.45 (broad
m, 10H), 1.36 (m, 5H), 1.24 - 0.96 (m, 9H), 0.89 (m, 10H), 0.83
(s, 3H), 0.77 (m, 2H), 0.67 (s, 3H).
N-(benzyloxy carbonyl)-5-~-6-~-amino-3-~-hydroxycholestane (4):
The amine 3 (465 mg. 1.15 mmol) and anhydrous K2CO3(457 mg, 3.3
mmol) were stirred in dry THF (4 mL), and cooled to 0~C in an
ice-water bath. Benzyl-chloroformate (0.16 mL, 1.15 mmol) was
added dropwise, and the reaction was stirred at 0~C for 15 min,
then at room temperature for 6h. The reaction was diluted with
water and poured into saturated NaHCO3 (15 mL). A thick,
flocculent white precipitate ~ormed. The a~ueous mixture was
extracted with CHCl3 (20 mL), and the aqueous layer was saturated
with NaC1 and extracted with CHCl3 (5x20 mL). The organic
extracts were washed with brine (40 mL), dried over Na2SO4,
filtered, and concentrated. ~The yield of crude material was 649
mg of a tan solid. The material was flash chromatographed on
silica in Hex/EtOAc (1:1), (using a little CHC13 to help dissolve
material) to give 546 mg (88~) of a white solid. 1H NMR (CDC13):
~7.88 (m, 4H), 7.23 (s, lH), 5.08 (d, J = 3.08Hz, 2H), 4.86 (d,
J = 9.46Hz, lH), 3.53 (m, lH), 3.50 (s, lH; overlaps with the
multiplet at 3.53), 2.00 (d, J = 12.35Hz, 2H), 1.85 (m, 3H), 1.53
(m, 4H), 1.49 - 1.22 (broad m, 9H), 1.20 - 0.98 (broad m, 10H),
o.9o (m, 9H), 0.87 (s, 3H), 0.77 (m, 2H), 0.66 (s, 3H). IR (neat
film): 3346 (broad), 1691, 1544, 1022 cm~l.
N-(benzyloxy carbonyl)-5-~-6-~-amino-3-~-mono-t-butyl malonyl
cholestane (5): The amine 4 (144.4 mg, 0.27 mmol), mono-t-butyl
malonate (107.7 mg, 0.67 mmol), and DMAP were dissolved in dry
CH2Cl2 (1.2 mL), and stirred efficiently at room temperature.
DCC (67.4 dmg, 0.33 mmol) was added in one portion under a stream
of Ar. The reaction was stirred at room temperature for 23h,
then diluted with ether, and filtered through a pad of Celite (a
white solid remains on the pad). The filtrate was washed with
10~ citric acid (20 mL), saturated NaHCO3 (20 mL), and brine (20
mL). The organic layer was dried over Na2SO4, filtered and
CA 022327~0 1998-03-23
W O 97/10836 PCTAJS96/15098
concentrated. The yield of crude material was 200 mg. The
material was flash chromatographed in Hex/EtOAc (6.7:1) to give
168.7 mg (92~) of a yellow-gold residue lH NMR (CDC13): ~7.36
(s, 4H), 7.29 (s, lH), 5.08 (q, J = 12.25, 27.09Hz, 2H), 4.70
(m, lH), 4.38 (d, J = 9.56Hz, lH), 3.27 (s, 2H), 2.00 (m, 2H),
1.85 (m, 4H), 1.54 (m, 3H), 1.48 (s, 9H), 1.35 (M, 8H), 1.11 (m,
llH), 0.89 9(m, 12H), 0.75 (m, 2H), 0.66 (s, 3H).
The N-CBZ group of 5 was cleanly removed by hydrogenolysis
using 10~ palladium on carbon under a hydrdogen atmosphere to
give 6 in 93~ yield. Trials employing 1,4-cyclohexadiene as the
hydrogen source [Felix et al. (1978) J. Org. Chem. 43:4194]
produced 6 in only 23~ yield, even when a large excess of the
diene was used. Longer reaction time did not improve the yield.
5-~-6-~-amino-3-~-mono-t-butyl malonyl cholestane (6): 5 (200.7
mg, 0.3 mmol) and 10~ palladium on carbon (194 mg) were stirred
vigorously in abs. EtOH (1.8 mL). The system was flushed with a
balloon of hydrogen, and an atmosphere of hydrogen was maintained
by 2 balloons of hydrogen. The reaction was stirred for 20h at
room temperature, then vented with Ar, and suction-filtered
through a tighly packed pad o~ Celite. The flask and filter cake
were thoroughly washed with 1:1 EtOH/THF. The filtrate was
concentrated in vacuo to give 167.8 mg o~ material. The crude
product was flash chromatographed first in Hex/EtOAc (3:2) to
elute off minor byproducts, then CHCl3/MeOH (9:1) to give the
product in a yield of 152.3 mg (93~) of a golden residue. 1H NMR
(CDCl3): ~4.76 (m, lH), 3.27 (s, 2H), 2.60 (m, lH), 2.22 (m, lH),
2.05 - 1.71 (broad m, 6H), 1.51 (s, 3H), 1.49 (s, 9H), 1.34
(broad m, 8H), 1.24 - 0.97 (broad m, 12H), 0.92 (d, J = 6.55Hz,
3H), 0.88 (d, J = 6.64Hz, 6H), 0.85 (s, 3H), 0.75 (m, 2H), 0.67
(s, 3H), IR (neat film): 3368, 2946, 2868, 1747, 1729, 1144, 1008
cm-l
Reductive amination of N(BOC) aminoacetaldehyde [Buchardt
et al. (1993) Org. Prep. Proc. Int. 25:457] is promoted by the
use of NaBH3CN and catalytic acetic acid. t-Butylester and BOC
groups are removed. The general plan is given in Scheme III.
CA 02232750 1998-03-23
PCTAJS96/15098
W O 97/10836
Scheme III
Na2H C~
6 + ~E~OC~HN 4, OE HoA
H 25:1 M~oH/~HF RO H ~
HN N~I~BOC)
R ~ tBu02C S: H2C Q
- HO2C c ~c o
~ r
r ~
R~o ~ _
--NH~"X-
CA 02232750 1998-03-23
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52
~UU~N - ~ LISTING
(1) GENERAL INFORMATION:
ti) APPLICANT: INNAP~ARMA, INC.
(ii) TITLE OF lNV NllON: Peptides and Pepti~o~i~tics Inhibiting
the Oncogenic Action o~ P21 Ras
(iii) NUMBER OF ~u N~S: 52
(iV) ~O~ ?T~.Cp~NIll.:N~ ~nnT~ q
rA ~nD~.~S~: Greenlee, Winner and Sullivan, P.C.
~BJ STREET: 5370 Manhattan Circle, Suite 201
C~ CITY: Boulder
~D~ STATE: Colorado
E~ ~Y: US
~F) ZIP: 80303
(v) COMPUTER ~n~RT.T.' FORM:
(A~ MEDIUM TYPE: Floppy disk
~B~ CCM~U1~:K: IBM PC compatible
iC OPERATING SYSTEM: PC-DOS/MS-DOS
~D) SOFTWARE: PatentIn Release ~1.0, Version ~1.30
(~i) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: PCT/US96/15098
(B) FILING DATE: 20-SEP-1996
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NU~BER: US 08/531,525
(B) FILING DATE: 21-SEP-1995
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/004,091
(B) FILING DATE: 21-SEP-1995
(~iii) AlLO~N~Y/AGENT INFORMATION:
(A) NAME: Ferber, Donna M.
(B) REGISTRATION N~MBER: 33,878
(C) REFERENCE/DOCKET NU~E3ER: 37-94A WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (303) 499-8080
(B) TELEFAX: (303) 499-8089
(2) INFORMATION FOR SEQ ID NO:l:
(i) ~Qu~ CHARACTERISTICS:
~A LENGTH: 6 amino acids
(B~ TYPE: amino acid
~ C I S TR.~ N I ) t~: I ] N ~: .C:
~D~ TOPOLOGY: linear
(ii~ MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO .
I
S~J~ 111 ~JTE SHEET (RULE 26)
CA 022327S0 1998-03-23
W O 97/10836 PCT~US96/15098
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Ile Lys Arg Val Lys Asp
l 5
(2) INFORMATION FOR SEQ ID NO:2:
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Lys Cys Asp Leu Ala
l 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS:
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOT~ETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Cys Asp Leu Ala Ala Arg Thr
l 5
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRAN~SS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Asp Leu Ala Ala
(2) INFORMATION FOR SEQ ID NO:5:
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WO 97/10836 PCT~US96/15098
54
( i ) S~U~N~ CHARACTERISTICS:
(A) LENGTH: 188 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys
Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr
Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly
Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr
Ser Ala Met Ary Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys
Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His Gln Tyr
Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val
100 105 110
Leu Val Gly Asn Lys Cys Asp Leu Ala Ala Thr Val Glu Ser Arg Gln
115 120 125
Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr Ser
130 135 140
Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val Arg
145 150 155 160
Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp Glu Ser
165 170 175
Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser
180 185
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CA 022327~0 l998-03-23
W O 97/10836 PCT~US96/15098
Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp
1 5 10
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Tyr Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro
1 5 10 15
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Gly Asn Lys Cys Asp Leu Ala Ala Arg Thr Val Glu
1 5 lO
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(C) sTR~NnFDNF-~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Cys Asp Leu Ala Ala Arg Thr
1 5
(2) INFORMATION FOR SEQ ID NO:10:
(i) ~U~N~ CHARACTERISTICS:
(A) LENGTH: 215 amino acids
CA 022327~0 l998-03-23
WO 97/10836 PCT~US96/l5098
56
(B) TYPE: amino acid
(C) STR~Nn~n~.~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Met Ala Ala Pro Pro Ala Arg Ala Arg Ala Asp Tyr Asp Tyr Leu Ile
1 5 10 15
Lys Leu Leu Leu Ile Gly Asp Ser Gly Val Gly Lys Ser Cys Leu Leu
Leu Arg Phe Ser Asp Gly Ser Phe Thr Thr Ser Phe Ile Thr Thr Ile
Gly Ile Asp Phe Lys Ile Arg Thr Ile Glu Leu Asp Gly Lys Arg Ile
Lys Leu Gln Ile Trp Asp Thr Ala Gly Gln Glu Arg Arg Thr Ile Thr
Thr Ala Tyr Tyr Arg Gly Ala Met Gly Ile Leu Leu Val Tyr Asp Val
Thr Asp Glu Ser Ser Phe Asn Asn Ile Arg Asn Trp Ile Arg Asn Ile
100 105 110
Glu Gln His Ala Ser Asp Asn Val Asn Lys Ile Leu Val Gly Asn Lys
115 120 125
Ala Asp Met Asp Glu Ser Lys Arg Ala Val Pro Thr Ala Lys Gly Gln
130 135 140
Ala Leu Ala Asp Glu Tyr Gly Ile Lys Phe Phe Glu Thr Ser Ala Lys
145 150 155 160
Thr Asn Leu Asn Val Glu Glu Val Phe Phe Ser Ile Gly Arg Asp Ile
165 170 175
Lys Gln Arg Leu Ser Asp Thr Asp Ser Arg Ala Glu Pro Ala Thr Ile
180 185 190
Lys Ile Ser Gln Thr Asp Gln Ala Ala Gly Ala Gly Gln Ala Thr Gln
195 200 205 ':
Lys Ser Ala Cys Cys Gly Thr
210 215
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 amino acids .
