Department of Biochemistry and Molecular Pharmacology , University of Massachusetts Medical School , Worcester , Massachusetts 01605 , United States.
Department of Biochemistry and Biophysics and the UNC Center for AIDS Research , University of North Carolina at Chapel Hill , Chapel Hill , North Carolina 27599 , United States.
ACS Chem Biol. 2019 Nov 15;14(11):2441-2452. doi: 10.1021/acschembio.9b00370. Epub 2019 Aug 13.
Drug resistance continues to be a growing global problem. The efficacy of small molecule inhibitors is threatened by pools of genetic diversity in all systems, including antibacterials, antifungals, cancer therapeutics, and antivirals. Resistant variants often include combinations of active site mutations and distal "secondary" mutations, which are thought to compensate for losses in enzymatic activity. HIV-1 protease is the ideal model system to investigate these combinations and underlying molecular mechanisms of resistance. Darunavir (DRV) binds wild-type (WT) HIV-1 protease with a potency of <5 pM, but we have identified a protease variant that loses potency to DRV 150 000-fold, with 11 mutations in and outside the active site. To elucidate the roles of these mutations in DRV resistance, we used a multidisciplinary approach, combining enzymatic assays, crystallography, and molecular dynamics simulations. Analysis of protease variants with 1, 2, 4, 8, 9, 10, and 11 mutations showed that the primary active site mutations caused ∼50-fold loss in potency (2 mutations), while distal mutations outside the active site further decreased DRV potency from 13 nM (8 mutations) to 0.76 μM (11 mutations). Crystal structures and simulations revealed that distal mutations induce subtle changes that are dynamically propagated through the protease. Our results reveal that changes remote from the active site directly and dramatically impact the potency of the inhibitor. Moreover, we find interdependent effects of mutations in conferring high levels of resistance. These mechanisms of resistance are likely applicable to many other quickly evolving drug targets, and the insights may have implications for the design of more robust inhibitors.
耐药性仍是一个日益严重的全球性问题。小分子抑制剂的疗效受到所有系统（包括抗菌药物、抗真菌药物、癌症治疗药物和抗病毒药物）中遗传多样性的影响。耐药变体通常包括活性位点突变和远端“次要”突变的组合，这些突变被认为可以补偿酶活性的丧失。HIV-1 蛋白酶是研究这些组合和耐药潜在分子机制的理想模型系统。达芦那韦（DRV）与野生型（WT）HIV-1 蛋白酶的结合效力<5 pM，但我们已经鉴定出一种对 DRV 失去效力 150,000 倍的蛋白酶变体，其在活性位点内外有 11 个突变。为了阐明这些突变在 DRV 耐药性中的作用，我们采用了多学科方法，结合酶学测定、晶体学和分子动力学模拟。对具有 1、2、4、8、9、10 和 11 个突变的蛋白酶变体进行分析表明，主要活性位点突变导致效力降低约 50 倍（2 个突变），而活性位点外的远端突变使 DRV 效力进一步降低至 13 nM（8 个突变）至 0.76 μM（11 个突变）。晶体结构和模拟表明，远端突变会引起微小的变化，这些变化会通过蛋白酶动态传播。我们的研究结果表明，远离活性位点的变化会直接、显著地影响抑制剂的效力。此外，我们发现突变赋予高耐药水平的相互依存效应。这些耐药机制可能适用于许多其他快速进化的药物靶点，并且这些发现可能对设计更稳健的抑制剂具有重要意义。
