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

开发作为杀微生物剂的 HIV-1 进入抑制剂前药

• 批准号: 8714598
• 负责人: Min Lu
• 金额: $ 3.11万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-15 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8714598
• 关键词: Development; HIV; entry; inhibitor; pre

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

描述（由申请人提供）：在看不见的疫苗中，紧急的公共卫生需要开发有效的局部菌心，可以减少女性新的HIV-1感染数量。已经清楚地鉴定出病毒细胞融合抑制剂在防止HIV-1的粘膜传播中的潜在作用。但是，据报道的GP41融合抑制剂均未在临床试验方面取得重大进展。 HIV-1的感染需要融合病毒和细胞膜，这是由GP41胞外域的两个七鸟脉区域的关联驱动的，以形成高度稳定的六螺旋束结构。尽管包含天然N36和C34肽的一个后灌注后基序没有抑制活性，但分离的肽通过在GP41上与其同源位点结合而抑制HIV-1进入。我们在此MIP VI应用中的目标是开发一种廉价，有效，结构化的“药物”形式的N-和C肽融合抑制剂，该抑制剂在原位使用时表现出显着的杀生活性。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，该数据抑制了低纳摩尔IC50值的原代HIV-1分离株的体外感染。我们提出了一种全面的，跨学科的方法，结合了高分辨率的结构确定，重组蛋白质的产生和诱变分析，病毒学和动物模型效果研究。在这个项目中，我们试图在体外和基于动物模型的体外和体内临床前的研究，旨在促进新的HIV-1 GP41 GP41肽融合抑制剂作为实用的菌心。具体目的是：1。优化和识别HIV-1肽融合抑制剂作为阴道杀菌剂的发育。 （a）识别并结合特定的氨基酸残基取代，以优化融合抑制剂肽的效力和溶解度。 （b）开发和优化可鲁棒的程序，以进行大规模的细菌表达和精选融合抑制剂肽的纯化。 （c）研究对肽抑制剂耐药性的机制，以避免引起抗性。 2。表征优化肽融合抑制剂的特异性，效力和毒性及其与CCR5抑制剂CMPD167和入口抑制剂BMS-378806的体外协同相互作用。 （a）确定优化的融合抑制剂肽对一组多种原代HIV-1分离株的病毒活性。 （b）评估其在兔模型中的毒性，免疫原性和药物稳定性。 （c）在体外研究抗病毒协同作用，以便对体内疗效测试的铅抑制剂组合进行合理的预测。 3。测试融合抑制剂肽的有效性以防止粘膜HIV-1感染。 （a）表征在人宫颈和阴道组织的HIV-1感染模型中有效肽抑制剂的特异性和效力。 （b）使用点头/scid-hu blt小鼠阴道传播模型来评估单独有效的肽抑制剂的体内效力和活动的广度，并与小分子CCR5抑制剂CMPD167和小分子进入抑制剂BMS-378806相结合。 1