(B) TYPE: amino acid
(C) STRP~nF.n~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
!
CA 022327~0 1998-03-23
W O 97/10836 PCT~US96/15098
57
(iii) ~YPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:
Met Ala Gly Tyr Ala Asp Glu Glu Tyr Asp Tyr Leu Phe Lys Leu Val
1 5 10 15
Leu Ile Gly Asp Ser Gly Val Gly Lys Ser Asn Leu Leu Ser Arg Phe
Thr Lys Asn Phe Asn Leu Glu Ser Lys Ser Thr Ile Gly Val Glu Phe
Ala Thr Lys Thr Thr Lys Val Glu Gly Lys Val Val Lys Ala Gln Ile
Trp Asp Thr Ala Gly Gln Glu Arg Tyr Arg Ala Ile Thr Ser Ala Tyr
Tyr Arg Gly Ala Val Gly Ala Leu Leu Ile Tyr Asp Val Thr Arg His
Ala Thr Phe Glu Asn Ala Ala Arg Trp Leu Arg Glu Leu Arg Gly His
100 105 110
Thr Asp Pro Asn Ile Val Val Met Leu Ile Gly Asn Lys Cys Asp Leu
115 120 125
Arg His Leu Val Ala Val Lys Thr Glu Glu Ala Lys Ala Phe Ala Glu
130 135 140
Arg Glu Ser Leu Tyr Phe Met Glu Thr Ser Ala Leu Asp Ala Thr Asn
145 150 155 160
Val Glu Asn Ala Phe Thr Glu Val Leu Thr Gln Ile His Lys Ile Val
165 170 175
Ser Lys Arg Ser Val Asp Gly Gly Gly Ser Ala Asp Leu Pro Gly Lys
180 185 190
Gly Glu Thr Ile Asn Val Lys Glu Asp Gly Ser Val Leu Lys Arg Met
195 200 205
Gly Cys Cys Ser Asn
210
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 amino acids
(B) TYPE: amino acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOT~ETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Arabidopsis thaliana
CA 022327~0 l998-03-23
WO 97/10836 PCTAJS96/1~098
58
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Ser Ser Asp Asp Glu Gly Arg Glu Glu Tyr Phe Lys Ile Val Val
1 5 10 15
Ile Gly Asp Ser Ala Val Gly Lys Ser Asn Leu Leu Ser Arg Tyr Ala
Arg Asn Glu Phe Ser Ala Asn Ser Lys Ala Thr Ile Gly Val Glu Phe
Gln Thr Gln Ser Met Ile Glu Gly Lys Glu Val Lys Ala Gln Ile Trp
Asp Thr Ala Gly Gln Glu Phe Arg Ala Val Thr Ser Tyr Tyr Arg Gly
Ala Val Gly Ala Leu Val Val Tyr Asp Ile Thr Arg Arg Thr Thr Phe
Glu Ser Val Gly Arg Trp Leu Asp Glu Leu Lys Ile His Ser Asp Thr
100 105 110
Thr Val Ala Arg Met Leu Val Gly Asn Lys Cys Asp Leu Glu Asn Ile
115 120 125
Arg Ala Val Ser Val Glu Glu Gly Lys Ala Leu Ala Glu Glu Glu Gly
130 135 140
Leu Phe Phe Val Glu Thr Ser Ala Leu Asp Ser Thr Asn Val Lys Thr
145 150 - 155 160
Ala Phe Glu Met Val Ile Leu Asp Ile Tyr Asn Asn Val Ser Arg Lys
165 170 175
Gln Leu Asn Ser Asp Thr Tyr Lys Asp Glu Leu Thr Val Arg Val Ser
180 185 190
Leu Val Lys Asp Asp Asn Ser Ala Ser Lys Gln Ser Ser Gly Phe Ser
195 200 205
Cys Cys Ser Ser Thr
210
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 201 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nN~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Discopyge ommata
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Asn Pro Glu Tyr Asp Tyr Leu Phe Lys Leu Leu Leu Ile Gly Asp
1 5 10 15
Ser Gly Val Gly Lys Ser Cys Leu Leu Leu Arg Phe Ala Asp Asp Thr
CA 022327~0 l998-03-23
W O 97/10836 PCTAUS96/15098
Tyr Thr Glu Ser Tyr Ile Ser Thr Ile Gly Val Asp Phe Lys Ile Arg
Thr Ile Glu Leu Asp Gly Lys Thr Ile Lys Leu Gln Ile Trp Asp Thr
Ala Gly Gln Glu Arg Phe Arg Thr Ile Thr Ser Ser Tyr Tyr Arg Gly
Ala His Gly Ile Ile Val Val Tyr Asp Val Thr Asp Gln Glu Ser Phe
Asn Asn Val Lys Gln Trp Leu Gln Glu Ile Asp Arg Tyr Ala Ser Glu
100 105 110
Asn Val Asn Lys Leu Leu Val Gly Asn Lys Cys Asp Leu Thr Thr Lys
115 120 125
Lys Val Val Asp Tyr Thr Thr Lys Glu Phe Ala Asp Ser Leu Gly Ile
130 135 140
Pro Phe Leu Glu Thr Ser Ala Lys Asn Ala Thr Asn Val Glu Gln Ala
145 150 155 160
Phe Met Thr Met Ala Ala Glu Ile Lys Lys Arg Met Gly Pro Gly Ala
165 170 175
Thr Ser Gly Gly Ser Glu Lys Ser ~sn Val Asn Ile Gln Ser Thr Pro
180 - 185 190
Val Lys Ser Ser Gly Gly Gly Cys Cys
195 200
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 202 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Lymnea stagnalis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Met Ser Thr Met Asn Pro Asp Tyr Asp Tyr Leu Phe Lys Leu Leu Leu
l 5 10 15
Ile Gly Asp Ser Gly Val Gly Lys Ser Cys Leu Leu Leu Arg Phe Ala
Asp Asp Thr Tyr Thr Glu Ser Tyr Ile Ser Thr Ile Gly Val Asp Phe
Lys Ile Arg Thr Ile Glu Leu Asp Gly Lys Thr Ile Lys Leu Gln Ile
Trp Asp Thr Ala Gly Gln Glu Arg Phe Arg Thr Ile Thr Ser Ser Tyr
CA 022327~0 l998-03-23
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Tyr Arg Gly Ala His Gly Ile Ile Val Val Tyr Asp Val Thr Asp Gln
Glu Ser Phe Asn Asn Val Lys Gln Trp Leu Gln Glu Ile Asp Arg Tyr
100 105 110
Ala Ser Glu Asn Val Asn Lys Leu Leu Val Gly Asn Lys Ser Asp Leu
115 120 125
Thr Thr Lys Lys Val Asp Phe Thr Thr Ala Lys Glu Tyr Ala Asp Gln
130 135 140
Leu Gly Ile Pro Phe Leu Glu Thr Ser Ala Lys Asn Ala Thr Asn Val
145 150 155 160
Glu Gln Ala Phe Met Thr Met Ala Ala Glu Ile Lys Asn Arg Met Gly
165 170 175
Pro Ile Thr Ala Ser Asp Ser Lys Pro Ser Val Lys Ile Asn Ser Ser
180 185 190
Thr Pro Ser Ala Asn Lys Gly Gly Cys Cys
195 200
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid
(C) STR~n~n~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Ala Tyr Ala Tyr Leu Phe Lys Tyr Ile Ile Ile Gly Asp Thr Gly
1 5 10 15
Val Gly Lys Ser Cys Leu Leu Leu Gln Phe Thr Asp Lys Arg Phe Gln
Pro Val His Asp Leu Thr Ile Gly Val Glu Phe Gly Ala Arg Met Ile
g5
Thr Ile Asp Gly Lys Gln Ile Lys Leu Gln Ile Trp Asp Thr Ala Gly
Gln Glu Ser Phe Arg Ser Ile Thr Arg Ser Tyr Tyr Arg Gly Ala Ala
Gly Ala Leu Leu Val Tyr Asp Ile Thr Arg Arg Asp Thr Phe Asn His
Leu Thr Thr Trp Leu Glu Asp Ala Arg Gln His Ser Asn Ser Asn Met
100 105 110
Val Ile Met Leu Ile Gly Asn Lys Ser Asp Leu Glu Arg Arg Glu Val
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115 120 125
Lys Lys Glu Glu Gly Glu Ala Phe Ala Glu His Gly Leu Ile Phe Met
130 135 140
Glu Thr Ala Lys Thr Ala Ser Val Glu Glu Ala Phe Ile Asn Thr Ala
145 150 155 160
Lys Glu Ile Tyr Glu Lys Ile Gln Glu Gly Val Phe Asp Ile Asn Asn
165 170 175
Glu Ala Asn Gly Ile Lys Ile Gly Pro Gln His Ala Ala Thr Asn Ala
180 185 190
Thr His Ala Gly Asn Gln Gly Gly Gln Gln Ala Gly Gly Gly Cys Cys
195 200 205
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 210 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(~i) ORIGINAL SOURCE:
(A) ORGANISM: Lymnea stagnalis
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Met Ser Tyr Ala Tyr Leu Phe Lys Tyr Ile Ile Ile Gly Asp Thr Gly
1 5 10 15
Val Gly Lys Ser Cys Leu Leu Leu Gln Phe Thr Asp Lys Arg Phe Gln
Pro Val His Asp Leu Thr Ile Gly Val Glu Phe Gly Ala Arg Met Ile
Thr Ile Asp Gly Lys Gln Ile Lys Leu Gln Ile Trp Asp Thr Ala Gly
Gln Glu Ser Phe Arg Ser Ile Thr Arg Ser Tyr Tyr Arg Gly Ala Ala
Gly Ala Leu Leu Val Tyr Asp Ile Thr Arg Arg Asp Thr Phe Asn His
Leu Thr Thr Trp Leu Glu Asp Ala Arg Gln His Ser Asn Ser Asn Met
100 105 110
Val Ile Met Leu Ile Gly Asn Lys Ser Asp Leu Glu Ala Arg Arg Val
