Multidrug resistance to HIV-1 protease inhibition requires cooperative coupling between distal mutations
Multidrug resistance to HIV-1 protease inhibition requires cooperative coupling between distal mutations
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DOI：
10.1021/bi0350405
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发表时间：
2003-11-25
期刊：
影响因子：
2.9
通讯作者：
Freire, E
中科院分区：
文献类型：
--
作者：
Ohtaka, H;Schön, A;Freire, E
对蛋白酶抑制剂具有耐药性的病毒株的出现是HIV-1/AIDS化疗中最严重的问题之一。最普遍的耐药突变是那些在临床使用中影响所有抑制剂的突变。在这篇文章中，我们鉴定了HIV-1蛋白酶的一个多药耐药突变体，它影响依地那韦、奈非那韦、沙奎那韦、利托那韦、氨丙那韦和洛比那韦。该突变株(MDR-HM)含有位于酶活性部位内外的6个氨基酸突变(L10I/M46I/I54V/V82A/I84V/L90M)。微量热法和酶动力学测量表明，该突变体使所有抑制剂的亲和力降低了2-3个数量级。相比之下，多重耐药突变体仅使底物的K-m增加了2倍，表明底物能够适应突变引起的变化，并保持其结合亲和力。为了了解耐药性的来源，我们还研究了三个含有特定区域突变的亚突变体，即活性部位(V82A/I84V)、翻盖区(M46I/I54V)和二聚化区(L10I/L90M)。这些突变本身都不会使抑制剂的亲和力降低超过I个数量级，此外，每组突变的影响总和并不是总体影响的总和，这表明存在协同效应。一个只包含四个活性位点突变(V82A/I84V/M46I/I54V)的突变体仅表现出很小的协同效应，这表明二聚体界面(L10I/L90M)的突变在引发协同反应中起着主要作用。这些研究表明，协同作用对总抗性的贡献率平均为1.2+/-0.7kcal/mol，其中大部分协同效应(0.8+/-0.7kcal/mol)是由二聚界面的突变介导的。并不是所有临床使用的抑制剂都受到突变之间的远程协同作用的影响。这些相互作用可以放大单个突变的影响，根据抑制物的不同，因子在2到40之间。将耐药性的能量学分解为焓和熵成分，提供了对抑制剂反应的定量解释，并为设计对这种类型突变敏感性较低的抑制剂提供了一套热力学指南。
The appearance of viral strains that are resistant to protease inhibitors is one of the most serious problems in the chemotherapy of HIV-1/AIDS. The most pervasive drug-resistant mutants are those that affect all inhibitors in clinical use. In this paper, we have characterized a multiple-drug-resistant mutant of the HIV-1 protease that affects indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, and lopinavir. This mutant (MDR-HM) contains six amino acid mutations (L10I/M46I/I54V/V82A/I84V/L90M) located within and outside the active site of the enzyme. Microcalorimetric and enzyme kinetic measurements indicate that this mutant lowers the affinity of all inhibitors by 2-3 orders of magnitude. By comparison, the multiiple-drug-resistant mutant only increased the K-m of the substrate by a factor of 2, indicating that the substrate is able to adapt to the changes caused by the mutations and maintain its binding affinity. To understand the origin of resistance, three submutants containing mutations in specific regions were also studied, i.e., the active site (V82A/I84V), flap region (M46I/I54V), and dimerization region (L10I/L90M). None of these sets of mutations by themselves lowered the affinity of inhibitors by more than I order of magnitude, and additionally, the sum of the effects of each set of mutations did not add up to the overall effect, indicating the presence of cooperative effects. A mutant containing only the four active site mutations (V82A/I84V/M46I/I54V) only showed a small cooperative effect, suggesting that the mutations at the dimer interface (L10I/L90M) play a major role in eliciting a cooperative response. These studies demonstrate that cooperative interactions contribute an average of 1.2 +/- 0.7 kcal/mol to the overall resistance, most of the cooperative effect (0.8 +/- 0.7 kcal/mol) being mediated by the mutations at the dimerization interface. Not all inhibitors in clinical use are affected the same by long-range cooperative interactions between mutations. These interactions can amplify the effects of individual mutations by factors ranging between 2 and 40 depending on the inhibitor. Dissection of the energetics of drug resistance into enthalpic and entropic components provides a quantitative account of the inhibitor response and a set of thermodynamic guidelines for the design of inhibitors with a lower susceptibility to this type of mutations.