Chemical probes for lymphoid tyrosine phosphatase

Lymphoid tyrosine phosphatase (LYP) is an intriguing therapeutic target for the treatment of several autoimmune disorders including anaphylaxis. Despite this, its precise biological functions in signaling cascades and cellular physiology are still poorly understood. Chemical probes can be beneficial for addressing biological questions pertaining to LYP by creating chemical knockdowns as well as for therapeutic inhibition of LYP. However, to be useful, these chemical probes need to be selective for LYP. Crucial insights about selective targeting of LYP can be gained from studying how LYP demonstrates substrate specificity in vivo. The substrate specificity of LYP in vivo can be attributed to the interactions of the substrates with the nonconserved features of LYP and to the biological regulation of LYP. Exploiting these interactions with nonconserved features of the LYP catalytic domain as well as mimicking the regulatory mechanisms can help in targeting LYP selectively over other homologous protein tyrosine phosphatases (PTPs). Peptides, being the closest mimics of biological substrates of LYP, can target both the conserved and nonconserved features of LYP. We have taken advantage of these desirable features of peptides by using a peptide substrate in screening for potent and selective LYP inhibitors. When used in inhibitor screens, the inhibitor potencies against this peptide substrate were found to correlate more strongly with the biological activity than those against a traditional small molecule substrate. From this iv inhibitor screening exercise, we identified epigallocatechin-3,5-digallate as the most potent LYP inhibitor reported so far (IC50 = 50 nM). We have also exploited one of the regulatory mechanisms of LYP, oxidation of the catalytic cysteine residue, to identify thiuram disulfides as a new class of covalent inhibitors of LYP. The best hit from a library of thiuram disulfides screened, compound 13, was shown to inhibit LYP with Ki of 1.1 ± 0.4 μM and kinact of 0.0040 ± 0.0007 s-1 in a pseudo-irreversible manner by forming mixed disulfide with the LYP. We have also developed peptide-based covalent inhibitors of LYP that take advantage of the selectivity offered by the peptide sequences and combine it with the utility of an electrophilic, cysteine-reactive, phosphotyrosine (pY) mimetic, warhead. We have separately optimized the electrophilic warhead and the peptide sequence to identify LDLL-(VsN)-SDDD as the best peptide-based covalent LYP inhibitor. This optimized inhibitor inhibited LYP with an IC50 of 20 μM and showed selective inhibition of LYP over other homologous PTPs. In summary, we have developed a toolkit of chemical probes for studying the activities of LYP.