Biochemical Mechanisms of Drug Resistance in HIV-1 RT

说明(申请人提供)：核苷逆转录酶(RT)抑制剂在HIV-1感染和艾滋病的治疗中发挥着重要作用；然而，这些药物的长期使用受到耐药突变株选择的限制。这些化合物产生链终止核苷酸，这些核苷酸被HIV-1RT结合并阻止DNA链的进一步延长。该实验室的工作有助于表明HIV-1 RT可以通过转移到核苷酸受体来催化体外切割末端核苷酸，并且HIV-1的一类主要核苷抗性突变体编码RT具有更高的核苷酸依赖切割活性。研究的目的是：(A)确定决定体外切割反应效率的因素，包括受体底物的结构特征、链末端残基的结构和引物-模板中的序列元件；(B)研究焦磷酸的结构类似物磷甲酸钠(PFA)抑制切割和DNA合成的机制，并确定PFA抗性突变体的抗性机制；(C)研究非核苷类RT抑制剂对切割和dNTP切割的抑制作用，以及(D)确定决定哪些细胞内化合物作为体内切除的受体底物的因素--即在未感染和HIV-1感染的个体的淋巴细胞中。此外，研究还建议扩展这些研究，以确定体内控制切除的因素，使用酶分析来检测切除的产物。这项研究的目标是确定切除活动在HIV-1感染中的作用，这对于开发更有效的抗HIV药物和治疗策略至关重要，特别是针对耐药病毒的出现。

DESCRIPTION (provided by applicant): Nucleoside reverse transcriptase (RT) inhibitors play an important role in therapy for HIV-1 infection and AIDS; however, long-term use of these drugs is limited by the selection of resistant mutants. These compounds give rise to chain-terminating nucleotides that are incorporated by HIV-1 RT and block further DNA chain elongation. Work from this laboratory was instrumental in showing that HIV-1 RT can catalyze the excision in vitro of chain-terminating nucleotides by transfer to a nucleotide acceptor and that a major class of nucleoside resistant mutants of HIV-1 encodes RT with increased nucleotide-dependent excision activity. Research is proposed to (a) define factors that determine the efficiency of the excision reaction in vitro including structural features of the acceptor substrate, structure of the chain-terminating residue, and sequence elements in the primer-template, (b) investigate the mechanism of inhibition of excision and DNA synthesis by foscarnet (PFA), a structural analog of pyrophosphate, and to determine the mechanism of resistance to PFA by PFA-resistant mutants, (c) investigate the effect of non-nucleoside RT inhibitors on excision and on dNTP inhibition of excision, and (d) to identify factors that determine which intracellular compounds serve as acceptor substrates for excision in vivo -- i.e., in lymphocytes from uninfected and HIV-1 infected individuals. In addition, research is proposed to extend these studies to identify factors that control excision in vivo using an enzymatic assay to detect the products of excision. The goal of this research is to define the role of the excision activity in HIV-1 infection, which will be crucial for the development of more effective drugs and therapeutic strategies against HIV, particularly against the emergence of resistant virus.