Fragment screening and assembly: A highly efficient approach to a selective and cell active protein tyrosine phosphatase 1B inhibitor

Article abstract of DOI:10.1021/jm034122o

Using an NMR-based fragment screening and X-ray crystal structure-based assembly, starting with millimolar ligands for both the catalytic site and the second phosphotyrosine binding site, we have identified a small-molecule inhibitor of protein tyrosine phosphatase 1B with low micromolar inhibition constant, high selectivity (30-fold) over the highly homologous T-cell protein tyrosine phosphatase, and good cellular activity in COS-7 cells.

Full text of DOI:10.1021/jm034122o

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232
J . Med. Chem. 2003, 46, 4232-4235
Letters
F r a gm en t Scr een in g a n d Assem bly: A
High ly Efficien t Ap p r oa ch to a Selective
a n d Cell Active P r otein Tyr osin e
P h osp h a ta se 1B In h ibitor
,
†
†
†
Gang Liu,* Zhili Xin, Zhonghua Pei,
‡
‡
Philip J . Hajduk, Cele Abad-Zapatero,
Charles W. Hutchins, Hongyu Zhao,
Thomas H. Lubben, Stephen J . Ballaron,
Deanna L. Haasch, Wiweka Kaszubska,
‡
†
†
†
†
†
†
†
F igu r e 1. Previously reported oxamic acid based selective
PTP1B inhibitor 1.
Cristina M. Rondinone, J ames M. Trevillyan, and
Michael R. J irousek†,§
Metabolic Disease Research and Advanced Technology,
Global Pharmaceutical Research and Development,
Abbott Laboratories, 100 Abbott Park Road,
Abbott Park, Illinois 60064-6098
ported, although their exact mechanisms of action are
questionable.
9
We envisioned that selective and cell-permeable PTP1B
inhibitors interacting with the catalytic site must
therefore possess smaller molecular size and no more
than one carboxylic acid. Targeting a second phospho-
tyrosine binding pocket (site 2) near the catalytic site
has been proposed as one possible strategy for gaining
Received J une 3, 2003
Abstr a ct: Using an NMR-based fragment screening and
X-ray crystal structure-based assembly, starting with milli-
molar ligands for both the catalytic site and the second
phosphotyrosine binding site, we have identified a small-
molecule inhibitor of protein tyrosine phosphatase 1B with low
micromolar inhibition constant, high selectivity (30-fold) over
the highly homologous T-cell protein tyrosine phosphatase, and
good cellular activity in COS-7 cells.
1
0
additional potency and selectivity. Such strategy has
been validated with the identification of potent and
selective, yet highly charged PTP1B inhibitors from this
11,12
laboratory.
We now report the discovery of a mod-
erately potent, highly selective, and cellular active
PTP1B inhibitor using an NMR-based fragment screen-
1
3
ing and an X-ray crystal structure based assembly
approach. This approach offers a highly efficient alter-
native to traditional high-throughput screening (HTS)
for identifying novel ligands that preferentially bind to
PTP1B and possess favorable physiochemical properties.
