Allele-specific inhibition of divergent protein tyrosine phosphatases with a single small molecule

Article abstract of DOI:10.1016/j.bmc.2008.07.053

A central challenge of chemical biology is the development of small-molecule tools for controlling protein activity in a target-specific manner. Such tools are particularly useful if they can be systematically applied to the members of large protein families. Here we report that protein tyrosine phosphatases can be systematically 'sensitized' to target-specific inhibition by a cell-permeable small molecule, Fluorescein Arsenical Hairpin Binder (FlAsH), which does not inhibit any wild-type PTP investigated to date. We show that insertion of a FlAsH-binding peptide at a conserved position in the PTP catalytic-domain's WPD loop confers novel FlAsH sensitivity upon divergent PTPs. The position of the sensitizing insertion is readily identifiable from primary-sequence alignments, and we have generated FlAsH-sensitive mutants for seven different classical PTPs from six distinct subfamilies of receptor and non-receptor PTPs, including one phosphatase (PTP-PEST) whose three-dimensional catalytic-domain structure is not known. In all cases, FlAsH-mediated PTP inhibition was target specific and potent, with inhibition constants for the seven sensitized PTPs ranging from 17 to 370 nM. Our results suggest that a substantial fraction of the PTP superfamily will be likewise sensitizable to allele-specific inhibition; FlAsH-based PTP targeting thus potentially provides a rapid, general means for selectively targeting PTP activity in cell-culture- or model-organism-based signaling studies.

Full text of DOI:10.1016/j.bmc.2008.07.053

Bioorganic & Medicinal Chemistry 16 (2008) 8090–8097
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Allele-specific inhibition of divergent protein tyrosine
phosphatases with a single small molecule
*
Xin-Yu Zhang, Vincent L. Chen, Mari S. Rosen, Elizabeth R. Blair, Anna Mari Lone, Anthony C. Bishop
Amherst College, Department of Chemistry, Amherst, MA 01002, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A central challenge of chemical biology is the development of small-molecule tools for controlling protein
activity in a target-specific manner. Such tools are particularly useful if they can be systematically applied
to the members of large protein families. Here we report that protein tyrosine phosphatases can be sys-
tematically ‘sensitized’ to target-specific inhibition by a cell-permeable small molecule, Fluorescein
Arsenical Hairpin Binder (FlAsH), which does not inhibit any wild-type PTP investigated to date. We show
that insertion of a FlAsH-binding peptide at a conserved position in the PTP catalytic-domain’s WPD loop
confers novel FlAsH sensitivity upon divergent PTPs. The position of the sensitizing insertion is readily
identifiable from primary-sequence alignments, and we have generated FlAsH-sensitive mutants for
seven different classical PTPs from six distinct subfamilies of receptor and non-receptor PTPs, including
one phosphatase (PTP-PEST) whose three-dimensional catalytic-domain structure is not known. In all
cases, FlAsH-mediated PTP inhibition was target-specific and potent, with inhibition constants for the
seven sensitized PTPs ranging from 17 to 370 nM. Our results suggest that a substantial fraction of the
PTP superfamily will be likewise sensitizable to allele-specific inhibition; FlAsH-based PTP targeting thus
potentially provides a rapid, general means for selectively targeting PTP activity in cell-culture- or
model-organism-based signaling studies.
Received 24 June 2008
Revised 18 July 2008
Accepted 19 July 2008
Available online 24 July 2008
Keywords:
Protein tyrosine phosphatases
Allele-specific inhibitors
Inhibitor sensitization
FlAsH
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
tially any eukaryotic protein kinase can be rendered sensitive to
a known ATP-competitive inhibitor through a simple site-directed
Engineering of protein/small-molecule interfaces has emerged
as a powerful means of generating small-molecule ligands that
control target-protein activity with high selectivity.1,2 Such protein
‘sensitization’ strategies are appealing because many of the poten-
tial bottlenecks of inhibitor discovery (e.g., compound synthesis
and screening) are bypassed: in principle, sensitivity to a known
small molecule can simply be ‘programmed’ into a target protein
of interest through genetic mutation. Several key criteria must be
met, however, for protein-sensitization strategies to be biologically
and generally useful: mutagenesis must confer to the target pro-
tein small-molecule sensitivity that is not present in the wild-type
protein or other members of the same protein family; the muta-
tion(s) employed should be functionally silent (i.e., the mutant
protein must retain its activity in the absence of the small-mole-
cule ligand); and, ideally, the amino acid residue(s) identified for
sensitization in a prototype target should be present in other mem-
bers of the protein family, eliminating the need to redesign a pro-
tein/inhibitor interface for each new target.3 To date, the protein
kinases have proven to be the enzyme family most amenable to
protein/small-molecule engineering: with a few caveats, essen-
mutation in the kinase active site.4 Attempts to systematically
engineer inhibitor sensitivity in other important enzyme families
such as the protein methyltransferases5 and phosphatidyl-inosi-
tol-3 kinases6 have also met with some success.
Tight control of protein-phosphorylation states is, arguably, the
most important signaling-regulation mechanism in mammalian
biology, and the mammalian protein tyrosine phosphatases (PTPs),
which catalyze the dephosphorylation of phosphotyrosine residues
in protein substrates, are an attractive family of targets for small-
molecule inhibitor discovery.7,8 PTPs, roughly 100 of which are en-
coded in the human genome, make up a large and diverse set of sig-
naling enzymes, and small molecules that specifically inhibit
individual PTPs would aid in the development of a full understand-
ing of PTP function in complex signaling cascades. We have made
significant progress in the development of a method for engineering
novel inhibitor sensitivity into PTP active sites.9–11 Recent attempts
at applying our active-site-targeting strategy across the PTP family,
however, have suggested that active-site sensitization will be some-
what limited in scope, presumably due to significant differences in
the detailed topology of individual PTP active sites.12 Moreover, ac-
tive-site-directed PTP inhibitor discovery is complicated by the low
cellular permeability of many known competitive PTP inhibitors,
most of which are negatively charged phosphotyrosine mimetics.8
* Corresponding author. Tel.: +1 413 542 8316; fax: +1 413 542 2735.