115 120 125
Lys Lys Glu Glu Gly Glu Ala Phe Arg Glu ~is Gly Leu Ile Phe Met
130 135 140
Glu Thr Ser Ala Lys Thr Ala Ala Asn Val Glu Glu Ala Phe Ile Asn
145 150 155 160
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Thr Ala Lys Glu Ile Tyr Gln Lys Ile Gln Asp Gly Val Phe Asp Ile
165 170 175
Asn Asn Glu Ala Asn Gly Ile Lys Ile Gly Pro Gln His Ser Pro Ala
180 185 l90
Ser Gln Ser Leu Asn Val Gly Gly Ser Gly Gly Asn Gln Gly Gly Asn
195 200 205
Cys Cys
210
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid
(C) STRANn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Oryctolagus cuniculus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Met Ala Tyr Ala Tyr Leu Phe Lys Tyr Ile Ile Ile Gly Asp Thr Gly
1 5 10 15
Val Gly Lys Ser Cys Leu Leu Leu Gln Phe Thr Asp Lys Arg Phe Gln
Pro Val His Asp Leu Thr Ile Gly Val Glu Phe Gly Ala Arg Met Ile
Thr Ile Asp Gly Lys Gln Ile Lys Leu Gln Ile Trp Asp Thr Ala Gln
Glu Ser Phe Arg Ser Ile Arg Ser Tyr Tyr Arg Gly Ala Gly Ala Leu
Leu Val Tyr Asp Ile Thr Arg Arg Asp Thr Phe Asn His Leu Thr Thr
Trp Leu Glu Asp Ala Arg Gln His Ser Asn Ser Asn Met Val Ile Met
100 105 110
Leu Ile Gly Asn Lys Ser Asp Leu Glu Ser Arg Arg Glu Val Lys Lys
115 120 125
Glu Glu Gly Glu Ala Phe Ala Arg Glu His Gly Leu Ile Phe Met Glu
130 135 140
Thr Ser Ala Lys Thr Ala Ser Asn Val Glu Glu Ala Phe Ile Asn Thr
145 150 155 160
Ala Lys Glu Ile Tyr Glu Lys Ile Gln Glu Gly Val Phe Asp Ile Asn
165 170 175
Asn Glu Ala Asn Gly Ile Lys Ile Gly Pro Gln His Gly Ala Thr Asn
180 185 190
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Ala His Ala Gly Asn Gln Gly Gly Gln Gln Ala Gly Gly Gly Cys Cys
195 200 205
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
~ (A) LENGTH~ 212 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus norvegicus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Met Ala Tyr Ala Tyr Leu Phe Lys Tyr Ile Ile Ile Gly Asp Thr Gly
1 5 10 15
Val Gly Lys Ser Cys Leu Leu Leu Gln Phe Thr Asp Lys Arg Phe Gln
Pro Val His Asp Leu Thr Met Gly Val Glu Phe Gly Ala Arg Met Ile
Thr Ile Asp Gly Lys Gln Ile Lys Leu Gln Ile Trp Asp Thr Ala Gly
Gln Glu Ser Phe Arg Ser Ile Thr Arg Ser Tyr Tyr Arg Gly Ala Ala
Gly Ala Leu Leu Val Tyr Asp Ile Thr Arg Arg Asp Thr Phe Asn His
Leu Thr Thr Trp Leu Glu Asp Ala Arg Gln His Ser Asn Ser Asn Met
100 105 110
Val Ile Met Leu Ile Gly Asn Lys Ser Asp Leu Glu Ser Arg Arg Glu
115 120 125
Val Lys Lys Glu Glu Gly Glu Ala Phe Ala Arg Glu His Gly Leu Ile
130 135 140
Phe Met Glu Thr Ser Ala Lys Thr Ala Ser Asn Val Glu Glu Ala Phe
145 150 155 160
Ile Asn Thr Ala Lys Glu Ile Tyr Glu Lys Ile Gln Glu Gly Val Phe
165 170 175
Asp Ile Asn Asn Glu Ala Asn Gly Ile Lys Ile Gly Pro Gln His Ala
180 185 190
Ala Thr Asn Ala Ser His Gly Gly Asn Gln Gly Gly Gln Gln Ala Gly
195 200 205
Gly Gly Cys Cys
210
(2) INFORMATION FOR SEQ ID NO:19:
-
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 218 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Drosophila melanogaster
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Met Ala Gly Gly Asp Pro Lys Trp Gln Lys Asp Ala Ala Asp Gln Asn
1 5 10 15
Phe Asp Tyr Met Phe Lys Leu Leu Ile Ile Gly Asn Ser Ser Val Gly
Lys Thr Ser Phe Leu Phe Arg Tyr Ala Asp Asp Ser Phe Thr Ser Ala
Phe Val Ser Thr Val Gly Ile Asp Phe Lys Val Lys Thr Val Phe Arg
His Asp Lys Arg Val Lys Leu Gln Ile Trp Asp Thr Ala Gly Gln Glu
Arg Tyr Arg Thr Ile Thr Thr Ala Tyr Tyr Arg Gly Ala Met Gly Phe
Ile Leu Met Tyr Asp Val Thr Asn Glu Asp Ser Phe Asn Ser Val Gln
100 105 110
Asp Trp Val Thr Gln Ile Lys Thr Tyr Ser Trp Asp Asn Ala Gln Val
115 120 125
Ile Leu Val Gly Asn Lys Cys Asp Met Glu Asp Gln Arg Val Ile Ser
130 135 140
Phe Glu Arg Gly Arg Gln Leu Ala Asp Gln Leu Gly Val Glu Phe Phe
Glu Thr Ser Ala Lys Glu Asn Val Asn Val Lys Ala Val Phe Glu Arg
165 170 175
Leu Val Asp Ile Ile Cys Lys Met Ser Glu Ser Leu Asp Ala Asp Pro
180 185 190
Thr Leu Val Gly Gly Gly Gln Lys Gly Gln Arg Leu Thr Asp Gln Pro
195 200 205
Gln Gly Thr Pro Asn Ala Asn Cys Asn Cys
210 215
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CXARACTERISTICS:
(A) LENGTH: 208 amino acids
(B) TYPE: amino acid
(C) STR~N~:SS: single
(D) TOPOLOGY: linear
-
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(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Rattus norvegicus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met Ser Glu Thr Tyr Asp Phe Leu Lys Phe Leu Val Ile Gly Asn Ala
1 5 10 15
Gly Thr Gly Lys Ser Cys Leu Leu His Gln Phe Ile Glu Lys Lys Phe
Lys Asp Asp Ser Asn His Thr Ile Gly Val Glu Phe Gly Gln Lys Ile
Ile Asn Val Gly Gly Lys Tyr Val Lys Leu Gln Ile Trp Asp Thr Ala
Gly Gln Glu Arg Phe Arg Val Thr Thr Ser Tyr Arg Gly Ala Ala Gly
Ala Leu Leu Val Tyr Asp Ile Thr Ser Arg Glu Thr Tyr Asn Ala Leu
Thr Asn Trp Leu Thr Asp Ala Arg Met Leu Ala Ser Gln Asn Ile Val
100 105 110
Ile Cys Gly Asn Lys Lys Asp Leu Asp Ala Asp Arg Glu Val Thr Phe
115 120 125
Leu Glu Ala Ser Arg Phe Ala Gln Glu Asn Glu Leu Met Phe Leu Glu
130 135 140
Thr Ser Ala Leu Thr Gly Glu Asn Val Glu Glu Ala Phe Met Gln Cys
145 150 155 160
Ala Arg Lys Ile Leu Asn Lys Ile Glu Ser Gly Glu Leu Asp Pro Glu
165 170 175
Arg Met Gly Ser Gly Ile Gln Tyr Gly Asp Ala Ala Leu Arg Gln Leu
180 185 190
Arg Ser Pro Arg Arg Thr Gln Ala Pro Ser Ala Gln Glu Cys Gly Cys
195 200 205
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS: ¦
(A) LENGTH: 203 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Caenorhabditis elegans
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Met Ala Asp Phe Thr Asn Asn Ala Leu Lys Lys Phe Lys Leu Val Phe
1 5 10 15
Leu Gly Glu Gln Ser Val Gly Lys Thr Ser Ile Ile Thr Arg Phe Met
Tyr Asp Ser Phe Asp Asn Thr Tyr Gln Ala Thr Ile Gly Ile Asp Phe
Leu Ser Lys Thr Met Tyr Leu Glu Asp Arg Thr Ile Arg Leu Gln Leu
Trp Asp Thr Ala Gly Gln Glu Arg Phe Arg Ser Leu Ile Pro Ser Tyr
Ile Arg Asp Ser Ser Val Ala Val Val Val Tyr Asp Ile Thr Asn Ala
Asn Ser Phe His Gln Thr Thr Lys Trp Val Asp Asp Val Arg Asn Glu
100 105 110
Arg Gly Cys Asp Val Ile Ile Val Leu Val Gly Asn Lys Thr Asp Leu
115 120 125
Ala Asp Lys Arg Gln Val Ser Thr Glu Asp Gly Glu Lys Lys Ala Arg
130 135 140
Asp Leu Asn Val Met Phe Ile Glu Thr Ser Ala Lys Ala Gly Tyr Asn
145 150 155 160
Val Lys Gln Leu Phe Arg Lys Ile Ala Leu Pro Gly Ile Val Gln Glu
165 170 175
Glu Thr Pro Glu Gln Pro Asn Ile Val Ile Met Asn Pro Pro Lys Asp
180 185 190
Ala Glu Glu Ser Gln Gly Arg Gln Cys Pro Cys
195 200
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 207 amino acids
(B) TYPE: amino acid
(C) STR~Nn~n~S single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
. i
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Met Ser Thr Gly Gly Asp Phe Gly Asn Pro Leu Arg Lys Phe Lys Leu
1 5 10 15