The general idea of this approach has been illustrated
by the discovery of oxamic acid based PTP1B inhibi-
Protein tyrosine phosphatase 1B (PTP1B), an intra-
cellular protein tyrosine phosphatase (PTPase), has
been implicated in negative regulation of insulin and
leptin signal transduction pathways among other bio-
1
logical functions. Mice lacking functional PTP1B ex-
hibit the phenotypes of increased insulin sensitivity,
improved glucose tolerance, and resistance to diet-
induced obesity.2 A synthetic small molecule that
selectively inhibits PTP1B action is therefore expected
to have similar beneficial effect in human and might
be developed into a therapeutic for the treatment of type
II diabetes and obesity.
1
1
tors. While potent, these highly charged inhibitors
were not cell-permeable. The lack of cellular perme-
ability and activity by this series of inhibitors had to
be circumvented through an ester prodrug approach, as
,3
1
4
exemplified by inhibitor 1 (Figure 1). To ensure
improved cell permeability of a new generation of
PTP1B inhibitors without resorting to a prodrug ap-
proach, we decided to screen only monocarboxylic acid
or non-carboxylic acid-based fragments for potential
catalytic site ligands. Since N-phenyloxamic acid ap-
pears to be the most potent non-phosphorus-containing
PTPases are highly specific for the doubly charged
phosphotyrosine (pTyr) residue. The crystal structures
of over a dozen PTPases (mostly catalytic domains) have
4
been determined. Besides the highly conserved key
structural features, it was noted that the PTP1B
catalytic site and vicinal binding sites are mostly
surface-exposed, highly hydrophilic, and homologous to
1
5
pTyr mimetic, we designed a series of heterocycle
carboxylic acids as potential N-phenyloxamic acid mi-
metics with reduced pKa. Some of them (2-5) are shown
in Figure 2. These compounds, when tested at 300 µM,
were found to be inactive in a colorimetry-based PTP1B-
mediated p-nitrophenyl phosphate (pNPP) hydrolysis
assay. The inhibition constants (Ki values) of these
compounds were estimated to be greater than 900 µM
by extrapolation of the Michaelis-Menten plots.
5
,6
other PTPases. As a result, known potent PTP1B
inhibitors tend to be large (MW > 500), highly charged,
and cell-impermeable, restricting the ability of the
7
,8
inhibitors to reach the intracellular location of PTP1B.
Several less charged PTP1B inhibitors have been re-
*
To whom correspondence should be addressed. Phone: 847-935-
224. Fax: 847-938-1674. E-mail: gang.liu@abbott.com.
Metabolic Disease Research.
Advanced Technology.
Current Address: Pfizer Global R&D, La J olla Laboratories, 10770
Science Center Drive, San Diego, CA 92121-1187.
1
†
These compounds were also subject to the more
sensitive NMR-based screening for possible interaction
with the catalytic site. Human PTP1B (residues 1-288)
‡
§
1
0.1021/jm034122o CCC: $25.00 © 2003 American Chemical Society
Published on Web 09/03/2003

