U.S. patent application number 10/788564 was filed with the patent office on 2004-07-29 for compounds that modulate the activity of ptp-1b and tc-ptp.
Invention is credited to Barr, Kenneth, Fahr, Bruce, Hansen, Stig, Wiesmann, Christian.
Application Number | 20040147596 10/788564 |
Document ID | / |
Family ID | 27791680 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040147596 |
Kind Code |
A1 |
Barr, Kenneth ; et
al. |
July 29, 2004 |
Compounds that modulate the activity of PTP-1B and TC-PTP
Abstract
The present invention relates to a new and improved method for
treating diabetes and or its associated complications by modulating
the activity of protein tryosin phosphatase 1B ("PTP-1B"). The
inventive compounds modulate the activity PTP-1B by binding to a
novel binding site referred herein as the PTP-1B exosite that is
distal to the active site of PTP-1B. The present invention also
relates to a new and improved method of treating immune system
disorders by modulating the activity of T-cell protein tyrosine
phosphatase ("TC-PTP"). The inventive compound modulate the
activity of TC-PTP by binding to a novel binding site referred
herein as the TC-PTP exosite that is distal to the active site of
PTP-1B.
Inventors: |
Barr, Kenneth; (San
Francisco, CA) ; Fahr, Bruce; (Foster City, CA)
; Hansen, Stig; (El Cerrito, CA) ; Wiesmann,
Christian; (Brisbane, CA) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
27791680 |
Appl. No.: |
10/788564 |
Filed: |
February 27, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10788564 |
Feb 27, 2004 |
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10374539 |
Feb 25, 2003 |
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60361475 |
Mar 1, 2002 |
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Current U.S.
Class: |
514/469 ;
549/436 |
Current CPC
Class: |
C07D 307/80 20130101;
C07D 405/12 20130101; A61K 31/635 20130101; A61P 3/10 20180101;
A61P 7/00 20180101; A61P 13/12 20180101; A61P 29/00 20180101; A61K
31/343 20130101; A61P 27/02 20180101; A61P 43/00 20180101; A61P
3/04 20180101; A61P 9/00 20180101; C07D 417/12 20130101; A61P 37/00
20180101 |
Class at
Publication: |
514/469 ;
549/436 |
International
Class: |
A61K 031/545; A61K
031/343 |
Claims
What is claimed is:
1. A compound that inhibits PTP-1B and that interacts with at least
one of the PTP-1B exosite-forming residues.
2. A compound that inhibits TC-PTP and that interacts with at least
one of the TC-PTP exosite-forming residues.
3. A compound having the structure having the structure 28wherein:
R.sup.1 is hydrogen, methyl, ethyl, or propyl; R.sup.2 is hydrogen,
--S(O.sub.2)R.sup.3, --NH(C(.dbd.O)R.sup.3,
--NH(C(.dbd.O)CH.sub.2(C.dbd.- O)OR.sup.3,
--S(O.sub.2)NR.sup.4R.sup.5, or --NR.sup.4S(O.sub.2)R.sup.3 where
R.sup.3 is C.sub.1-C.sub.5 alkyl, R.sup.4 is hydrogen,
C.sub.1-C.sub.5 alkyl, unsubstituted cyclic moiety, or substituted
cyclic moiety, and R.sup.5 is either hydrogen or R.sup.5 and
R.sup.4 together form an unsubstituted cyclic moiety or a
substituted cyclic moiety; R.sup.6 is hydrogen or alternatively
when R is --NR.sup.4S(O.sub.2)NR.sup- .3; then R.sup.6 and R.sup.4
together form an unsubstituted cyclic moiety or substituted cyclic
moiety; and, L is --NHS(O.sub.2)-- or
--S(O.sub.2)NR.sup.7CH.sub.2-- where R.sup.7 is hydrogen or
C.sub.1-C.sub.5 alkyl.
4. The compound of claim 3 wherein the one or more substituents on
the substituted cyclo group are each independently selected from
the group consisting of: C.sub.1-C.sub.5 alkyl, phenyl, benzyl, F,
Cl, I, Br, --OH; --NO.sub.2; --CN; --CF.sub.3; --CH.sub.2CF.sub.3;
--CH.sub.2Cl; --CH.sub.2OH; --CH.sub.2CH.sub.2OH;
--CH.sub.2NH.sub.2; --CH.sub.2SO.sub.2CH.sub.3; --OR.sup.8;
--C(O)R.sup.8; --COOR.sup.8; --C(O)NR.sup.8R.sup.9; --OC(O)R.sup.8;
--OCOOR.sup.8; --OC(O)NR.sup.8R.sup.9; --NR.sup.8R.sup.9;
--S(O).sub.2R.sup.8; and --NR.sup.8C(O)R.sup.9 where R.sup.8 and
R.sup.9 are each independently hydrogen, C.sub.1-C.sub.5 alkyl,
phenyl or benzyl.
5. The compound of claim 3 wherein R.sup.2 and R.sup.6 are both
hydrogen.
6. The compound of claim 3 wherein R.sup.2 is --S(O.sub.2)NHR.sup.5
where R.sup.5 is an unsubstituted cyclic moiety or substituted
cyclic moiety, and R.sup.6 is hydrogen.
7. The compound of claim 3 wherein R.sup.2 is --S(O.sub.2)R.sup.3
where R.sup.3 is methyl, ethyl, or propyl, and R.sup.6 is
hydrogen.
8. The compound of claim 3 wherein R.sup.2 is --NH(C(.dbd.O)R.sup.3
where R.sup.3 is methyl, ethyl, or propyl, and R.sup.6 is
hydrogen.
9. The compound of claim 3 wherein R.sup.2 is
--NH(C(.dbd.O)CH.sub.2(C.dbd- .O)OR.sup.3 where R.sup.3 is methyl,
ethyl, or propyl, and R.sup.6 is hydrogen.
10. The compound of claim 3 wherein R.sup.2 is
--NR.sup.4S(O.sub.2)R.sup.3 wherein R.sup.3 is methyl and R.sup.4
and R.sup.6 together form an unsubstituted heterocyclo or a
substituted heterocyclo.
11. A compound having the structure 29wherein: R.sup.10 is
C.sub.1-C.sub.5 alkyl or NHR.sup.11 where R.sup.11 is hydrogen,
C.sub.1-C.sub.10 alkyl or aryl; and, L is --NHS(O.sub.2)-- or
--S(O.sub.2)N(CH.sub.2).sub.3CH.sub.2--.
12. The compound of claim 11 wherein R.sup.10 is methyl, ethyl or
propyl.
13. The compound of claim 11 wherein R.sup.10 is NHR.sup.11 and
R.sup.11 is hydrogen.
14. The compound of claim 11 wherein R.sup.10 is NHR.sup.11 and
R.sup.11 is aryl.
15. The compound of claim 19 wherein R.sup.11 is phenyl.
16. The compound of claim 19 wherein R.sup.11 is heteroaryl.
17. An exosite mutant of PTP-1B.
18. An exosite mutant of TC-PTP.
19. A pharmaceutical composition comprising an effective amount of
a compound of any one of claims 1-3, and 11, or a prodrug or
pharmaceutically acceptable derivative thereof, in admixture with a
pharmaceutically acceptable carrier.
20. A method of identifying an exosite inhibitor of PTP-1B
comprising a) contacting a test compound with PTP-1B; b) contacting
the test compound with an exosite mutant of PTP-1B; and c)
comparing the activity of PTP-1B in the presence of the test
compound with the activity of the exosite mutant of PTP-1B in the
presence of the test compound.
21. A method of identifying an exosite inhibitor of TC-PTP
comprising a) contacting a test compound with TC-PTP; b) contacting
the test compound with an exosite mutant of TC-PTP; and c)
comparing the activity of TC-PTP in the presence of the test
compound with the activity of the exosite mutant of TC-PTP in the
presence of the test compound.
22. A method for treating type 2 diabetes, or a pathologic
condition associated with type 2 diabetes, comprising administering
to a subject in need thereof a therapeutically effective amount of
a PTP-1B exosite inhibitor of claim 1.
23. The method of claim 22 wherein the pathologic condition
associated with type 2 diabetes is insulin resistance.
24. A method for treating inflammation is provided comprising
administering to a subject in need thereof a therapeutically
effective amount of a TC-PTP exosite inhibitor of claim 2.
25. A method for treating an immune system disorder comprising
administering to a subject in need thereof a therapeutically
effective amount of a TC-PTP exosite inhibitor of claim 2.
26. A method for treating a hematopoiesis disorder comprising
administering to a subject in need thereof a therapeutically
effective amount of a TC-PTP exosite inhibitor of claim 2.
Description
BACKGROUND
[0001] Diabetes mellitus is a major risk factor for potentially
debilitating diseases such as cardiovascular disease and stroke,
and is the leading cause of blindness, renal failure and lower limb
amputations in adults. Type 2 or noninsulin-dependent diabetes
mellitus accounts for over ninety percent of all diabetes cases.
Although current treatments for type 2 diabetes result in lower
levels of blood sugar, side effects include weight gain,
hyperglycemia, edema, and liver toxicity. Obesity, a condition that
is often strongly correlated with diabetes further complicates
treatment options. In particular, obesity further exacerbates the
insulin resistance that is a hallmark of diabetes so that current
treatments for diabetes typically lose their efficacy after a few
years. As a result, a need exists for new and improved methods for
treating diabetes and/or its associated complications.
DESCRIPTION OF THE FIGURES
[0002] FIG. 1 is a sequence alignment of the first 298 residues of
human PTP-1B (SEQ ID NO.1) and the first 296 residues of human
TC-PTP (hTC-ptp; SEQ ID NO.2).
[0003] FIG. 2 is the exosite region of PTP-1B complexed with
compound 5. The accessible surface of the exosite-forming residues
is shown.
[0004] FIG. 3 shows the amino acids that comprise the exosite
region of PTP-1B.
[0005] FIG. 4 is the same exosite region of PTP-1B as in FIG. 3 but
in the absence of an exosite ligand. The accessible surface of the
exosite-forming residues is shown. The structure that traverses and
occludes the bulk of the exosite region is a helix formed by
residues 283-298.
[0006] FIG. 5 is a ribbon diagram of PTP-1B in the absence of an
exosite ligand where Tyr-152, Asn-193, and Trp-291 are
highlighted.
[0007] FIG. 6 is a ribbon diagram of PTP1B in the presence of
exosite ligand 5 where Tyr-152, Asn-1,93, and Tryp-291 are
highlighted.
[0008] FIG. 7 is a sequence alignment of the first 298 residues of
human PTP-1B (SEQ ID NO. 1).and the first 297 residues of human LAR
(SEQUENCE ID NO. 3).
[0009] FIG. 8 is a plot of the reaction velocity of PTP-1B versus
increasing concentration of substrate in the presence of varying
concentrations of compound 5.
[0010] FIG. 9 is a dose response curve for insulin receptor
phosphorylation as a function of increasing concentration of
compound 5.
[0011] FIG. 10 is a Western blot showing the selectivity of
compound 5 in cells. The 95 kDa band corresponds to the insulin
receptor. DMSO is the negative control and vanadate (the lane
marked "Van"), a nonspecific phosphatase inhibitor, is the positive
control. In FIG. 10, "3892" corresponds to compound 5 and "Ins"
corresponds to insulin.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0012] A. Definitions
[0013] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention belongs.
References, such as Singleton et al., Dictionary of Microbiology
and Molecular Biology 2nd ed., John Wiley & Sons (New York,
N.Y. 1994), and March, Advanced Organic Chemistry Reactions,
Mechanisms and Structure 4th ed., John Wiley & Sons (New York,
N.Y. 1992), provide one skilled in the art with a general guide to
many of the terms used in the present application.
[0014] The terms "type 2 diabetes," "type II diabetes," type 2
diabetes mellitus," "type II diabetes mellitus,"
"non-insulin-dependent diabetes," and "non-insulin-dependent
diabetes mellitus (NIDDM)" are used interchangeably, and refer to a
chronic diseases characterized by insulin resistance at the level
of fat and muscle cells and resultant hyperglycemia.
[0015] The term "pathologic condition associated with type 2
diabetes" is used to refer to any condition that results, at least
partially, from the long-term effects of type 2 diabetes. Such
conditions include, without limitation, diabetic retinopathy,
diabetic neuropathy, hypertension, atherosclerosis, diabetic
ulcers, and in general damage caused to blood vessels, nerves and
other internal structures by elevated blood sugar levels.