E-mail address: acbishop@amherst.edu (A.C. Bishop).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.053

X.-Y. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8090–8097
8091
While active-site sensitization has proven extremely powerful
for the study of protein-kinase function, it is possible that the ki-
nases represent a ‘special case.’ Many, or most, enzyme families
may not be readily amenable to analogous strategies: the require-
ment for finding a sensitizing mutation in the active site that does
not disrupt enzyme activity may simply be too constraining. How-
ever, a method for generating small-molecule-sensitized enzymes
that avoids the pitfalls of active-site engineering has recently been
described: insertion of a small-molecule-binding peptide into a
protein domain has been used to generate sensitized variants of
two enzymes, TEM-1 (a b-lactamase)13 and T-cell PTP (TCPTP).14
These studies utilized a cell-permeable small molecule, Fluorescein
Arsenical Hairpin Binder (FlAsH, Fig. 1A),15,16 to specifically inhibit
TEM-1 and TCPTP mutants that contain the optimized FlAsH-bind-
ing peptide, CCPGCC, embedded in their respective catalytic do-
mains.13,14 Here we show that insertion of the CCPGCC motif can
be widely used for the generation of FlAsH-sensitized PTPs. Guided
by PTP primary-sequence alignments, we demonstrate that site-
specific insertion of CCPGCC confers novel FlAsH sensitivity upon
seven classical PTPs from six distinct subfamilies. Importantly,
the observed inhibition of the FlAsH-sensitized mutants is highly
specific; FlAsH does not inhibit any wild-type PTP investigated to
date. Our results demonstrate that a substantial fraction of the
classical PTP superfamily can be readily sensitized to a single
small-molecule inhibitor, and suggest that other PTPs will be like-
wise sensitizable to allele-specific inhibition by FlAsH.
2. Results and discussion
2.1. Design strategy
Recently, we demonstrated that insertion of a tetra-cysteine-
containing hexapeptide (CCPGCC) in the catalytic domain of TCPTP
is sufficient for sensitization of that phosphatase to inhibition by
FlAsH.14 We found that, for TCPTP, the most highly sensitizing site
of insertion (187 in human TCPTP numbering) is near a canonical
PTP loop that is known to close upon PTP-substrate binding (the
WPD loop, Fig. 1B).17,18 FlAsH binding presumably precludes prop-
er closing (or, possibly, opening) of the mutant’s WPD loop and
specifically attenuates the activity of the engineered TCPTP. It
has been surmised from the basis of sequence homology and struc-
tural evidence that almost every classical PTP similarly utilizes the
conserved WPD region as a critical activity determinant (39 classi-
cal PTPs are encoded in the human genome).19,20 Therefore, we
hypothesized that insertion of CCPGCC at the position correspond-
ing to 187 of TCPTP could represent a general strategy for the site-
specific incorporation of inhibition sites in many different PTPs
(Fig. 1C).
Figure 1. (A) Chemical structure of FlAsH. (B) Structure-based-design of CCPGCC-
insertion mutants. The crystal structures of PTP1B (cyan), a close homolog of TCPTP,
in the ‘open’ (PDB: 2HNQ)34 and ‘closed’ (PDB: 1C83)35 forms were superimposed
and the WPD loops of the two PTP1B structures were colored green and red,
respectively. The position of the CCPGCC-insertion corresponds to Glu186 in PTP1B.
The side chain of the catalytically important aspartate residue, Asp181, is shown to
highlight the movement of the WPD loop. For perspective, PTP1B’s active-site
catalytic cysteine residue (Cys215) is shown in space-filling representation. This
image was generated using the Chimera software package (http://www.cgl.ucs-
f.edu/chimera). (C) WPD-loop alignment of the PTPs discussed in this study. (See
Ref. 20 for a sequence alignment of all 39 human classical PTP domains.) The amino
acid residues corresponding to Asp181 and Glu186 of PTP1B are noted in bold type.
CCPGCC-insertion positions are indicated by the vertical red line. The numbers of
the insertion positions vary widely due to the diversity in size and structure of the
PTP family outside of the conserved PTP domains.36
To test the scope of FlAsH-based PTP targeting, we selected six
biologically important PTPs (in addition to TCPTP) from six distinct
subfamilies that are well spread across the human family of both
non-transmembrane (NT) and receptor-like (R) classical PTPs:
PTP1B, PTP-PEST, PTPH1, FAP-1, HePTP, and PTPa 20
PTP1B, sub-
.
type NT1, is a close homolog of TCPTP (69% PTP-domain identity)
and an important type-II-diabetes drug target.21 PTP-PEST (sub-
type NT4, 34% PTP-domain identity with TCPTP) is a wide-ranging
and ubiquitously expressed signaling molecule, which is involved
in the regulation of the actin cytoskeleton and has recently been
shown to be cleaved by caspase-3 during apoptosis.22,23 PTPH1
and FAP-1 (subtypes NT5 and NT7, 41% and 37% PTP-domain iden-
tity with TCPTP, respectively) have been found mutated in human
HePTP (subtype R7, 34% PTP-domain identity with TCPTP) is a T-
cell-specific phosphatase that can be overexpressed in preleukemic
myeloproliferative diseases.26
When the PTP-domain primary sequences of the six PTPs de-
scribed above are aligned with that of TCPTP, the position corre-
sponding to TCPTP’s residue 187 is readily identifiable, as it lies
immediately to the C-terminal side of an invariant proline residue
colorectal cancers.24 PTP
a (subtype R4, 38% PTP-domain identity
with TCPTP) is a receptor PTP that dephosphorylates and activates
Src-family kinases, affecting a host of downstream pathways,
including cell proliferation and neuronal differentiation.25 And

8092
X.-Y. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8090–8097
that marks the end of the conserved portion of the WPD loop (Fig.
