Punthasee Puminan, Laciak Adrian R, Cummings Andrea H, Ruddraraju Kasi Viswanatharaju, Lewis Sarah M, Hillebrand Roman, Singh Harkewal, Tanner John J, Gates Kent S
Department of Chemistry, University of Missouri , 125 Chemistry Building, Columbia, Missouri 65211, United States.
Department of Biochemistry, University of Missouri , 117 Schweitzer Hall, Columbia, Missouri 65211, United States.
Biochemistry. 2017 Apr 11;56(14):2051-2060. doi: 10.1021/acs.biochem.7b00151. Epub 2017 Apr 3.
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.
蛋白酪氨酸磷酸酶1B（PTP1B）是一个经过验证的药物靶点，但事实证明，开发对该酶具有药用价值的可逆抑制剂很困难。在此，我们探索了一种使PTP1B失活的共价策略，该策略使用一种共轭物，其由一个活性位点导向的5-芳基-1,2,5-噻二唑烷-3-酮1,1-二氧化物抑制剂通过一个短连接子与一个亲电α-溴乙酰胺部分相连组成。抑制剂-亲电试剂共轭物5a导致PTP1B活性随时间依赖性丧失，这与共价失活机制一致。失活发生的二级速率常数为(1.7±0.3)×10 M⁻¹ min⁻¹。对失活酶的质谱分析表明，修饰的主要位点是C121，这是一个远离活性位点的残基。先前的研究提供了证据，表明变构残基C121的共价修饰可导致PTP1B失活[汉森，S.K.，坎西拉，M.T.，肖，T.P.，孔，J.，陈，T.，和埃尔兰森，D.A.（2005年）《生物化学》44，7704 - 7712]。总体而言，我们的结果与一个不同寻常的酶失活过程一致，即抑制剂-亲电试剂共轭物与PTP1B活性位点的非共价结合保护亲核催化性C215残基不被共价修饰，从而允许通过变构残基C121的选择性修饰使酶失活。