[0016] The term "obesity" is used to describe an excessive amount
of body fat. Typically, a person is considered obese if he or she
has a body mass index (BMI) of 30 kg/m.sup.2 or greater.
[0017] The term "treatment" refers to both therapeutic treatment
and prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the
disorder as well as those prone to have the disorder or those in
whom the disorder is to be prevented. Thus, in the case of obesity,
the term "treatment" includes the treatment of obese subjects as
well as preventative treatment of subjects a risk of developing
obesity. Similarly, in the case of type 2 diabetes, "treatment"
refers both to treating subjects diagnosed with type 2 diabetes and
those at risk of developing type 2 diabetes.
[0018] The term "mammal" for purposes of treatment refers to any
animal classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, cats,
cattle, horses, sheep, pigs, goats, rabbits, etc. Preferably, the
mammal is human.
[0019] B. Detailed Description
[0020] The present invention relates to new and improved methods
for treating diabetes and/or its associated complications by
modulating the activity of protein tyrosine phosphatase 1B
("PTP-1B"). The inventive compounds modulate the activity of PTP-1B
by binding to a novel binding site (referred herein as "the PTP-1B
exosite") that is distal to the active site of PTP-1B. Thus, in one
aspect of the present invention, compounds are provided that bind
to the exosite of PTP-1B. In another aspect of the present
invention, methods are provided for using these compounds to
modulate the activity of PTP-1B. In another aspect of the present
invention, methods are provided of using an exosite inhibitor of
PTP-1B to treat various disease conditions including diabetes,
insulin resistance, and obesity.
[0021] The present invention also relates to methods for treating
immune system disorders by modulating the activity of T-cell
protein tyrosine phosphatase ("TC-PTP"). The inventive compounds
modulate TC-PTP by binding to a novel binding site (referred herein
as "the TC-PTP exosite") that is distal to the active site of
TC-PTP. Thus, in one aspect of the present invention, compounds are
provided that bind to the exosite of TC-PTP. In another aspect of
the present invention, methods are provided for using these
compounds to modulate the activity of TC-PTP. In another aspect of
the present invention, methods are provided of using an exosite
inhibitor of TC-PTP to treat various disease conditions including
inflammation, immune system disorders, and hematopoietic
disorders.
[0022] PTP-1B and TC-PTP are protein tyrosine phosphatases.
Tyrosine phosphorylation is reversible and dynamic, and the
equilibrium between phosphorylated and unphosphorylated protein is
governed by the opposing activities of protein tyrosine kinases
("PTKs") that catalyze the addition of a phosphate group and
protein tyrosine phosphatases ("PTPs") that catalyze the reverse
activity or the removal of the added phosphate group.
[0023] The signature motif of a PTP is (H/V)C(X).sub.5R(S/T) where
X is any amino acid residue. See Zhang, Current Opinions in
Chemical Biology 5: 416-423 (2001); and Zhang, Annual Review of
Pharmacology and Toxicology 42: 209-234 (2002). The PTP signature
motif is found in a critical loop (termed the PTP loop or P-loop)
in the active site of the catalytic PTP domain and includes two
(cysteine and arginine) of the three essential catalytic residues.
The third catalytic residue is aspartic acid and is found in the
WPD loop (also known as the flexible loop). In addition, all PTPs
are characterized by their ability to hydrolyze p-nitrophenyl
phosphate without the presence of a metal ion, sensitivity to
vanadate, and insensitivity to okadaic acid.
[0024] The three-dimensional structures of the PTP domains are
remarkably similar despite the variation in amino acid sequences
and the differences in substrate specificity between the
tyrosine-specific PTPs and the dual-specificity PTPs. The PTP
domains, whose structures have been solved to date, are
.alpha./.beta. domains that are composed of a highly twisted mixed
.beta.-sheet flanked by .alpha.-helices on both sides. Because of
the similarity in the tertiary fold of PTP domains, the functional
diversity of PTPs generally is a consequence of the presence of
diverse noncatalytic regulatory and targeting domains that are
found in the N and C termini of the PTP catalytic domain.
[0025] The active site of the PTP domain is located within a
crevice on the molecular surface and is formed by several critical
loops. Most of the 27 invariant residues in the sequences of the
PTP domains are found on these loops. Invariant. residues more
distally located from the catalytic sites are buried and are
believed to stabilize the protein domain's tertiary fold.
[0026] The mechanism, for the hydrolysis reaction catalyzed by PTPs
is believed to be follows. The phosphate group of pTyr is
coordinated within the active site by main-chain amide groups and
the Arg side chain of the PTP loop. The binding of pTyr to the
active site induces a major conformational change of the WPD loop
from the open form to the closed form that shifts as much as 8
Angstroms so that the aspartic acid within the WPD loop becomes
properly positioned to act as a general acid in the hydrolysis
reaction. The catalytic reaction begins with a nucleophilic attack
by the cysteine (in the PTP loop) forming a cysteinyl-phosphate
intermediate. Cleavage of the scissile phosphate-oxygen bond is
facilitated by protonation of the phenolic oxygen by the aspartic
acid in the WPD loop. The closed or the catalytically competent
form (wherein the WPD loop occludes the active site) is believed to
prevent non-specific phosphoryl transfer reactions to extraneous
phosphoryl acceptors. In addition, the closed WPD loop is believed
to play a role in activating a nucleophilic water molecule that
ultimately hydrolyzes the cysteinyl-phosphate intermediate.
[0027] The discovery of the exosite in both PTP-1B and TC-PTP opens
up an alternative route to modulating these important phosphatases.
To date, all of the known drug programs targeting PTP-1B and TC-PTP
have focused on identifying active site inhibitors. Although these
programs generally have succeeded in identifying potent active site
inhibitors against the target enzymes, achieving cell penetration
(in view of the highly charged nature of most p-Tyr mimetics),
selectivity and in vivo efficacy have been problematic. However,
because the exosite is more hydrophobic in nature than the active
site and generally is not conserved among phosphatases, cell
penetration and selectivity can be attained much more readily.
Moreover, because the exosite inhibitors do not have to compete
with endogenous substrates, in vivo efficacy can be achieved with
less potent compounds than the active site PTP inhibitors.
[0028] PTP-1B
[0029] PTP-1B has been shown to play a major role in modulating
metabolism rates and insulin sensitivity. One of the most
compelling studies implicating PTP-1B to diabetes and obesity is
the knock-out study in mice. Elchebly et al., Science 283:1544-1548
(1999) and Klaman et al, Mol. Cell. Biol. 20: 5479-5489 (2000).
Disruption of the mouse homolog of the gene encoding PTP-1B
resulted in healthy mice. In the fasted state, no difference was
detected between the PTP-1B deficient mice and the control mice in
the concentrations of glucose or insulin. However, in the fed
state, the PTP-1B deficient mice had slightly lower concentrations
of glucose and approximately half the concentration of circulating
insulin than those of the control mice. Moreover, when fed a high
fat diet, the PTP-1B deficient mice were resistant to weight gain
and remained insulin sensitive whereas the control mice gained
weight rapidly and became insulin resistant. This and other
subsequent studies have demonstrated PTP-1B as a validated target
for the treatment of various metabolic disorders including diabetes
and obesity.
[0030] Human PTP-1B is a 435 amino acid protein. The sequence
comprises a short N-terminal sequence, a PTP domain and an ER
anchor. Because the full-length protein is insoluble in bacteria,
studies of PTP-1B typically have been on a 298 amino acid form or
on a 321 amino acid form that was first isolated from human
placenta. These truncated forms are fully functional in activity
studies and is consistent with the crystallographic findings that
only the first 298 residues are ordered.
[0031] TC-PTP
[0032] Human TC-PTP is a 415 amino acid protein that among other
things is involved in hematopoiesis, and T- and B-cell activation.
Two isoforms or splice variants of TC-PTP are expressed and differ
only at their extreme C-termini. The splice variants appear to be a
means for controlling the subcellular location of TC-PTP. The 48 kD
variant contains a hydrophobic sequence of 34 residues as the
C-terminus tail and is localized in the endoplasmic reticulum. Both
residues 346-358 as well as the hydrophobic C-terminus tail are
required to target this form of TC-PTP to the endoplasmic
reticulum. In contrast, the 45 kD variant contains a hydrophilic
sequence of 6 amino acids and is found primarily in the nucleus.
Residues 350-358 and 377-381. are believed to form a bipartite
nuclear localization signal.
[0033] FIG. 1 is a sequence alignment of human PTP-1B and TC-PTP.
The sequences show a 68% identity between the first 298 residues of
PTP-1B and the first 296 residues of TC-PTP. When referring to the
TC-PTP sequence herein, following convention, it will be referred
to using the PTP-1B based residue number system. For example, the
catalytic cysteine in the active site of PTP-1B is Cys-215. The
corresponding cysteine in TC-PTP is also referred to Cys-215 based
on the sequence alignment with PTP-1B even though the active site
cysteine is the 214.sup.th amino acid if it were numbered
consecutively.
[0034] The Exosite
[0035] In one aspect of the present invention, compounds are
provided that bind to the exosite of PTP-1B. The exosite is an
adaptive binding site on PTP-1B comprising at least one (more
preferably at least two residues) selected from the group
consisting of: Glu-186; Ser-187; Pro-188; Ala-189; Leu-192;
Asn-193; Phe-196; Lys-197; Arg-199; Glu-200; Leu-272; Glu-276;
Gly-277; Lys-279; Phe-280; Ile-281; and Met-282. In the presence of
a suitable ligand, one or more of these residues form an adaptive
binding site that is not normally present. For the purposes of
illustration, the formation of this adaptive binding site that is
referred herein as the exosite will be described with reference to
the following compound 1
[0036] This compound binds to and inhibits PTP-1B with an IC.sub.50
of about 30 .mu.M. In the presence of compound 5 and as shown in
FIG. 2, PTP-1B creates an exosite that is distal to the active
site. The image in FIG. 2 is based upon the crystal complex of
PTP-1B and compound 5. To highlight the presence of the crevice
which forms the exosite binding site, the surface accessible
surfaces are shown of residues Glu-186; Ser-187; Pro-188; Ala-189;
Leu-192; Asn-193; Phe-196; Lys-197; Glu-200; Leu-272; Glu-276'
Gly-277; Lys-279; Phe-280; Ile-281; and Met-282 (Arg-199 is not
pictured in this view of the exosite). FIG. 3 is an illustration of
the same region showing only the carbon and heteroatoms of residues
Glu-186; Ser-187; Pro-188; Ala-189; Leu-192; Asn-193; Phe-196;
Lys-197; Glu-200; Leu-272; Glu-276' Gly-277; Lys-279; Phe-280;
Ile-281; and Met-282.
[0037] The exosite is referred to as an adaptive binding site
because the presence of a suitable ligand induces major
conformational rearrangement in the enzyme that creates the exosite
binding site. In the absence of such a ligand, the presence of the
exosite cannot be discerned or predicted given that these same
residues do not form a contiguous surface accessible region. FIG. 4
is a crystal structure of residues 1-298 of PTP-1B in the absence
of an exosite ligand. As it can be seen, the majority of the
exosite region is no longer surface accessible as it is occluded by
the presence of a helix formed by residues 283-298.
[0038] The large conformational change that occurs in the presence
of an exosite ligand is mediated by the interactions of at least
three residues: Tyr-152, Asn-193, and Trp-291 and is believed to be
part of a regulatory mechanism for PTP-1B. FIG. 5 illustrates a few
of the key interactions of these residues in the absence of an
exosite ligand. The N.sub..delta.2 of Asn-193 makes a hydrogen bond
with the O.eta. of Tyr-152 and the helix formed by residues 283-298
is maintained in position at least in part from the non-bonded
interactions of the indole ring of Trp-291 with the phenyl rings of
Phe-280 and Phe-196 (not pictured). A fourth residue, Lys-197 (not
pictured), is also believed to participate in maintaining the
hydrogen bond interaction between the N.sub..delta.2 of Asn-193 and
O.eta. of Tyr-152.
