Generation of inhibitor-sensitive protein tyrosine phosphatases via active-site mutations

Abstract

Protein tyrosine phosphatases (PTPs) catalyze the dephosphorylation of phosphotyrosine, a central control element in mammalian signal transduction. Small-molecule inhibitors that are specific for each cellular PTP would be valuable tools in dissecting phosphorylation networks and for validating PTPs as therapeutic targets. However, the common architecture of PTP active sites impedes the discovery of selective PTP inhibitors. Our laboratory has recently used enzyme/inhibitor-interface engineering to generate selective PTP inhibitors. The crux of the strategy resides in the design of "inhibitor-sensitized" PTPs through protein engineering of a novel binding pocket in the target PTP. "Allele-specific" inhibitors that selectively target the sensitized PTP can be synthesized by modifying broad-specificity inhibitors with bulky chemical groups that are incompatible with wild-type PTP active sites; alternatively, specific inhibitors that serendipitously recognize the sensitized PTP's nonnatural pocket may be discovered from panels of "non-rationally" designed compounds. In this review, we describe the current state of the PTP-sensitization strategy, with emphases on the methodology of identifying PTP-sensitizing mutations and synthesizing the compounds that have been found to target PTPs in an allele-specific manner. Moreover, we discuss the scope of PTP sensitization in regard to the potential application of the approach across the family of classical PTPs.

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

“…PTPs that were expressed from pET21-based plasmids (PTPH1, TCPTP, PTP-PEST, PTPα) contained His 6 -tags and were purified using SwellGel Nickel Chelated Discs (Pierce) according to the manufacturer's instructions and as described,12 with one change: IPTG inductions were carried out at 26°C for 16 hours. GST-tagged PTP1B was purified as described 31…”

“…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 somewhat limited in scope, presumably due to significant differences in the detailed topology of individual PTP active sites 12. Moreover, active-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…”

Approaches to Study Phosphatases

“…applied the “bump-hole” approach of allele-specific kinase inhibition to PTPs. They sensitized PTPs to biarsenical compounds creating via mutagenesis strategies non-natural allosteric-inhibition sites or changing the active sites, thereby controlling the function of each cellular phosphatase by designing selective mutant/inhibitor pairs. , Interestingly, recently they were able to apply that approach to create a SHP2 mutant that is activatable through binding of biarsenical compounds, showing the versatility of this approach.…”

Target-specific control of lymphoid-specific protein tyrosine phosphatase (Lyp) activity

“…For a mutant enzyme/inhibitor pair to be useful in chemical biology it is imperative that the mutated protein retains activity in the absence of the drug 25, 42. To investigate the possible effect of the CCPGCC mutation on Lyp’s PTP activity, we determined the Michaelis-Menten kinetic parameters for wild-type and Lyp-CCPGCC with the artificial PTP substrate para -nitrophenyl phosphate ( p NPP).…”