Targeting a cryptic allosteric site for selective inhibition of the oncogenic protein tyrosine phosphatase Shp2

Abstract

Protein tyrosine phosphatases (PTPs) have been the subject of considerable pharmaceutical-design efforts because of the ubiquitous connections between misregulation of PTP activity and human disease. PTP-inhibitor discovery has been hampered, however, by the difficulty in identifying cell-permeable compounds that can selectively target PTP active sites, and no PTP inhibitors have progressed to the clinic. The identification of allosteric sites on target PTPs therefore represents a potentially attractive solution to the druggability problem of PTPs. Here we report that the oncogenic PTP Shp2 contains an allosteric-inhibition site that renders the enzyme sensitive to potent and selective inhibition by cell-permeable biarsenical compounds. Because Shp2 contains no canonical tetracysteine biarsenical-binding motif, the enzyme's inhibitor-binding site is not readily predictable from its primary or three-dimensional structure. Intriguingly, however, Shp2's PTP domain does contain a cysteine residue (C333) at a position that is removed from the active site and is occupied by proline in other classical PTPs. We show that Shp2's unusual cysteine residue constitutes part of a Shp2-specific allosteric-inhibition site, and that Shp2's sensitivity to biarsenicals is dependent on the presence of the naturally occurring C333. The determinative role of this residue in conferring inhibitor sensitivity is surprising because C333's side chain is inaccessible to solvent in Shp2 crystal structures. The discovery of this cryptic Shp2 allosteric site may provide a means for targeting Shp2 activity with high specificity and suggests that buried-yet-targetable allosteric sites could be similarly uncovered in other protein families.

Figure 1

Shp2 is potently and selectively inhibited by FlAsH. (A and B) After incubation (120 min) at the indicated FlAsH concentrations at pH 7.0, phosphatase activities of the indicated PTPs were measured with pNPP and normalized to a DMSO-only control for the corresponding enzyme.

Figure 2

FlAsH-induced inhibition of Shp2 requires the presence of organic arsenical moieties, is time-dependent, and is substrate-independent. (A) After incubation (120 min) with the indicated compounds, Shp2’s PTP activity was measured with pNPP and normalized to no-inhibitor controls. (B) Shp2’s rates of dephosphorylating pNPP in the presence of 1 μM FlAsH over 1 min windows were normalized to a DMSO-only control. The results of three independent experiments (grayscale) were averaged, and the averaged data (not shown) were fit as a single-variable exponential decay. (C) Phosphatase activity was measured by continuous absorbance at 282 nm with a phosphopeptide substrate (DADEpYLIPQQG) at pH 7.0 after incubation (150 min) with FlAsH (225 nM).

Figure 3

Shp2’s backdoor cysteines constitute a buried allosteric site. (A) Partial primary sequence alignment (human Shp2 numbering) of the PTPs that appear in Figure 1A. (B) Three-dimensional structure of the Shp2 catalytic domain (Protein Data Bank entry 3B7O). The PTP active site is colored magenta. The enzyme’s solvent-accessible surface is shown but rendered transparently so that the buried C333 and C367 side chains (colored by element) can be visualized.

Figure 4

Backdoor cysteine 333 is required for the FlAsH sensitivity of Shp2. (A) After incubation with the indicated FlAsH concentrations, PTP activities of the catalytic domains of wild-type and C333P Shp2 were measured with pNPP and normalized to DMSO-only controls. (B) After incubation with 500 nM FlAsH and varying concentrations of β-mercaptoethanol (β-ME) (white bars, 0 mM; light gray bars, 250 μM; dark gray bars, 500 μM; black bars, 1 mM), PTP activities of the catalytic domains of wild-type and C333P Shp2 were measured with pNPP and normalized to no-FlAsH controls. (C) Target-specific inhibition of Shp2 in a complex proteomic mixture. After incubation with the indicated FlAsH concentrations, PTP activities of lysates of cells expressing wild-type or C333P Shp2 were measured with pNPP and normalized to DMSO-only controls.

Figure 5

Protection of C333 and C367 by FlAsH. (A) Sequence of the Shp2 catalytic-domain construct used in the cysteine-protection assays, with the sequences of peptides identified in the MS experiments (1–11) highlighted. Wild-type (B) or C333P (C) Shp2 (2.7 μM) was incubated with DMSO or FlAsH (27 μM), followed by addition of iodoacetic acid (50 mM). The labeled proteins were trypsinized, and the abundances of the resulting peptides were quantitated by LC–MS/MS. Relative abundances indicate the normalized intensities of the indicated peptides in a FlAsH-treated sample as compared to its no-FlAsH control. Peptides containing positions 333 and 367 are colored orange. Peptides containing other carboxymethylated cysteines are colored blue. Other non-cysteine-containing peptides are colored gray.

Figure 6

FlAsH inhibits full-length Shp2 in a dose- and time-dependent manner. PTP activities of full-length wild-type and C333P Shp2 were measured with DiFMUP at pH 7.0 after incubation with the indicated FlAsH concentrations for 150 min (A) or with FlAsH (1 μM) for the indicated time intervals (B). Data points represent the rates of dephosphorylation over a 10 min time window (centered at the indicated time point) normalized to a DMSO-only control for the corresponding enzyme.