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4233
Sch em e 1. Preparation of PTP1B Inhibitors 8 and 9a
F igu r e 2. Selected heterocycle carboxylic acids designed to
mimic N-phenyloxamic acid as potential pTyr mimetics.
F igu r e 3. Site 2 ligand and PTP1B inhibitors discovered by
fragment screening and assembly approach.
a
Reagents and conditions: (a) dimethyl oxalate, NaOMe,
MeOH, room temp; (b) NH2OH‚HCl, p-TsOH, MeOH, room temp;
(c) 3-tributylstannyl prop-2-en-1-ol, Pd2(dba)3, CuI, P(2-furyl)3,
DMF, room temp; (d) NBS, BPO, CCl4, 70 °C; (e) DMSO, solid
NaHCO3, 120 °C; (f) (EtO)2POCH2CO2Et, NaH, THF, room temp;
selectively labeled with 13C at the methyl groups of
isoleucine residues (δ1 only) was used,16 and isoxazole
carboxylic acid 5 (Figure 2) was identified as a weak
binder with a dissociation constant (Kd) of 800 µM
through monitoring of the chemical shift change of
catalytic site residue Ile219.
(g) DIBAL-H, CH2Cl2, 0 °C to room temp; (h) 5-tributylstannyl-
isoxazole-3-carboxylic acid ethyl ester, Pd2(dba)3, CuI, P(2-furyl)3,
DMF, room temp; (i) methyl 2,6-dihydroxybenzoate, DEAD, Ph3P,
THF, room temp; (j) NaOH, THF/MeOH, room temp.
Previously, salicylic acid 6 (Figure 3) has been identi-
fied as a site 2 ligand (Kd ) 1.2 mM) through an NMR-
mize the π-stacking interaction with Tyr46. Among the
analogues examined, an equally potent inhibitor 9
based screening using PTP1B selectively 13C-labeled at
(
Figure 3, Ki ) 6.9 ( 2.3 µM) was identified by
fluorinating the 6-position of the phenyl ring of 8. 8 and
represent a new class of monocarboxylic acid based
the methyl groups of methionine residues. Structure-
based linking with a catalytic site directed oxamic acid
based pharmacophore led to the identification of potent
PTP1B inhibitor 1 with 4-fold selectivity over the highly
homologous T-cell PTPase (TCPTP).1 The binding mode
of the neutral methyl salicylate based site 2 ligand was
established through X-ray crystallography.
9
PTP1B inhibitors.
4
Both 8 and 9 demonstrated greater than 30-fold
selectivity (Ki of 202 ( 26 and 164 ( 1 µM, respectively)
1
7
over TCPTP, the most homologous PTPase, and showed
no inhibitory activity against LAR, CD45, cdc25, and
SHP-2 at the highest concentration tested (300 µM).
Interestingly, for the first time, inhibitor 7, which
interacts only with catalytic site, also showed a smaller
degree of selectivity (4-fold) against the highly homolo-
gous TCPTP (Ki ) 634 µM). The exact origin of such
selectivity is unclear, although the minor amino acid
residue differences in the catalytic domain between
To link the catalytic site ligand 5 and site 2 methyl
salicylate ligand effectively, the binding mode of cata-
lytic site ligand 5 in PTP1B needs to be determined.
However, the crystal structure of 5 complexed with
PTP1B could not be solved because of its weak binding
affinity. Fortunately, a related analogue 7 (Figure 3)
was potent enough against PTP1B (Ki ) 148 µM) for
X-ray crystal structure determination of the complex.
The X-ray structure of 7 bound to PTP1B (data not
shown) suggested that a four-atom linker off the meta
position of the phenyl ring should be optimal for linking
with the methyl salicylate based site 2 ligand.
1
8
PTP1B and TCPTP could affect the intrinsic recogni-
tion capability for different substrates and inhibitors.
From the structure-activity relationship of these com-
pounds, site 2 binding moiety contributes about 10-fold
1
9
The orientation of methyl salicylate in site 2 was
selectivity over TCPTP. To the best of our knowledge,
1
4
modeled according to the X-ray crystal structure of 1.
8 and 9 represent the most selective PTP1B inhibitors
2
0
Similar to inhibitor 1, linking catalytic site binder 5 to
the 6-position of methyl salicylate through an ether
linkage was determined to be the most effective way for
assembly. Therefore, a C2 symmetrical methyl 2,6-
dihydroxybenzoate was used for linking. Improvement
of the initially linked molecules by conformational
modification of the linker led to the identification of
inhibitor 8 (Figure 3), which showed a Ki of 5.7 ( 0.9
µM against PTP1B. Electron-withdrawing groups were
then introduced into the central phenyl ring to maxi-
over TCPTP reported to date.
The synthesis of 8 and 9 is outlined in Scheme 1.
2
1
Briefly, Stille coupling between the readily available
isoxazole ester 8a and tributylstannylpropenol yielded
the allyl alcohol 8b. Subsequent Mitsunobu reaction
with methyl 2,6-dihydroxybenzoate and saponification
of the resultant ester 8c yielded inhibitor 8. Similarly,
Horner-Emmons olefination of aldehyde 9a with
phosphonoacetate provided a cinnamate, which was
then reduced to allyl alcohol 9b. Stille coupling of iodide