Val Phe Leu Gly Glu Gln Ser Val Gly Lys Thr Ser Leu Ile Thr Arg
Phe Met Tyr Asp Ser Phe Asp Asn Thr Tyr Gln Ala Thr Ile Gly Ile
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Asp Phe Leu Ser Lys Thr Met Tyr Leu Glu Asp Arg Thr Val Arg Leu
Gln Leu Trp Asp Thr Ala Gly Gln Glu Arg Phe Arg Ser Leu Ile Pro
Ser Tyr Ile Arg Asp Ser Thr Val Ala Val Val Val Tyr Asp Ile Thr
Asn Val Asn Ser Phe Gln Gln Thr Thr Lys Trp Ile Asp Asp Val Arg
100 105 110
Thr Glu Arg Gly Ser Asp Val Ile Ile Met Leu Val Gly Asn Lys Thr
115 120 125
Asp Leu Ala Asp Lys Arg Gln Val Ser Ile Glu Glu Gly Glu Arg Lys
130 135 140
Ala Lys Glu Leu Asn Val Met Phe Ile Glu Ser Ala Lys Ala Gly Tyr
145 150 155 160
Asn Val Lys Gln Leu Phe Arg Arg Val Ala Ala Ala Leu Pro Gly Met
165 170 175
Glu Ser Thr Gln Asp Arg Ser Arg Glu Asp Met Ile Asp Ile Lys Leu
180 185 190
Glu Lys Pro Gln Glu Gln Pro Val Ser Glu Gly Gly Cys Ser Cys
195 200 205
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 203 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Canis ~amiliaris
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Met Thr Ser Arg Lys Lys Val Leu Leu Lys Val Ile Ile Leu Gly Asp
1 5 10 15
Ser Gly Val Gly Lys Thr Ser Leu Met Asn Gln Tyr Val Asn Lys Lys
Phe Ser Asn Gln Tyr Lys Ala Thr Ile Gly Ala Asp Phe Leu Thr Lys
Glu Val Met Val Asp Asp Ary Leu Val Thr Met Gln Ile Trp Asp Thr
Ala Gly Gln Glu Arg Phe Gln Ser Leu Gly Val Phe Tyr Arg Gly Ala
Asp Cys Cys Val Leu Val Phe Asp Val Thr Ala Pro Asn Thr Phe Lys
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Thr Leu Asp Ser Trp Arg Asp Glu Phe Leu Ile Gln Ala Ser Pro Arg
100 105 110
Asp Pro Glu Asn Phe Pro Phe Val Val Leu Gly Asn Lys Ile Asp Leu
115 120 125
Glu Asn Arg Gln Val Ala Thr Lys Arg Ala Gln Ala Trp Cys Tyr Ser
130 135 140
Lys Asn Asn Ile Pro Tyr Phe Glu Thr Ser Ala Lys Glu Ala Ile Asn
145 150 155 160
Val Glu Gln Ala Phe Gln Thr Ile Ala Arg Asn Ala Leu Lys Gln Glu
165 170 175
Thr Glu Val Glu Leu Tyr Asn Glu Phe Pro Glu Pro Ile Lys Leu Asp
180 185 190
Lys Asp Ala Lys Thr Ser Ala Glu Cys Ser Cys
195 200
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 202 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nM~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Dictyostelium discoideum
(Xi) ~QU~N~ DESCRIPTION: SEQ ID NO:24:
Met Thr Lys Lys Lys Val Leu Leu Lys Val Ile Ile Leu Gly Asp Ser
1 5 10 15
Gly Val Gly Lys Thr Ser Leu Met Asn Gln Tyr Val Asn Lys Lys Phe
Ser Asn Gln Tyr Lys Ala Thr Ile Gly Ala Asp Phe Leu Thr Lys Glu
Leu Met Val Asp Asp Arg Val Val Thr Met Gln Ile Trp Asp Thr Ala
Gly Gln Glu Arg Phe Gln Ser Leu Gly Val Ala Phe Tyr Arg Gly Ala
Asp Cys Cys Val Leu Cys Tyr Asp Val Asn Val Ala Lys Thr Phe Glu
Asn Leu Asp Ser Trp Arg Asp Glu Phe Leu Ile Gln Ala Gly Pro Arg
100 105 110
Asp Pro Asp Asn Phe Pro Phe Val Val Leu Gly Asn Lys Ile Asp Leu
115 120 125
Glu Asn Gln Arg Val Val Ser Gln Lys Arg Ala Ala Ser Trp Cys Gln
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130 135 140
Ser Lys Gly Asn Ile Pro Tyr Phe Glu Thr Ser Ala Lys Glu Ala Ile
145 150 155 160
Asn Val Glu Gln Ala Phe Gln Thr Ile Ala Arg Asn Ala Ile Lys Leu
165 170 175
Glu Asp Gly Leu Val Phe Pro Ile Pro Thr Asn Ile Gln Val Ile Pro
180 185 190
Glu Pro Gln Pro Ala Ly5 Ser Gly Cys Cys
195 200
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Canis ~m; 1 l~is
(xi) ~U~N~ DESCRIPTION: SEQ ID NO:25:
Met Lys Thr Tyr Asp Tyr Leu Phe Lys Leu Leu Leu Ile Gly Asp Ser
1 5 10 15
Gly Val Gly Lys Thr Cys Val Leu Phe Arg Phe Ser Glu Asp Ala Phe
Asn Ser Thr Phe Ile Ser Thr Ile Gly Ile Asp Phe Lys Ile Arg Thr
Ile Glu Leu Asp Gly Lys Arg Ile Lys Leu Gln Ile Trp Asp Thr Ala
Gly Gln Glu Arg Phe Arg Thr Ile Thr Thr Ala Tyr Tyr Arg Ala Met
Gly Ile Met Leu Val Tyr Asp Ile Thr Asn Glu Lys Ser Phe Asp Asn
Ile Arg Asn Trp Ile Arg Asn Ile Glu Glu His Ala Ser Ala Asp Val
100 105 110
Glu Lys Met Ile Leu Gly Asn Lys Cys Asp Val Asn Asp Lys Arg Gln
115 120 125
Val Ser Lys Glu Arg Gly Glu Lys Leu Ala Leu Asp Tyr Gly Ile Lys
130 135 140
Phe Met Glu Thr Ser Ala Lys Ala Asn Ile Asn Val Glu Asn Ala Phe
145 150 155 160
Phe Thr Leu Ala Arg Asp Ile Lys Ala Lys Met Asp Lys Lys Leu Glu
165 170 175
Gly Asn Ser Pro Gln Gly Ser Asn Gln Gly Val Lys Ile Thr Pro Asp
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180 185 190
Gln Gln Lys Arg Ser Ser Phe Phe Arg Cys Val Leu Leu
195 200 205
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 191 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Dictyostelium discoideum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Met Glu Glu Glu Ile Leu Tyr Lys Ile Ile Leu Val Gly Glu Ser Gly
1 5 10 15
Val Gly Lys Ser Ser Ile Leu Val Arg Phe Thr Asp Asn Thr Phe Ser
Gln His Phe Ala Pro Thr Leu Gly Val Phe Val Lys Thr Ile Arg Asn
Lys Glu Thr Gly Gln Thr Val Lys Leu Gln Leu Trp Asp Thr Ala Gly
Gln Glu Arg Phe Lys Ser Ile Thr Gln Phe Tyr Arg Gly Ser His Gly
Val Ile Val Val Tyr Asp Val Thr Asp Pro Lys Ser Phe Glu Arg Leu
Lys Asn Trp Val Glu Asp Ile Asn Gln Tyr Thr Gln Asp Gly Met Ile
100 105 110
Ile Ile Leu Val Gly Asn Lys Ser Asp Met Val Ala Gln Arg Lys Val
115 120 125
Thr Phe Glu Gln Gly Gln Glu Met Ala Glu Gln Leu Lys Thr Lys Phe
130 135 1~0
Leu Glu Val Ser Ala Lys Glu Asn Asn Gly Val Thr Gln Val Phe Asp
145 150 155 160
Leu Leu Val Gln Asp Ile Glu Ala Thr Met Lys Asn Ser Lys Val Ala
165 170 175
Gln Asn Gln Leu Asn Leu Ser Val Gly Gln Glu Arg Gly Cys Cys
180 185 190
(2) INFORMATION FOR SEQ ID NO:27: ~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 189 amino acids
(B) TYPE: amino acid
(C) sTR~ND~nN~s: single
(D) TOPOLOGY: linear
I
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(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Caenorhabditis elegans
.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Met Gln Ala Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys
1 5 10 15
Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Ala Phe Pro Gly Glu Tyr
Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Val Asp Gly
Arg Pro Ile Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln Asp Tyr Asp
Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Val Cys
Phe Ala Leu Asn Asn Pro Ala Ser Phe Glu Asn Val Arg Ala Lys Trp
Tyr Pro Glu Val Ser His His Cys Pro Asn Thr Pro Ile Ile Leu Val
100 105 110
Gly Thr Lys Ala Asp Leu Arg Glu Asp Asp Thr Val Glu Arg Leu Arg
115 120 125
Glu Arg Arg Leu Gln Pro Val Ser Gln Thr Gln Gly Tyr Val Met Ala
130 135 140
Lys Glu Ile Lys Ala Val Lys Tyr Leu Glu Cys Ser Ala Leu Thr Gln
145 150 155 160
Arg Gly Leu Lys Gln Val Phe Asp Glu Ala Ile Arg Ala Val Val Thr
165 170 175
Pro Pro Gln Arg Ala Lys Lys Ser Lys Cys Thr Val Leu
180 185
(2) INFORMATION FOR SEQ ID NO:28:
(i) ~u~ CHARACTERISTICS:
