Small-molecule inhibitors of gp41-mediated fusion as HIV-1 topical microbicides

说明(申请人提供)：在持续缺乏有效疫苗的情况下，局部杀微生物剂提供了一种可靠的替代预防策略，以减少艾滋病毒-1的性传播。几种病毒融合和进入抑制剂已被证明可以通过阴道和/或直肠途径防止SIV感染恒河猴，并作为杀微生物剂候选药物处于临床前和早期临床开发阶段。HIV-1膜融合是由gp41包膜糖蛋白的一系列大规模结构转变所介导的。有证据表明，一种被称为发夹前中间体的暂时性gp41物种是抑制HIV-1进入的药物的潜在靶点。这项研究计划的长期目标是使用现代分子和结构方法来鉴定和开发一种新的小分子gp41融合抑制剂，用于局部使用的HIV-1杀菌剂。为了实现这一目标，我们将利用我们最近对gp41自主折叠的三聚螺旋线圈亚域以及通过创新的基于结构的药物设计技术开发的小分子先导化合物的结构测定所揭示的特定表面特征，该亚域为假定的前发夹构象提供了一个原子模型。我们为本项目的R21部分提出了以下具体目标：1)通过靶向gp41前发夹中间体来鉴定和优化两系列新型小分子化合物，这些小分子化合物通过靶向gp41前发夹中间体来抑制HIV-1膜融合。我们将设计和合成两组活性三氮酮和联苯类化合物，表征与N-三聚体盘状线圈相互作用的平衡性质，并评价它们的抗HIV-1活性和作用机制。结合的抑制剂将通过X射线结晶学可视化，以允许结合亲和力的精炼。该项目R33阶段的具体目标是：2)表征具有增强的gp41抑制活性的改良小分子化合物的特异性、效力和毒性。我们将在原代细胞和人宫颈阴道组织外植体中进行体外研究，以确定选定的小分子gp41抑制化合物对不同的HIV-1初级分离株的杀毒活性，以及它们潜在的毒性或炎症效应。我们还将使用兔阴道刺激模型来评估融合抑制剂的刺激潜力。3)采用NOD/SCID-Hu BLT小鼠阴道传播模型，评价优化的小分子融合抑制剂单独及与针对HIV-1gp120(BMS-378806)和CCR5(CMPD167)的进入抑制剂联合应用的体内效力和广度。我们将评估单独使用小分子融合抑制剂以及与BMS-378806和CMPD167协同使用时，人源化BLT小鼠免受多种HIV-1变异株阴道攻击的保护作用。我们的重点是确定一类新的有效的HIV-1融合抑制剂，适合作为杀微生物剂配方的一种成分进行开发。

DESCRIPTION (provided by applicant): In the continuing absence of an effective vaccine, topical microbicides offer a credible alternative preventive strategy to reduce sexual transmission of HIV-1. Several viral fusion and entry inhibitors have been shown to prevent SHIV infection of rhesus macaques by the vaginal and/or rectal routes and are in preclinical and early clinical development as microbicide candidates. HIV-1 membrane fusion is mediated by a series of large-scale structural transitions in the gp41 envelope glycoprotein. Evidence indicates that a transient gp41 species known as the prehairpin intermediate is a potential target for drugs that inhibit HIV-1 entry. The long-term goal of this research plan is to use modern molecular and structural methods to identify and develop a novel small-molecule gp41 fusion inhibitor for inclusion in a topical HIV-1 microbicide. To achieve this, we will capitalize on specific surface features revealed by our recent structure determination of an autonomously folded, trimeric coiled-coil subdomain of gp41 that provides an atomic model for the putative prehairpin conformation, as well as small-molecule lead compounds developed by means of an innovative structure-based drug design technology. We propose the following specific aim for the R21 component of this project: 1) To identify and optimize two series of novel small-molecule compounds that inhibit HIV-1 membrane fusion by targeting the gp41 prehairpin intermediate. We will design and synthesize two sets of analogs of active triazinone and biphenyl compounds, characterize the equilibrium properties of interactions with the N-trimer coiled coil, and evaluate their anti-HIV-1 activity and mechanism of action. Bound inhibitors will be visualized by x-ray crystallography in order to allow refinement of binding affinity. The specific aims of the R33 phase of the project are: 2) To characterize the specificity, potency and toxicity of improved small-molecule compounds with enhanced gp41 inhibitory activity. We will conduct in vitro studies in primary cells and human cervicovaginal tissue explants to determine the virucidal activity of select small-molecule gp41 inhibitory compounds against diverse primary HIV-1 isolates, and their potentially toxic or inflammatory effects. We will also use the rabbit vaginal irritation model to evaluate the irritation potential of the fusion inhibitors. 3) To assess the in vivo potency and breadth of activity of optimized small-molecule fusion inhibitors alone and in combination with entry inhibitors targeting HIV-1 gp120 (BMS-378806) and CCR5 (CMPD167) using the NOD/SCID-hu BLT mouse vaginal transmission model. We will evaluate the protection of humanized BLT mice from vaginal challenge with multiple HIV-1 variants by small-molecule fusion inhibitors alone and in synergistic combination with BMS-378806 and CMPD167. Our emphasis is to identify a new class of potent HIV-1 fusion inhibitors suitable for development as a component of a microbicide formulation.