Developing Cyclopeptide Nef Inhibitors to Facilitate HIV-1 Eradication

开发环肽 Nef 抑制剂以促进 HIV-1 根除

基本信息

批准号：
10652729
负责人：
Rudi Fasan
金额：
$ 53.86万
依托单位：
UNIVERSITY OF MASSACHUSETTS DARTMOUTH
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10652729
关键词：
Acquired Immunodeficiency Syndrome Adaptor Signaling Protein Adopted Affinity Amino Acids Animal Disease Models Anti-Retroviral Agents Antigen Presentation Binding Biochemical Biological Assay Biological Availability Biological Models Biophysics CD4 Positive T Lymphocytes Cell membrane Cell physiology Cell surface Cells Cellular biology Clathrin Clathrin Adaptors Complex Crystallization Cyclic Peptides Cyclization Cytotoxic T-Lymphocytes Data Defense Mechanisms Detection Development Disease Doctor of Philosophy Down-Regulation Epitopes Goals HIV-1 Host Defense Mechanism Immune Immunologic Receptors In Vitro Infection Knowledge Laboratories Lead Major Histocompatibility Complex Mediating Modality Molecular Conformation Patients Peptides Permeability Phage Display Pharmaceutical Preparations Play Posture Property Proteins Resolution Role Series Site Structure Surface T-Lymphocyte Tail Testing Therapeutic Toxic effect Transcription Factor AP-1 Viral Viral Pathogenesis Viral Physiology Virus Virus Replication Work analog antibody-dependent cell cytotoxicity base design drug discovery enhancer-binding protein AP-2 env Gene Products experience high throughput screening improved in vivo inhibitor insight mimetics nef Genes nef Protein novel protein aminoacid sequence protein function protein protein interaction rational design recruit screening small molecule small molecule libraries success

项目摘要

PROJECT SUMMARY: While currently available antiretrovirals block viral replication and thus control HIV-1 infection, they do not cure the disease; latent reservoirs of replication-competent virus persist. To eradicate HIV-1 infection, novel an- tiretrovirals must be developed. These drugs would ideally induce the killing of infected cells once latency is reversed. An attractive direction in developing such antiretrovirals is the inhibition of the HIV-1 Nef protein. By modulating surface-levels of immune receptors, Nef enables infected cells to evade host defense mechanisms. Among the many functions of Nef, surface downregulations of CD4 and major histocompatibility complex class I (MHC-I) are the most prominent and presumably most relevant in antiretroviral drug discovery. By downregu- lating CD4 from the cell surface, Nef enables CD4-induced epitopes of the viral Env protein to remain con- cealed, which renders infected cells less sensitive to antibody-dependent cellular cytotoxicity (ADCC). By downregulating MHC-I, Nef disrupts host antigen presentation so that infected cells are protected from killing by cytotoxic T lymphocytes (CTLs). Conceivably, therapeutic inhibition of these Nef functions may restore the activities of ADCC and CTLs, thus facilitating the detection and clearance of infected cells. Crystal structures solved by us showed that Nef-mediated downregulations of CD4 and MHC-I involve a common site on Nef. In each case, however, this site is remodeled by Nef’s association with target-specific, hijacked clathrin adaptor proteins (APs) to uniquely accommodate the intended substrate. Furthermore, when bound to Nef, both the CD4 cytosolic tail and the MHC-I cytosolic tail adopt curved, near-circular postures, which suggests that this multifunctional site of Nef is structurally poised to bind curved peptide sequences. We therefore hypothesize that cyclic peptides mimicking these cytosolic tails may function efficiently as inhibitors to block the correspond- ing cellular activities of Nef. Cyclic peptides (or cyclopeptides) are a promising, novel class of therapeutics, which are uniquely capable of disrupting protein-protein interactions. Importantly, high-affinity cyclopeptide in- hibitors can be developed efficiently using recently established strategies. In this project, we will develop such cyclopeptide-based Nef inhibitors. Our specific aims are: 1) use our established MOrPH-PhD platform to screen and select CD4-mimetic cyclopeptide inhibitors that bind to the Nef/AP2 complex in high affinity, and solve high-resolution crystal structures of the cyclopeptide-bound Nef/AP2 complexes to enable structure- based derivatization of the inhibitors; 2) use the same work flow to develop and optimize MHC-I-mimetic cyclo- peptides into potent inhibitors, which block recruitment of MHC-I into the Nef/AP1 complex; 3) use cell-based assays to characterize the identified Nef inhibitors for their efficacy, cell permeability, and cellular toxicity, and then use the knowledge learned to guide further derivatizations for improved properties. Successful completion of this work should yield cyclic peptide-based Nef inhibitors with high affinity in vitro and significant efficacy in cells, which could ideally be developed into novel antiretrovirals with unique therapeutic potentials.