(A) LENGTH: 191 amino acids
(B) TYPE: amino acid
(C) STR~n~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SO~RCE:
(A) ORGANISM: Dictyostelium discoideum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Met Gln Ala Ile Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys
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1 5 10 15
Thr Cys Leu Leu Ile Ser Tyr Thr Thr Asn Ala Phe Pro Gly Glu Tyr
Ile Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Val Asp Gly
Lys Pro Ile Asn Leu Gly Leu Trp Asp Thr Ala Gly Gln Glu Asp Tyr
Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Ile
Cys Phe Ser Ile Ile Ser Pro Ser Ser Phe GlU Asn Val Asn Gly Lys
Trp His Pro Glu Ile Cys Xis His Pro Asn Val Pro Ile Leu Val Gly
100 105 110
Thr Lys Leu Asp Met Arg Asp Lys Glu Thr Gln Asp Arg Leu Lys Glu
115 120 125
Lys Lys Leu Tyr Pro Ile Ser Tyr Glu Gln Gly Leu Ala Lys Met Lys
130 135 140
Glu Ile Asn Ala Val Lys Tyr Leu Glu Cys Ser Ala Leu Thr Glu Lys
145 150 155 160
Gly Leu Lys Thr Val Phe Asp Glu Ala Ile Arg Ala Val Ile Asn Pro
165 170 175
Pro Leu Ser Lys Lys Lys Lys Ser Ser Gly Gly Cys Asn Ile Leu
180 185 190
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 192 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Dictyostelium discoideum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Met Gln Ser Ile Lys Leu Val Val Val Gly Asp Gly Ala Val Gly Lys
1 5 10 15
Thr Cys Leu Leu Ile Ser Tyr Thr Ser Asn Ser Phe Pro Thr Glu Tyr
Val Pro Thr Val Phe Asp Asn Tyr Ser Ala Asn Val Met Val Asp Asn
Lys Thr Val Ser Leu Gly Leu Trp Asp Thr Ala Gly Gln Glu Asp Tyr
Asp Arg Leu Arg Pro Leu Ser Tyr Pro Gln Thr Asp Val Phe Leu Ile
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Cys Phe Ala Ile Ile Ser Gln Ser Tyr Thr Asn Val Lys Ser Lys Trp
Trp Pro GlU Val Thr His His Cys Pro Asn Cys Thr Ile Leu Val Gly
100 105 110
Thr Lys Cys Asp Leu Arg Asp Lys Glu Ser Leu Glu Lys Leu Arg Glu
115 120 125
Lys His Gln Gln Pro Leu Thr Phe Gln Gln Gly Glu Gln Met Ala Lys
130 135 140
Glu Ile Lys Ala Phe Cys Tyr Met Glu Cys Ser Ala Leu Thr Gln Lys
145 150 155 160
Gly Leu Lys Gln Val Phe Asp Glu Ala Ile Lys Ala Val Ile Phe Pro
165 170 175
Asp Arg Asp Lys Ala Thr Asn Lys Lys Asn Ser Lys Cys Ser Ile Leu
180 185 190
(2) INFORMATION EOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 185 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~.~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOUR OE :
(A) ORGANISM: Dictyostelium discoideum
(xi) ~yu~ DESCRIPTION: SEQ ID NO:30:
Met Ser Ala Ala Glu Val Ile Lys Leu Val Val Ile Gly Gly Ala Val
1 5 10 15
Gly Lys Thr Cys Leu Leu Ile Tyr Ala Asn Asn Arg Phe Pro Glu Asp
Tyr Ile Pro Thr Val Phe Asp Asn Tyr Val Val Asn Leu Thr Ala Gly
~5
Asp Arg Asn Ile Glu Leu Gly Leu Trp Asp Thr Ala Gly Glu Tyr Asp
Lys Leu Arg Pro Leu Ser Tyr Ala Asn Asn Val Phe Leu Ile Cys Phe
Ser Ile Asn Pro Val Ser Phe Glu Asn Val Tyr Thr Lys Trp Tyr Pro
Glu Val Met His Phe Cys Pro Glu Val Gln Ile Leu Val Gly Thr Lys
100 105 110
Leu Asp Thr Arg Asp Asp Arg Gly Val Leu Asp Lys Leu Gln Gln Thr
115 120 125
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Gly His Lys Pro Ile Thr Thr Glu Gln Gly Asn Asp Leu Ala Arg Arg
130 135 140
Ile Lys Ala Ile Lys Tyr Met Glu Cys Ser Ala Lys Thr Ser Gln Asn
145 lS0 155 160
Leu Lys Gln Val Phe Asp Glu Ala Ile Lys Ser Val Leu Phe Ile Lys
165 170 175
Lys Lys Lys Ser Lys Cys Ile Val Met
180 185
(2) INFORM~TION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 205 amino acids
(B) TYPE: amino acid
(C) STR~NT~nl~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Met Ala Ala Asn Lys Pro Lys Gly Gln Asn Ser Leu Ala Leu His Lys
1 5 10 15
Val Ile Met Val Gly Ser Gly Gly Val Gly Lys Ser Ala Leu Thr Leu
Gln Phe Met Tyr Asp Glu Phe Val Glu Asp Tyr Glu Pro Thr Lys Ala
Asp Ser Tyr Arg Lys Lys Val Val Leu Asp Gly Glu Glu Val Gln Ile
Asp Ile Leu Asp Thr Ala Gly Gln Glu Asp Tyr Ala Ala Ile Arg Asp
Asn Tyr Phe Arg Ser Gly Glu Gly Phe Leu Cys Val Phe Ser Ile Thr
Glu Met Glu Ser Phe Ala Ala Thr Ala Asp Phe Arg Glu Gln Ile Leu
lO0 105 110
Arg Val Lys Glu Asp Glu Asn Val Pro Phe Leu Leu Val Gly Asn Lys
115 120 125
Ser Asp Leu Glu Asp Lys Arg Gln Val Ser Val Glu Glu Ala Lys Asn
130 135 140
Arg Ala Glu Gln Trp Asn Val Asn Tyr Val Glu Thr Ser Ala Lys Thr
145 150 155 160
Arg Ala Asn Val Asp Lys Val Phe Phe Asp Leu Met Arg Glu Ile Arg
165 170 175
Ala Arg Lys Met Glu Asp Ser Lys Lys Asn Gly Lys Lys Lys Arg Lys
180 185 190
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Ser Leu Ala Lys Arg Ile Arg Glu Arg Cys Cys Ile Leu
195 200 205
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 amino acids
(B) TYPE: amino acid
(C) STRPNn~nNR-~S: single
(D) TOPOLOGY: linear
(ii) MOT.T~rTJT.~ TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Met Ala Ala Asn Lys Ser Ly3 Gly Gln Ser Ser Leu Ala Leu His Lys
1 5 10 15
Val Ile Met Val Gly Ser Gly Gly Val Gly Lys Ser Ala Leu Thr Leu
Gln Phe Met Tyr Asp Glu Phe Val Glu Asp Tyr Glu Pro Thr Lys Ala
Asp Ser Tyr Arg Lys Lys Val Val Leu Asp Gly Glu Glu Val Ile Asp
Ile Leu Asp Thr Ala Gly Gln Glu Asp Tyr Ala Ile Arg Asp Asn Tyr
Phe Arg Ser Gly Glu Gly Phe Leu Leu Val Phe Ser Ile Thr Glu His
Glu Ser Phe Thr Ala Thr Ala Glu Phe Arg Glu Gln Ile Leu Arg Val
100 105 110
Lys Ala Glu Glu Asp Lys Ile Pro Leu Leu Val Val Gly Asn Lys Ser
115 120 125
Asp Leu Glu Glu Arg Arg Gln Val Pro Val Glu Glu Ala Arg Ser Lys
130 135 140
Ala Glu Glu Trp Gly Val Gln Tyr Val Glu Thr Ser Ala Lys Thr Arg
145 150 155 160
Ala Asn Val Asp Lys Val Phe Phe Asp Leu Met Arg Glu Ile Arg Thr
165 170 175
Lys Lys Met Ser Glu Asn Lys Asp Lys Asn Gly Lys Lys Ser Ser Lys
~ 180 185 190
Asn Lys Lys Ser Phe Lys Glu Arg Cys Cys Leu Leu
195 200
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 200 amino acids
(B) TYPE: amino acid
(C) sTRpNnT~nN~s single
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Discopyge ommata
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Met Ala Ala A5n Lys Asn Lys Asn Gln Ser Ser Leu Leu Lys Val Ile
1 5 10 15
Met Val Gly Ser Gly Gly Val Gly Lys Ser Ala Leu Thr Leu Gln Phe
Met Tyr Asp Glu Phe Val Glu Asp Tyr Glu Pro Thr Lys Ala Asp Ser
Tyr Arg Lys Lys Val Val Leu Asp Gly Glu Val Gln Ile Asp Ile Leu
Asp Thr Ala Gly Gln Glu Asp Tyr Ala Ile Arg Asp Asn Tyr Phe Arg
Ser Gly Glu Gly Phe Leu Cys Val Phe Ser Ile Glu Gln Glu Ser Phe
Thr Ala Thr Val Glu Phe Arg Glu Gln Ile Leu Arg Val Lys Glu Glu
100 105 110
Asp Lys Ile Pro Leu Leu Leu Val Gly Asn Lys Ser Asp Leu Glu Asp
115 120 125
Arg Arg Gln Val Ser Ile Glu Glu Ala Arg Ser Lys Ala Glu Glu Trp
130 135 140
Gly Val Gln Tyr Val Glu Thr Ser Ala Lys Thr Arg Ala Asn Val Asp
145 150 155 160
Lys Val Phe Phe Asp Leu Met Arg Glu Val Arg Ala Lys Lys Met Ser
165 170 175
