Developing Cyclopeptide Nef Inhibitors to Facilitate HIV-1 Eradication

开发环肽 Nef 抑制剂以促进 HIV-1 根除

• 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
• 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

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阻断病毒复制，从而控制HIV-1感染，但它们不能治愈 这种疾病;具有复制能力的病毒的潜伏库持续存在。为了根除HIV-1感染，新的和- 必须开发逆转录病毒药物。一旦潜伏期延长，这些药物将理想地诱导杀死受感染的细胞。 颠倒了开发这种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。通过 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的许多功能中，CD4和主要组织相容性复合物类的表面下调是Nef的主要功能之一。 MHC-I是抗逆转录病毒药物发现中最突出和可能最相关的。通过downregu- Nef从细胞表面除去CD4，使病毒Env蛋白的CD4诱导的表位保持与细胞表面的CD4结合。 细胞，这使得感染的细胞对抗体依赖性细胞毒性（ADCC）不太敏感。通过 Nef下调MHC-I，破坏宿主抗原呈递，从而保护受感染的细胞免受杀伤 细胞毒性T淋巴细胞（CTL）。可以想象，这些Nef功能的治疗性抑制可以恢复这些功能。 ADCC和CTL的活性，从而促进感染细胞的检测和清除。晶体结构 结果表明Nef介导的CD4和MHC-I的下调涉及Nef上的一个共同位点。在 然而，在每一种情况下，Nef与靶特异性的、被劫持的网格蛋白接头的关联都会重塑该位点。 蛋白质（AP），以独特地适应预期的基板。此外，当与Nef绑定时， CD4胞质尾和MHC-I胞质尾采取弯曲的、接近圆形的姿势，这表明这可能是由于细胞内的CD4细胞的凋亡。 Nef的多功能位点在结构上准备结合弯曲的肽序列。因此我们假设 模拟这些胞质尾部的环肽可以作为抑制剂有效地阻断相应的- Nef的细胞活性。环肽（或环肽）是一类有前途的新型治疗剂， 其独特地能够破坏蛋白质-蛋白质相互作用。重要的是，高亲和力环肽在- 使用最近建立的策略可以有效地开发抑制剂。在这个项目中，我们将开发这样的 基于环肽的Nef抑制剂。我们的具体目标是：1）利用我们建立的MOrPH-PhD平台， 筛选和选择以高亲和力结合Nef/AP 2复合物的CD4模拟环肽抑制剂，和 解析环肽结合的Nef/AP 2复合物的高分辨率晶体结构， 基于抑制剂的衍生化; 2）使用相同的工作流程开发和优化MHC-I模拟环- 肽转化为有效的抑制剂，其阻断MHC-I募集到Nef/AP 1复合物中; 3）使用基于细胞的 用于表征所鉴定的Nef抑制剂的功效、细胞渗透性和细胞毒性的测定，以及 然后使用所学习的知识来指导进一步的衍生以改进性质。成功完成 这项工作的结果应该会产生基于环肽的Nef抑制剂，其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。