1C). To test the hypothesis that a FlAsH-binding peptide at the 187
position could sensitize multiple PTPs to inhibition, we cloned, ex-
pressed, and purified the catalytic domains of the six PTPs de-
scribed above, as well as their corresponding CCPGCC-insertion
mutants: PTP1B-186, PTP-PEST-204, PTPH1-816, FAP1-2364,
PTPa-406, and HePTP-211 (insertion position is indicated by the
red line in Figure 1C; numbering is according to the human en-
zymes and corresponds to the position of the first cysteine residue
in the insert).
2.2. PTP activity of insertion mutants
It is, of course, quite possible that insertion of a hexapeptide
into a structured loop could seriously disrupt the fold and/or activ-
ity of an enzyme. In previous work on TCPTP, however, we found
that its PTP domain is remarkably tolerant to peptide insertions.
In particular, the TCPTP-187 mutant does retain inherent PTP activ-
ity, albeit with a catalytic efficiency (kcat/KM) that is an order of
magnitude lower than that of wild-type TCPTP.14
Figure 2. FlAsH-dependent inhibition of PTP-insertion mutants. The indicated PTP
enzymes (2.5 M) were incubated in the absence or presence of FlAsH (10 M),
diluted and assayed for activity with the artificial PTP substrate para-nitrophenyl
l
l
phosphate (pNPP) at pH 7.0. ‘% Activity’ represents the PTP catalytic efficiency (kcat
KM
/
In good agreement with the TCPTP-187 precedent, all six of the
analogous PTP-insertion mutants could be expressed from Esche-
richia coli, and they were readily purified as affinity-tagged pro-
teins (either His6 or GST, see Section 4). When assayed for
activity, four of the six insertion mutants demonstrated significant
reductions in inherent catalytic efficiency when assayed in the ab-
sence of FlAsH (Table 1). These diminutions in PTP activity, com-
pared to the corresponding wild-type PTP, were similar in scale
to that of TCPTP-187 (TCPTP-187: 7-fold14; PTP1B-186: 19-fold;
HePTP: 9-fold; FAP1-2364: 14-fold; and PEST-204: 7-fold). We
cannot predict a priori what level of decrease in PTP enzymatic effi-
ciency is tolerable for a mutant to complement the function of the
wild-type. And it is possible that the reductions in catalytic activity
noted above are of sufficient magnitude to limit the cellular-com-
plementation ability of the CCPGCC-insertion mutants. There does
exist, however, a potential mitigation of this obstacle: we have re-
cently shown that much less radical mutations in the WPD loop (as
few as two cysteine point mutations, with no insertions) can be
used to confer strong FlAsH sensitivity upon TCPTP.27 These more
subtly sensitized TCPTP mutants retain wild-type-like activity in
the absence of FlAsH, and the homology of the WPD loop (Fig.
1C) suggests that similar ‘rescuing’ strategies may work for other
PTPs—such as PTP1B, HePTP, FAP-1, and PTP-PEST—that lose some
activity upon hexapeptide insertion.
)
in the presence of FlAsH divided by the control (vehicle only) catalytic
efficiency of the same enzyme.
responding wild-type PTPs, PTPH1 and PTP
a
. These results suggest
that a substantial fraction of PTPs (2 out 7 in the current panel) will
be fully enzymatically tolerant of the CCPGCC insertion and need
no rescuing as described above.
2.3. Novel FlAsH sensitivity of insertion mutants
We next investigated the effects of FlAsH addition on our panel
of wild-type and insertion-mutant PTPs. Importantly, the wild-
type activities of all seven PTPs were essentially unaffected by
the addition of FlAsH (Fig. 2 and Table 2). [The DMSO vehicle had
little to no effect on any PTP’s activity (compare ‘(ꢀ)’ entries in Ta-
Table 2
Kinetic constants of wild-type and CCPGCC-insertion PTPs in the absence (ꢀ) and
presence (+) of FlAsH when assayed with pNPP
Enzyme
kcat (sꢀ1
)
KM (mM)
kcat/KM (mMꢀ1 sꢀ1
)
PTP1B (ꢀ)
4.4 0.16
4.4 0.16
2.4 0.16
2.4 0.12
1.9 0.060
1.8 0.020
PTP1B (+)
PTP1B 4C-186 (ꢀ)
0.12 0.0062
0.0038 0.000061
0.91 0.080
1.3 0.043
0.13 0.0057
0.0030 0.000082
Remarkably, two of the CCPGCC-insertion mutants, PTPH1-816
and PTPa-406, retain their full catalytic activities in the absence of
FlAsH (Table 1). There is no significant difference in the catalytic-
rate constants of either of these proteins with respect to their cor-
PTP1B 4C-186 (+)
HePTP (ꢀ)
3.0 0.10
2.8 0.029
6.2 0.37
6.6 0.16
0.49 0.013
0.42 0.0055
HePTP (+)
HePTP-211 (ꢀ)
0.16 0.020
0.039 0.0033
5.3 0.92
5.2 0.68
0.031 0.0017
0.0076 0.00035
HePTP-211 (+)
FAP-1 (ꢀ)
1.0 0.061
0.85 0.033
1.1 0.13
1.1 0.10
0.94 0.057
0.80 0.045
Table 1
FAP-1 (+)
Kinetic constants of wild-type and CCPGCC-insertion PTPs assayed with pNPP
FAP-1-2364 (ꢀ)
0.20 0.014
0.028 0.00082
4.0 0.39
4.5 0.15
0.050 0.0013
0.0062 0.00011
Enzyme
kcat (sꢀ1
)
KM (mM)
kcat/KM (mMꢀ1 sꢀ1
)
FAP-1-2364 (+)
PTP1B
PTP1B-186
5.5 0.24
0.11 0.0021
2.4 0.25
0.94 0.042
2.3 0.15
0.12 0.0050
PTP-PEST (ꢀ)
0.46 0.00057
0.45 0.038
4.9 0.29
4.7 0.80
