Structure-based design and discovery of protein tyrosine phosphatase inhibitors incorporating novel isothiazolidinone heterocyclic phosphotyrosine mimetics

Article abstract of DOI:10.1021/jm0504555

Structure-based design led to the discovery of novel (S)-isothiazolidinone ((S)-IZD) heterocyclic phosphotyrosine (pTyr) mimetics that when incorporated into dipeptides are exceptionally potent, competitive, and reversible inhibitors of protein tyrosine phosphatase 1B (PTP1B). The crystal structure of PTP1B in complex with our most potent inhibitor 12 revealed that the (S)-IZD heterocycle interacts extensively with the phosphate binding loop precisely as designed in silico. Our data provide strong evidence that the (S)-IZD is the most potent pTyr mimetic reported to date.

Full text of DOI:10.1021/jm0504555

6544
J. Med. Chem. 2005, 48, 6544-6548
Letters
strong binding have limited the cell permeability and
oral bioavailability of inhibitors that contain them.
Structure-Based Design and Discovery of
Protein Tyrosine Phosphatase Inhibitors
Incorporating Novel Isothiazolidinone
Heterocyclic Phosphotyrosine Mimetics
Protein tyrosine phosphatase 1B (PTP1B) is a para-
digm for protein phosphatase research and drug dis-
covery. PTP1B was the first PTP purified3,4 in the 1980s
and has been extensively studied within academia and
the pharmaceutical industry. It is regarded by many as
one of the best validated drug targets for intervention
in type 2 diabetes and obesity. PTP1B knockout and
antisense studies have shown lower blood glucose levels
and improved insulin responsiveness in normal and
diabetic mice through enhanced IR signaling in periph-
eral tissues.5-7 These mice are also protected from high-
fat-diet-induced obesity. Disappointingly, nearly all
medicinal chemistry efforts to date have been severely
hindered because of the lack of suitable druglike lead
compounds for optimization to drug candidates. High-
throughput screening efforts have been ineffective,
typically yielding numerous very weak noncompetitive
inhibitors presumably due to the oxidation or alkylation
of the active site Cys2158 or to compound aggregation
effects.9 In contrast, the designed inhibitors incorporat-
ing pTyr mimetics have provided potent competitive
PTP inhibitors. However, none of these compounds
containing highly charged nonhydrolyzable phosphonate
or carboxylic acid pTyr mimetics have shown significant
cell membrane permeability and oral bioavailability. In
our initial efforts to identify lead compounds that
address these key issues, we utilized structure-based
design methods to derive novel heterocyclic pTyr mi-
metics that strongly bind and effectively replace the
undesired highly charged phosphonate and carboxylic
acid moieties in known inhibitors of PTP1B.
Our efforts toward the design of PTP1B inhibitors
started with a comprehensive review of the X-ray crystal
structures of PTP1B/inhibitor complexes available in the
Protein Data Bank (PDB). As is well documented in the
literature, nearly all competitive inhibitors of PTP1B
derive the majority of their binding energy through
multiple hydrogen-bonding interactions with the cata-
lytic residues in the deep tyrosine phosphate binding
pocket. The most potent of these PTP1B inhibitors
incorporate highly charged anions that mimic the pTyr
substrate I, such as difluoromethylphosphonates (DFMP)
II, carboxymethylsalicyclic acids (CMS) III, and oxaly-
laminobenzoic acids (OBA) IV that strongly bind the
highly polarized pTyr binding site residues10 (Figure 1).
Andrew P. Combs,* Eddy W. Yue, Michael Bower,
Paul J. Ala, Brian Wayland, Brent Douty,
Amy Takvorian, Padmaja Polam, Zelda Wasserman,
Wenyu Zhu, Matthew L. Crawley, James Pruitt,
Richard Sparks, Brian Glass, Dilip Modi,
Erin McLaughlin, Lori Bostrom, Mei Li,
Laurine Galya, Karl Blom, Milton Hillman,
Lucie Gonneville, Brian G. Reid, Min Wei,
Mary Becker-Pasha, Ronald Klabe, Reid Huber,
Yanlong Li, Gregory Hollis, Timothy C. Burn,
Richard Wynn, Phillip Liu, and Brian Metcalf
Discovery Chemistry, Incyte Corporation,
Experimental Station, E336/132A, Route 141 and
Henry Clay Road, Wilmington, Delaware 19880
Received May 12, 2005
Abstract: Structure-based design led to the discovery of novel
(S)-isothiazolidinone ((S)-IZD) heterocyclic phosphotyrosine
(pTyr) mimetics that when incorporated into dipeptides are
exceptionally potent, competitive, and reversible inhibitors of
protein tyrosine phosphatase 1B (PTP1B). The crystal struc-
ture of PTP1B in complex with our most potent inhibitor 12
revealed that the (S)-IZD heterocycle interacts extensively with
the phosphate binding loop precisely as designed in silico. Our
data provide strong evidence that the (S)-IZD is the most
potent pTyr mimetic reported to date.
Protein tyrosine phosphatases (PTPs) and protein
tyrosine kinases (PTKs) in concert tightly control the
phosphorylation state of many proteins involved in signal
transduction pathways. The aberrant regulation of these
signaling pathways is known to be involved in numerous
disease states, including diabetes, cancer, and rheuma-
toid arthritis.1 The recent FDA approvals of PTK inhib-
itors, such as Iressa, Gleevec, and Tarceva, have dem-
onstrated that drug modulation of the phosphorylation
state of proteins involved in specific signal transduction
pathways is an effective means of treating disease.2 In
contrast to the success of PTK inhibitors, the discovery
of drug candidates for PTP drug targets has proven less
tractable. While kinases contain a deep hydrophobic
pocket in the ATP binding site suitable for discovery of
orally bioavailable small-molecule drugs, the active
closed form of the catalytic site of PTPs contain a highly
polar phosphotyrosine (pTyr) binding site contiguous to
a shallow peptide substrate binding region. To date, all
potent and competitive inhibitors of PTPs have incor-
porated various charged pTyr mimetics to achieve
strong binding to the phosphonate binding site. Unfor-
tunately, these same polar pTyr mimetics necessary for
Detailed analysis of the crystal structures of apoPTP1B
and PTP1B complexed with DFMP, CMS, and OBA
containing inhibitors revealed that each pTyr mimetic
selectively displaces one or two of the three tightly
bound water molecules in the tyrosine phosphate bind-
ing site. In the crystal structures of PTP1B with
inhibitors bearing a DFMP pTyr mimetic, the bis-anion-
ic phosphorus moiety is stabilized by binding to back-
bone NH groups of the loop (C215-R221), located at the
N-terminus of helix R4 (Figure 2a). Specifically, one
* To whom correspondence should be addressed. Phone: (302) 498-
6832. Fax: (302) 425-2708. E-mail: acombs@incyte.com.
10.1021/jm0504555 CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/23/2005