[0039] In contrast, FIG. 6 illustrates the situation in the
presence of exosite ligand 5. As it can be seen, the benzofuran
moiety of compound 5 displaces the indole ring of Trp-291 causing
the helix formed by residues 283-298 to become displaced and/or
disordered. The carbonyl oxygen of compound 5 makes a hydrogen bond
with N.sub..delta.2 of Asn-193 so that the N.sub..delta.2 of
Asn-193 is no longer available for hydrogen bonding to O.eta. of
Tyr-152. The disruption of the hydrogen bond between Asn-193 and
Tyr-152 in part mediates a conformation change in the phenolic ring
of Tyr-152. The rotation of the phenolic ring of Tyr-152 propagates
a conformational change in the active site of PTP-1B that
functionally inactivates the enzyme.
[0040] The importance of the three key residues, particularly the
interaction between Asn-193 and Tyr-152, is supported by the
ability (or lack thereof) of compound 5 to bind to the exosite of
and inhibit other phosphatases. In TC-PTP, many of the
exosite-forming residues of PTP-1B are conserved including Asn-193,
Lys-197 and Tyr-152. Not surprising, compound 5 also inhibits
TC-PTP, although with a slightly lower potency than that observed
for PTP-1B (IC.sub.50 of about 130 .mu.M). In contrast, compound 5
does not inhibit tyrosine phosphatase leukocyte common antigen
related protein ("LAR") where the exosite-forming residues of
PTP-1B generally are not conserved including Asn-193, Lys-197, and
Tyr-152. A sequence alignment of PTP-1B and LAR is shown in FIG.
7.
[0041] Exosite Inhibitors and Methods of Identifying the Same
[0042] In one aspect of the present invention, compounds are
provided that interact with at least one residue (preferably at
least two residues and more preferably at least three residues)
selected from the group consisting of: Glu-186; Ser-187; Pro-188;
Ala-189; Leu-192; Asn-193; Phe-196; Lys-197; Arg-199; Glu-200;
Leu-272; Glu-276; Gly-277; Lys-279; Phe-280; Ile-281; and Met-282
of PTP-1B (collectively referred to as "PTP-1B exosite-forming
residues"). The resulting exosite ligand-PTP-1B complex is
considered another aspect of the present invention. In one
embodiment embodiment, compounds are provided that interact with at
least one residue (preferably at least two residues and more
preferably at least three residues) selected from the group
consisting of Asn-193, Phe-196, Lys-197, Arg-199; Glu-276, and
Phe-280 of PTP-1B. In another embodiment, the interaction between
the compound and the PTP-1B exosite-forming residues comprises an
interaction between the compound and Asn-193 and Phe-196. In yet
another embodiment, the interaction between the compound and the
PTP-1B exosite forming residues comprises an interaction between
the compound and Asn-193 and Phe-280.
[0043] In another aspect of the present invention, compounds are
provided that interact with at least one residue (preferably at
least two residues and more preferably at least three residues)
selected from the group consisting of: Glu-186; Ser-187; Pro-188;
Ala-189; Leu-192; Asn-193; Phe-196; Lys-197; Arg-199; Glu-200;
Met-272; Glu-276; Gly-277; Lys-279; Cys-280; Ile-281; and Lys-282
of TC-PTP (collectively TC-PTP exosite-forming residues). The
resulting exosite ligand-TC-PTP complex is considered another
aspect of the present invention. In one embodiment, compounds are
provided that interact with at least one residue (preferably at
least two residues and more preferably at least three residues)
selected from the group consisting of Asn-193; Phe-196; Lys-197;
Arg-199; Glu-276; and Cys-280 of TC-PTP. In another embodiment, the
interaction between the compound and the TC-PTP exosite-forming
residues comprises an interaction between the compound and Asn-193
and Phe-196. In yet another embodiment, the interaction between the
compound and the TC-PTP exosite-forming residues comprises an
interaction between the compound and Asn-193 and Cys-280.
[0044] A compound is said to interact with an exosite-forming
residue (whether PTP-1B or TC-PTP) if the compound forms a hydrogen
bond, a salt bridge, or a van der Waals contact with an
exosite-forming residue. A compound is considered to form a
hydroxyl-hydroxyl or hydroxyl-carbonyl hydrogen bond if the
distance between the donor and acceptor atom is between about 2.5
Angstroms and about 3.0 Angstroms. A compound is considered to form
an amide-carbonyl, amide-hydroxyl, or amide-imidazole hydrogen bond
if the distance between the donor and acceptor atom is about 2.7
Angstroms and 3.3 Angstroms. A compound is considered to form a
amide-sulfur hydrogen bond if the distance between the donor and
acceptor atom is between about 3.3 Angstroms and 3.9 Angstroms.
[0045] A compound is considered to form a salt bridge with an
exosite-forming residue if the distance between an amino (ionized)
group and a carboxylic acid (ionized) group is about 2.5 Angstroms
to about 4.0 Angstroms.
[0046] A compound is considered to make a van der Waals contact if
the distance between a carbon or a heteroatom in the compound and a
carbon or a heteroatom in an exosite-forming residue is between
about 2.0 Angstroms to about 5.0 Angstroms (preferably between
about 2.5 Angstroms to about 4.0 Angstroms, and more preferably
between about 2.5 and about 3.5 Angstroms).
[0047] In another embodiment, the compound (whether it is an
exosite ligand of PTP-1B or TC-PTP) is an "isolated" compound. As
used herein, the term "isolated" means purified (at least 80% pure,
preferably at least 90% pure, more preferably at least 95% pure,
and most preferably at least 99% pure as measured by weight). The
term "isolated" with respect to naturally occurring compounds such
as polypeptides includes any state that is not naturally occurring.
Examples of a state that is not naturally occurring with respect to
a polypeptide are purified or recombinant forms of that
polypeptide. In another embodiment, the compound is not a
polypeptide. In yet another embodiment, the compound does not
include an amino acid residue.
[0048] In another aspect of the present invention, methods are
provided for identifying compounds that bind to the exosite of
PTP-1B and inhibit the activity of PTP-1B. In one embodiment, the
method comprise:
[0049] a) contacting a test compound with PTP-1B;
[0050] b) contacting the test compound with an exosite mutant of
PTP-1B; and
[0051] c) comparing the activity of PTP-1B in the presence of the
test compound with the activity of the exosite mutant of PTP-1B in
the presence of the test compound.
[0052] PTP-1B for the purposes of these methods is wild-type PTP-1B
or any functional truncated form thereof (e.g., a form that is
capable of dephosphorylating a phosphotyrosine and includes all of
the native exosite-forming residues). In one embodiment, the PTP-1B
is human PTP-1B. In another embodiment, the PTP-1B comprises SEQ
ID. NO. 1.
[0053] An exosite mutant of PTP-1B is a PTP-1B wherein at least one
of the PTP-1B exosite-forming residues has been modified to a
different amino acid such that the resulting PTP-1B is no longer
capable of being inhibited through the exosite site or displays a
diminished capacity (less than about 75%/o inhibition compared to
SEQ ID NO.1 for a known exosite inhibitor such as compound 5;
preferably less than about 50%, more preferably less than about
25%) of being inhibited through the exosite. Exosite mutants of
PTP-1B comprise another aspect of the present invention.
[0054] In one embodiment, the exosite mutant of PTP-1B is PTP-1B
wherein Asn-193 has been mutated to another amino acid. In another
embodiment, the exosite mutant of PTP-1B is PTP-1B wherein Asn-193
has been mutated to alanine. In another embodiment, the exosite
mutant of PTP-1B is PTP-1B wherein Lys-197 has been mutated to
another amino acid. In another embodiment, the exosite mutant of
PTP-1B is PTP-1B wherein Lys-197 has been mutated to cysteine. In
another embodiment, the exosite mutant of PTP-1B is PTP-1B wherein
Asn-193 and Phe-196 have been mutated. In another embodiment, the
exosite mutant of PTP-1B is PTP-1B wherein Asn-193 has been mutated
to alanine and Lys-197 has been mutated to cysteine. In another
embodiment, the exosite mutant of PTP-1B is PTP-1B wherein Asn-193
has been mutated to alanine and Phe-196 has been mutated to
arginine. In another embodiment, the exosite mutant of PTP-1B is
PTP-1B wherein Asn-193, Phe-196, and Phe-280 have been mutated. In
another embodiment, the exosite mutant of PTP-1B is PTP-1B wherein
Asn-193 is mutated to alanine, Phe-196 has been mutated to arginine
and Phe-280 has been mutated to cysteine.
[0055] Test compounds that inhibit PTP-1B but not an exosite mutant
of PTP-1B are potential exosite inhibitors. Compounds that inhibit
PTP-1B in a non-competitive manner with active site ligands and
that inhibit PTP-1B in a competitive manner with known exosite
ligands such as compound 5 are PTP-1B exosite inhibitors.
[0056] In another aspect of the present invention, methods are
provided for finding compounds that bind to the exosite of TC-PTP
and inhibit the activity of TC-PTP. In one embodiment, the method
comprise:
[0057] a) contacting a test compound with TC-PTP;
[0058] b) contacting the test compound with an exosite mutant of
TC-PTP; and
[0059] c) comparing the activity of TC-PTP in the presence of the
test compound with the activity of the exosite mutant of TC-PTP in
the presence of the test compound.
[0060] TC-PTP for the purposes of these methods is wild-type TC-PTP
or any functional truncated form thereof (e.g., a form that is
capable of dephosphorylating a phosphotyrosine and includes all of
the native exosite-forming residues). In one embodiment, the TC-PTP
is human TC-PTP. In another embodiment, the TC-PTP comprises SEQ
ID. NO.2.
[0061] An exosite mutant of TC-PTP is a TC-PTP wherein at least one
of the TC-PTP exosite-forming residues has been modified to a
different amino acid such that the resulting TC-PTP is no longer
capable of being inhibited through the exosite site or displays a
diminished capacity (less than about 75% inhibition compared to SEQ
ID NO.2 for a known exosite inhibitor such as compound 5;
preferably less than about 50%; and more preferably less than about
25%) of being inhibited through the exosite. Exosite mutants of
TC-PTP comprise another aspect of the present invention.
[0062] In one embodiment, the exosite mutant of TC-PTP is TC-PTP
wherein Asn-193 has been mutated to another amino acid. In another
embodiment, the exosite mutant of TC-PTP is TC-PTP wherein Asn-193
has been mutated to alanine. In another embodiment, the exosite
mutant of TC-PTP is TC-PTP wherein Lys-197 has been mutated to
another amino acid. In another embodiment, the exosite mutant of
TC-PTP is TC-PTP wherein Lys-197 has been mutated to a cysteine. In
another embodiment, the exosite mutant of TC-PTP is TC-PTP wherein
Asn-193 and Phe-196 have been mutated. In one embodiment, the
exosite mutant of TC-PTP is TC-PTP wherein Asn-193 has been mutated
to alanine and Phe-196 has been mutated to arginine. In another
embodiment, the exosite mutant of TC-PTP is TC-PTP wherein Asn-193
and Lys-197 have been mutated. In another embodiment, the exosite
mutant of TC-PTP is TC-PTP wherein Asn-193 has been mutated to
alanine and Lys-197 has been mutated to cysteine.
[0063] Test compounds that inhibit TC-PTP but not mutant TC-PTP are
potential TC-PTP exosite inhibitors. Compounds that inhibit TC-PTP
in a non-competitive manner with active site ligands and that
inhibit TC-PTP in a competitive manner with exosite ligands-are
TC-PTP exosite inhibitors.
[0064] Compounds of the Present Invention
[0065] In another aspect of the present invention, compounds are
provided having the structure 2
[0066] wherein:
[0067] R.sup.1 is hydrogen, methyl, ethyl, or propyl;
[0068] R.sup.2 hydrogen --S(O.sub.2)R.sup.3, --NH(C(.dbd.O)R.sup.3,
--NH(C(.dbd.O)CH.sub.2(.dbd.O)OR.sup.3,
--S(O.sub.2)NR.sup.4R.sup.5, or --NR.sup.4S(O.sub.2)R.sup.3 where
R.sup.3 is C.sub.1-C.sub.5 alkyl, R.sup.4 is hydrogen
C.sub.1-C.sub.5 alkyl, unsubstituted cyclic moiety, or substituted
cyclic moiety, and R.sup.5 is either hydrogen or R.sup.5 and
R.sup.4 together form an unsubstituted cyclic moiety or a
substituted cyclic moiety;
[0069] R.sup.6 is hydrogen or alternatively when R.sup.2 is
--NR.sup.4S(O.sub.2)NR.sup.3, then R.sup.6 and R.sup.4 together
form an unsubstituted cyclic moiety or substituted cyclic moiety;
and,
[0070] L is --NHS(O.sub.2)-- or --S(O.sub.2)NR.sup.7CH.sub.2--
where R.sup.7 is hydrogen or C.sub.1-C.sub.5 alkyl.