0.096 0.0058
0.097 0.011
PTP-PEST (+)
HePTP
HePTP-211
3.1 0.045
0.22 0.0081
6.9 0.19
4.4 0.24
0.45 0.014
0.049 0.00084
PTP-PEST-204 (ꢀ)
0.077 0.0016
0.014 0.0015
4.2 0.10
5.0 0.92
0.018 0.00020
0.0028 0.00020
PTP-PEST-204 (+)
FAP-1
FAP-1-2364
0.82 0.0036
0.24 0.0033
0.94 0.032
3.8 0.10
0.87 0.026
0.062 0.0011
PTPH1 (ꢀ)
5.3 0.075
4.8 0.13
0.56 0.027
0.53 0.032
9.4 0.36
9.0 0.30
PTPH1 (+)
PTP-PEST
PTP-PEST-204
0.59 0.018
0.081 0.0012
3.9 0.072
3.5 0.13
0.15 0.0057
0.023 0.00061
PTPH1-816 (ꢀ)
5.4 0.20
0.53 0.0074
0.84 0.075
1.0 0.025
6.4 0.32
0.53 0.0064
PTPH1-816 (+)
PTPH1
PTPH1-816
5.2 0.18
5.3 0.28
0.49 0.038
0.74 0.064
11 0.47
7.1 0.24
PTP
PTP
a
a
(ꢀ)
1.6 0.092
1.6 0.010
8.3 0.81
7.8 0.26
0.19 0.0077
0.21 0.0059
(+)
PTP
PTP
a
a
1.7 0.15
2.2 0.28
8.0 0.90
2.7 0.60
0.21 0.0052
0.83 0.075
PTP
PTP
a
a
-406 (ꢀ)
2.0 0.20
0.10 0.014
2.6 0.50
5.1 1.0
0.79 0.073
0.021 0.0015
-406
-406 (+)

X.-Y. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8090–8097
8093
ble 2 with the corresponding Table 1 entries).] These data demon-
strate that the FlAsH ligand has no significant non-specific affinity
for PTP domains, implying that FlAsH could be used in a cellular
context to target a single engineered PTP, without substantial
off-target PTP inhibition. Consistent with this hypothesis, all of
the CCPGCC-insertion mutants are strongly inhibited by FlAsH
(Fig. 2 and Table 2). Upon incubation with a 4-fold molar excess
of FlAsH, PTP1B-186 catalytic efficiency drops 43-fold (0.13 to
mediated inhibition of PTP1B-186, PTP-PEST-204, PTPH1-816,
FAP1-2364, PTP -406, and HePTP-211 is comparable in scale and
kind to that observed with TCPTP-187, implying a conserved mech-
a
anism of action across the classical PTP family.
2.4. Potency of FlAsH-induced inhibition
To determine the potency of FlAsH’s inhibitory activity on all of
the PTP-insertion mutants, we assayed their activities after incuba-
tion with varying concentrations of FlAsH. Under the conditions of
the assay, none of the wild-type PTPs showed any significant inhi-
bition at any FlAsH concentration (Fig. 3). By contrast, the activities
of all seven CCPGCC-insertion mutants dropped dramatically in a
dose-dependent manner when pre-incubated with FlAsH. From
0.0030 mMꢀ1 ꢀ1
s ); HePTP-211 activity decreases 4-fold (0.031 to
0.0076 mMꢀ1 sꢀ1); FAP-1-2364 activity drops 8-fold (0.050
to 0.0062 mMꢀ1 sꢀ1); PTP-PEST-204 activity drops 6-fold (0.018
to 0.0028 mMꢀ1 sꢀ1); PTPH1-816 activity drops 12-fold (6.4 to
0.53 mMꢀ1 sꢀ1); and PTP
a-406 activity drops 38-fold (0.79 to
0.021 mMꢀ1 sꢀ1). The magnitude of these FlAsH-induced reduc-
tions in sensitized-PTP activity ranges from roughly 3-fold lower
to 3-fold higher than that of the previously reported 12-fold drop
observed for TCPTP-187.14 And as with TCPTP-187, the FlAsH-in-
duced inhibition of the current mutants appears to be noncompet-
itive in nature, as the losses of catalytic efficiency are due
predominately to decreases in kcat (Table 2). In sum, the FlAsH-
these data we estimated the apparent inhibition constants ðKIapp
Þ
of FlAsH with the seven sensitized mutants (Table 3). With the
exception of FAP-1-2364 ðKIapp ¼ 370 nMÞ, all of the insertion mu-
tants demonstrated low-nanomolar sensitivity to FlAsH, with the
KaI pp values for the more potently inhibited mutants ranging from
17 nM (TCPTP-187) to 89 nM (HePTP-211).
Figure 3. Dose-dependent inhibition of wild-type and CCPGCC-insertion mutant PTPs. PTP activity of each wild-type and mutant was measured at the indicated FlAsH
concentrations, and normalized to uninhibited (no-FlAsH) controls. Enzyme concentrations used were as follows: 100 nM for TCPTP and PTPH1; 200 nM for PTP1B, HePTP,
and FAP-1; 500 nM for PTP-PEST; and 50 nM for PTPa.

8094
X.-Y. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8090–8097
Table 3
Apparent inhibition constants ðKaI ppÞ of FlAsH for CCPGCC-insertion PTPs
Enzyme
KaI pp (nM)
TCPTP-187
PTP1B-186
HePTP-211
FAP-1-2364
PTP-PEST-204
PTPH1-816
17 3.3
46 5.9
89 12
370 140
53 6.8
38 1.7
39 1.7
PTPa-406
2.5. Phosphopeptide dephosphorylation by PTPH1-816 and
PTP -406
a
We selected PTPH1-816 and PTPa-406 as candidates for further
characterization. These two mutants are particularly attractive as
inhibitor-sensitized enzymes in that they demonstrate kinetic
parameters that are essentially indistinguishable (or marginally
improved, in the case of PTPa-406) from the corresponding wild-
type enzymes in the absence of an allele-specific inhibitor. (Even
sensitized protein kinases generally demonstrate substantially de-
creased kcat/KM values with ATP.28,29) Thus, of the seven sensitized
PTPs described above, PTPH1-816 and PTPa-406 are the most
likely to meet the critical criterion for cellular signaling studies
using engineered proteins: that a targeted mutant protein retains
the cellular function of the wild-type in the absence of ligand.
