Covalent Allosteric Inactivation of Protein Tyrosine Phosphatase 1B (PTP1B) by an Inhibitor–Electrophile Conjugate

Abstract

Protein tyrosine phosphatase 1B (PTP1B) is a validated drug target, but it has proven difficult to develop medicinally useful, reversible inhibitors of this enzyme. Here we explored covalent strategies for the inactivation of PTP1B using a conjugate composed of an active site-directed 5-aryl-1,2,5-thiadiazolidin-3-one 1,1-dioxide inhibitor connected via a short linker to an electrophilic α-bromoacetamide moiety. Inhibitor-electrophile conjugate 5a caused time-dependent loss of PTP1B activity consistent with a covalent inactivation mechanism. The inactivation occurred with a second-order rate constant of (1.7 ± 0.3) × 10 M min. Mass spectrometric analysis of the inactivated enzyme indicated that the primary site of modification was C121, a residue distant from the active site. Previous work provided evidence that covalent modification of the allosteric residue C121 can cause inactivation of PTP1B [Hansen, S. K., Cancilla, M. T., Shiau, T. P., Kung, J., Chen, T., and Erlanson, D. A. (2005) Biochemistry 44, 7704-7712]. Overall, our results are consistent with an unusual enzyme inactivation process in which noncovalent binding of the inhibitor-electrophile conjugate to the active site of PTP1B protects the nucleophilic catalytic C215 residue from covalent modification, thus allowing inactivation of the enzyme via selective modification of allosteric residue C121.

Potential Therapeutic Target Protein Tyrosine Phosphatase-1B for Modulation of Insulin Resistance with Polyphenols and Its Quantitative Structure–Activity Relationship

“…The docking result of silydianin was also compared with some of the reported inhibitors (PDB ID: 5T19, 2QBP, 1T49 and 4I8N) and it was observed to be interacting with the residues of the active site. Arg221 and Gln266 were observed to be common interacting residues in silydianin and the reported inhibitors [ 60 , 61 , 62 , 63 ].…”

Covalent inhibition of protein tyrosine phosphatases

“…Recently, new covalent allosteric inhibition of PTPs was also reported where covalent modification of cysteines present in pockets close to the active site led to loss of phosphatase activity. 151 Fluorogenic reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABDF, compound 48) (Fig. 22) was reported as an allosteric inhibitor of PTP1B.…”

Cysteine modifiers suggest an allosteric inhibitory site on the CAL PDZ domain

“…There is a small but growing literature on the development of covalent allosteric enzyme inhibitors. For example, covalent modification on allosteric residue Cys 121 can inactivate protein tyrosine phosphatase 1B [48]. Non-catalytic residues Cys 296 and Cys 310 of AKT were targeted by effective inhibitors that stabilize the inactive conformation of the kinase [49].…”