[0071] In one embodiment, the compounds are of structure I wherein
the one or more substituents on the substituted cyclo group are
each independently selected from the group consisting of:
C.sub.1-C.sub.5 alkyl, phenyl, benzyl, F, Cl, I, Br, --OH;
--NO.sub.2; --CN; --CF.sub.3; --CH.sub.2CF.sub.3; --CH.sub.2Cl;
--CH.sub.2OH; --CH.sub.2CH.sub.2OH; --CH.sub.2NH.sub.2;
--CH.sub.2SO.sub.2CH.sub.3; --OR.sup.8; --C(O)R.sup.8;
--COOR.sup.8; --C(O)NR.sup.8R.sup.9; --OC(O)R.sup.8; --OCOOR.sup.8;
--OC(O)NR.sup.8R.sup.9; --NR.sup.8R.sup.9; --S(O).sub.2R.sup.8; and
--NR.sup.8C(O)R.sup.9 where R.sup.8 and R.sup.9 are each
independently hydrogen, C.sub.1-C.sub.5 alkyl, phenyl or
benzyl.
[0072] In another embodiment, the compounds are of structure I
wherein R.sup.1 is ethyl.
[0073] In another embodiment, the compounds are of structure I
wherein R.sup.2 and R.sup.6 are both hydrogen.
[0074] In another embodiment, the compounds are of structure I
wherein R.sup.2 is --S(O.sub.2)NHR.sup.5 where R.sup.5 is an
unsubstituted cyclic moiety or substituted cyclic moiety, and
R.sup.6 is hydrogen. In another embodiment the cyclic moiety is
morpholine or pipendine. In another embodiment, the cyclic moiety
is an aryl group. In another embodiment, the cyclic moiety is
phenyl. In another embodiment, the cyclic moiety is a 5-membered
heteroaryl. In another embodiment, the cyclic moiety is an
imidazole, oxazole, or thiazole. In another embodiment, the cyclic
moiety is a 6-membered heteroaryl. In another embodiment, the
cyclic moiety is pyridine, pyrimidine, or pyrazine.
[0075] In another embodiment, the compounds are of structure I
wherein R.sup.2 is --S(O.sub.2)R.sup.3 where R.sup.3 is methyl,
ethyl, or propyl, and R.sup.6 is hydrogen.
[0076] In another embodiment, the compounds are of structure I
wherein R.sup.2 is --NH(C(.dbd.O)R.sup.3 where R.sup.3 is methyl,
ethyl, or propyl, and R.sup.6 is hydrogen.
[0077] In another embodiment, the compounds are of structure I
wherein R.sup.2 is --NH(C(.dbd.O)CH.sub.2(C.dbd.O)OR.sup.3 where
R.sup.3 is methyl, ethyl, or propyl, and R.sup.6 is hydrogen.
[0078] In another embodiment, the compounds are of structure I
wherein R.sup.2 is --NR.sup.4S(O.sub.2)R.sup.3 wherein R.sup.3 is
methyl and R.sup.4 and R.sup.6 together form an unsubstituted
heterocyclo or a substituted heterocyclo. In another embodiment
R.sup.4 and R.sup.6 together form an unsubstituted pyrrolidine or a
substituted pyrrolidine.
[0079] In another embodiment, the compounds are of structure I
wherein L is --NHS(O.sub.2)--.
[0080] In another embodiment, the compounds are of structure I
wherein L is --S(O.sub.2)NR.sup.7CH.sub.2-- and R.sup.7 is
methyl.
[0081] In another embodiment, the compounds are of structure I
wherein L is --S(O.sub.2)NR.sup.7CH.sub.2-- and R.sup.7 is
ethyl.
[0082] In another embodiment, the compounds are of structure I
wherein L is --S(O.sub.2)NR.sup.7CH.sub.2-- and R.sup.7 is
propyl.
[0083] In another aspect of the present invention, compounds are
provided having the following structure: 3
[0084] wherein:
[0085] R.sup.10 is C.sub.1-C.sub.5 alkyl or NHR.sup.11 where
R.sup.11 is hydrogen, C.sub.1-C.sub.10 alkyl or aryl; and,
[0086] L is NHS(O.sub.2)-- or
--S(O.sub.2)N(CH.sub.2).sub.3CH.sub.2--.
[0087] In one embodiment, the compounds are of structure II and
R.sup.10 is methyl, ethyl or propyl.
[0088] In another embodiment, the compounds are of structure II and
R.sup.10 is NHR.sup.11. In another embodiment, R.sup.11 is
hydrogen. In another embodiment R.sup.11 is aryl. In another
embodiment, R.sup.11 is a heteroaryl. In another embodiment,
R.sup.11 is phenyl. In another embodiment, R.sup.11 is thiazole. In
another embodiment, R.sup.11 is pyrimidine.
[0089] It will be appreciated by one of ordinary skill in the art
that compounds of the present invention include one or more
asymmetric centers. The inventive compounds may be in the form of
an individual enantiomer, diasteromer or geometric isomer, or may
be in the form of a mixture of stereoisomers unless otherwise
indicated. In the case of compounds containing double bonds, these
double bonds can be either Z or E or a mixture thereof, unless
otherwise indicated.
[0090] As used herein "aliphatic" or "unsubstituted aliphatic"
refers to a straight, branched, cyclic, or polycyclic hydrocarbon
and includes alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and
cycloalkynyl moieties.
[0091] The term "alkyl" or "unsubstituted alkyl" refers to a
saturated hydrocarbon.
[0092] The term "alkenyl" or "unsubstituted alkenyl" refers to a
hydrocarbon with at least one carbon-carbon double bond.
[0093] The term "alkynl" or "unsubstituted alkynl" refers to a
hydrocarbon with at least one carbon-carbon triple bond.
[0094] The term "aryl" or "unsubstituted aryl" refers to a mono or
polycyclic unsaturated moieties having at least one aromatic ring.
The term includes heteroaryls that include one or more heteroatoms
-within the at least one aromatic ring. Illustrative examples of
aryl include: phenyl, naphthyl, tetrahydronaphthyl, indanyl,
indenyl, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,
imidazolyl, thiazolyl, oxazolyl, isooxazoly, thiadiazolyl,
oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and
the like.
[0095] The term "alkylaryl" or "unsubstituted alkylaryl" refers to
an aryl moiety that is substituted with at least one aliphatic
group.
[0096] The term "cyclo" or "cyclic moiety" refers to a mono or
polycyclic aliphatic or aromatic groups. The cyclic aliphatic
groups include partially unsaturated cyclic moieties (having one or
more double or triple bonds). The term includes heterocyclic and
heteroaryl groups.
[0097] The term "substituted moiety" refers to a substituted
version of the moiety where at least one hydrogen atom is
substituted with another group including but not limited to:
aliphatic; aryl, alkylaryl, F, Cl, I, Br, --OH; --NO.sub.2; --CN;
--CF.sub.3; --CH.sub.2CF.sub.3; --CH.sub.2Cl; --CH.sub.2OH;
--CH.sub.2CH.sub.2OH; --CH.sub.2NH.sub.2;
--CH.sub.2SO.sub.2CH.sub.3; --OR.sup.x; --C(O)R.sup.x;
--COOR.sup.x; --C(O)NR.sup.xR.sup.y; --OC(O)R.sup.x; --OCOOR.sup.x;
--OC(O)NR.sup.xR.sup.y; --NR.sup.xR.sup.y; --S(O).sub.2R.sup.x; and
--NR.sup.xC(O)R.sup.y where R.sup.x and R.sup.y are each
independently hydrogen, substituted aliphatic, unsubstituted
aliphatic, substituted aryl, or unsubstituted aryl. Additionally,
substitutions at adjacent groups on a moiety can together form a
cyclic group.
[0098] Pharmaceutical Compositions
[0099] The present invention provides compounds useful among other
things for treating obesity, diabetes and pathological conditions
associated with type 2 diabetes, such as insulin resistance, in the
case of PTP-1B inhibitors, and for treating inflammation, and
hematopoietic and immunological disorders in the case of TC-PTP.
Hematopietic disorders include, without limitation, disorders
associated with impaired B cell lymphopoiesis and/or
erythropoiesis.
[0100] In one aspect of the present invention, pharmaceutical
compositions are provided, which comprise any one of the compounds
described herein (or a prodrug, pharmaceutically acceptable salt or
other pharmaceutically acceptable derivative thereof), and
optionally comprise a pharmaceutically acceptable carrier. In
certain embodiments, these compositions optionally further comprise
one or more additional therapeutic agents. These therapeutic agents
can include other anti-diabetes drugs in the case of PTP-1B
inhibitors and can include other agents that modulate the immune
response in the case of TC-PTP. Alternatively, the additional
therapeutic agents can be those that alleviate or mitigate any side
effect of the compounds of the present invention.
[0101] The term "pharmaceutically acceptable derivative" is any
pharmaceutically acceptable salt, ester, or salt of such ester, of
a compound (or any other adduct or derivative) that, upon
administration to a patient, is capable of providing (directly or
indirectly) the desired compound. Pharmaceutically acceptable
derivatives includes among other things, prodrugs--a derivative of
a compound that typically contains an additional moiety that is
removed in vivo yielding the parent molecule as the
pharmacologically active species.
[0102] The term "pharmaceutically acceptable salt" refers to those
salts which are, within the scope of sound medical judgment,
suitable for use in contact with the tissues of humans and lower
animals without undue toxicity, irritation, allergic response and
the like, and are commensurate with a reasonable benefit/risk
ratio. Pharmaceutically acceptable salts of amines, carboxylic
acids, and other types of compounds, are well known in the art. For
example, S. M. Berge, et al. describe pharmaceutically acceptable
salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977),
incorporated herein by reference. The salts can be prepared in situ
during the final isolation and purification of the compounds of the
invention, or separately by reacting a free base or free acid
function with a suitable reagent, as described generally below. For
example, a free base function can be reacted with a suitable acid.
Furthermore, where the compounds of the invention carry an acidic
moiety, suitable pharmaceutically acceptable salts thereof may,
include metal salts such as alkali metal salts, e.g. sodium or
potassium salts; and alkaline earth metal salts, e.g. calcium or
magnesium salts. Examples of pharmaceutically acceptable, nontoxic
acid addition salts are salts of an amino group formed with
inorganic acids such as hydrochloric acid, hydrobromic acid,
phosphoric acid, sulfuric acid and perchloric acid or with organic
acids such as acetic acid, oxalic acid, maleic acid, tartaric acid,
citric acid, succinic acid or malonic acid or by using other
methods used in the art such as ion exchange. Other
pharmaceutically acceptable salts include adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,
borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hernisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, parnoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Representative alkali or alkaline earth metal salts include sodium,
lithium, potassium, calcium, magnesium, and the like. Further
pharmaceutically acceptable salts include, when appropriate,
nontoxic ammonium, quaternary ammonium, and amine cations formed
using counterions such as halide, hydroxide, carboxylate, sulfate,
phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.
[0103] The term "pharmaceutically acceptable ester" refers to
esters that hydrolyze in vivo and include those that break down
readily in the human body to leave the parent compound or a salt
thereof Suitable ester groups include, for example, those derived
from pharmaceutically acceptable aliphatic carboxylic acids,
particularly alkanoic, alkenoic, cycloalkanoic and alkanedioic
acids, in which each alkyl or alkenyl moiety advantageously has not
more than 6 carbon atoms. Examples of particular esters include
formates, acetates, propionates, butyrates, acrylates and
ethylsuccinates.
[0104] The term "pharmaceutically acceptable prodrugs" as used
herein refers to those prodrugs of the compounds of the present
invention which are, within the scope of sound medical judgment,
suitable for use in contact with the issues of humans and lower
animals with undue toxicity, irritation, allergic response, and the
like, commensurate with a reasonable benefit/risk ratio, and
effective for their intended use, as well as the zwitterionic
forms, where possible, of the compounds of the invention. The term
"prodrug" refers to compounds that are rapidly transformed in vivo
to yield the parent compound of the above formula, for example by
hydrolysis in blood. A discussion is provided in T. Higuchi and V.
Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S.
Symposium Series, and in Edward B. Roche, ed., Bioreversible
Carriers in Drug Design, American Pharmaceutical Association and
Pergamon Press, 1987, both of which are incorporated herein by
reference.
