Mechanism of Action of Novel Dual Actiing Pyrimidinediones

项目描述（由申请人提供）：本项目旨在明确小分子嘧啶二酮类HIV抑制剂IQP-0410的抗HIV活性机制。iip -0410通过两种不同的机制抑制HIV复制，作为非核苷类逆转录酶抑制剂，同时也有效抑制病毒进入。与其他nnrti不同，IQP-0410同时抑制HIV-1和HIV-2。IQP-0410阻止HIV进入靶细胞的能力是通过一种独特的机制发生的，并且已经发现该化合物对临床HIV毒株比实验室衍生的病毒更有效。拟议的研究将利用体外试验来选择和基因型和表型鉴定对iip -0410的抗性病毒。该项目将重点研究病毒gag、pol和包膜基因中产生耐药性的突变。将设计一系列嵌合病毒，其中包含野生型HIV-1和HIV-2序列，以及在选择过程的早期、中期和后期阶段来自抗性病毒的序列，以检查单个结构域对病毒的IQP-0410抗性和适应性的贡献。伪病毒拥有来自野生型和抗iqc -0410的HIV-1和HIV-2病毒的包膜糖蛋白，将评估它们在存在和不存在iqc -0410的情况下感染靶细胞的能力，并量化环境突变对iqc -0410抗病毒活性的影响。在抗性选择鉴定的包膜gp120和gp41结构域中，具有位点特异性突变的伪病毒将被用来确定iqc -0410在进入抑制中靶向的包膜结构内的构象表位。此外，实验将进一步了解gag基因突变的作用，并评估IQP-0410对感染细胞中病毒成熟的潜在影响。抗性突变对病毒RT和蛋白酶活性的影响也将采用生化分析进行检测。公共卫生相关性本拨款提案的目标是确定人类免疫缺陷病毒（HIV）小分子抑制剂的作用机制。已知HIV抑制剂（IQP-0410）可抑制病毒感染和复制的两个基本阶段（逆转录和病毒进入），因此具有提供与单药联合治疗的潜力。拟议的研究将确定病毒内化合物的分子靶标，化合物抑制病毒复制的机制，以及病毒中发生的使其对化合物产生抗性的突变以及这些突变对病毒复制和传染性的影响。

DESCRIPTION (provided by applicant): The goal of this project is to define the mechanism of anti-HIV activity of the small molecule pyrimidinedione HIV inhibitor IQP-0410. IQP-0410 inhibits HIV replication by two distinct mechanisms, acting as a nonnucleoside reverse transcriptase inhibitor while also potently suppressing virus entry. Unlike other NNRTIs, IQP-0410 inhibits both HIV-1 and HIV-2. The ability of IQP-0410 to prevent HIV entry into target cells occurs through a unique mechanism and the compound has been found to be more potent against clinical strains of HIV than against laboratory-derived viruses. The proposed research will utilize in vitro assays for selection and genotypic and phenotypic characterization of viruses resistant to IQP-0410. The program will focus on resistance engendering mutations within the viral gag, pol and envelope genes. A series of chimeric viruses containing wild type HIV-1 and HIV-2 sequences and sequences from resistant viruses at early, mid, and late stages of the selection process will be engineered to examine the contribution of the individual domains to IQP-0410 resistance and fitness of the virus. Pseudotype viruses possessing envelope glycoproteins from wild type and IQP-0410-resistant HIV-1 and HIV-2 viruses will be evaluated for their ability to infect target cells in the presence and absence of IQP-0410 and to quantify the effects of env mutations on the antiviral activity of IQP-0410. Pseudotype viruses with site-specific mutations engineered into envelope gp120 and gp41 domains identified by resistance selection will be utilized to define the conformational epitope within the envelope structure targeted by IQP-0410 in entry inhibition. Additionally, experiments will be performed to further understand the role of mutations in the gag gene and evaluate potential effects of IQP-0410 on virus maturation from infected cells. The impact of resistance mutations on the activity of viral RT and protease will also be examined using biochemical analysis. PUBLIC HEALTH RELEVANCE The goal of this grant proposal is to define the mechanism of action of a small molecule inhibitor of human immunodeficiency virus (HIV). The HIV inhibitor (IQP-0410) is known to inhibit two essential stages of virus infection and replication (reverse transcription and virus entry), and thus has the potential for providing a combination therapy with administration of a single drug. The proposed research will define the molecular targets of the compound within the virus, the mechanism by which the compound inhibits virus replication, and mutations that occur in the virus that make it resistant to the compound and the effects of these mutations on virus replication and infectivity.