Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106, USA.
Biochemistry. 2010 Jan 26;49(3):601-10. doi: 10.1021/bi901579y.
Efavirenz (EFV) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 reverse transcriptase (RT) used for the treatment of AIDS. RT is a heterodimer composed of p66 and p51 subunits; p51 is produced from p66 by C-terminal truncation by HIV protease. The monomers can form p66/p66 and p51/p51 homodimers as well as the p66/p51 heterodimer. Dimerization and efavirenz binding are coupled processes. In the crystal structure of the p66/p51-EFV complex, the drug is bound to the p66 subunit. The binding of efavirenz to wild-type and dimerization-defective RT proteins was studied by equilibrium dialysis, tryptophan fluorescence, and native gel electrophoresis. A 1:1 binding stoichiometry was determined for both monomers and homodimers. Equilibrium dissociation constants are approximately 2.5 microM for both p66- and p51-EFV complexes, 250 nM for the p66/p66-EFV complex, and 7 nM for the p51/p51-EFV complex. An equilibrium dissociation constant of 92 nM for the p66/p51-EFV complex was calculated from the thermodynamic linkage between dimerization and inhibitor binding. Binding and unbinding kinetics monitored by fluorescence were slow. Progress curve analyses revealed a one-step, direct binding mechanism with association rate constants k(1) of approximately 13.5 M(-1) s(-1) for monomers and heterodimer and dissociation rate constants k(-1) of approximately 9 x 10(-5) s(-1) for monomers. A conformational selection mechanism is proposed to account for the slow association rate. These results show that efavirenz is a slow, tight-binding inhibitor capable of binding all forms of RT and suggest that the NNRTI binding site in monomers and dimers is similar.
依非韦伦（EFV）是一种 HIV-1 逆转录酶（RT）的非核苷逆转录酶抑制剂（NNRTI），用于治疗艾滋病。RT 是一种由 p66 和 p51 亚基组成的异二聚体；p51 由 HIV 蛋白酶通过 C 端截断从 p66 产生。单体可以形成 p66/p66 和 p51/p51 同源二聚体以及 p66/p51 异源二聚体。二聚化和依非韦伦结合是偶联过程。在 p66/p51-EFV 复合物的晶体结构中，药物与 p66 亚基结合。通过平衡透析、色氨酸荧光和天然凝胶电泳研究了依非韦伦与野生型和二聚化缺陷 RT 蛋白的结合。确定了单体和同源二聚体的 1:1 结合化学计量比。对于 p66-和 p51-EFV 复合物，平衡解离常数约为 2.5 μM，对于 p66/p66-EFV 复合物，平衡解离常数约为 250 nM，对于 p51/p51-EFV 复合物，平衡解离常数约为 7 nM。从二聚化和抑制剂结合的热力学联系计算出 p66/p51-EFV 复合物的平衡解离常数为 92 nM。通过荧光监测的结合和去结合动力学较慢。进展曲线分析显示，单体和异源二聚体的结合机制为一步直接结合，结合速率常数 k1 约为 13.5 M-1 s-1，解离速率常数 k-1 约为 9 x 10-5 s-1。提出了构象选择机制来解释缓慢的结合速率。这些结果表明，依非韦伦是一种缓慢、紧密结合的抑制剂，能够结合所有形式的 RT，并表明单体和二聚体中 NNRTI 结合位点相似。