4
234 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20
Letters
F igu r e 5. Reversal of dephosphorylation of STAT3 in COS-7
cells by PTP1B inhibitor 9.
F igu r e 4. X-ray crystal structure of PTP1B complexed with
. Carbon is in orange and green for 9, oxygen is in red,
9
nitrogen is in blue, sulfur is in yellow, and fluorine is in dark-
green.
F igu r e 6. Reversal of PTP1B-dependent dephosphorylation
of STAT3 in COS-7 cells by PTP1B inhibitor 9.
9
b with tributylstannylisoxazole yielded 9c. Following
the standard Mitsunobu and hydrolysis sequence, ester
In a more mechanism-specific way of assaying cellular
PTP1B inhibition by 9, COS-7 cells were transiently
transfected with exogenous PTP1B. The inhibition of
STAT3 phosphorylation caused by the overexpression
of PTP1B was partially reversed by 9 dose-dependently
(10-100 µM), similar to the effect exerted by a nonspe-
9
d was converted to the desired inhibitor 9.
The X-ray crystal structure of PTP1B (residues
-322)/9 complex was solved at a resolution of 2.6 Å,
1
2
2
which is shown in Figure 4. The isoxazole carboxylic
acid binds to the active site of PTP1B with the WPD
loop in the closed conformation. Bidentate-type hydro-
gen bonds between carboxylic acid and Arg221 provided
the critical interactions. The isoxazole and phenyl rings
sit snugly in the hydrophobic pocket normally occupied
by the phenyl ring of pTyr. The hydrogen bond network
between 9 and PTP1B remained essentially the same
as that between 1 and PTP1B. The ether oxygen of 9
hydrogen-bonds with Gln262. The salicylate carboxylate
group adapts an out-of-plane conformation and is in
hydrogen-bond contact with Arg254. The hydroxyl group
of 9, which should remain as a neutral group at
physiological pH (∼7.4), hydrogen-bonds with Arg24 and
Arg254. The aromatic portion of the salicylate lies on
top of the hydrophobic side chain of Met258, providing
complementary van der Waals interaction.
2
5
cific phosphatase inhibitor BpV(pic) at 10 µM (Figure
6). Consistent with the cellular activity, 9 showed
-
6
moderate cellular permeability (Papp > 1 × 10 cm/s)
2
6
in a Caco-2 cell membrane assay. Interestingly, 8
-
6
showed low Caco-2 permeability (Papp < 1 × 10 cm/s)
and much reduced cellular activity in COS-7 cells (data
not shown). This beneficial fluorine effect originated
most likely from the increased lipophilicity of 9.27 In the
literature, there are only two examples of PTP1B
inhibitors with cellular activity without resorting to a
2
8,29
prodrug approach.
However, in both accounts, no
selectivity data against TCPTP were reported on those
peptidomimetic PTP1B inhibitors.
In conclusion, the extremely sensitive NMR-based
screening allowed the identification of weak ligands for
both the catalytic site and a second phosphotyrosine
binding site. Structure-based assembly of these simple
ligands led to the efficient discovery of moderately
potent, highly selective, and cellular active PTP1B
inhibitor 9. Apart from the general applicability in the
early stages of drug development, an advantage of this
approach is that it starts from screening a small number
of compounds with predefined properties. The identified
ligands thus allow for expansion to generate molecules
with optimized affinity, selectivity, and cellular activity,
minimizing the immediate risk of obtaining lead mol-
ecules with disadvantageous pharmacokinetic proper-
Leptin causes tyrosine phosphorylation of the signal
transducer and activator of transcription 3 (STAT3) via
activation of the J anus kinase 2 (J AK2). PTP1B has
been shown to negatively regulate leptin pathways by
2
3
24
dephosphorylating J AK2 both in vitro and in vivo.
was analyzed in a COS-7 cellular system for its ability
9
to reverse PTP1B-dependent inhibition of STAT3 phos-
phorylation. Initially, 9 was found to enhance the
steady-state phosphorylation of STAT3 in COS-7 cells
in a dose-dependent manner (10-100 µM) through a
mechanism of inhibiting the endogenous level of J AK2-
regulating PTPases, including PTP1B (Figure 5).

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 20 4235
ties. Therefore, structure-based fragment screening and
assembly provide valuable tools for quick identification
of novel ligands with specific binding properties, and
the results compared favorably to the hits from HTS.
(13) Shuker, S. B.; Hajduk, P. J .; Meadows, R. P.; Fesik, S. W.
Discovering High-Affinity Ligands for Proteins: SAR by NMR.
Science 1996, 274, 1531-1534.
(14) Liu, G.; Xin, Z.; Liang, H.; Abad-Zapatero, C.; Hajduk, P. J .;
J anowick, D. A.; Szczepankiewicz, B. G.; Pei, Z.; Hutchins, C.
W.; Ballaron, S. J .; Stashko, M. A.; Lubben, T. H.; Berg, C. E.;
Rondinone, C. M.; Trevillyan, J . M.; J irousek, M. R. Selective
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Ack n ow led gm en t. We thank Drs. Elizabeth Everitt
and Mark Schurdak for determining the Caco-2 perme-
ability of inhibitors 8 and 9.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails and analytical and spectral data of key compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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J M034122O