Developing Cyclopeptide Nef Inhibitors to Facilitate HIV-1 Eradication

项目总结： 虽然目前可用的抗逆转录病毒药物可以阻止病毒复制，从而控制艾滋病毒-1感染，但它们并不能治愈 这种疾病；具有复制能力的病毒的潜在储存库仍然存在。为了根除HIV-1感染，新的和 必须开发抗逆转录病毒药物。这些药物最理想的情况是，一旦潜伏期延长，就会导致感染细胞的死亡 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-1Nef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防御机制。 在Nef的众多功能中，CD4和主要组织相容性复合体类的表面下调 I(MHC-I)是抗逆转录病毒药物发现中最突出的，也可能是最相关的。通过降级- Nef使CD4诱导的病毒包膜蛋白的表位保持连接，从而使细胞表面的CD4结合后的抗原表位保持不变。 CELAD，这使得感染细胞对抗体依赖的细胞毒性(ADCC)不那么敏感。通过 下调MHC-I，Nef扰乱宿主抗原提呈，从而保护受感染的细胞免受杀戮 通过细胞毒性T淋巴细胞(CTL)。可想而知，对这些Nef功能的治疗抑制可能会恢复 ADCC和CTL的活性，从而有助于检测和清除受感染的细胞。晶体结构 我们的研究表明，Nef介导的CD4和MHC-I的下调涉及Nef上的一个共同位点。在……里面 然而，每一种情况下，这个位置都被Nef与靶标特异的、被劫持的笼状蛋白适配器联系在一起 蛋白质(AP)，以独特地适应预期的底物。此外，当绑定到Nef时， CD4胞浆尾巴和MHC-I胞浆尾巴采用弯曲的、近圆形的姿势，这表明这 Nef的多功能位点在结构上准备与弯曲的多肽序列结合。因此，我们假设 模拟这些胞浆尾巴的环肽可能有效地发挥抑制作用，以阻断相应的- Nef的ING细胞活性。环肽(或环肽)是一类很有前途的新型治疗药物， 它们是唯一能够破坏蛋白质之间相互作用的物质。重要的是，高亲和力的环肽- 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 以环肽为基础的Nef抑制剂。我们的具体目标是：1)利用我们建立的Morph-Phd平台 筛选和选择与Nef/AP2复合体高亲和力结合的CD4模拟环肽抑制剂，以及 解决环肽结合的Nef/AP2络合物的高分辨晶体结构，以使结构- 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-模拟环状化合物。 多肽转化为有效的抑制剂，阻止MHC-I重新进入Nef/AP1复合体；3)使用基于细胞的 对已鉴定的Nef抑制剂的有效性、细胞渗透性和细胞毒性进行表征的分析，以及 然后使用所学的知识来指导进一步的派生以改进属性。成功完成 这项工作应该产生环肽为基础的Nef抑制剂，在体外具有高亲和力和显著的疗效 细胞，理想情况下可以发展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 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.