All of the initial analyses of PTPH1-816 and PTPa-406 were car-
ried out with an artificial small-molecule PTP substrate, pNPP, and
it is possible that a CCPGCC mutant could demonstrate wild-type-
level kinetics with pNPP yet fail to demonstrate robust kinetics
with a more physiologically relevant substrate. To further confirm
the suitability of PTPH1-816 and PTPa-406 as inhibitor-sensitized
enzyme variants, we measured their activities with a phosphopep-
tide substrate DADEpYLIPQQG, the sequence of which corresponds
to the auto-phosphorylation site of the epidermal growth factor
receptor (EGFR988–998).30 We found that, in the absence of FlAsH,
the kinetic parameters of DADEpYLIPQQG dephosphorylation by
PTPH1-816 and PTPa-406 are unchanged with respect to the corre-
sponding wild-type values (Table 4, e.g., compare ‘PTPH1 (ꢀ)’ to
‘PTPH1-816 (ꢀ)’). These data show that the catalytic activity of
these CCPGGCC mutants is ‘wild-type-like’ regardless of choice of
PTP substrate and further support that the proposition that
PTPH1-816 and PTP
in a cellular context.
Along similar lines, it is important that FlAsH, the target-specific
inhibitor of PTPH1-816 and PTP -406, acts in a substrate-indepen-
a-406 would function like their parent PTPs
a
dent manner, as PTPs will encounter a variety of phosphorylated-
protein substrates in a cellular context. Indeed, the engineered
FlAsH sensitivities of PTPH1-816 and PTPa-406 also proved to be
substrate-independent (Fig. 4 and Table 4). When incubated with
FlAsH, the activities of both mutants dropped dramatically and
Table 4
Kinetic constants of wild-type and CCPGCC-insertion PTPH1 and PTP
(ꢀ) and presence (+) of FlAsH when assayed with DADEpYLIPQQG
a
in the absence
Mꢀ1 sꢀ1
)
Enzyme
kcat (sꢀ1
)
KM
(
lM)
kcat/KM
(l
Figure 4. Specific inhibition of PTPH1-816 and PTP
phosphopeptide substrate DADEpYLIPQQG. PTP enzymes (2.5
in the absence (black lines) or presence (gray lines) of FlAsH (10
final enzyme concentration noted for each panel, and assayed at pH 7.0. (A) Wild-
type PTPH1 (36 nM). (B) PTPH-816 (36 nM). (C) Wild-type PTP (140 nM). (D)
PTP -406 (180 nM).
a
-406 when assayed with the
M) were incubated
M), diluted to the
PTPH1 (ꢀ)
8.0 0.48
6.9 0.80
3.1 0.89
2.7 0.14
2.8 0.76
2.6 0.20
l
PTPH1 (+)
l
PTPH1-816 (ꢀ)
9.8 0.65
1.8 0.070
3.5 0.34
7.7 0.62
2.8 0.25
0.23 0.0093
a
PTPH1-816 (+)
a
PTP
PTP
a
a
(ꢀ)
4.2 0.27
4.2 0.40
82 9.4
77 1.8
0.052 0.0029
0.055 0.0045
(+)
specifically. The magnitude of these inhibition events is, within er-
ror, precisely in line with what was observed when pNPP was used
as substrate (PTPH1-816: 12-fold catalytic efficiency drop with
PTP
PTP
a
-406 (ꢀ)
3.4 0.33
0.12 0.027
84 15
100 12
0.041 0.0043
0.0012 0.00021
a-406 (+)

X.-Y. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8090–8097
8095
pNPP and 12-fold drop with DADEpYLIPQQG; PTP
a
-406: 38-fold
Collectively, the data above show that FlAsH can rapidly, po-
tently, and specifically target engineered WPD loops via a con-
served mechanism that is independent of the particular PTP that
has been sensitized to inhibition. There is no correlation between
drop with pNPP and 34-fold drop with DADEpYLIPQQG). As with
pNPP, the FlAsH-induced inhibition of DADEpYLIPQQG dephospho-
rylation is potent and specific to the engineered CCPGCC mutants;
moreover, FlAsH appears to operate by a conserved mechanism
that is independent of the particular PTP and substrate being
investigated.
PTP subfamily and FlAsH potency. For example, PTPH1 and PTPa,
which belong to different subfamilies (and neither of which be-
longs to the same subfamily as the model phosphatase, TCPTP)
are among the most potently inhibited in our panel of sensitized
PTPs. Moreover, the PTPs whose activities drop most dramatically
at a fixed FlAsH concentration include both non-transmembrane
2.6. Time dependence of PTPH1-816 and PTPa-406 inhibition
by FlAsH
(PTP1B) and receptor-like (PTPa) phosphatases.
One of the advantages of chemical, as opposed to genetic, ap-
proaches for studying protein function is that, generally, chemical
inhibitors act rapidly (seconds to minutes) while DNA- or RNA-
based approaches take much longer (hours to days) to reduce lev-
els of a target protein’s activity. To ensure that FlAsH acts on a
timescale that is consistent with its potential use in chemical-ge-
netic experiments, we measured the time course of FlAsH-induced
inhibition for PTPH1-816 and PTPa-406. In these experiments, the
FlAsH-responsive mutants were assayed after incubation with
FlAsH for various amounts of time. As shown in Figure 5, the activ-
ities of both PTPH1-816 and PTPa-406 drop immediately upon
FlAsH addition, with maximum inhibition being achieved in
approximately 30 min. These experiments show that the majority
of FlAsH-sensitive PTP inhibition occurs within minutes and sug-
gest that FlAsH may be able to rapidly ‘knockout’ PTP activity in
a cell or lysate that expresses a sensitized PTP.