[0105] As described above, the pharmaceutical compositions of the
present invention additionally can comprise a pharmaceutically
acceptable carrier, which, as used herein, includes any and all
solvents, diluents, or other liquid vehicle, dispersion or
suspension aids, surface active agents, isotonic agents, thickening
or emulsifying agents, preservatives, solid binders, lubricants and
the like, as suited to the particular dosage form desired.
Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.
Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various
carriers used in formulating pharmaceutical compositions and known
techniques for the preparation thereof. Except insofar as any
conventional carrier medium is incompatible with the compounds of
the invention, such as by producing any undesirable biological
effect or otherwise interacting in a deleterious manner with any
other component(s) of the pharmaceutical composition, its use is
contemplated to be within the scope of this invention. Some
examples of materials which can serve as pharmaceutically
acceptable carriers include, but are not limited to, sugars such as
lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatine; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil, sesame oil; olive oil; corn oil and soybean
oil; glycols; such as propylene glycol; esters such as ethyl oleate
and ethyl laurate; agar; buffering agents such as magnesium
hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water;
isotonic saline; Ringer's solution; ethyl alcohol, and phosphate
buffer solutions, as well as other non-toxic compatible lubricants
such as sodium lauryl sulfate and magnesium stearate, as well as
coloring agents, releasing agents, coating agents, sweetening,
flavoring and perfuming agents, preservatives and antioxidants can
also be present in the composition, according to the judgment of
the formulator.
[0106] Methods of Use and Dosage Forms
[0107] In another aspect of the present invention, a method for
treating diabetes is provided comprising administering to a subject
in need thereof a therapeutically effective amount of a PTP-1B
exosite inhibitor.
[0108] In another aspect of the present invention, a method for
treating insulin resistance is provided comprising administering to
a subject in need thereof a therapeutically effective amount of a
PTP-1B exosite inhibitor.
[0109] In another aspect of the present invention, a method for
treating diabetes is provided comprising administering to a subject
in need thereof a therapeutically effective amount of a PTP-1B
exosite inhibitor.
[0110] In another aspect of the present invention, a method for
treating inflammation is provided comprising administering to a
subject in need thereof a therapeutically effective amount of a
TC-PTP exosite inhibitor.
[0111] In another aspect of the present invention, a method for
treating immune system disorders is provided comprising
administering to a subject in need thereof a therapeutically
effective amount of a TC-PTP exosite inhibitor.
[0112] In yet another aspect of the present invention, a method for
treating a hematopoiesis disorder is provided comprising
administering to a subject in need thereof a therapeutically
effective amount of a TC-PTP exosite inhibitor.
[0113] The term subject refers to a mammal, more preferably a
human.
[0114] The term "effective amount" refers to an amount of a
compound that will elicit the biological or medical response of
subject that is being sought by a researcher or clinician. The term
"therapeutically effective amount" means any amount which, as
compared to a corresponding subject who has not received such
amount, results in improved treatment, healing, prevention, or
amelioration of a disease or disorder, or a decrease in the rate of
advancement of a disease or disorder, and also includes amounts
effective to enhance normal physiological function. The exact
amount required will vary from subject to subject, depending on the
species, age, and general condition of the subject, the severity of
the infection, the particular therapeutic agent, its mode of
administration, and the like.
[0115] The compounds of the invention are preferably formulated in
dosage unit form for ease of administration and uniformity of
dosage. The expression "dosage unit form" as used herein refers to
a physically discrete unit of therapeutic agent appropriate for the
patient to be treated. It will be understood, however, that the
total daily usage of the compounds and compositions of the present
invention will be decided by the attending physician within the
scope of sound medical judgment. The specific therapeutically
effective dose level for any particular patient or organism will
depend upon a variety of factors including the disorder being
treated and the severity of the disorder; the activity of the
specific compound employed; the specific composition employed; the
age, body weight, general health, sex and diet of the patient; the
time of administration, route of administration, and rate of
excretion of the specific compound employed; the duration of the
treatment; drugs used in combination or coincidental with the
specific compound employed; and like factors well known in the
medical arts.
[0116] Furthermore, after formulation with an appropriate
pharmaceutically acceptable carrier in a desired dosage, the
pharmaceutical compositions of this invention can be administered
to humans and other animals orally, rectally, parenterally,
intracistemally, intravaginally, intraperitoneally, topically (as
by powders, ointments, or drops), bucally, as an oral or nasal
spray, or the like, depending on the severity of the infection
being treated. In certain embodiments, the compounds of the
invention may be administered at dosage levels of about 0.001 mg/kg
to about 50 mg/kg, from about 0.01 mg/kg to about 25 mg/kg, or from
about 0.1 mg/kg to about 10 mg/kg of subject body weight per day,
one or more times a day, to obtain the desired therapeutic effect.
It will also be appreciated that dosages smaller than 0.001 mg/kg
or greater than 50 mg/kg (for example 50-100 mg/kg) can be
administered to a subject. In certain embodiments, compounds are
administered orally or parenterally.
[0117] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active compounds, the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0118] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0119] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0120] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of the drug from subcutaneous or
intramuscular injection. This may be accomplished by the use of a
liquid suspension or crystalline or amorphous material with poor
water solubility. The rate of absorption of the drug then depends
upon its rate of dissolution that, in turn, may depend upon crystal
size and crystalline form. Alternatively, delayed absorption of a
parenterally administered drug form is accomplished by dissolving
or suspending the drug in an oil vehicle. Injectable depot forms
are made by forming microencapsule matrices of the drug in
biodegradable polymers such as polylactide-polyglycolide. Depending
upon the ratio of drug to polymer and the nature of the particular
polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include (poly(orthoesters)
and poly(anhydrides). Depot injectable formulations are also
prepared by entrapping the drug in liposomes or microemulsions
which are compatible with body tissues.
[0121] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carriers'such as cocoa butter, polyethylene glycol or a
suppository wax which are solid at ambient temperature but liquid
at body temperature and therefore melt in the rectum or vaginal
cavity and release the active compound.
[0122] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar--agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0123] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
that can be used include polymeric substances and waxes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polethylene
glycols and the like.
[0124] The active compounds can also be in micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose and starch. Such dosage forms may also
comprise, as in normal practice, additional substances other than
inert diluents, e.g., tableting lubricants and other tableting aids
such as magnesium stearate and microcrystalline cellulose. In the
case of capsules, tablets and pills, the dosage forms may also
comprise buffering agents. They may optionally contain opacifying
agents and can also be of a composition that they release the
active ingredient(s) only, or preferentially, in a certain part of
the intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions which can be used include polymeric
substances and waxes.
[0125] Dosage forms for topical or transdermal administration of a
compound of this invention include ointments, pastes, creams,
lotions, gels, powders, solutions, sprays, inhalants or patches.
The active component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, ear drops, and
eye drops are also contemplated as being within the scope of this
invention. Additionally, the present invention contemplates the use
of transdermal patches, which have the added advantage of providing
controlled delivery of a compound to the body. Such dosage forms
are made by dissolving or dispensing the compound in the proper
medium. Absorption enhancers can also be used to increase the flux
of the compound across the skin. The rate can be controlled by
either providing a rate controlling membrane or by dispersing the
compound in a polymer matrix or gel.
[0126] It will also be appreciated that the compounds and
pharmaceutical compositions of the present invention can be
formulated and employed in combination therapies, that is, the
compounds and pharmaceutical compositions can be formulated with or
administered concurrently with, prior to, or subsequent to, one or
more other desired therapeutics or medical procedures. The
particular combination of therapies (therapeutics or procedures) to
employ in a combination regimen will take into account
compatibility of the desired therapeutics and/or procedures and the
desired therapeutic effect to be achieved. It will also be
appreciated that the therapies employed may achieve a desired
effect for the same disorder (for example, an inventive compound
may be administered concurrently with another anticancer agent for
example), or they may achieve different effects (e.g., control of
any adverse effects).
EXAMPLE 1
[0127] Cloning of PTP-1B
[0128] Truncated versions of wildtype human PTP-1B were made as
follows. A cDNA encoding the first 321 amino acids of human PTP-1B
(SEQ ID NO: 4) was isolated from human fetal heart total RNA
(Clontech).
1 MEMEKEFEQIDKSGSWAAIYQDIRHEASDFPCRVAKLPKNKNRNRYRDVSPFDH SEQ ID NO.
4: SRIKLHQEDNDYINASLIKMEEAQRSYILTQGPLPNTCGHFWEMVWEQKSRGV- V
MLNRVMEKGSLKCAQYWPQKEEKEMIFEDTNLKLTLISEDIKSYYTVRQLELEN
LTTQETREILHFHYTTWPDFGVPESPASFLNFLFKVRESGSLSPEHGPVVVHCSAG
IGRSGTFCLADTCLLLMDKRKDPSSVDIKKVLLEMRKFRMGLIQTADQLRFSYLA
VIEGAKFIMGDSSVQDQWKELSHEDLEPPPEHIPPPPRPPKRILEPH
[0129] Oligonucleotide primers corresponding to nucleotides 91 to
114 (For) and complementary to nucleotides 1030 to 1053 (Rev) of
the PTP-1B cDNA (Genbank M31724.1; Chernoff, J. et. al., Proc.
Natl. Acad. Sci. U.S.A. 87: 2735-2739 (1990)) were synthesized and
used to generate a DNA using the polymerase chain reaction.
2 Forward: GCCATATGGAGATGGAAAAGGAGTTG GAG (SEQ ID NO: 5) Reverse:
GCGACGCGAATTCTTAATTGTGTGGCTCCAGGATTCGTTT (SEQ ID NO: 6)
[0130] The primer Forward incorporates an NdeI restriction site at
the first ATG codon and the primer Rev inserts a UAA stop codon
followed by an EcoRI restriction site after nucleotide 1053. cDNAs
were digested with restriction nucleases NdeI and EcoRI and cloned
into pRSETc (Invitrogen) using standard molecular biology
techniques. The identity of the isolated cDNA was verified by DNA
sequence analysis.
[0131] A shorter cDNA, PTP-1B 298, encoding amino acid residues
1-298 was generated using oligonuclotide primers Forward and Rev2
and the clone described above as a template in a polymerase chain
reaction.
[0132] Reverse 2: TGCCGGAATTCCTTAGTCCTCGTGGGAAAGCTCC (SEQ ID NO:
7)
EXAMPLE 2
[0133] PTP-1B Mutants
[0134] The following mutants were made as follows. The 321 amino
acid form of PTP-1B (SEQ ID NO: 3) in pRSETc (Invitrogen) was used
as a template and T7 and RSETrev primers were used as "outside"
primers.
3 PTP-1B[321; N193A; F196R]: Fwd primer:
TTCTTGGCGTTTCTTCGCAAAGTCCGA SEQ ID. NO: 8 Rev primer:
GACTTTGCGAAGAAACGCCAAGAATGA SEQ ID. NO: 9 PTP-1B[321; F280C] Fwd
primer: GGTGCCAAATGCATCATGGGG SEQ ID. NO: 10 Rev primer:
CCCGATGATGCATTTGGCACC SEQ ID. NO: 11
[0135] PTP-1B[321; N193A; F196R; F280C] was generated by joining an
NdeI-PstI fragment from PTP-1B[321; N193A; F196R], corresponding to
residues 1-215, with a PstI-EcoRI fragment from PTP-1B[321; F280C],
corresponding to residues 216-321.
[0136] PTP-1B[298; N193A; F196R; F280C] was generated by PCR using
PTP-1B[321; N193A; F196R; F280C] as a template. T7 vector primer
was used as forward primer and truncation at residue 298 was
generated using the primer:
[0137] TGCCGGAATTCCTTAGTCCTCGTGCGAAAGCTCC (SEQ ID. NO: 12).
[0138] The following mutants were made using Kunkel mutagenesis and
PTP-1B[298] as a template.
