Characterization of JT-4-173, a Potent Antiviral that Inhibits HIV-1 by a Novel Mechanism of Action

JT-4-173 的表征，一种通过新颖作用机制抑制 HIV-1 的强效抗病毒药物

基本信息

批准号：
10762518
负责人：
Xin Wen
金额：
$ 4.77万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-11 至 2027-07-10
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10762518
关键词：
Acquired Immunodeficiency Syndrome Active Sites Adherence Affect Anti-Retroviral Agents Antiviral Agents Base Sequence Behavior Binding Binding Sites Biochemical Biological Assay Biology Catalytic Domain Cessation of life Clinical Complementary DNA Complex Cryoelectron Microscopy DNA DNA Binding DNA biosynthesis DNA-Directed DNA Polymerase Data Detection Development Drug Targeting Drug resistance Effectiveness Enzymes Epidemic FDA approved Foundations Future Generations Genome HIV HIV-1 Health Services Accessibility Highly Active Antiretroviral Therapy Hydrophobicity In Vitro Individual Infection Length Life Ligation Long Terminal Repeats Molecular Molecular Conformation Molecular Mechanisms of Action Mutate Nucleic Acids Oral Pancreatic ribonuclease Patients Persons Pharmaceutical Preparations Pharmacotherapy Physiological Polymerase Polymers Primer Extension Process Provirus Integration RNA RNA-Directed DNA Polymerase Recombinants Reporting Reverse Transcription Ribonuclease H Role Scientist Site Structure Techniques Therapeutic Training Treatment Protocols Viral Viral Load result Virus Virus Replication Work X-Ray Crystallography common treatment curative treatments design drug resistant virus ds-DNA endonuclease experimental study improved inhibitor insight medical specialties mutant nanopore non-nucleoside reverse transcriptase inhibitors novel polymerization prevent standard care training opportunity transmission process viral DNA viral RNA

项目摘要

ABSTRACT As of 2021, over 38 million individuals are living with human immunodeficiency virus type 1 (HIV-1). Currently, there is no cure for HIV, and these individuals are subjected to life-long, oral drug therapy. Highly active antiretroviral therapy (HAART) is the standard treatment regimen comprising a cocktail of several antiretroviral drugs. HAART has made remarkable strides in controlling viral loads, reducing transmission, and preventing the onset of acquired immunodeficiency syndrome (AIDS) for patients with access to treatment. However, the long- term effectiveness of HAART remains threatened due to drug resistance. Therefore, developing novel antiretroviral drugs with different mechanisms of action (MOA) is required to improve therapeutic options. Of all approved antiretrovirals, the HIV-1 reverse transcriptase (RT) is one of the most frequently targeted enzymes in the virus. The primary role of RT is to convert the single-strand viral RNA genome into double-strand DNA (dsDNA) for host-genome integration. Non-nucleoside RT inhibitors (NNRTI) are one of the FDA-approved classes of antiretroviral often included in HAART. It stops the formation of the viral dsDNA by binding to RT at a distinct hydrophobic pocket (NNIBP) in proximity but not overlapping the enzyme’s polymerase active site. JT- 4-173 (JT) is a novel potent HIV-1 specific inhibitor previously identified in the Sarafianos lab. A recent structural study in our lab revealed that JT binds to the NNIBP, yet it does not inhibit normal polymerization. Our preliminary data showed that JT has no significant impact on early nor late-stage RT products, but the number of integrated proviruses significantly reduced. This behavior was not observed in any of the currently known NNRTIs. I hypothesize that JT inhibits the polymerization activity required for strand displacement synthesis, an essential step in late-stage reverse transcription for the complete formation of the dsDNA. In the proposal, I aim to determine the impact of JT on early-, intermediate- and late-reverse transcription products along with 1- and 2- long terminal repeat (LTR) circles and integrated provirus (Aim 1.1), the exact stopping site(s) of reverse transcription by JT using sequencing techniques (Aim 1.2), and the impact of JT on HIV-1 RT in strand displacement synthesis using a primer-extension biochemical assay (Aim 1.3). To determine the molecular mechanism of JT, I will use cryogenic electron microscopy (cryo-EM) to solve the HIV-1 RT/JT structures in complex with dsDNA substrates relevant to sites of strand displacement (Aim 2.1), then identify and mutate the key interactive residues on RT to confirm its effect on strand displacement synthesis using the primer-extension assay (Aim 2.2). These studies will reveal JT’s exact impact on HIV-1 reverse transcription and elucidate the unique MOA of a novel NNRTI. I will take full advantage of our lab’s background in RT biology and our collaborators’ specialties in structural, sequencing, and biochemical studies to complete these proposed experiments and advance my training. These planned studies will not only lay the foundation for developing a novel antiretroviral but will also improve our understanding of HIV-1 RT biology.

抽象的截至2021年，超过3800万人患有人类免疫缺陷病毒1型（HIV-1）。现在，艾滋病毒无法治愈，这些人受到终身口服药物治疗。高度活跃抗逆转录病毒疗法（HAART）是完成几种抗逆转录病毒鸡尾酒的标准治疗方案毒品。 Haart在控制病毒载荷，减少传播并防止可获得治疗患者获得的免疫缺陷综合征（AIDS）的发作。但是，长期 HAART的期限有效性仍然受到耐药性的威胁。因此，发展小说具有不同作用机理（MOA）的抗逆转录病毒药物需要改善治疗选择。所有批准的抗逆转录病毒，HIV-1逆转录酶（RT）是最常见的靶向酶之一病毒。 RT的主要作用是将单链病毒RNA基因组转化为双链DNA （DSDNA）用于宿主基因组整合。非核苷RT抑制剂（NNRTI）是FDA批准的一种 HAART经常包括抗逆转录病毒类。它通过在A处与RT结合来阻止病毒dsDNA的形成近端的独特疏水口袋（NNIBP），但不会重叠酶的聚合酶活性位点。 jt- 4-173（JT）是先前在Sarafianos实验室中鉴定出的一种新型潜在的HIV-1特异性抑制剂。最近的结构在我们的实验室中的研究表明，JT与NNIBP结合，但并未抑制正常聚合。我们的初步数据表明，JT对早期或晚期RT产品没有重大影响，但综合的数量预科病毒大大减少。在任何当前已知的NNRTI中都没有观察到这种行为。我假设JT抑制链置换合成所需的聚合活性，这是必不可少的在晚期逆转录中逐步完成DSDNA的完整形成。在提案中，我的目标是确定JT对早期，中间和晚期转录产物以及1-和2-的影响长时间重复（LTR）电路和集成的病毒（AIM 1.1），确切的停止位点反向 JT使用测序技术（AIM 1.2）和JT对HIV-1 RT在链中的影响使用引物扩展生化测定法（AIM 1.3）的位移合成。确定分子 JT的机理，我将使用低温电子显微镜（Cryo-EM）求解HIV-1 RT/JT结构与dsDNA底物相关的复合物与链位位点相关（AIM 2.1），然后识别并突变 RT上的关键交互式残基，以确认其对使用引物延伸的影响链位移合成的影响测定（AIM 2.2）。这些研究将揭示JT对HIV-1逆转录的确切影响，并阐明新颖的NNRTI的独特MOA。我将充分利用我们实验室的RT生物学背景和我们的背景合作者在结构，测序和生化研究方面的专业，以完成这些建议实验并推进我的培训。这些计划的研究不仅将为发展一个基础新型的抗逆转录病毒，但也将提高我们对HIV-1 RT生物学的理解。