Glu Asn Lys Asp Lys Asn Gly Lys Lys Ser Ser Arg Asn Lys Lys Ser
180 185 190
Leu Arg Glu Arg Cys Cys Ile Leu
195 200
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 194 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Discopyge ommata
-
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Met Ala Lys Lys Thr Tyr Asp Leu Leu Phe Lys Leu Leu Leu Ile Gly
1 5 10 15
Asp Ser Gly Val Gly Lys Thr Cys Val Leu Phe Arg Phe Ser Asp Asp
Ala Phe Asn Thr Thr Phe Ile Ser Thr Ile Gly Ile Asp Phe Lys Ile
Lys Thr Val Glu Leu His Gly Lys Lys Ile Lys Leu Gln Ile Trp Asp
Thr Ala Gly Gln Glu Arg Phe His Thr Ile Thr Ser Tyr Tyr Arg Gly
Ala Met Gly Ile Met Leu Val Tyr Asp Ile Thr Asn Ala Lys Ser Phe
Glu Asn Ile Ser Lys Trp Leu Arg Asn Ile Asp Glu His Ala Asn Glu
100 105 110
Asp Val Glu Arg Met Leu Leu Gly Asn Lys Asp Met Glu Asp Lys Arg
115 120 125
Val Val Leu Lys Ser Lys Gly Gln Ile Ala Glu His Ala Ile Arg Phe
130 135 140
Phe Glu Thr Ser Ala Lys Ala Asn Ile Asn Ile Glu Lys Ala Phe Leu
~45 1~0 155 169
Thr Leu Ala Glu Asp Ile Leu Gln Lys Thr Pro Val Lys Glu Pro Asp
165 170 175
Arg Glu Asn Val Asp Ile Ser Thr Gly Gly Gly Gly Leu Lys Lys Cys
180 185 190
Cys Ser
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CH~RACTERISTICS-
(A) LENGTH: 207 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS- single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE
(A) ORGANISM: Discopyge ommata
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Met Lys Thr Tyr Asp Tyr Leu Phe Lys Leu Leu Leu Ile Gly Asp Ser
Gly Val Gly Lys Thr Cys Leu Leu Phe Arg Phe Ser Glu Asp Ala Phe
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Asn Thr Thr Phe Ile Ser Thr Ile Gly Ile Asp Phe Lys Ile Arg Thr
Val Glu Leu Asp Gly Lys Lys Ile Lys Leu Gln Ile Trp Asp Thr Ala
Gly Gln Glu Arg Phe Arg Thr Ile Thr Ala Tyr Tyr Arg Gly Ala Met
Gly Ile Met Ly9 Val Asp Ile Thr Asn Glu Lys Ser Phe Asp Asn Ile
Lys Asn Trp Ile Arg Asn Ile Glu Glu His Ala Ser Ser Asp Val Glu
100 105 110
Arg Met Ile Leu Gly Asn Lys Cys Asp Met Asn Glu Lys Arg Gln Val
115 120 125
Ser Lys Glu Arg Gly Glu Lys Leu Ala Ile Asp Tyr Gly Ile Lys Phe
130 135 140
Leu Glu Thr Ser Ala Lys Ser Ser Ile Asn Val Glu Glu Ala Phe Ile
145 150 155 160
Thr Leu Ala Arg Asp Ile Met Thr Lys Leu Asn Lys Lys Met Asn Glu
165 170 175
Asn Ser Leu Gln Glu Ala Val Asp Lys Leu Lys Ser Pro Pro Lys Lys
180 185 190
Pro Ser Gln Lys Lys Lys Gln Leu Ser Phe Arg Cys Ser Leu Leu
195 200 205
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 213 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Discopyge ommata
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
Met Gly Thr Arg Asp Asp Glu Tyr Asp Tyr Leu Phe Lys Val Val Leu
1 5 10 15
Ile Gly Asp Ser Gly Val Gly Lys Ser Asn Leu Leu Ser Arg Phe Thr
20 25 30 ~ ;
Arg Glu Phe Asn Leu Glu Ser Lys Ser Thr Ile Gly Val Glu Phe Ala
Thr Arg Ser Ile Gln Val Asp Gly Lys Thr Ile Lys Gln Ile Trp Asp
Thr Gly Gln Glu Arg Tyr Arg Ala Ile Thr Ser Ala Tyr Tyr Arg Gly
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Ala Val Gly Ala Leu Leu Val Tyr Asp Ile Ala Lys His Leu Thr Tyr
Glu Asn Val Glu Arg Trp Leu Lys Glu Leu Arg Asp His Ala Asp Asn
100 105 110
Asn Ile Val Ile Met Leu Val Gly Asn Lys Ser Asp Leu Arg His Leu
115 120 125
Arg Val Pro Thr Asp Ala Arg Ala Phe Ala Glu Lys Asn Asn Leu Ser
130 135 140
Phe Ile Glu Thr Ser Ala Leu Asp Ser Thr Asn Val Glu Glu Ala Phe
145 150 155 160
Lys Asn Ile Leu Thr Glu Ile Tyr Arg Ile Val Ser Gln Lys Gln Ile
165 170 175
Ser Asp Arg Ser Ala His Asp Glu Ser Pro Gly Asn Asn Val Val Asp
180 185 190
Ile Ser Val Pro Pro Thr Thr Asp Gly Gln Lys Ser Asn Lys Leu Gln
195 200 205
Cys Cys Gln Asn Met
210
(2) INFORMATION FOR SEQ ID NO:37:
(i) S~U~ CHARACTERISTICS:
(A) LENGTH: 184 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nN~s: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Dictyostelium discoideum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Met Pro Leu Arg Phe Lys Ile Val Val Leu Gly Ser Gly Gly Val Gly
1 5 10 15
Lys Ser Ala Leu Thr Val Gln Phe Val Gln Gly Ile Phe Val Glu Lys
Tyr Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Glu Val Asp
Ser Asn Gln Cys Met Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln
Phe Thr Met Arg Asp Leu Tyr Met Lys Asn Gly Gln Gly Phe Val Leu
Val Tyr Ser Ile Ile Ser Asn Ser Thr Phe Asn Glu Leu Pro Asp Leu
9o 95
Arg Glu Gln Ile Leu Arg Val Lys Asp Cys Glu Asp Val Pro Met Val
100 105 110
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Leu Val Gly Asn Lys Cys Asp Leu His Asp Gln Arg Val Ile Ser Thr
115 120 125
Glu Gln Gly Glu GlU Leu Ala Arg Lys Phe Gly Asp Cys Tyr Phe Leu
130 135 140
Glu Ala Ser Ala Lys Asn Lys Val Asn Val Glu Gln Ile Phe Tyr Asn
145 150 155 160
Leu Ile Arg Gln Ile Asn Ary Lys Asn Pro Val Gly Pro Pro Ser Lys
165 170 175
Ala Lys Ser Lys Cys Ala Leu Leu
180
(2) INFORMATION FOR SEQ ID NO:38:
(i) ~U~ CHARACTERISTICS:
(A) LENGTH: 179 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Met Arg Glu Tyr Lys Val Val Val Leu Gly Ser Gly Gly Val Gly Lys
1 5 10 15
Ser Ala Leu Thr Val Gln Phe Val Thr Gly Thr Phe Ile Glu Lys Tyr
Asp Pro Thr Ile Glu Asp Phe Tyr Arg Lys Glu Ile Glu Val Asp Ser
Ser Pro Ser Val Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln Phe
Ala Ser Arg Asp Leu Tyr Ile Lys Asn Gly Gln Gly Phe Ile Leu Val
Tyr Ser Leu Val Asn Gln Gln Phe Gln Asp Ile Lys Pro Met Arg Asp
Gln Ile Ile Arg Val Lys Tyr Glu Lys Val Pro Val Ile Leu Val Gly
100 105 110
Asn Lys Val ASp Leu Glu Ser Glu Arg Glu Val Ser Ser Ser Glu Gly
115 120 125
Arg Ala Leu Ala Glu Glu Trp Gly Cys Pro Phe Met Glu Thr Ser Ala
130 135 140 ,
Lys Ser Lys Thr Met Val Asp Glu Leu Phe Ala Glu Ile Val Arg Gln
145 150 155 160
Met Asn Tyr Ala Ala Gln Pro Asp Lys Asp Asp Pro Cys Cys Ser Ala
165 170 175
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Cys Asn Gln
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 183 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Met Arg Glu Tyr Lys Val Val Val Leu Gly Ser Gly Gly Val Gly Lys
Ser Ala Leu Thr Val Gln Phe Val Thr Gly Ser Phe Ile Glu Lys Tyr
Asp Pro Thr Ile Glu Asp Phe Tyr Arg Lys Glu Ile Glu Val Asp Ser
Ser Pro Ser Val Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln Phe
Ala Ser Met Arg Asp Leu Tyr Ile Lys Asn Gly Gln Gly Phe Ile Leu
Val Tyr Ser Leu Val Asn Gln Gln Ser Phe Gln Asp Ile Lys Pro Met
Arg Asp Gln Ile Ile Arg Val Lys Arg Tyr Glu Arg Val Pro Met Ile
100 105 110
Leu Val Gly Asn Lys Val Asp Leu Glu Gly Glu Arg Glu Val Ser Tyr
115 120 125
Gly Glu Gly Lys Ala Leu Ala Glu Glu Trp Ser Cys Pro Phe Met Glu
130 135 140
Thr Ser Ala Lys Asn Lys Ala Ser Val Asp Glu Leu Phe Ala Glu Ile
145 150 155 160
Val Arg Gln Met Asn Tyr Ala Ala Gln Ser Asn Gly Asp Glu Gly Cys