4 E186C GAATGAGGCTGGTGAGCAAGGGACTCCAAAG SEQ ID NO: 13 S187C
GAATGAGGCTGGGCATTCAGGGACTCC SEQ ID NO: 14 A189C
GTTCAAGAATGAGCATGGTGATTCAGG SEQ ID NO: 15 K197C
CTGACTCTCGGACGCAGAAAAGAAAGTTC SEQ ID NO: 16 E200C
GAGTGACCCTGAGCATCGGACTTTGAAAAG SEQ ID NO: 17 C215S
GATGCCTGCACTGGAGTGGACCACAAC SEQ ID NO: 18 M258C
CTGGATCAGCCCACACCGAAACTTCCT SEQ ID NO: 19 Q262C
CTGGTCGGCTGTACAGATCAGCCCCAT SEQ ID NO: 20 L272C
CTTCGATCACAGCGCAGTAGGAGAAGCG SEQ ID NO: 21 E276C
GAATTTGGCACCGCAGATCACAGCCAG SEQ ID NO: 22 I281C
AGAGTCCCCCATGCAGAATTTGGCACC SEQ ID NO: 23 V287C
CCACTGATCCTGGCAGGAAGAGTCCCC SEQ ID NO: 24
[0139] Besides mutations to cysteines, mutations removing naturally
occurring cysteines (referred to as "scrubs") can also be made. For
example, naturally occurring cysteines at positions 92 and 121 can
be mutated to other residues such as alanine or serine.
Illustrative examples of oligos for this purpose include:
5 C92A CCAAAAGTGACCGGCTGTGTTAGGCAA SEQ ID NO: 25 C121A
CCAGTATTGTGCGGCTTTTAACGAACC SEQ ID NO: 26 C121S
CCAGTATTGTGCGCTTTTTAACGAACC SEQ ID NO: 27
[0140] Sequencing of PTP-1B clones was accomplished as follows. The
concentration of plasmid DNA was quantitated by absorbance at 280
nm. 1000 ng of plasmid was mixed with sequencing reagents (1 .mu.g
DNA, 6 .mu.l water, 1 .mu.l sequencing primer at 3.2 pm/.mu.l, 8
.mu.l sequencing mixture with Big Dye [Applied Biosystems]). The
sequencing primers are SEQ ID NO: 28 and SEQ ID NO: 29.
6 Forward primer, "T7" AATACGACTCACTATAG SEQ ID NO: 28 Reverse
primer, "RSET REV" TAGTTATTGCTCAGCGGTGG SEQ ID NO: 29
[0141] The mixture was then run through a PCR cycle (96.degree. C.,
10 s; 50.degree. C., 5 s; 60.degree. C., 4 minutes; 25 cycles) and
the DNA reaction products were precipitated (20 .mu.l mixture, 80
.mu.l 75% isopropanol; incubated 20 minutes at room temperature
then pelleted at 14 K rpm for 20 minutes; wash with 250 .mu.l 75%
isopropanol; heat 1 minute at 94.degree. C.). The precipitated
products were then resuspended in 20 .mu.l TSB (Applied Biosystems)
and the sequence read and analyzed by an Applied Biosystems 310
capillary gel sequencer. In general, 1/4 of the plasmids contained
the desired mutation.
[0142] Expression of Cysteine Mutants of PTP-1B
[0143] Mutant proteins were expressed as follows. PTP-1B clones
were transformed into BL21 codon plus cells (Stratagene) (1 .mu.l
double-stranded DNA, 2 .mu.l 5.times.KCM, 7 .mu.l water, 10 .mu.l
DMSO competent cells; incubate 20 minutes at 4.degree. C., 10
minutes at room temperature), plated onto LB/agar containing 100
.mu.g/ml ampicillin, and incubated at 37.degree. C. overnight. 2
single colonies were picked off the plates or from frozen glycerol
stocks of these mutants and inoculated in 100 ml 2YT with 50
.mu.g/ml carbenicillin and grown overnight at 37.degree. C. 50 ml
from the overnight cultures were added to 1.5 L of
2YT/carbenicillin (50 .mu.g/ml) and incubated at 37.degree. C. for
3-4 hours until late-log phase (absorbance at 600 nm
.about.0.8-0.9). At this point, protein expression was induced with
the addition of IPTG to a final concentration of 1 mM. Cultures
were incubated at 37.degree. C. for another 4 hours and then cells
were harvested by centrifugation (7K rpm, 7 minutes) and frozen at
-20.degree. C.
[0144] PTP-1B proteins were purified from the frozen cell pellets
as described in the following. First, cells were lysed in a
microfluidizer in 100 ml of buffer containing 20 mM MES pH 6.5, 1
mM EDTA, 1 mM DTT, and 10% glycerol buffer (with 3 passes through a
Microfluidizer [Microfluidics 110S]) and inclusion bodies were
removed by centrifugation (10K rpm, 10 minutes). Purification of
all PTP-1B mutants was performed at 4.degree. C. The supernatants
from the centrifugation were filtered through 0.45 .mu.m cellulose
acetate (5 .mu.l of this material was analyzed by SDS-PAGE) and
loaded onto an SP Sepharose fast flow column (2.5 cm
diameter.times.14 cm long) equilibrated in Buffer A (20 mM MES pH
6.5, 1 mM EDTA, 1 mM DTT, 1% glycerol) at 4 ml/min.
[0145] The protein was then eluted using a gradient of 0-50% Buffer
B over 60 minutes (Buffer B: 20 mM MES pH 6.5, 1 mM EDTA, 1 mM DTT,
1% glycerol, 1 M NaCl). Yield and purity was examined by SDS-PAGE
and, if necessary, PTP-1B was further purified by hydrophobic
interaction chromatography (HIC). Protein was supplemented with
ammonium sulfate until a final concentration of 1.4 M was reached.
The protein solution was filtered and loaded onto an HIC column at
4 ml/min in Buffer A2: 25 mM Tris pH 7.5, 1 mM EDTA, 1.4 M
(NH.sub.4).sub.2SO.sub.4, 1 mM DTT. Protein was eluted with a
gradient of 0-100% Buffer B over 30 minutes (Buffer B2: 25 mM Tris
pH 7.5, 1 mM EDTA, 1 mM DTT, 1% glycerol). Finally, the purified
protein was dialyzed at 4.degree. C. into the appropriate assay
buffer (25 mM Tris pH 8, 100 mM NaCl, 5 mM EDTA, 1 mM DTT, 1%
glycerol). Yields varied from mutant to mutant but typically were
within the range of 3-20 mg/L culture.
EXAMPLE 3
[0146] This example describes one illustrative method for
determining the IC.sub.50 of the compounds of the present invention
against PTP-1B. Substrate, pNPP (Sigma), was dissolved at 4 mM in
1.times.HN buffer (50 mM HEPES pH 7.0; 100 mM NaCl; 1 mM DTT) and
83 ul was mixed with 2 ul DMSO or 2 ul compound in DMSO. The
reaction was started by addition of PTP-1B (750 .eta.g in standard
assay conditions) in 15 .mu.l 1.times.HN buffer. The rate of
product formation (OD405 nm minus OD655 nm, BioRad Benchmark or
Molecular Devices Spectramax 190) was measured every 30 seconds for
15 minutes at 25 degrees C., and data were analyzed by linear
regression. For endpoint assays, the reaction was stopped after 15
min. with 50 .mu.l 3M NaOH and OD405 nm-OD655 nm was measured. For
IC.sub.50 determination, rates normalized relative to uninhibited
controls were plotted against, compound concentration and fitted
using a 4 parameter non-linear regression curve fit
(y=[(A-D)/(1+{x/C}{circumflex over ( )}B)]+D, Spectramax Software
package).
EXAMPLE 4
[0147] This example describes some of the methods that were used to
validate that the compounds of the present invention bind to PTP-1B
at the exosite region.
[0148] The compounds of the present invention, typified by compound
5 (IC.sub.50=30 .mu.M), behave as if PTP-1B had an allosteric site.
When the reaction velocity is plotted against substrate
concentration (PNPP) in the presence of varying concentrations of
compound 5, a sigmoidal dependence is shown instead of the
hyperbolic plot predicted by Michaelis-Menton. Moreover, as typical
with allosteric inhibitors and as shown by FIG. 8, there is a
decrease in V.sub.max with increasing inhibitor concentration but
with no significant effect on K.sub.m.
[0149] To the assess whether the truncation of PTP-1B affected the
results, enzymatic studies were performed on both the 298 residue
version and the 403 amino acid version which only lacks the
terminal hydrophobic 35 amino acids. Both forms of PTP-1B showed
similar results although the potency of the exosite inhibitors
against the 403 amino -acid form of PTP-1B tended to be slightly
more potent.
[0150] To assess whether the inhibition of PTP-1B with the exosite
compounds were due to some artifact such as the formation of a
covalent complex, protein denaturation, aggregation or
precipitation, a time dependence experiment was performed.
Increasing incubation time from 5 minutes to over an hour had no
impact on inhibition. Direct binding kinetics showed a fast on-rate
and a relatively fast off-rate. In addition, biacore data showed
that compound 5 bound to PTP-1B with a 1:1 binding stoichiometry.
This was further substantiated by a protein titration experiment in
which the concentration of PTP-1B was increased from 210 nM to 21
.mu.M by addition of catalytically inactive PTP-1B mutations (C215S
or D181C versions of SEQ ID NO. 1). In the presence of 21 .mu.M of
PTP-1B mutants, the lC.sub.50 of compound 5 was determined to be 77
.mu.M and 81 .mu.M respectively, which was consistent with the
IC.sub.50 using IR peptide substrate (71 .mu.M) and the biacore
K.sub.d (75 .mu.M). In addition, a crystal structure of PTP-1B
bound to compound 5 was solved showing the compound 5 bound to the
exosite region.
EXAMPLE 5
[0151] This example describes an illustrative assay for quantifying
the degree of insulin receptor phosphorylation in cells. Any
suitable cells can be used. In this example, CHO--IR cells were
grown in DMEM w/10% fetal bovine serum supplemented with penicillin
and streptomycin. Cells were plated at a density of 40,000 cells
per well in a 96 well plate. The following day, the medium was
changed to DMEM without serum and the cells were serum starved for
16 hours. 1 hour prior to harvesting, varying concentrations of a
test compound diluted in DMBM were added to the cells. Where
indicated, human insulin was added to cells 10 minutes prior to
harvesting. Cell were lysed in 30 mM HEPES, 150 mM NaCl, 1% triton
X-100, and 0.02% sodium azide. Insulin receptor phosphorylation was
quantitated in an ELISA assay as described previously (Jongsoon
Lee). FIG. 9 shows the dose response curve for compound 5. As it
can be seen, the level of insulin receptor phosphorylation is a
function of the concentration of compound 5.
EXAMPLE 6
[0152] This example describes a typical western blot experiment to
assess the specificity of the compounds of the present invention to
inhibit PTP-1B as opposed to other phosphatases. In this example,
CHO--IR cells were used. CHO--IR cells were grown in complete
medium as stated above in the cell-based assay. Cells were then
plated at a density of 300,000 cells per well in a 6-well plate.
The following day cells were serum starved for 16 hours. One hour
prior to lysis, cells were treated with either DMSO, 2 mM SP3892,
or 25 uM pervanadate. Ten minutes prior to lysis, 1 uM human
insulin was added to the wells indicated. Cells were lysed with the
same buffer stated above in the cell-based assay with the addition
of 0.1% SDS. Following lysis, total cell lysate was loaded onto a
4-12% Bis-Tris gel, and proteins were then transferred onto a PVDF
membrane. The membrane was blocked in 5% milk/TBST for 1 hour at
room temperature and then incubated with an anti-IR/IGF-1R [pYpY
1162/1163] primary antibody overnight. The following day, the
membrane was washed in TBST and incubated with a goat-anti-rabbit
secondary antibody for 1 hour at room temperature followed by ECL
development. FIG. 10 shows a western blot for compound 5. The 95
kDa band corresponds to the insulin receptor. DMSO is the negative
control; vanadate (the lane marked "Van"), a nonspecific
phosphatase inhibitor, is the positive control; "3892" corresponds
to compound 5; and "Ins" corresponds to insulin.
EXAMPLE 7
[0153] This example describes additional screening methods for
potential exosite inhibitors. These assays are used to distinguish
between true exosite inhibitors from those compounds that inhibit
through some non-specific interaction.
[0154] In one method, a candidate for an exosite inhibitor is
tested using a range of enzyme concentrations that is capable of
allosteric regulation (referred herein as the catalytically
competent enzyme). If the compound is inhibiting solely through the
allosteric mechanism, then the observed inhibition should be
predictable. For example, the fraction of saturable inhibition or
the theoretical inhibition of an enzyme can be calculated as
follows:
f[Ki+E(tot)+I(tot)-sqrt(((Ki+E(tot)+I(tot)){circumflex over (
)}2)-4* E(tot)*I(tot))]/(2*E(tot))
[0155] where:
[0156] f is the fraction saturable inhibition;
[0157] Ki is the inhibition constant or IC.sub.50;
[0158] E(tot) is the concentration of catalytically competent
enzyme; and
[0159] I(tot) is the inhibitor concentration.