The key advantage of using protein engineering for targeting
large protein families is that, by introducing chemical diversity
into the target protein (through mutagenesis), novel and specific li-
gand/receptor pairs can be designed efficiently. Our current data
support this assertion: due to the conserved structure and mecha-
nism of the WPD loop, FlAsH sensitization was systematically ap-
plied to seven divergent classical PTPs for which ‘traditional’
inhibitors have not been identified. Importantly, WPD-loop sensiti-
zation requires no detailed structural information for a target PTP.
Of the seven PTPs investigated to date, only one (PTP1B) has been
crystallized in both its open and closed WPD-loop structures (Fig.
1B). Moreover, no crystal structure has been reported for the cata-
lytic domain of PTP-PEST, which, like other members of our PTP pa-
nel, is readily sensitized through primary-sequence-based CCPGCC
insertion. These findings suggest a large fraction of the classical
PTPs will be similarly susceptible to FlAsH sensitization, including
other PTPs for which no three-dimensional structures are known.
3. Conclusion
Few systematic means for chemically targeting members of
large protein families have been described. In the present work,
we show that protein tyrosine phosphatases, a critical family of
cell-signaling enzymes, can be readily sensitized to inhibition by
FlAsH, a small molecule that has no significant effect on the activity
of wild-type PTPs. We have identified a site in the conserved WPD
loop of PTP domains that can be modified to display a FlAsH-bind-
ing peptide, CCPGCC; and we show that CCPGCC insertion system-
atically confers strong FlAsH sensitivity to enzymes from six
different PTP subfamilies. Since essentially all known classical PTPs
utilize the WPD-loop mechanism targeted in this study, it is likely
that other PTPs can be sensitized through an analogous approach,
and, therefore, that FlAsH can be widely used for chemically target-
ing PTPs in cell-signaling studies.
4. Experimental
4.1. Materials, equipment, and general procedures
FlAsH was synthesized as described14–16 and dissolved in
DMSO. The phosphopeptide substrate DADEpYLIPQQG was pur-
chased from EMD. All DNA sequencing was carried out at the Cor-
nell Biotechnology Resource Center. All PTP assays were performed
in triplicate, and all error bars and ‘ ’ values represent the standard
deviations of at least three independent experiments. Sequences of
all cloning and mutagenic primers are provided as Supplementary
material.
4.2. Cloning of PTP catalytic domains
Figure 5. Time-dependent inhibition of PTPH1-816 and PTP
a-406. PTP enzymes
(250 nM) were incubated with FlAsH (1 M). At the indicated time points after
l
PTP1B and HePTP: The plasmids encoding glutathione-S-trans-
ferase (GST)-tagged PTP1B31 and six-histidine (His6)-tagged
HePTP32 were generous gifts from Professor Zhong-Yin Zhang
(Indiana University School of Medicine) and Professor Rebecca
FlAsH addition, pNPP was added to initiate PTP activity. Percentage activity at each
time point was normalized to uninhibited (no-FlAsH) controls. Data were fitted
according to two-phase exponential decay using SigmaPlot 10.0. (A) PTPH1-816. (B)
PTPa-406.

8096
X.-Y. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8090–8097
Page (Brown University), respectively. TCPTP and PTPH1: The plas-
mids encoding His6-tagged TCPTP (pHEH042) and PTPH1
(pERB047) have been previously described.10,11 FAP-1: a fragment
encoding FAP-1’s PTP domain (residues 2160–2481) was amplified
(template: Open Biosystems Clone ID 6718945) with Pfu DNA
polymerase (Stratagene). The PCR product and pET21b vector
DNA were cut with HindIII and gel purified, and then re-cut with
4.5. PTP activity assays
All PTP activity assays with para-nitrophenyl phosphate (pNPP)
were carried out at 22 °C in a total reaction volume of 200 lL con-
taining pNPP (0.25–20 mM) and the appropriate phosphatase (10–
500 nM) in 1ꢁ PTP buffer (50 mM 3,3-dimethylglutarate at pH 7.0,
1 mM EDTA, and 50 mM NaCl). Reactions were quenched by the
BamH1 and gel purified again. Ligation reactions (1
gase, 2
L 10ꢁ ligase buffer (New England Biolabs), ꢂ10 ng of in-
sert and ꢂ10 ng of vector in a 20 L reaction) were performed
overnight at 15 °C. Ligation products were transformed into com-
petent DH5 E. coli and plated on LB/Agar containing 100 g/mL
l
L T4 DNA li-
addition of 40 lL of 5 M NaOH. The reaction mixtures (200 lL)
l
were loaded onto a 96-well plate, and the absorbances at 405 nm
were measured. Kinetic constants were determined by fitting the
data to the Michaelis–Menten equation. To measure the effect of
l
a
l
FlAsH on PTP activity, PTP solutions (2.5
incubated in the absence (DMSO vehicle only) or presence of FlAsH
(10 M). After 2.5 h at room temperature, PTP activity in the pres-
lM) in 1ꢁ PTP buffer were
ampicillin. A single colony was isolated, and the presence of the in-
sert in the resulting plasmid (pAML001) was confirmed by restric-
l
tion digests and by DNA sequencing. PTP-PEST:
a
fragment
ence and absence of FlAsH was assayed as described above. For
inhibition-constant measurements, PTP assays were carried out
as described above, but with varying concentrations of FlAsH at a
pNPP concentration equal to the KM for the enzyme. The percent-
age activity was obtained by dividing the absorbance in the pres-
ence of FlAsH by the absorbance of the no-FlAsH control.