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6545
backbone of S216 and A217 and the side chain of R221
(position O2). Interestingly, in these structures the
binding site is larger than the substrate and retains a
water molecule at position O3, which is bound to the
backbone amide of F182 and the side chain of Q266, in
a lobe of the binding site directly beneath the phenyl
ring of F182. This water molecule is not believed to be
the water that hydrolyzes the phosphocysteinyl inter-
mediate in the second step of PTP1B catalysis11 but
instead appears to be involved in positioning and
bonding to the third terminal oxygen and the tyrosine
phenolic oxygen in the substrate. This water molecule
is present in crystal structures of PTP1B in complex
with vanadate, DFMP, and pTyr, suggesting that it is
tightly bound and integral to the structure of the
catalytic site. In contrast, crystal structures in complex
with compounds bearing the CMS III pTyr mimetic
show that the charged carboxylic acid displaces only the
water molecule at position O3 while maintaining the
two additional water molecules at positions O1 and O2
(Figure 2b). Thus, DFMP pTyr mimetics replace water
molecules at positions O1 and O2 while retaining O3,
whereas CMS pTyr mimetics replace water molecules
at position O3 while retaining O1 and O2.
Figure 1. Known pTyr mimetics shown in ionized form
expected at physiological pH.
Graphical overlay of the DFMP and CMS containing
inhibitors illustrated their complementary, yet distinctly
different modes of binding (Figure 3). Closer inspection
and chemical intuition suggested that five-membered
heterocycles V, such as the thiadiazolidinone (TDZ, VI)
and isothiazolidinones (IZD, VII and VIII), could ef-
fectively mimic the interactions of DFMP and CMS lig-
ands, thus displacing all three water molecules while
realizing all of the corresponding hydrogen bonds (Fig-
ure 4).12 We anticipated that these heterocycles would
have significantly increased binding affinity because the
entropic gain from displacing a single structural water
molecule has been estimated to be as high as 2 kcal/
mol.13 In addition, the rigid heterocycle has significantly
less conformational freedom than that of the acyclic
pTyr mimetics DFMP and CMS, thus reducing the
entropic cost of binding to the protein. Similar design
concepts had been utilized in the discovery of cyclic urea
HIV protease inhibitors where the urea oxygen was
designed to constrain the original acyclic inhibitor while
simultaneously replacing a highly conserved structural
water molecule in the protein.14 Since our initial re-
search, several companies have disclosed the discovery
of PTP1B inhibitors containing the TDZ heterocycle in
the patent literature.15-18 Just recently, a fragment-
based design of compounds containing TDZ pTyr mi-
Figure 2. Crystal structures of DFMP and CMS mimetics
bound in the PTP1B pTyr active site. The inhibitors are
depicted with gray carbon atoms, a purple phosphorus atom,
red oxygen atoms, and aqua fluorine atoms. The protein is
depicted with green carbon atoms, blue nitrogen atoms, and
red oxygen atoms, and the water molecule is depicted as a red
sphere. The three water molecule binding sites are labeled O1,
O2, and O3. Hydrogen bonds are shown as dashed yellow lines.
(a) Two water molecules (O1 and O2) have been replaced by
the DFMP oxygen atoms (PDB code 1N6W). (b) The water
molecule at O3 has been replaced by the CMS carboxylate
oxygen (PDB code 1G7G).
terminal oxygen atom is held in place by interactions
with the backbone amides of I219 and G220 (position
O1) and a second terminal oxygen is bound to the
Figure 3. Stepwise sequence of crystal structures used in the structure-based design of the unsaturated IZD heterocyclic pTyr
mimetic VII. The pTyr mimetics are depicted with green carbon atoms bound in the pTyr binding site depicted as a gray surface.
Red spheres indicate water molecules: (a) structure of a DFMP inhibitor and water molecule O3 bound to the active site of
PTP1B in structure 1N6W; (b) structure of a CMS inhibitor with water molecules in the O1 and O2 sites in the same reference
frame, from structure 1G7G;9 (c) overlay of the two structures, showing the genesis of the five-membered ring heterocycle; (d)
designed IZD heterocycle VII overlaying the two original structures; (e) our designed IZD heterocycle VII alone.