[0160] For example, if IC.sub.50 of an allosteric inhibitor is 3
.mu.M, the inhibitor concentration is 3 .mu.M, and the
concentration of catalytically competent enzyme is 0.01 .mu.M, then
the theoretical inhibition is 49.94%. If the concentration of
catalytically competent enzyme is increased to 1.29 .mu.M, under
the same conditions, then the theoretical inhibition is 44.69%.
Thus, if the candidate for an exosite inhibitor is a true
allosteric inhibitor, than increasing the enzyme concentration from
0.01 .mu.M to 1.29 .mu.M will not result in a significant change in
the observed inhibition. Thus, if a dramatic change were observed
when the enzyme concentration is varied from 0.01 .mu.M and 1.29
.mu.M, than the candidate compound is not an exosite inhibitor but
is likely to be inhibiting the enzyme through a non-specific
interaction.
[0161] In another method, the non-specific interaction is
eliminated by assaying the candidate compound against the same
concentration of enzyme that is capable of allosteric regulation
but in the presence of increasing concentrations of inactivated
enzyme, typically between 0.25 and 1 times the putative IC.sub.50
of the candidate exosite inhibitor. For example, an inactivated
PTP-1B or TC-PCP is one that is no longer capable of
dephosphorylating a tyrosine. In this manner, the expected
inhibition for an exosite inhibitor remains constant despite the
increasing overall protein concentration. An illustrative example
of an inactivated PTP-1B and TC-PCP is where the active site
cysteine (Cys215) of each respective phosphatase is mutated to a
serine. Other methods for eliminating compounds that inhibit in a
non-specific manner also can be used such as those described by
McGovern et al., J. Med. Chem., 45: 1712-1722 (2002) which is
incorporated herein by reference.
EXAMPLE 8
[0162] This example describes the synthesis of the following
compound 4
[0163] which was prepared according to Scheme A and the protocol
below. 5
[0164] Compound 1. Benzbromarone (compound 1) was purchase from the
Sigma-Aldrich chemical company.
[0165] Compound 2. A heterogeneous mixture of 1 (26.8 g, 60.0
mmol), K.sub.2CO.sub.3 (33.5 g, 240 mmol), and methyl iodide (12.9
g, 90.0 mmol) in acetone (200 mL) was heated to reflux for 16
hours. Ethyl acetate (300 mL) was added, and the mixture was
extracted sequentially with water (200 mL), 2.5 M NaOH (2.times.100
mL), and brine (100 mL). The organic portion was dried over
Na.sub.2SO.sub.4 and concentrated to give 26.3 g (100 % yield) of
the title compound as a white solid. .sup.1H NMR (CDCl.sub.3):
.delta. 1.36 (t, J=7.5 Hz, 3 H), 2.90 (q, J=7.5 Hz, 2 H), 3.98 (s,
3 H), 7.24 (app t, J=7.4 Hz, 1 H), 7.32 (app t, J=7.2 Hz, 1 H),
7.42 (d, J=7.2 Hz, 1 H), 7.50 (d, J=7.9 Hz, 1 H), 7.99 (s, 2 H).
LRMS: ion 438 (M+).
[0166] Compound 3. A clear, pale yellow solution of 2 (5.28 g, 12.0
mmol) in anhydrous dioxane (12 mL) was cooled to 0.degree. C. and
treated dropwise with neat chlorosulfonic acid (42.4 g, 360 mmol)
over two hours with vigorous mixing. The reaction mixture was
carefully transferred dropwise into a rapidly stirred slurry of 1 M
HCl (200 mL) and crushed ice (600 mL). The cloudy mixture was
extracted with ether (2.times.300 mL). The combined organic
extracts were dried over Na.sub.2SO.sub.4 and concentrated to give
a viscous oil. Flash column chromatography (96:4 hexane/ethyl
acetate)afforded 3.10 g (48% yield) of the title compound as a
white solid. .sup.1H NMR (CDCl.sub.3): .delta. 1.41 (t, J=7.4 Hz, 3
H), 2.95 (q, J=7.4 Hz, 2 H), 4.00 (s, 3 H), 7.70 (d, J=8.5 Hz, 1
H), 7.96 (d, J=8.5 Hz, 1 H), 7.97 (s, 2 H), 8.22 (s, 1 H). LRMS:
ion 536 (M+).
[0167] Compound 4. To a stirring mixture of
4-amino-benzenesulfonamide (0.25 g, 1.45 mmoL), anhydrous pyridine
(0.3 mL, 3.80 mmoL) and DMAP ("dimethylaminopyridine") (0.006 g,
0.049 mmoL) in 10 mL of anhydrous THF ("tetrahydrofuran") was added
6-chlorosulfonyl benzbromarone methyl ether (compound 3) (0.5 g,
0.938 mmoL) in 5 mL of THF via syringe dropwise. After stirred at
room temperature for 6 h, the reaction mixture was diluted with
ethyl acetate (200 mL). The organic layer was washed with 1.0 N HCl
aq. solution (2.times.60 mL) and brine, dried over
Na.sub.2SO.sub.4, and concentrated under reduced pressure. The
crude product was purified on a silica gel flash column (10-20%
EtOAc in Hexane as eluent ) to give 0.412 g (0.615 mmoL, 65.6%) as
a light yellow solid.
[0168] Compound 5. To a stirring solution of compound 4 in 15 mL of
anhydrous DCE at -78.degree. C. was added 1.0 M BBr.sub.3 in DCM
(3.1 mL, 3.08 mmoL) slowly. After completion of addition, the
resulting mixture was allowed warm up to room temperature and stir
for 4 h. The reaction was quenched with 1.0 N HCl aq. solution. The
reaction mixture was extracted with DCM (2.times.100 mL), the
combined organic layer was washed with brine, dried over
Na.sub.2SO.sub.4, concentrated under reduced pressure. The residue
was purified on preparative HPLC to give 0.192 g (0.293 mmoL,
47.6%) as a white solid. .sup.1H NMR (DMSO-d.sub.6, 400 MHz)
.delta. 8.03 (s, 1H), .delta. 7.89 (s, 2H), .delta. 7.71 (d,
J=8.61, 3H), .delta. 7.53 (d, J=8.37, 1H), .delta. 7.26 (d, J=8.67,
2H), .delta. 2.85 (q, J=7.52, 2H), .delta. 1.31 (t, J=7.50, 3H); MS
(API-ES.sup.+) at m/z 656, 658, 660.
EXAMPLE 9
[0169] This example describes the synthesis of compounds of the
structure 6
[0170] where Et is ethyl and Ar.sup.1 is unsubstituted aryl or
substituted aryl (where the term aryl includes heteroaryl). These
compounds were made according to Example 8 except that
Ar.sup.1NH.sub.2 was used instead of 4-amino-benzenesulfonamide.
Illustrative examples of specific embodiments of Ar.sup.1NH.sub.2
and their resulting products are shown in Table 1.
7TABLE 1 Ar.sup.1NH.sub.2 Product 7 8 9 10 11 12 13 14 15 16 17 18
19 20
EXAMPLE 10
[0171] This example describes the synthesis of the following
compound 21
[0172] which was made according to Scheme B and the protocol below.
22
[0173] Compound 6. To a mixture of benzbromarone methyl ether (0.3
g, 0.696 mmoL) and hexamethylenetetramine ("HMET") (0.483 g, 3.45
mmoL) was added slowly 20 mL of TFA ("trifluoracetic acid"). The
reaction mixture was heated at 80.degree. C. for 48 hours. The
reaction mixture was cooled to room temperature, pooled to 60 mL of
ice-water, and strongly stirred. The mixture was extracted with
EtoAc (3.times.80 mL), and the combined organic solution was washed
with water and brine, dried over Na.sub.2SO.sub.4, filtered and
concentrated. Flash chromatography over silica gel (10% ethyl
acetate in hexanes) provided the desired aldehyde as light yellow
oil (0.180 g, 55.7%). .sup.1H NMR (CDCl.sub.3, 400 MHz) .delta.
10.2 (s, 1H), .delta. 7.98 (s, 1H), .delta. 7.94 (s, 2H), .delta.
7.76 (d, J=8.13 Hz, 1H), .delta. 7.54 (d, J=8.09 Hz, 1H), .delta.
3.94 (s, 3H), .delta. 2.92 (q, J=7.50, 7.52, 7.52 Hz, 2H), 61.35
(t, J=7.50, 7.52 Hz, 3H); MS (API-ES.sup.+) at m/z 463, 465,
467.
[0174] Compound 7. Compound 6 (0.100 g, 0.216 mmoL) was dissolved
in THF/DCM (1:1). Propylamine (0.047 mL, 0.648 mmoL) was added,
followed by addition of catalytic amount of concentrated acetic
acid. After stirring for 5 minutes, the sodium
triacetoxyboronhydride (0.090 g, 0.432 mmoL) was added in one
portion at room temperature. The reaction mixture was stirred for 2
hours and then the solvent portion of the reaction mixture was
removed under reduced pressure. The residue was partitioned between
50 mL of EtOAc and 50 mL of saturated NaHCO.sub.3 aqueous solution.
The aqueous solution was extracted with EtOAc (2.times.30 mL), and
the combined organic solution was washed with brine, dried over
Na.sub.2SO.sub.4, filtered, concentrated. The crude product was
used for next step without purification. .sup.1H NMR,
(DMSO-d.sub.6, 400 MHz) .delta. 8.00 (s, 2H), .delta. 7.80(s, 1H),
.delta. 7.45 (d, J=8.13 Hz, 1H), .delta. 7.38 (d, J=8.09 Hz, 1H),
.delta. 4.25 (s, 2H), .delta. 3.89 (s, 3H), .delta. 2.87 (m, 4H),
.delta. 1.62 (m, 2H), .delta. 1.25 (t, J=7.47, 7.40 Hz, 3H),
.delta. 0.87 (t, J=7.48, 7.45 Hz, 3H); MS (API-ES.sup.+) at m/z
508, 510, 512.
[0175] Compound 8. To a stirring mixture of compound 7 (0.050 g,
0.0986 mmoL), triethylamine (0.110 mL, 0.788 mmoL), and DMAP (0.006
g, 0.049 mmoL) in 2 mL of anhydrous THF was added dropwise by
syringe 4-methanesulfonyl-benzenesulfonyl chloride (0.075 g, 0.295
mmoL) in 1 mL of THF. After stirring at room temperature for 3
hours, the reaction mixture was diluted with EtOAc (30 mL). The
organic solution was washed with 1.0 N HCl aqueous solution
(1.times.20 mL), water and brine, dried over Na.sub.2SO.sub.4, and
concentrated. The crude product was purified on a silica gel flash
column (20% EtOAc in Hexane) to give 0.061 g (0.083 mmoL, 81.1%) as
a light yellow solid. MS (API-ES.sup.+) at m/z 725, 727, 729.
[0176] Compound 9. To a stirring solution of compound 8 (0.061 g,
0.083 mmol) in 5 mL of anhydrous DCE at -78.degree. C. was added
slowly 1.0 M BBr.sub.3 in DCM (0.250 mL, 0.250 mmoL). The resulting
mixture was allowed to warm up to room temperature and was stirred
for 4 hours. The reaction was quenched with 1.0 N HCl aqueous
solution. The resulting reaction mixture was extracted with DCM
(2.times.30 mL), and the combined organic solution was washed with
brine, dried over Na.sub.2SO.sub.4, and concentrated. The residue
was purified on preparative HPLC to give 0.030 g (0.043 mmoL,
51.8%) as awhite solid. .sup.1H NMR (DMSO-d.sub.6, 400 MHz) d 8.13
(s, 4H), .delta. 7.91 (s, 2H), .delta. 7.60(s, 1H), .delta. 7.45
(d, J=8.13 Hz, 1H), .delta. 7.25 (d, J=8.09 Hz, 1H), .delta. 4.50
(s, 2H), .delta. 3.55 (s, 3H), .delta. 3.18 (m, 2H), .delta. 2.75
(m, 2H), .delta.1.25 (m, 5H), .delta. 0.63 (t, J=7.27, 7.36 Hz,
3H); MS (API-ES.sup.+) at m/z 713, 715, 717.