165 170 175
Cy8 Ser Ala Cys Val Ile Leu
180
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 184 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
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(iii) HYPOTXETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Discopyge ommata
(xi) SEQUENCE DESCRIPTION: SEQ ID MO:40:
Met Arg Glu Tyr Lys Leu Val Val Leu Gly Ser Gly Gly Val Gly Lys
1 5 10 15
Ser Ala Leu Thr Val Gln Phe Val Gln Gly Ile Phe Val Glu Lys Tyr
Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Glu Val Asp Cys
Gln Pro Cys Met Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln Phe
Thr Ala Met Arg Asp Leu Tyr Met Lys Asn Gly Gln Gly Phe Ala Leu
Val Tyr Ser Ile Thr Ala Gln Ser Thr Phe Asn Asp Leu Gln Asp Leu
Arg Glu Gln Ile Leu Arg Val Lys Asp Thr Glu Asp Val Pro Met Ile
100 105 110
Leu Val Gly Asn Lys Cys Asp Leu Glu Asp Glu Arg Val Val Gly Lys
115 120 125
Glu Gln Gly Gln Asn Leu Ala Arg Gln Trp Asn Asn Cys Ala Phe Leu
130 135 140
Glu Ser Ser Ala Lys Ser Lys Ile Asn Val Asn Glu Ile Phe Tyr Asp
145 150 155 160
Leu Val Arg Gln Ile Asn Arg Lys Ala Pro Val Glu Lys Cys Lys Lys
165 170 175
Lys Lys Ser Gln Cys Thr Leu Leu
180
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 180 amino acids
(B) TYPE: amino acid
(C) STR~Nn~n~S single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Met Arg Glu Tyr Lys Leu Val Val Gly Ser Gly Gly Val Gly Lys Ser
1 5 10 15
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~la Leu Thr Val Gln Phe Val Gln Gly Phe Val Glu Lys Tyr Asp Pro
Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Glu Val Asp Cys Gln Gln
Cy8 Met Leu Glu Asp Thr Ala Gly Thr Glu Gln Phe Thr Ala Met Arg
Asp Leu Tyr Met Lyg Asn Gly Gln Gly Phe Ala Leu Val Tyr Ser Ile
Thr Ala Gln Ser Thr Phe Asn Asp Leu Gln Asp Leu Arg Glu Gln Ile
Leu Arg Val Lys Asp Thr Glu Asp Val Pro Met Ile Leu Val Gly Asn
100 105 110
Lys Cys Asp Leu Glu Asp Glu Arg Val Val Gly Lys Glu Gln Gly Gln
115 120 125
Asn Leu Ala Arg Gln Trp Cys Asn Cys Ala Phe Leu Glu Ser Ser Ala
130 135 140
Lys Ser Lys Ile Asn Val Asn Glu Ile Phe Tyr Asp Leu Val Arg Gln
145 150 155 160
Ile Asn Arg Lys Thr Pro Val Glu Lys Lys Lys Pro Lys Lys Lys Ser
165 170 175
Cys Leu Leu Leu
180
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 179 amino acids
(B) TYPE: amino acid
(C) STRA~n~nNF~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Met Arg Glu Tyr Lys Leu Val Val Leu Gly Ser Gly Gly Val Gly Lys
1 5 10 15
Ser Ala Leu Thr Val Gln Phe Val Gln Gly Ile Phe Val Glu Lys Tyr
Agp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Glu Val Asp Ala
Gln Gln Cys Met Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln Phe
Thr Ala Met Arg Asp Leu Tyr Met Lys Asn Gly Gln Gly Phe Ala Leu
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Val Tyr Ser Ile Thr Ala Gln Ser Thr Phe Asn Asp Leu Gln Asp Leu
Arg Glu Gln Ile Leu Arg Val Lys Asp Thr Asp Asp Val Pro Met Ile
100 105 110
Leu Val Gly Asn Lys Cys Asp Leu Glu Asp Glu Arg Val Val Gly Lys
115 120 125
Glu Gln Gly Gln Asn Leu Ala Arg Gln Trp Asn Asn Cys Ala Phe Leu
130 135 140
Glu Ser Ser Ala Lys Ser Lys Ile Asn Val Glu Ile Phe Tyr Asp Leu
145 150 155 160
Val Arg Gln Ile Asn Arg Lys Thr Pro Val Pro Gly Lys Ala Arg Lys
165 170 175
Lys Ser Ser
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 184 amino acids
(B) TYPE: amino acid
(C) STR~NDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Drosophila melanogaster
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Met Arg Glu Tyr Lys Ile Val Val Leu Gly Ser Gly Gly Val Gly Lys
1 5 10 15
Ser Ala Leu Thr Val Gln Phe Val Gln Cys Ile Phe Val Glu Lys Tyr
Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Glu Val Asp Gly
Gln Gln Cys Met Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln Phe
Thr Ala Met Arg Asp Leu Tyr Met Lys Asn Gly Gln Gly Phe Val Leu
Val Tyr ser Ile Thr Ala Gln Ser Thr Phe Asn Asp Leu Gln Asp Leu
Arg Glu Gln Ile Leu Arg Val Lys Asp Thr Asp Asp Val Pro Met Val
100 105 110
Leu Val Gly Asn Lys Cys Asp Leu Glu Glu Glu Arg Val Val Gly Lys
115 120 125
Glu Leu Gly Lys Asn Leu Ala Thr Gln Phe Asn Cys Ala Phe Met Glu
130 135 140
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Thr Ser Ala Lys Ala Lys Val Asn Val Asn Asp Ile Phe Tyr Asp Leu
145 150 155 160
Val Arg Gln Ile Asn Lys Lys Ser Pro Glu Lys Lys Gln Lys Lys Pro
165 170 175
Lys Lys Ser Leu Cys Val Leu Leu
180
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTE: 182 amino acids
(B) TYPE: amino acid
(C) STR~Nn~n~s: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Dictyostelium discoideum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Met Thr Glu Tyr Lys Leu Val Ile Val Gly Gly Gly Gly Val Gly Lys
1 5 10 15
Ser Leu Thr Ile Gln Leu Ile Gln Asn His Phe Asp Glu Tyr Asp Pro
Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Ser Ile Asp Asp Glu Thr
Cys Leu Leu Ile Leu Asp Thr Ala Gly Gln Glu Glu Ser Ala Met Arg
Asp Gln Tyr Met Arg Thr Gly Gln Gly Phe Leu Cys Val Tyr Ser Ile
Thr Ser Arg Ser Ser Tyr Asp Glu Ile Ala Ser Phe Arg Glu Gln Ile
Leu Arg Val Lys Asp Lys Asp Arg Val Pro Leu Ile Leu Val Gly Asn
100 105 110
Lys Ala Asp Leu Asp His Glu Arg Gln Val Ser Val Asn Glu Gly Gln
115 120 125
Glu Leu Ala Lys Asp Ser Leu Ser Phe His Glu Ser Ser Ala Lys Ser
130 135 140
Arg Ile Asn Val Glu Glu Ala Phe Tyr Ser Leu Val Arg Glu Ile Arg
145 150 155 160
Lys Glu Leu Lys Gly Asp Gln Ser Ser Gly Lys Ala Gln Lys Lys Lys
165 170 175
Lys Gln Cys Leu Ile Leu
180
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
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(A) LENGTH: 190 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPO~ L~AL: NO
(vi) ORIGINAL SOVRCE:
(A) ORGANISM: Dictyostelium discoideum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Met Ser Val Ser Asn Glu Tyr Lys Leu Val Val Gly Gly Gly Gly Val
1 5 10 15
Gly Lys Ser Ala Leu Thr Ile Gln Phe Gln Asn His Phe Ile Glu Glu
Tyr Asp Pro Thr Ile Glu Asp Ser Tyr Arg Arg Gln Cys Gln Val Asp
Glu Asp Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Asp Asp
Tyr Ser Met Arg Asp Gln Tyr Met Arg Thr Gly Gln Gly Phe Leu Val
Tyr A~p Val Ser Arg Thr Ser Phe Glu Glu Ile Asn Val Val Glu Gln
Ile Arg Val Lys Asp Asn Asp Lys Val Pro Ile Val Leu Val Gly Asn
100 105 110
Lys Cys Asp Leu Glu Asn Leu Arg Glu Val Thr Glu Gly Glu Gly Ser
115 120 125
Glu Leu Ala Lys Ser Phe Ser Val Pro Phe Leu Glu Thr Ser Ala Lys
130 135 140
Lys Arg Leu Asn Val Asp Glu Cys Phe Phe Glu Val Val Arg Glu Ile
145 150 155 160
Lys Lys Ser Leu Lys Glu Pro Gly Arg Ser Lys Lys Asp Lys Lys Gly
165 170 175
Gly Ile Leu Lys Lys Phe Lys Gly Gly Asp Cys Leu Ile Leu
180 185 190
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 188 amino acids
(B) TYPE: amino acid .
