Developing Cyclopeptide Nef Inhibitors to Facilitate HIV-1 Eradication

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，新型抗逆转录病毒药物- 必须开发病毒。一旦潜伏期逆转，这些药物将理想地诱导杀死感染细胞。 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。通过调制 Nef是免疫受体的表面水平，它使受感染的细胞能够逃避宿主的防御机制。中 Nef的许多功能，CD 4和主要组织相容性复合物I类（MHC-I）的表面下调 是抗逆转录病毒药物发现中最突出的，也可能是最相关的。通过下调CD 4 从细胞表面看，Nef使病毒Env蛋白的CD 4诱导表位保持隐蔽， 使感染的细胞对抗体依赖性细胞毒性（ADCC）较不敏感。通过下调 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细胞的杀伤 淋巴细胞（CTL）。可以想象，这些Nef功能的治疗性抑制可以恢复 ADCC和CTL，从而促进感染细胞的检测和清除。我们解决的晶体结构 表明Nef介导的CD 4和MHC-I的下调涉及Nef上的共同位点。在每种情况下， 然而，这个位点被Nef与靶特异性的、被劫持的网格蛋白衔接蛋白（AP）的结合所重塑。 以唯一地容纳预期的衬底。此外，当与Nef结合时，CD 4胞质尾 和MHC-I胞质尾部采取弯曲，近圆形的姿势，这表明，这个多功能的网站， Nef可以被环肽靶向，这是一类很有前途的新疗法，非常适合破坏蛋白质- 蛋白质相互作用在有希望的初步数据的支持下，该项目旨在开发小型macrocy- 环状肽，能够模拟CD 4和MHC-I的胞质尾部，从而阻断细胞活性 通过抑制Nef介导的蛋白质-蛋白质相互作用。高亲和力环肽Nef抑制剂将 通过最近在我们的实验室中建立的使能战略来开发。特别是一种强大的噬菌体 展示平台将用于优化可与Nef/AP 2结合的CD 4模拟环肽抑制剂 具有高亲和力的复合物。同时，将应用类似的工作流程来开发和优化MHC-I-模拟物 将MHC-I环肽转化为有效的抑制剂，其可以阻断MHC-I募集到Nef/AP 1复合物中。高分辨率 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。使用一组基于细胞的测定，将表征这些化合物在以下方面的功效： 阻断Nef在细胞中的功能、细胞渗透性和细胞毒性，这些知识将被用来 指导进一步衍生化以提高细胞活性。成功完成这项工作应产生循环 基于肽的Nef抑制剂在体外具有高亲和力，在细胞中具有显著功效，其可以理想地 开发成具有独特治疗潜力的新型抗逆转录病毒药物。

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.