项目概要：虽然目前可用的抗逆转录病毒药物可阻止病毒复制，从而控制 HIV-1 感染，但它们无法治愈为了根除 HIV-1 感染，需要采取新的方法来消除这种疾病；必须开发出逆转录病毒药物，一旦潜伏期结束，这些药物将理想地诱导杀死受感染的细胞。开发此类抗逆转录病毒药物的一个有吸引力的方向是抑制 HIV-1 Nef 蛋白。 Nef 调节免疫受体的表面水平，使受感染的细胞能够逃避宿主的防御机制。在 Nef 的众多功能中，CD4 和主要组织相容性复合物类的表面下调 I (MHC-I) 是抗逆转录病毒药物发现中最突出且可能最相关的。 Nef 将 CD4 从细胞表面分离出来，使 CD4 诱导的病毒 Env 蛋白表位保持不变。被封闭，这使得受感染的细胞对抗体依赖性细胞毒性（ADCC）不太敏感。 Nef 下调 MHC-I，破坏宿主抗原呈递，从而保护受感染的细胞免遭杀死可以想象，治疗性抑制这些 Nef 功能可能会恢复细胞毒性 T 淋巴细胞 (CTL) 的作用。 ADCC 和 CTL 的活性，从而促进受感染细胞的检测和清除。我们解决的结果表明，Nef 介导的 CD4 和 MHC-I 下调涉及 Nef In 上的一个共同位点。然而，在每种情况下，该位点都会通过 Nef 与特定目标、被劫持的网格蛋白适配器的关联进行重塑蛋白质（AP）独特地适应预期底物此外，当与 Nef 结合时，两者都可以。 CD4胞质尾部和MHC-I胞质尾部采取弯曲的、接近圆形的姿势，这表明这 Nef 的多功能位点在结构上准备结合弯曲的肽序列。模拟这些胞质尾部的环肽可以有效地作为抑制剂来阻断相应的 Nef 的细胞活性是一种有前景的新型治疗剂，重要的是，高亲和力环肽具有独特的破坏蛋白质-蛋白质相互作用的能力。可以使用最近制定的策略有效地开发抑制剂。在这个项目中，我们将开发这样的抑制剂。基于环肽的 Nef 抑制剂。我们的具体目标是：1) 使用我们已建立的 MOrPH-PhD 平台来筛选并选择以高亲和力与 Nef/AP2 复合物结合的 CD4 模拟环肽抑制剂，以及解析环肽结合的 Nef/AP2 复合物的高分辨率晶体结构，以实现结构- 基于抑制剂的衍生化；2) 使用相同的工作流程来开发和优化 MHC-I-模拟环- 将肽转化为有效的抑制剂，阻止 MHC-I 募集到 Nef/AP1 复合物中 3) 使用基于细胞的方法；表征已鉴定的 Nef 抑制剂的功效、细胞通透性和细胞毒性的测定，以及然后利用学到的知识指导进一步的衍生化以改进性能。这项工作应该会产生基于环肽的 Nef 抑制剂，在体外具有高亲和力，并且在细胞，理想情况下可以开发成具有独特治疗潜力的新型抗逆转录病毒药物。