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Dictyostelium discoideum
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Met Ser Lys Leu Leu Lys Leu Val Ile Val Gly Asp Gly Gly Val Gly
1 5 10 15
Lys Ser Ala Leu Thr Ile Gln Leu Thr Gln Asn Gln Phe Ile Ala Glu
Tyr Asp Pro Thr Ile Glu Asn Ser Tyr Arg Lys Gln Val Asn Ile Asp
Glu Glu Val Tyr Met Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu
Tyr Ser Ala Met Arg Asp Gln Tyr Ile Arg Ser Gly Arg Gly Phe Leu
Ile Val Tyr Ser Ile Ile Ser Arg Ala Ser Phe Glu Ala Val Thr Thr
Phe Arg Glu Gln Ile Leu Arg Val Lys Asp Leu Ser Thr Tyr Pro Ile
100 105 110
Val Ile Ile Gly Asn Lys Ala Asp Leu Pro Asp Lys Asp Arg Lys Val
115 120 125
Pro Pro Met Glu Gly Lys Glu Leu Ala Lys Phe Gly Ala Pro Phe Leu
130 135 140
Glu Thr Ser Ala Lys Ser Arg Val Asn Val Glu Glu Ala Phe Phe Thr
145 150 155 160
Leu Val Arg Glu Ile Lys Arg Trp Asn Gln Asn Pro Gln Asn Glu Glu
165 170 175
Met Leu Pro Pro Lys Lys Arg Gly Cys Ile Ile Leu
180 185
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CEARACTERISTICS:
(A) LENGTH: 188 amino acids
(B) TYPE: amino acid
(C~ STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Dictyostelium discoideum
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
Met Glu Tyr Lys Leu Val Ile Val Gly Gly Gly Gly Val Gly Lys Ser
1 5 10 15
Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Ile Asp Glu Tyr Asp
Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Thr Ile Asp Glu Glu
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Thr Cy9 Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr Ser
Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Gln Gly Phe Leu Cys Val
Tyr Ser Ile Thr Ser Arg Ser Ser Phe Asp Glu Ile Ala Ser Phe Arg
Glu Gln Ile Leu Arg Val Lys Asp Lys Asp Arg Val Pro Met Ile Val
100 105 110
Val Gly Asn Lys Cys Asp Leu Glu Ser Asp Arg Gln Val Thr Thr Gly
115 120 125
Glu Gly Gln Asp Leu Ala Lys Ser Phe Gly Ser Pro Phe Leu Glu Thr
130 135 140
Ser Ala Lys Ile Arg Val Asn Val Glu Glu Ala Phe Tyr Ser Leu Val
145 150 155 160
Arg Glu Ile Arg Lys Asp Leu Lys Gly Asp Ser Lys Pro Glu Lys Gly
165 170 175
Lys Lys Lys Ary Pro Leu Lys Ala Cys Thr Leu Leu
180 185
t2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 204 amino acids
(B) TYPE: amino acid
(C) sTR~Mn~nN~s: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Caenorhabditis elegans
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
Met Ser Ser Ser Leu Gln Ser Asn Arg Gln Ser Leu Asn Arg Lys Val
1 5 10 15
Ala Val Met Gly Tyr Pro His Val Gly Lys Ser Ala Leu Val Leu Arg
Phe Thr Gln Asn Ile Phe Pro Glu Arg Tyr Glu Ser Thr Ile Glu Asp
Gln His Ser Lys His Ile Ala Ala Phe His Arg Asp Tyr His Leu Arg
Val Thr Asp Thr Ala Gly Gln Gln Glu Tyr Thr Val Phe Pro Arg Ser
Cys Ser Leu Asp Ile Asn Gly Phe Ile Leu Val Tyr Ala Ile Asp Asp
Ary Lys Ser Phe Glu Met Cys Ser Asn Ile Tyr Glu Lys Ile Val Arg
100 105 110
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89
Thr Tyr Gly Asp Thr Ser Ile Pro Ile Val Ile Val Gly Lys Thr Asp
115 120 125
Leu Ser Thr Gln Val Val Arg Ala Glu Glu Gly Glu Glu Leu Ala Arg
130 135 140
Gln Trp Asp Ala Lys Phe Val Glu Ile Thr Ala Arg Glu Ser Asn Arg
145 150 155 160
Val His Glu Val Phe Glu Leu Leu Leu Arg Glu Ile Glu Ile Ser Arg
165 170 175
Gly Asn Leu Ser Pro Thr Glu Arg Pro Asn Gly Asn Ser Pro Lys Arg
180 185 190
Pro Phe Lys Asp Asp Gly Lys Pro Cys Ser Ile Ser
195 200
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 215 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Coprinus cinereus
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
Met Ala Ala Arg Ala Gln Phe Leu Arg Glu Tyr Lys Leu Val Val Val
1 5 10 15
Gly Gly Gly Gly Val Gly Lys Ser Ala Leu Thr Ile Gln Phe Ile Gln
Ser His Phe Val Asp Glu Tyr Asp Pro Thr Ile Glu Asp Ser Tyr Arg
Lys Gln Cys Ile Ile Asp Asp Glu Val Ala Leu Leu Asp Val Leu Asp
Thr Ala Gly Gln Glu Glu Tyr Gly Ala Met Arg Glu Gln Tyr Met Arg
Thr Gly Glu Gly Phe Leu Leu Val Tyr Ser Ile Thr Ser Arg Asn Ser
Phe Glu Glu Ile Ser Ile Phe His Gln Gln Ile Leu Arg Val Lys Asp
100 105 110
Gln Asp Ser Phe Pro Val Ile Val Val Ala Asn Lys Cys Asp Leu Glu
115 120 125
Tyr Glu Arg Gln Val Gly Met Asn Glu Gly Arg Asp Leu Ala Lys His
130 135 140
Phe Gly Cys Lys Phe Ile Glu Thr Ser Ala Lys Gln Arg Ile Asn Val
145 150 155 160
CA 022327~0 l998-03-23
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Asp Glu Ala Phe Ser Asn Leu Val Arg Glu Ile Arg Lys Tyr Asn Arg
165 170 175
Glu Gln Gln Thr Gly Arg Pro Ala Ile Ala Ala Gly Gly Gly Gly Pro
180 185 190
Ala Gly Ser Tyr Thr Gln Asp Arg His His Asp Glu Ala Pro Gly Cys
195 200 205
Cys Ala Gly Cys Val Ile Ala
210 215
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 206 amino acids
(B) TYPE: amino acid
tC) STR~Nn~nN~S: single
(D) TOPO~OGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Geodia cydonium
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
Met Thr Glu Tyr Lys Ile Val Val Gly Gly Gly Leu Val Gly Lys Ser
1 5 10 15
Ala Leu Thr Leu Gln Leu Val Gln Val Cys Ile Lys Asp Gln Tyr Tyr
Leu Ile Glu Phe Gln Asn Asn Gln Phe Gln Phe Glu Asn Leu Gln Asn
His Tyr Ile Asp Tyr Asp Pro Thr Val Glu Asp Ser Arg Arg Glu Val
Ser Ile Asp Asp Gln Thr Cys Leu Asn Ile Leu Asp Thr Ala Gly Gln
Gln His Ser Asn Ala Gln Ser Met Asp Ala His Trp Ser Thr Val Phe
Val Cys Leu Phe Asn Tyr Phe Asn Ile Thr Ser Met Tyr Asp Glu Ile
100 105 110
Ala Ser Phe Arg Glu Gln Ile Leu Arg Val Lys Asp Gly Ala Lys Asp
115 120 125
Leu Val Pro Leu Ile Leu Ile Ile Asn Lys Ala Asp Leu Asp His Glu
130 135 140
Ser Gln Gly Ser Gly Asn Glu Gly Gln Leu Ala Lys Asp Ser Leu Ser
145 150 155 160 ,
Phe His Gln Ser Ser Ala Lys Ser Arg Ile Asn Leu Glu Glu Ile Pro
165 170 175
Tyr Ser Leu Val Arg Glu Leu Arg Lys Glu Leu Lys Leu Asp Gln Ser
180 185 190
CA 022327~0 1998-03-23
WO 97/10836 PCT~US96/15098
91
Ser Gly Lys Ala Gln Lys Lys Lys Lys Gln Cys Leu Ile Ile
195 200 205
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 198 amino acids
(B) TYPE: amino acid
(C) STR~Mn~N~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Canis ~m; 1 i~is
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Met Lys Lys Thr Tyr Asp Leu Leu Phe Lys Leu Leu Leu Ile Gly Asp
1 5 10 15
Ser Gly Val Gly Lys Thr Cys Val Leu Phe Arg Phe Ser Asp Asp Ala
Phe Asn Thr Thr Phe Ile Ser Ile Gly Ile Asp Phe Lys Ile Lys Thr
Val Glu Leu Gln Gly Lys Lys Ile Lys Leu Gln Ile Trp Asp Thr Ala
Gly Gln Glu Arg Phe His Thr Ile Thr Thr Ser Tyr Tyr Arg Gly Ala
Met Gly Ile Met Leu Val Tyr Asp Ile Thr Asn Gly Lys Ser Phe Glu
Asn Ile Ser Lys Trp Leu Arg Asn Ile Asp Glu His Ala Asn Glu Asp
100 105 110
Val Glu Arg Met Leu Leu Gly Asn Lys Cys Asp Met Asp Asp Lys Arg
115 120 125
Val Val Pro Lys Gly Lys Gly Glu Gln Ile Ala Arg Glu His Gly Ile
130 135 140
Arg Phe Phe Glu Thr Ser Ala Lys Val Asn Ile Asn Ile Glu Lys Ala
145 150 155 160
Phe Leu Thr Leu Ala Glu Asp Ile Leu Arg Lys Thr Pro Val Lys Glu
165 170 175
Pro Asn Ser Glu Asn Val Asp Ile Ser Ser Gly Gly Gly Val Thr Gly
180 185 190
Trp Lys Ser Lys Cys Cys
195
(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 214 amino acids
(B) TYPE: amino acid
(C) STR~Nn~M~.~s: single
CA 022327~0 l998-03-23
W O 97/10836 PCT~US96/15098
(D) TOPOLOGY: linear
(ii) MOLBCULE TYPE: protein
(iii) HYPOTHETICAL: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
Met Gly Thr Arg Asp Asp Glu Tyr Asp Tyr Leu Phe Lys Val Val Leu
1 5 10 15
Ile Gly Asp Ser Gly Val Gly Lys Ser Asn Leu Leu Ser Arg Phe Thr
Arg Asn Glu Phe Asn Leu Glu Ser Lys Ser Thr Ile Gly Val Glu Phe
Ala Thr Arg Ser Ile Gln Val Asp Gly Lys Thr Ile Lys Ala Gln Ile
Trp Asp Thr Ala Gly Gln Glu Arg Tyr Arg Ala Ile Thr Ser Ala Tyr
Tyr Arg Gly Ala Val Gly Ala Leu Leu Val Tyr Asp Ile Ala Lys His
Leu Thr Tyr Glu Asn Val Glu Arg Trp Leu Lys Glu Leu Arg Asp His
100 105 110
Ala Asp Ser Asn Ile Val Ile Met Leu Val Gly Asn Lys Ser Asp Leu
115 120 125
Arg His Leu Arg Ala Val Pro Thr Asp Glu Ala Arg Ala Phe Ala Glu
130 135 140
Lys Asn Gly Leu Ser Phe Ile Glu Thr Ser Ala Leu Asp Ser Thr Asn
145 15Q 155 160
Val Glu Ala Ala Phe Gln Thr Ile Leu Thr Glu Ile Tyr Arg Ile Val
165 170 175
Ser Gln Lys Gln Met Ser Asp Arg Glu Asn Asp Met Ser Pro Ser Asn
180 185 190
Asn Val Val Pro Ile His Val Pro Pro Thr Thr Glu Lys Pro Lys Val
195 200 205
Gln Cys Cys Gln Asn Ile
210