6546 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21
Letters
Table 1. Dipeptide Containing IZD Inhibitors of PTP1B
Figure 4. Designed heterocyclic pTyr mimetics shown in
ionized form expected at physiological pH.
Scheme 1. Synthesis of Dipeptides Containing IZD
Heterocyclesa
a PNPP enzyme assay. b Ki.
8. The heterocycle of common intermediate 9 was
reduced, isomers were separated, and each was further
elaborated to afford the diastereomers 11 and 12.
Unsaturated IZD heterocycle containing 8 and satu-
rated IZD heterocycle containing 11 and 12 were tested
for their enzymatic inhibitor activity toward PTP1B.
Gratifyingly, unsaturated IZD heterocyclic 8 and satu-
rated IZD heterocyclic 12 were potent inhibitors of
PTP1B with IC50 values of 3000 and 190 nM, respec-
tively (Table 1). Mode of inhibition studies unequivocally
established that both compounds were competitive and
reversible inhibitors of PTP1B (12, Ki ) 180 nM). Ab
initio calculations predicted that the (S)-IZD would be
the active isomer, based on comparison of calculated
conformations of (S)- and (R)-IZD heterocycle fragments
with the crystal structure of an inhibitor bearing the
unsaturated IZD (see Supporting Information). Indeed,
X-ray crystallography of the PTP1B/12 complex revealed
that the (S)-IZD isomer was active. Compound 12 was
also tested against the phosphatases TCPTP, SHP-1,
and SHP-2. As expected, 12 was shown to be an
equipotent inhibitor (IC50 ) 135 nM) of the highly
homologous (74% identity) phosphatase TCPTP while
maintaining complete selectivity (IC50 > 100 000 nM)
over the more distantly related PTPs.
As a comparator to other pTyr mimetics, an identical
dipeptide 13 containing a DFMP, arguably the most
potent pTyr mimetic reported to date, was synthesized
according to literature methods and assayed against
PTP1B. The (S)-IZD heterocyclic pTyr mimetic contain-
ing inhibitor 12 demonstrated an impressive 10-fold
more potent inhibition of PTP1B than the DFMP
derivative 13. As we predicted, the combined entropic
gains from eliminating the third structural water
molecule from the active site and the rigid conformation
of the (S)-IZD heterocyclic pTyr mimetic gave an
estimated 1.4 kcal/mol lowering in binding energy
compared to the DFMP.
a Reagents and conditions: (a) NH2CO3, (Boc2O), 97%; (b) 3,
Pd(dppf)2Cl2DCM, K2CO3, dioxane, 80 °C, 78%; (c) 4 M HCl/
dioxane, 98%; (d) BocPhe, EDC, HOAt, DIEA, DMF/DCM, 84%;
(e) H2, Pd/C, MeOH, 96%; (f) TFA/dioxane, (iPr)3SiH, 7 ) 65%, 10
) 79%; (g) AcOH, EDC, HOAt, DIEA, DMF/DCM, 8 ) 75%, 11/12
) 82%.
metics that inhibit PTP1B was published, albeit with
only micromolar activity.19
The isothiazolidinone (IZD) heterocycles (VII and
VIII) were chosen as our initial synthetic target because
the IZD heterocycle allows the two sulfonyl oxygens to
effectively mimic the oxygens of the DFMP inhibitor,
while the carbonyl mimics the CMS inhibitor carbonyl
and the ionized NH mimics the carboxylic anion or
DFMP anion. We postulated that non-peptide-contain-
ing inhibitors containing the highly delocalized anion
of the cyclic N-acylsulfonamide heterocycle containing
inhibitors may provide improved membrane perme-
ability compared to the hard charged phosphonates and
carboxylate anion containing inhibitors, since the analo-
gous acyclic acylsulfonamides are known cell-permeable
isosteres for carboxylic acids.20
Peptides containing the IZD heterocyclic pTyr mimet-
ics were synthesized in 10-11 linear steps from readily
available starting materials (Scheme 1). The key syn-
thetic reaction was a novel Suzuki coupling of chloro-
heterocycle 321,22 with 4-phenylalanineboronic acid 2 to
afford the fully protected scaffold 4. The N-terminus of
4 was subsequently elaborated via peptide couplings
and the heterocycle was deprotected to give inhibitor
The crystal structure of PTP1B in complex with 12
was solved to 2.0 Å resolution and confirmed that the
(S)-IZD heterocyclic pTyr mimetic binds to the active
closed form of the catalytic pocket in precisely the in
silico designed conformation (Figure 5). The (S)-IZD