Inhibition constants were estimated from these data using non-lin-
ear regression analysis as previously described.27 For time-course
encoding the catalytic domain of PTP-PEST (residues 1–307) was
amplified (template: Open Biosystems Clone ID 5268732) and
cloned into pET21b essentially as described above (resulting plas-
mid: pERB041). Restriction sites used were EcoRI and XhoI. PTP
a: a
fragment encoding the catalytic domain of PTP (residues 212–
a
503) was amplified (template: Open Biosystems Clone ID
3920094) and cloned into pET21b as above (resulting plasmid:
pERB043). Restriction sites used were SacI and HindIII.
inhibition experiments, PTPH1-816 or PTPa-406 (250 nM) and
FlAsH (1 M) were mixed and incubated for 1, 3, 5, 10, 15, 20,
l
4.3. Insertional mutagenesis
and 30 min. At the noted time points, pNPP (concentration equal
to KM for the PTP being assayed) was added to an aliquot of the
PTP/FlAsH mixture to initiate PTP activity. Reactions were
quenched after 30–60 s, and percentage activity at each time point
was obtained as described above.
PTP1B-186, HePTP-211, FAP1-2364, PTP-PEST-204, and PTPH1-
816: insertion of CCPGCC-encoding DNA into the relevant tem-
plates (see above) was carried out using Quikchange mutagenesis
essentially as previously described for the plasmid encoding
TCPTP-187.14 The resulting CCPGCC-insertion-encoding plasmids
are pXYZ092 (PTP1B-186), pACB148 (HePTP-211), pACB146
(FAP1-2364), pACB149 (PTP-PEST-204), and pMSR001 (PTPH1-
Phosphopeptide-dephosphorylation
assays
using
DAD-
EpYLIPQQG were carried out by measuring increasing absorbance
at 282 nm essentially as described.33 Assays were performed at
22 °C in a total volume of 140
50 mM 3,3-dimethylglutarate, pH 7.0, 125 mM NaCl, 1 mM EDTA,
70 M DADEpYLIPQQG, and the appropriate phosphatase pre-incu-
bated with FlAsH or DMSO vehicle. Pre-incubation mixtures con-
tained 2.5 enzyme and 10 FlAsH. Final enzyme
concentrations for the experiments shown in Figure 4 were
36 nM for PTPH1 and PTPH-816, 140 nM for PTP , and 180 nM
for PTP -406. Due to the strong inhibition of PTPH-816 and
PTP -406 in the presence of FlAsH, higher enzyme concentrations
were used to extract the relevant kinetic constants shown in Table
4: 180 nM for PTPH1-816 (+) and 1820 nM for PTP -406 (+). Ki-
lL and contained the following:
816). PTP
tion could not be achieved with the PTP
Quikchange-based approaches, and ‘inverse’ PCR13 was used in-
stead: plasmid DNA (pERB043, ꢂ50 ng in 1 L), cloned Pfu 10ꢁ
reaction buffer (5 L), primers (5 L of each at a concentration of
10 M), cloned Pfu DNA polymerase (1 L, 2.5 U, Stratagene),
2 mM dNTP mix (5 L), and water (28 L) were combined and
a-406: For reasons that are not clear, the 18-base inser-
a
-encoding template using
l
l
lM
lM
l
l
l
l
a
l
l
a
placed in a temperature cycler. The reaction mixture was subjected
to one cycle of 95 °C for 2 min, then 25 cycles of 95 °C for 1 min,
55 °C for 1 min, and 68 °C for 16 min. At the end of temperature cy-
a
a
cling, 10 U (1
l
L) of DpnI restriction enzyme was added, and the
netic constants were obtained by non-linear regression to the inte-
reaction mixture was incubated at 37 °C for 1 h. Following DpnI
grated Michaelis–Menten equation using SigmaPlot 10.0.
digestion of parental DNA, the reaction mixture was EtOH precip-
itated and re-suspended in 8
buffer, and 1 L of polynucleotide kinase (ABgene), and the reac-
tion mixture was incubated at 37 °C for 1 h. T4 DNA ligase (1 L)
was added and the mixture was incubated at 15 °C overnight.
The ligation mixture was used to transform DH5 competent cells,
and plasmids from ampicillin-resistant colonies were purified.
Upon DNA sequencing it was revealed that all of the mutated plas-
mids contained an extra base in the inserted section. One further
round of mutagenesis was carried out to delete the extra base
lL of water, 1 lL of T4 DNA ligase
Acknowledgments
l
l
This research was supported by the National Institutes of
Health (1 R15 GM071388-01A1) and Research Corporation
(CC6372).
a
Supplementary data
Sequences of all cloning and mutagenic primers. Supplementary
data associated with this article can be found, in the online version,
at doi:10.1016/j.bmc.2008.07.053.
using a standard Quikchange protocol. The resulting PTP
encoding plasmid is pXYZ290.
a-406-
4.4. Protein expression and purification
References and notes
PTPs that were expressed from pET21-based plasmids (PTPH1,
TCPTP, PTP-PEST, PTP ) contained His6-tags and were purified
using SwellGel Nickel Chelated Discs (Pierce) according to the
manufacturers’ instructions and as described,12 with one change:
IPTG inductions were carried out at 26 °C for 16 h. GST-tagged
PTP1B was purified as described.31
1. Walsh, D. P.; Chang, Y. T. Chem. Rev. 2006, 106, 2476.
a
2. Bishop, A.; Buzko, O.; Heyeck-Dumas, S.; Jung, I.; Kraybill, B.; Liu, Y.; Shah, K.;
Ulrich, S.; Witucki, L.; Yang, F.; Zhang, C.; Shokat, K. M. Annu. Rev. Biophys.
Biomol. Struct. 2000, 29, 577.
