Ohtaka Hiroyasu, Schön Arne, Freire Ernesto
Department of Biology and Biocalorimetry Center, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Biochemistry. 2003 Nov 25;42(46):13659-66. doi: 10.1021/bi0350405.
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 1 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.
对蛋白酶抑制剂产生抗性的病毒株的出现是HIV-1/AIDS化疗中最严重的问题之一。最普遍的耐药突变体是那些影响所有临床使用抑制剂的突变体。在本文中，我们对一种HIV-1蛋白酶的多重耐药突变体进行了表征，该突变体影响茚地那韦、奈非那韦、沙奎那韦、利托那韦、安普那韦和洛匹那韦。这个突变体（MDR-HM）在酶的活性位点内外包含六个氨基酸突变（L10I/M46I/I54V/V82A/I84V/L90M）。微量量热法和酶动力学测量表明，这个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，这表明底物能够适应由突变引起的变化并维持其结合亲和力。为了理解抗性的起源，还研究了在特定区域含有突变的三个亚突变体，即活性位点（V82A/I84V）、瓣区（M46I/I54V）和二聚化区域（L10I/L90M）。这些突变组单独一个都不会使抑制剂的亲和力降低超过1个数量级，此外，每组突变的效应之和也不等于总体效应，这表明存在协同效应。仅含有四个活性位点突变（V82A/I84V/M46I/I54V）的突变体仅表现出较小的协同效应，这表明二聚体界面处的突变（L10I/L90M）在引发协同反应中起主要作用。这些研究表明，协同相互作用对总体抗性的平均贡献为1.2±0.7千卡/摩尔，大部分协同效应（0.8±0.7千卡/摩尔）由二聚化界面处的突变介导。并非所有临床使用的抑制剂都受到突变之间远程协同相互作用的相同影响。这些相互作用可以使单个突变的效应放大2至40倍，具体取决于抑制剂。将耐药性的能量学分解为焓和熵成分，为抑制剂反应提供了定量描述，并为设计对这类突变敏感性较低的抑制剂提供了一套热力学指导原则。