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 21 6547
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Figure 5. Crystal structure of PTP1B/12 showing the (S)-
IZD heterocyclic pTyr mimetic VIII bound to the phosphate
binding loop of PTP1B. The protein is depicted in ribbon-and-
stick style, and the inhibitor is depicted in ball-and-stick style.
The (S)-IZD heterocycle makes all of the predicted polar
contacts and displaces all three active site water molecules.
Hydrogen bonds are indicated with dashed lines.
heterocycle binds at the center of the phosphate binding
loop (Cys215-Arg221), where each of the three oxygen
atoms of the (S)-IZD heterocycle displaces a structural
water molecule and forms the expected hydrogen-
bonding interactions with backbone amides and side
chain of Arg221. The aryl ring directly attached to the
(S)-IZD heterocycle participates in extensive van der
Waals interactions with Tyr46, Val49, Ile219, and F182
of the flap. The peptidic portion of 12 binds in an ex-
tended conformation, identical to other known peptide-
like structures bound to PTP1B (see Supporting Infor-
mation for full view of inhibitor 12 bound to PTP1B).
Novel heterocyclic pTyr mimetics were designed
through meticulous analysis and overlay of structural
information available in the PDB. A dipeptide contain-
ing the (S)-IZD pTyr mimetic was synthesized and
demonstrated competitive, reversible, and potent inhibi-
tion of PTP1B (12, Ki ) 180 nM). An X-ray crystal
structure of PTP1B/12 showed that the ligand bound
precisely as designed in silico. Furthermore, the (S)-IZD
pTyr mimetic was found to be a 10-fold more potent
inhibitor of PTP1B than an analogous peptidic com-
pound bearing a DFMP. These data support the conclu-
sion that the (S)-IZD heterocycle is the most potent pTyr
mimetic discovered to date. While the peptidic inhibitors
described herein are not cell-permeable or orally bio-
available, these diffusely anionic IZD heterocyclic pTyr
mimetics provide new opportunities for the discovery
of such PTP1B inhibitors when incorporated into suit-
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(19) Black, E.; Breed, J.; Breeze, A. L.; Embrey, K.; Garcia, R.; Gero,
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Acknowledgment. We thank Rakesh Kohli at the
University of Pennsylvania for providing HRMS data.
Supporting Information Available: Synthesis proce-
dures and analytical data for intermediates and final products
(1H NMR, 13C NMR, HRMS, HPLC), experimental procedures
for the biological assays, ab initio modeling, and crystal-
lographic methods and data. This material is available free of
charge via the Internet at http://pubs.acs.org.
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JM0504555