The Interdependence of Drug Resistance: HIV-1 Protease

描述（由申请人提供）：人类免疫缺陷病毒1型（HIV-1）蛋白酶（PR）是对3400万HIV感染者进行高效抗逆转录病毒治疗（HAART）的五种病毒靶点之一。病毒蛋白酶是关键靶标，因为它负责通过加工Gag-Pol多肽使病毒体成熟。患者对HAART方案的依从性至关重要，因为不依从会导致病毒持续复制。病毒复制逃逸和随后的反弹通过允许携带各种耐药突变的病毒群体的生长而阻碍HAART治疗。病毒药物靶点（包括病毒蛋白酶）内的突变允许抑制逃避和持续 生物功能。了解严重耐药的机制是抑制剂开发的主要障碍。突变数量的增加与抗性的严重程度不成正比，这表明抗性不是简单的相加，而是相互依赖的。我认为，物理氨基酸的性质，位置和耐药突变的组合的顶点是多药耐药的相互依赖的性质。为了探索潜在的模式，在病毒蛋白酶的相互依赖的耐药机制的基础上，我将使用一组五个多药耐药（MDR）蛋白酶来自患者。该组中的蛋白酶各自具有19-26个突变，并且甚至对最有效的蛋白酶抑制剂（PI）具有抗性。使用一系列的生物化学和生物物理技术，我将确定在体外抑制和热力学概况的每一个蛋白酶在面板。我将使用X射线晶体学和分子动力学模拟的结构和动态特性的物理方面的相互依存的阻力模式。除了蛋白酶变体之外，我还获得了它们从NC到Gag的p6（残基407-488）的同源底物。我将使用患者来源的蛋白酶及其相应的底物来确定底物识别和加工如何在抑制剂存在下使用上述技术继续进行。识别和利用病毒靶点的多药耐药性机制可以提供必要的工具，积极改善目前的治疗和HIV-1靶点的抑制剂设计。

DESCRIPTION (provided by applicant): The human immunodeficiency virus type 1 (HIV-1) protease (PR) is one of five viral targets of highly active anti-retroviral therapy (HAART) administered to the 34 million individuals living with HIV. The viral protease is a critical targetas it is responsible for virion maturation via processing of the Gag-Pol polypeptide. Patient adherence to HAART regimens is crucial, as non-adherence leads to continuous viral replication. Viral replication escape and subsequent rebound impedes HAART treatment by allowing for the growth of viral populations bearing various resistant mutations. The mutations within viral drug targets, including the viral protease, allow for inhibition evasion and continued biological function. Understanding the mechanisms underlying severe drug resistance is a major hindrance in inhibitor development. Increasing mutation number is not directly proportional to the severity of resistance, suggesting that resistance is not simply additive but that it is interdependent. I propose that the culmination of physical amino acid properties, locations, and combinations of resistant mutations underlie interdependent nature of multi-drug resistance. To probe potential patterns that underlie the interdependent mechanisms of resistance in the viral protease, I will use a panel of five multi-drug resistance (MDR) proteases derived from patients. The proteases in this panel bear between 19-26 mutations each and are resistant to even the most potent protease inhibitors (PIs). Using an array of biochemical and biophysical techniques, I will determine the in vitro inhibition and thermodynamic profiles for each of the proteases in th panel. I will use X-Ray crystallography and molecular dynamics simulations to structurally and dynamically characterize the physical aspects of interdependent resistance patterns. In addition to the protease variants, I have also obtained their cognate substrates from NC to p6 of Gag (residues 407-488). I will use the patient-derived proteases and their corresponding substrates to determine how substrate recognition and processing is allowed to continue in the presence of inhibitors using the techniques described above. Discerning and taking advantage of the mechanisms that underlie multi-drug resistance in viral targets could provide the tools necessary to proactively meliorate both current treatment and inhibitor design for HIV-1 targets.