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.

Direct Chemical Activation of a Rationally Engineered Signaling Enzyme

“…[11] We found, however, that incubation of either TC Shp2 or TC/C333P Shp2 with 1 μM FlAsH gives rise to a measurable increase in PTP activity (Figure 2), suggesting that FlAsH is indeed capable of disrupting the autoinhibitory inter-domain interactions in the TC mutants in accordance with our design strategy. The activation of TC Shp2 is detectable but modest (Figure 2A), consistent with a model in which enzyme activation by FlAsH binding at the EF loop is in competition with inhibition induced by FlAsH binding at Shp2’s naturally occurring PTP-domain allosteric site.…”

“…The activation of TC Shp2 is detectable but modest (Figure 2A), consistent with a model in which enzyme activation by FlAsH binding at the EF loop is in competition with inhibition induced by FlAsH binding at Shp2’s naturally occurring PTP-domain allosteric site. [11] By contrast, the activation of TC/C333P Shp2 is robust: a FlAsH-induced PTP-activity increase of approximately 3-fold is consistently observed over a range of substrate concentrations (Figure 2B). Based on TC Shp2’s low level of activation, as well as the potential complications deriving from simultaneous activating and inactivating FlAsH-binding events on the enzyme, we focused our further efforts on TC/C333P Shp2, which contains only a single FlAsH-binding site and demonstrates strong, unambiguous FlAsH-induced activation.…”

“…(We have previously found that, in contrast to other classical PTPs, Shp2’s PTP domain can be inhibited by biarsenicals due to the presence of C333, a non-conserved cysteine residue that constitutes part of a Shp2-specific allosteric-inhibition site. [11] )…”

EGF augments TGFβ-induced epithelial–mesenchymal transition by promoting SHP2 binding to GAB1

“…These and other findings motivate the ongoing search for specific inhibitors of SHP2. New strategies to develop SHP2 inhibitors that target allosteric binding sites unique to SHP2 have shown promise in achieving SHP2 specificity (Yu et al, 2013;Chio et al, 2015). Therapies developed using such strategies would have potential application in many diseases.…”

Targeting EPO and EPO receptor pathways in anemia and dysregulated erythropoiesis

“…PTPN11 inhibitors, however, are successfully being developed for candidate use in juvenile myelomonocytic leukemia and Noonan disease 129-131 . For such inhibitors attention should be paid to possible compromising effects on erythropoiesis.…”