The Interdependence of Drug Resistance: HIV-1 Protease

描述(申请人提供)：人类免疫缺陷病毒1型(HIV-1)蛋白酶(PR)是对3400万艾滋病毒携带者实施高效抗逆转录病毒疗法(HAART)的五个病毒靶点之一。病毒蛋白酶是一个关键的靶点，因为它负责通过加工Gag-Pol多肽来实现病毒粒子的成熟。患者对HAART方案的坚持至关重要，因为不坚持会导致病毒的持续复制。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种群的增长来阻碍HAART治疗。病毒药物靶点内的突变，包括病毒蛋白水解酶，允许逃避抑制并继续 生物功能。了解严重耐药的机制是抑制药物开发的主要障碍。突变数量的增加与抗药性的严重程度不成正比，这表明抗药性不是简单的相加，而是相互依赖的。我认为，物理氨基酸性质、位置和耐药突变组合的顶峰是多重耐药的相互依存性质的基础。为了探索病毒蛋白酶相互依赖的耐药机制的潜在模式，我将使用一组来自患者的五种多药耐药(MDR)蛋白酶。这个小组中的蛋白水解酶每个都有19-26个突变，甚至对最有效的蛋白水解酶抑制剂(PI)也有抵抗力。使用一系列生化和生物物理技术，我将确定TH小组中每一种蛋白酶的体外抑制和热力学曲线。我将使用X射线结晶学和分子动力学模拟来从结构和动态上描述相互依赖的阻力模式的物理方面。除了这些酶变异体外，我还获得了它们的同源底物，从Nc到P6的Gag(残基407-488)。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处理如何使用上述技术在抑制剂存在的情况下继续进行。识别和利用病毒靶点多重耐药的潜在机制可以提供必要的工具，以主动改善目前针对HIV-1靶点的治疗和抑制剂设计。

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.