Thiuram Disulfides as Pseudo‐irreversible Inhibitors of Lymphoid Tyrosine Phosphatase

The improper regulation of tyrosine phosphorylation is implicated in multiple human diseases including diabetes, cancer, hypertension and autoimmunity.[1] As a consequence, there is significant interest in identifying chemical inhibitors of the enzymes responsible for regulating tyrosine phosphorylation, namely the tyrosine kinases and protein tyrosine phosphatases (PTPs). One enzyme of particular interest in autoimmunity is the lymphoid tyrosine phosphatase (LYP, PTPN22), a negative regulator of early T cell receptor (TCR) signaling.[2] A single nucleotide polymorphism (SNP) C1858T in the PTPN22 gene results in a gain of function variant of LYP that has been associated with numerous autoimmune disorders including type I diabetes, rheumatoid arthritis, systemic lupus erythematosus, Graves’ disease, and myasthenia gravis.[3, 4] On the other hand, another SNP in the same PTPN22 gene, G788A, leads to a putative loss of function variant that is found to be protective against systemic lupus erythematosus.[5] LYP is also a positive regulator of anaphylaxis and inhibition of LYP helps to mitigate anaphylaxis in mice.[6] Taken together, these findings reinforce the potential of LYP inhibition for the treatment of immune system disorders. The catalytic domains of PTPs are highly homologous and all contain the PTP signature motif, (H/V)C(X)5R(S/T), in the conserved active site.[7] The local environment in the PTP active site lowers the pKa of the active site cysteine residue, making it highly reactive and susceptible to oxidation.[8] Therefore, several PTPs including LYP are inactivated by reversible or irreversible oxidation of thecatalytic cysteine residue upon exposure to oxidizing agents.[8–11] Our laboratory has previously shown that nitrosating agents such as sodium nitroprusside and the active site-directed NO-donating peptide, Ac-ARLIEDNE-(HCyNO)-TAREG-NH2, cause reversible, competitive and covalent oxidative inactivation of LYP.[12] Thiophilic heavy metals ions including zinc(II), gold(I) and mercury(II) readily inhibit LYP activity.[13–16] In addition, S-thiolation can inhibit PTP activity. For example, PTP1B is inhibited by hydrogen sulfide-mediated S-thiolation during ischemia and reperfusion.[17] Furthermore, in the presence of excess of H2O2 in vitro, the catalytic cysteine residue of LYP forms a disulfide bond with C129 to reversibly inactivate the enzyme.[18] These examples illustrate the diverse mechanisms by which PTP activity can be regulated by virtue of the reactivity of the catalytic cysteine residue. Thiuram disulfides are a chemically distinct class of disulfides that can inhibit enzymes containing low pKa thiols by forming mixed disulfides.[19] A prototype of this class of compounds, disulfiram (Figure 1, trade name Antabuse), is used clinically for the treatment of alcohol dependence.[20] Disulfiram inhibits aldehyde dehydrogenase, an enzyme involved in alcohol metabolism, through S-thiolation, leading to acetaldehyde build-up and unpleasant symptoms following the ingestion of alcohol.[21] Disulfiram, along with another thiuram disulfide, thiram (Figure 1), have also been shown to possess anticancer activity.[22–24] The anticancer activity of disulfiram has been attributed to S-thiolation of several biological targets including protein kinase C (PKC),[19] glutathione-S-transferase (GST),[25] nuclear factor-κB[26] and the caspases.[27] Based on the known reactivity of disulfiram towards biological thiolates and the knowledge that LYP is inactivated by cysteine oxidation and disulfide bond formation, we hypothesized that disulfiram and other thiuram disulfides can inhibit LYP by S-thiolation. Figure 1 Chemical Structures of disulfiram and thiram.