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）：由于目前还没有疫苗，公共卫生部门迫切需要开发一种有效的外用杀菌剂，以减少女性新发 HIV-1 感染的数量。基于病毒-细胞融合抑制剂的杀微生物剂在预防 HIV-1 粘膜传播方面的潜在作用已得到明确证实。然而，所报道的 gp41 融合抑制剂均未在临床试验方面取得重大进展。 HIV-1感染需要病毒和细胞膜的融合，由gp41胞外域中两个七肽重复区域的联合驱动，形成高度稳定的六螺旋束结构。虽然这种包含天然 N36 和 C34 肽的融合后基序没有抑制活性，但分离的肽通过与 gp41 上的同源位点结合来抑制 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 N 肽和 C 肽融合抑制剂“前药”形式，该抑制剂在原位使用时表现出显着的杀菌活性。我们的开发工作将基于通过截短的六螺旋束获得的初步数据，该六螺旋束可抑制具有低纳摩尔 IC50 值的初级 HIV-1 分离株的体外感染。我们提出了一种综合的、跨学科的方法，结合了高分辨率结构测定、重组蛋白生产和诱变分析、病毒学和动物模型功效研究。在这个项目中，我们寻求进行体外和体内临床前和基于动物模型的研究，旨在促进新型 HIV-1 gp41 肽融合抑制剂作为实用杀微生物剂的开发。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) 识别并纳入特定的氨基酸残基取代，以优化融合抑制肽的效力和溶解度。 (b) 开发和优化用于大规模细菌表达和纯化选定融合抑制肽的稳健程序。 (c) 研究肽抑制剂的耐药机制，以避免引发耐药性。 2. 表征优化的肽融合抑制剂的特异性、效力和毒性及其与 CCR5 抑制剂 CMPD167 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肽对多种主要 HIV-1 分离株的杀病毒活性。 (b) 评估其在兔模型中的毒性、免疫原性和药物稳定性。 (c) 研究体外抗病毒协同作用，以便对用于体内功效测试的先导抑制剂组合做出合理预测。 3. 测试融合抑制肽预防粘膜HIV-1感染的有效性。 (a) 在人类宫颈和阴道组织的 HIV-1 感染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使用以及与小分子 CCR5 抑制剂 CMPD167 和小分子进入抑制剂 BMS 组合的体内效力和活性广度-378806。 1