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：
10759561
负责人：
Rudi Fasan
金额：
$ 70.18万
依托单位：
UNIVERSITY OF MASSACHUSETTS DARTMOUTH
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-01 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10759561
关键词：
Acquired Immunodeficiency Syndrome Adaptor Signaling Protein Adopted Affinity Amino Acids Animal Model 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 Cyclic Peptides Cyclization Cytoprotection Cytotoxic T-Lymphocytes Data Defense Mechanisms Derivation procedure Detection Development Disease Doctor of Philosophy Dose Down-Regulation Epitopes Experimental Designs Goals HIV-1 Host Defense Mechanism Human Immune Immunologic Receptors In Vitro Infection Knowledge Laboratories Lead Major Histocompatibility Complex Measures Mediating Modality Molecular Conformation Patients Penetration Peptides Permeability Phage Display Pharmaceutical Preparations Play Posture 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 design drug discovery enhancer-binding protein AP-2 env Gene Products high throughput screening improved in vivo inhibitor insight mimetics nef Genes nef Protein novel peptidomimetics protein function protein protein interaction rational design recruit screening small molecule small molecule libraries success synergism unnatural amino acids

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 antiretro- virals 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 downregulating CD4 from the cell surface, Nef enables CD4-induced epitopes of the viral Env protein to remain concealed, 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 can be targeted by cyclic peptides, a promising new class of therapeutics well-suited to disrupt protein- protein interactions. Supported by promising preliminary data, this project aims to develop small-sized macrocy- clic peptides capable of mimicking the cytosolic tails of CD4 and MHC-I and thus blocking the cellular activities of Nef through inhibition of Nef-mediated protein-protein interactions. High-affinity cyclopeptide Nef inhibitors will be developed through enabling-strategies recently established in our laboratories. Specifically, a powerful phage display platform will be applied to optimize CD4-mimetic cyclopeptide inhibitors that can bind to the Nef/AP2 complex with high affinity. In parallel, a similar workflow will be applied to develop and optimize MHC-I-mimetic cyclopeptides into potent inhibitors that can block recruitment of MHC-I into the Nef/AP1 complex. High-resolu- tion structures of the cyclopeptide-Nef complexes will be obtained to enable structure-based optimization of the Nef inhibitors. Using a panel of cell-based assays, these compounds will be characterized for their efficacy in blocking Nef functions in cells, cell permeability, and cellular toxicity, and this knowledge will be leveraged to guide further derivatization for improved cellular activity. 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，新型抗逆转录病毒药物仍存在。必须开发出病毒。一旦潜伏期逆转，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通过调节抑制 HIV-1 Nef 蛋白。在免疫受体的表面水平，Nef 使受感染的细胞能够逃避宿主的防御机制。 Nef 的许多功能、CD4 和主要组织相容性复合体 I 类 (MHC-I) 的表面下调通过下调 CD4 是抗逆转录病毒药物发现中最突出且可能最相关的。从细胞表面来看，Nef 能够使 CD4 诱导的病毒 Env 蛋白表位保持隐藏，从而通过下调，使受感染的细胞对抗体依赖性细胞毒性 (ADCC) 不太敏感。 MHC-I、Nef 破坏宿主抗原呈递，从而保护受感染的细胞免受细胞毒性 T 的杀伤可以想象，治疗性抑制这些 Nef 功能可能会恢复淋巴细胞 (CTL) 的活性。 ADCC 和 CTL，从而有助于我们解决的受感染细胞的检测和清除。显示 Nef 介导的 CD4 和 MHC-I 下调涉及 Nef 上的共同位点。然而，该位点通过 Nef 与目标特异性、被劫持的网格蛋白衔接蛋白 (AP) 的关联而被重塑独特地容纳预期底物此外，当与 Nef 结合时，CD4 胞质尾部。 MHC-I 胞质尾部采取弯曲的、接近圆形的姿势，这表明该多功能位点 Nef 可以通过环肽作为目标，环肽是一种很有前景的新型疗法，非常适合破坏蛋白质 - 在有希望的初步数据的支持下，该项目旨在开发小尺寸的大环蛋白。 clic 肽能够模仿 CD4 和 MHC-I 的胞质尾部，从而阻断细胞活性高亲和力环肽 Nef 抑制剂将通过抑制 Nef 介导的蛋白质-蛋白质相互作用来发挥 Nef 的作用。通过我们实验室最近建立的启用策略来开发，具体来说，是一种强大的噬菌体。展示平台将用于优化可与 Nef/AP2 结合的 CD4 模拟环肽抑制剂同时，类似的工作流程将用于开发和优化 MHC-I 模拟物。环肽转化为有效的抑制剂，可以阻止 MHC-I 募集到高分辨率的 Nef/AP1 复合物中。将获得环肽-Nef复合物的结构，以实现基于结构的优化 Nef 抑制剂。将使用一组基于细胞的测定来表征这些化合物的功效。阻断 Nef 在细胞、细胞通透性和细胞毒性方面的功能，这些知识将被用来指导进一步衍生化以改善细胞活性。成功完成这项工作应该会产生循环。基于肽的 Nef 抑制剂在体外具有高亲和力，在细胞中具有显着疗效，理想情况下开发成具有独特治疗潜力的新型抗逆转录病毒药物。