EXAMPLE 11
[0177] This example describes the synthesis of compounds of the
structure 23
[0178] where Et is ethyl and Ar.sup.2 is unsubstituted aryl or
substituted aryl (where the term aryl includes heteroaryl). These
compounds were made according to Example 10 except that
Ar.sup.2SO.sub.2Cl was used instead of
4-methanesulfonyl-benzenesulfonyl chloride. Illustrative examples
of specific embodiments of Ar.sup.2SO.sub.2Cl and their resulting
products are shown in Table 2.
8TABLE 2 Ar.sup.2SO.sub.2Cl Product 24 25 26 27
[0179] All references cited throughout the specification are hereby
expressly incorporated herein by reference.
[0180] While the present invention has been described with
reference to the specific embodiments thereof, it would be
understood by those skilled in the art that various changes may be
made and equivalents may be substituted without departing from the
true spirit and scope of the invention. In addition, many
modifications may be made to adapt a particular situation,
material, composition of matter, process, process step or steps, to
the objection, spirit and scope of the present invention. All such
modifications are intended to be within the scope of the claims
appended hereto.
Sequence CWU 1
1
29 1 298 PRT Homo sapiens 1 Met Glu Met Glu Lys Glu Phe Glu Gln Ile
Asp Lys Ser Gly Ser Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg
His Glu Ala Ser Asp Phe Pro Cys 20 25 30 Arg Val Ala Lys Leu Pro
Lys Asn Lys Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe
Asp His Ser Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr
Ile Asn Ala Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80
Tyr Ile Leu Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85
90 95 Glu Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn
Arg 100 105 110 Val Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp
Pro Gln Lys 115 120 125 Glu Glu Lys Glu Met Ile Phe Glu Asp Thr Asn
Leu Lys Leu Thr Leu 130 135 140 Ile Ser Glu Asp Ile Lys Ser Tyr Tyr
Thr Val Arg Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Thr Thr Gln
Glu Thr Arg Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro
Asp Phe Gly Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe
Leu Phe Lys Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195 200 205
Gly Pro Val Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210
215 220 Phe Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys
Asp 225 230 235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu
Met Arg Lys Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln
Leu Arg Phe Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe
Ile Met Gly Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu
Ser His Glu Asp 290 295 2 296 PRT Homo sapiens 2 Met Pro Thr Thr
Ile Glu Arg Glu Phe Glu Glu Leu Asp Thr Gln Arg 1 5 10 15 Arg Trp
Gln Pro Leu Tyr Leu Glu Ile Arg Asn Glu Ser His Asp Tyr 20 25 30
Pro His Arg Val Ala Lys Phe Pro Glu Asn Arg Asn Arg Asn Arg Tyr 35
40 45 Arg Asp Val Ser Pro Tyr Asp His Ser Arg Val Lys Leu Gln Asn
Ala 50 55 60 Glu Asn Asp Tyr Ile Asn Ala Ser Leu Val Asp Ile Glu
Glu Ala Gln 65 70 75 80 Arg Ser Tyr Ile Leu Thr Gln Gly Pro Leu Pro
Asn Thr Cys Cys His 85 90 95 Phe Trp Leu Met Val Trp Gln Gln Lys
Thr Lys Ala Val Val Met Leu 100 105 110 Asn Arg Ile Val Glu Lys Glu
Ser Val Lys Cys Ala Gln Tyr Trp Pro 115 120 125 Thr Asp Asp Gln Glu
Met Leu Phe Lys Glu Thr Gly Phe Ser Val Lys 130 135 140 Leu Leu Ser
Glu Asp Val Lys Ser Tyr Tyr Thr Val His Leu Leu Gln 145 150 155 160
Leu Glu Asn Ile Asn Ser Gly Glu Thr Arg Thr Ile Ser His Phe His 165
170 175 Tyr Thr Thr Trp Pro Asp Phe Gly Val Pro Glu Ser Pro Ala Ser
Phe 180 185 190 Leu Asn Phe Leu Phe Lys Val Arg Glu Ser Gly Ser Leu
Asn Pro Asp 195 200 205 His Gly Pro Ala Val Ile His Cys Ser Ala Gly
Ile Gly Arg Ser Gly 210 215 220 Thr Phe Ser Leu Val Asp Thr Cys Leu
Val Leu Met Glu Lys Gly Asp 225 230 235 240 Asp Ile Asn Ile Lys Gln
Val Leu Leu Asn Met Arg Lys Tyr Arg Met 245 250 255 Gly Leu Ile Gln
Thr Pro Asp Gln Leu Arg Phe Ser Tyr Met Ala Ile 260 265 270 Ile Glu
Gly Ala Lys Cys Ile Lys Gly Asp Ser Ser Ile Gln Lys Arg 275 280 285
Trp Lys Glu Leu Ser Lys Glu Asp 290 295 3 296 PRT Homo sapiens 3
Pro Ile Thr Asp Leu Ala Asp Asn Ile Glu Arg Leu Lys Ala Asn Asp 1 5
10 15 Gly Leu Lys Phe Ser Gln Glu Tyr Glu Ser Ile Asp Pro Gly Gln
Gln 20 25 30 Phe Thr Trp Glu Asn Ser Asn Leu Glu Val Asn Lys Pro
Lys Asn Arg 35 40 45 Tyr Ala Asn Val Ile Ala Tyr Asp His Ser Arg
Val Ile Leu Thr Ser 50 55 60 Ile Asp Gly Val Pro Gly Ser Asp Tyr
Ile Asn Ala Asn Tyr Ile Asp 65 70 75 80 Gly Tyr Arg Lys Gln Asn Ala
Tyr Ile Ala Thr Gln Gly Pro Leu Pro 85 90 95 Glu Thr Met Gly Asp
Phe Trp Arg Met Val Trp Glu Gln Arg Thr Ala 100 105 110 Thr Val Val
Met Met Thr Arg Leu Glu Glu Lys Ser Arg Val Lys Cys 115 120 125 Asp
Gln Tyr Trp Pro Ala Arg Gly Thr Glu Thr Cys Gly Leu Ile Gln 130 135
140 Val Thr Leu Leu Asp Thr Val Glu Leu Ala Thr Tyr Thr Val Arg Thr
145 150 155 160 Phe Ala Leu His Lys Ser Gly Ser Ser Glu Lys Arg Glu
Leu Arg Gln 165 170 175 Phe Gln Phe Met Ala Trp Pro Asp His Gly Val
Pro Glu Tyr Pro Thr 180 185 190 Pro Ile Leu Ala Phe Leu Arg Arg Val
Lys Ala Cys Asn Pro Leu Asp 195 200 205 Ala Gly Pro Met Val Val His
Cys Ser Ala Gly Val Gly Arg Thr Gly 210 215 220 Cys Phe Ile Val Ile
Asp Ala Met Leu Glu Arg Met Lys His Glu Lys 225 230 235 240 Thr Val
Asp Ile Tyr Gly His Val Thr Cys Met Arg Ser Gln Arg Asn 245 250 255
Tyr Met Val Gln Thr Glu Asp Gln Tyr Val Phe Ile His Glu Ala Leu 260
265 270 Leu Glu Ala Ala Thr Cys Gly His Thr Glu Val Pro Ala Arg Asn
Leu 275 280 285 Tyr Ala His Ile Gln Lys Leu Gly 290 295 4 320 PRT
Homo sapiens 4 Met Glu Met Glu Lys Glu Phe Glu Gln Ile Asp Lys Ser
Gly Ser Trp 1 5 10 15 Ala Ala Ile Tyr Gln Asp Ile Arg His Glu Ala
Ser Asp Phe Pro Cys 20 25 30 Arg Val Ala Lys Leu Pro Lys Asn Lys
Asn Arg Asn Arg Tyr Arg Asp 35 40 45 Val Ser Pro Phe Asp His Ser
Arg Ile Lys Leu His Gln Glu Asp Asn 50 55 60 Asp Tyr Ile Asn Ala
Ser Leu Ile Lys Met Glu Glu Ala Gln Arg Ser 65 70 75 80 Tyr Ile Leu
Thr Gln Gly Pro Leu Pro Asn Thr Cys Gly His Phe Trp 85 90 95 Glu
Met Val Trp Glu Gln Lys Ser Arg Gly Val Val Met Leu Asn Arg 100 105
110 Val Met Glu Lys Gly Ser Leu Lys Cys Ala Gln Tyr Trp Pro Gln Lys
115 120 125 Glu Glu Lys Glu Met Ile Phe Glu Asp Thr Asn Leu Lys Leu
Thr Leu 130 135 140 Ile Ser Glu Asp Ile Lys Ser Tyr Tyr Thr Val Arg
Gln Leu Glu Leu 145 150 155 160 Glu Asn Leu Thr Thr Gln Glu Thr Arg
Glu Ile Leu His Phe His Tyr 165 170 175 Thr Thr Trp Pro Asp Phe Gly
Val Pro Glu Ser Pro Ala Ser Phe Leu 180 185 190 Asn Phe Leu Phe Lys
Val Arg Glu Ser Gly Ser Leu Ser Pro Glu His 195 200 205 Gly Pro Val
Val Val His Cys Ser Ala Gly Ile Gly Arg Ser Gly Thr 210 215 220 Phe
Cys Leu Ala Asp Thr Cys Leu Leu Leu Met Asp Lys Arg Lys Asp 225 230
235 240 Pro Ser Ser Val Asp Ile Lys Lys Val Leu Leu Glu Met Arg Lys
Phe 245 250 255 Arg Met Gly Leu Ile Gln Thr Ala Asp Gln Leu Arg Phe
Ser Tyr Leu 260 265 270 Ala Val Ile Glu Gly Ala Lys Phe Ile Met Gly
Asp Ser Ser Val Gln 275 280 285 Asp Gln Trp Lys Glu Leu Ser His Glu
Asp Leu Glu Pro Pro Pro Glu 290 295 300 His Ile Pro Pro Pro Pro Arg
Pro Pro Lys Arg Ile Leu Glu Pro His 305 310 315 320 5 29 DNA Homo
sapiens 5 gccatatgga gatggaaaag gagttcgag 29 6 40 DNA Homo sapiens
6 gcgacgcgaa ttcttaattg tgtggctcca ggattcgttt 40 7 34 DNA Homo
sapiens 7 tgccggaatt ccttagtcct cgtgggaaag ctcc 34 8 27 DNA Homo
sapiens 8 ttcttggcgt ttcttcgcaa agtccga 27 9 27 DNA Homo sapiens 9
gactttgcga agaaacgcca agaatga 27 10 21 DNA Homo sapiens 10
ggtgccaaat gcatcatggg g 21 11 21 DNA Homo sapiens 11 ccccatgatg
catttggcac c 21 12 34 DNA Homo sapiens 12 tgccggaatt ccttagtcct
cgtgcgaaag ctcc 34 13 31 DNA Homo sapiens 13 gaatgaggct ggtgagcaag
ggactccaaa g 31 14 27 DNA Homo sapiens 14 gaatgaggct gggcattcag
ggactcc 27 15 27 DNA Homo sapiens 15 gttcaagaat gagcatggtg attcagg
27 16 29 DNA Homo sapiens 16 ctgactctcg gacgcagaaa agaaagttc 29 17
30 DNA Homo sapiens 17 gagtgaccct gagcatcgga ctttgaaaag 30 18 27
DNA Homo sapiens 18 gatgcctgca ctggagtgca ccacaac 27 19 27 DNA Homo
sapiens 19 ctggatcagc ccacaccgaa acttcct 27 20 27 DNA Homo sapiens
20 ctggtcggct gtacagatca gccccat 27 21 28 DNA Homo sapiens 21
cttcgatcac agcgcagtag gagaagcg 28 22 27 DNA Homo sapiens 22
gaatttggca ccgcagatca cagccag 27 23 27 DNA Homo sapiens 23
agagtccccc atgcagaatt tggcacc 27 24 27 DNA Homo sapiens 24
ccactgatcc tggcaggaag agtcccc 27 25 27 DNA Homo sapiens 25
ccaaaagtga ccggctgtgt taggcaa 27 26 27 DNA Homo sapiens 26
ccagtattgt gcggctttta acgaacc 27 27 27 DNA Homo sapiens 27
ccagtattgt gcgcttttta acgaacc 27 28 17 DNA Homo sapiens 28
aatacgactc actatag 17 29 20 DNA Homo sapiens 29 tagttattgc
tcagcggtgg 20
* * * * *