Development of an HIV-1 entry inhibitor pre-drug as a microbicide

描述（由申请人提供）：由于看不到疫苗，迫切需要开发一种有效的局部杀微生物剂，以减少女性新发HIV-1感染的数量。基于病毒-细胞融合通道的杀微生物剂在预防HIV-1粘膜传播中的潜在作用已被明确确定。然而，没有一个报道的gp 41融合抑制剂在临床试验方面取得了重大进展。HIV-1感染需要病毒和细胞膜的融合，由gp 41胞外域中的两个七重重复区的结合驱动，以形成高度稳定的六螺旋束结构。尽管这种包含天然N36和C34肽的融合后基序没有抑制活性，但分离的肽通过与它们在gp 41上的同源位点结合来抑制HIV-1进入。我们在这项MIP VI应用中的目标是开发一种廉价、有效、结构化的“前药”形式的N-和C-肽融合抑制剂，在原位使用时表现出显着的杀微生物活性。我们的开发工作将基于用截短的六螺旋束获得的初步数据，该截短的六螺旋束抑制具有低纳摩尔IC 50值的主要HIV-1分离株的体外感染。我们提出了一个全面的，跨学科的方法，结合高分辨率的结构测定，重组蛋白的生产和诱变分析，病毒学和动物模型的疗效研究。在这个项目中，我们寻求进行体外和体内临床前和动物模型为基础的研究，旨在促进新的HIV-1 gp 41肽融合抑制剂作为一个实用的杀微生物剂的发展。具体目标是：1。优化和鉴定HIV-1肽融合抑制剂，以开发为阴道杀微生物剂。(a)鉴定并整合优化融合抑制剂肽效力和溶解度的特定氨基酸残基取代。(b)开发和优化用于大规模细菌表达和纯化所选融合抑制剂肽的稳健程序。(c)研究对肽抑制剂的耐药机制，以避免引发耐药。2.表征优化的肽融合抑制剂的特异性、效力和毒性及其与CCR 5抑制剂CMPD 167和进入抑制剂BMS-378806的体外协同相互作用。(a)确定优化的融合抑制剂肽对多种原代HIV-1分离株的杀病毒活性。(b)在家兔模型中评价其毒性、免疫原性和药物稳定性。(c)研究体外抗病毒协同作用，以便合理预测体内有效性测试的主要抑制剂组合。3.测试融合抑制剂肽保护免受粘膜HIV-1感染的有效性。(a)在人宫颈和阴道组织HIV-1感染的体外模型中表征有效肽抑制剂的特异性和效力。(b)使用NOD/SCID-hu BLT小鼠阴道传播模型评估高效肽抑制剂单独以及与小分子CCR 5抑制剂CMPD 167和小分子进入抑制剂BMS-378806组合的体内效力和活性宽度。1

DESCRIPTION (provided by applicant): With no vaccine in sight, there is an urgent public health need to develop an effective topical microbicide that can reduce the number of new HIV-1 infections in women. The potential role of virus-cell fusion inhibitor-based microbicides in preventing mucosal transmission of HIV-1 has been clearly identified. However, none of the reported gp41 fusion inhibitors has made significant progress toward clinical trials. HIV-1 infection requires fusion of the viral and cellular membranes, driven by association of two heptad-repeat regions in the gp41 ectodomain to form a highly stable six-helix bundle structure. Whereas this postfusion motif comprising native N36 and C34 peptides has no inhibitory activity, the isolated peptides inhibit HIV-1 entry by binding to their cognate sites on gp41. Our goal in this MIP VI application is to develop an inexpensive, potent, structured 'pro- drug' form of the N- and C-peptide fusion inhibitors that exhibits significant microbicidal activity upon use in situ. Our development effort will be based on preliminary data obtained with a truncated six-helix bundle that inhibits in vitro infection by primary HIV-1 isolates with low nanomolar IC50 values. We propose a comprehensive, interdisciplinary approach that combines high-resolution structural determination, recombinant protein production and mutagenic analyses, virology, and animal model efficacy studies. In this project we seek to conduct in vitro and in vivo preclinical and animal model-based research intended to facilitate the development of new HIV-1 gp41 peptide fusion inhibitor as a practical microbicide. The Specific Aims are: 1. To optimize and identify HIV-1 peptide fusion inhibitors for development as a vaginal microbicide. (a) To identify and incorporate specific amino-acid residue substitutions that optimize both potency and solubility of fusion inhibitor peptides. (b) To develop and optimize robust procedures for the large-scale bacterial expression and purification of select fusion inhibitor peptides. (c) Investigate the mechanisms of resistance to peptide inhibitors so as to avoid eliciting resistance. 2. To characterize the specificity, potency and toxicity of optimized peptide fusion inhibitors and their in vitro synergistic interactions with the CCR5 inhibitor CMPD167 and the entry inhibitor BMS-378806. (a) Determine the virucidal activity of optimized fusion inhibitor peptides against a diverse set of primary HIV-1 isolates. (b) Evaluate their toxicity, immunogenicity and drug stability in the rabbit model. (c) Study antiviral synergy in vitro in order to make rational predictions for lead inhibitor combinations for in vivo efficacy testing. 3. To test the effectiveness of the fusion inhibitor peptides to protect against mucosal HIV-1 infection. (a) Characterize the specificity and potency of effective peptide inhibitors in an in vitro model of HIV-1 infection of human cervical and vaginal tissue. (b) Use the NOD/SCID-hu BLT mouse vaginal transmission model to assess the in vivo potency and breadth of activity of highly effective peptide inhibitors alone and in combination with the small-molecule CCR5 inhibitor CMPD167 and the small-molecule entry inhibitor BMS-378806. 1