3. Bishop, A. C.; Buzko, O.; Shokat, K. M. Trends Cell Biol. 2001, 11, 167.
4. Elphick, L. M.; Lee, S. E.; Gouverneur, V.; Mann, D. J. ACS Chem. Biol. 2007, 2, 299.
5. Lin, Q.; Jiang, F.; Schultz, P. G.; Gray, N. S. J. Am. Chem. Soc. 2001, 123, 11608.

X.-Y. Zhang et al. / Bioorg. Med. Chem. 16 (2008) 8090–8097
8097
6. Alaimo, P.; Knight, Z. A.; Shokat, K. M. Bioorg. Med. Chem. 2005, 13, 2825.
7. Tonks, N. K. Nat. Rev. Mol. Cell Biol. 2006, 7, 833.
24. Wang, Z.; Shen, D.; Parsons, D. W.; Bardelli, A.; Sager, J.; Szabo, S.; Ptak, J.;
Silliman, N.; Peters, B. A.; van der Heijden, M. S.; Parmigiani, G.; Yan, H.; Wang,
T. L.; Riggins, G.; Powell, S. M.; Willson, J. K.; Markowitz, S.; Kinzler, K. W.;
Vogelstein, B.; Velculescu, V. E. Science 2004, 304, 1164.
25. Pallen, C. J. Curr. Top. Med. Chem. 2003, 3, 821.
26. Zanke, B.; Squire, J.; Griesser, H.; Henry, M.; Suzuki, H.; Patterson, B.; Minden,
M.; Mak, T. W. Leukemia 1994, 8, 236.
8. Bialy, L.; Waldmann, H. Angew. Chem. Int. Ed. 2005, 44, 3814.
9. Bishop, A. C.; Blair, E. R. Bioorg. Med. Chem. Lett. 2006, 16, 4002.
10. Blair, E. R.; Hoffman, H. E.; Bishop, A. C. Bioorg. Med. Chem. 2006, 14, 464.
11. Hoffman, H. E.; Blair, E. R.; Johndrow, J. E.; Bishop, A. C. J. Am. Chem. Soc. 2005,
127, 2824.
12. Bishop, A. C.; Zhang, X. Y.; Lone, A. M. Methods 2007, 42, 278.
13. Erster, O.; Eisenstein, M.; Liscovitch, M. Nat. Methods 2007, 4, 393.
14. Zhang, X. Y.; Bishop, A. C. J. Am. Chem. Soc. 2007, 129, 3812.
15. Griffin, B. A.; Adams, S. R.; Tsien, R. Y. Science 1998, 281, 269.
16. Adams, S. R.; Campbell, R. E.; Gross, L. A.; Martin, B. R.; Walkup, G. K.; Yao, Y.;
Llopis, J.; Tsien, R. Y. J. Am. Chem. Soc. 2002, 124, 6063.
27. Zhang, X.-Y.; Bishop, A. C. Biochemistry 2008, 47, 4491.
28. Bishop, A. C.; Ubersax, J. A.; Petsch, D. T.; Matheos, D. P.; Gray, N. S.; Blethrow,
J.; Shimizu, E.; Tsien, J. Z.; Schultz, P. G.; Rose, M. D.; Wood, J. L.; Morgan, D. O.;
Shokat, K. M. Nature 2000, 407, 395.
29. Blethrow, J.; Zhang, C.; Shokat, K. M.; Weiss, E. L. Curr. Protoc. Mol. Biol. 2004,
18. 18.11.1.
17. Jia, Z.; Barford, D.; Flint, A. J.; Tonks, N. K. Science 1995, 268, 1754.
18. Stuckey, J. A.; Schubert, H. L.; Fauman, E. B.; Zhang, Z. Y.; Dixon, J. E.; Saper, M.
A. Nature 1994, 370, 571.
19. Tabernero, L.; Aricescu, A. R.; Jones, E. Y.; Szedlacsek, S. E. FEBS J. 2008, 275, 867.
20. Andersen, J. N.; Mortensen, O. H.; Peters, G. H.; Drake, P. G.; Iversen, L. F.; Olsen,
O. H.; Jansen, P. G.; Andersen, H. S.; Tonks, N. K.; Moller, N. P. Mol. Cell. Biol.
2001, 21, 7117.
30. Zhang, Z. Y.; Thieme-Sefler, A. M.; Maclean, D.; McNamara, D. J.; Dobrusin, E.
M.; Sawyer, T. K.; Dixon, J. E. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 4446.
31. Shen, K.; Keng, Y. F.; Wu, L.; Guo, X. L.; Lawrence, D. S.; Zhang, Z. Y. J. Biol. Chem.
2001, 276, 47311.
32. Mustelin, T.; Tautz, L.; Page, R. J. Mol. Biol. 2005, 354, 150.
33. Zhang, Z. Y.; Maclean, D.; Thieme-Sefler, A. M.; Roeske, R. W.; Dixon, J. E. Anal.
Biochem. 1993, 211, 7.
21. Zhang, S.; Zhang, Z. Y. Drug Discovery Today 2007, 12, 373.
22. Hallé, M.; Liu, Y. C.; Hardy, S.; Théberge, J. F.; Blanchetot, C.; Bourdeau, A.;
Meng, T. C.; Tremblay, M. L. Mol. Cell. Biol. 2007, 27, 1172.
23. Côté, J. F.; Chung, P. L.; Théberge, J. F.; Hallé, M.; Spencer, S.; Lasky, L. A.;
Tremblay, M. L. J. Biol. Chem. 2002, 277, 2973.
34. Barford, D.; Flint, A. J.; Tonks, N. K. Science 1994, 263, 1397.
35. Andersen, H. S.; Iversen, L. F.; Jeppesen, C. B.; Branner, S.; Norris, K.; Rasmussen,
H. B.; Moller, K. B.; Moller, N. P. J. Biol. Chem. 2000, 275, 7101.
36. Alonso, A.; Sasin, J.; Bottini, N.; Friedberg, I.; Friedberg, I.; Osterman, A.; Godzik,
A.; Hunter, T.; Dixon, J.; Mustelin, T. Cell 2004, 117, 699.