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

描述（由申请人提供）：在没有疫苗的情况下，迫切需要开发一种有效的局部杀微生物剂，以减少妇女中新感染艾滋病毒-1的人数。基于病毒-细胞融合抑制剂的杀微生物剂在预防HIV-1粘膜传播中的潜在作用已被明确确定。然而，没有报道的gp41融合抑制剂在临床试验中取得重大进展。HIV-1感染需要病毒和细胞膜的融合，由gp41外结构域的两个七联体重复区域的结合驱动，形成高度稳定的六螺旋束结构。虽然这个融合后基序包括天然的N36和C34肽没有抑制活性，但分离的肽通过结合gp41上的同源位点来抑制HIV-1的进入。我们在MIP VI应用中的目标是开发一种廉价、有效、结构化的N-和c -肽融合抑制剂的“前药物”形式，这种抑制剂在原位使用时表现出显著的杀微生物活性。我们的开发工作将基于截断的六螺旋束获得的初步数据，该数据可抑制具有低纳摩尔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，旨在促进开发新的HIV-1 gp41肽融合抑制剂作为一种实用的杀微生物剂。具体目标是：1。优化和鉴定HIV-1肽融合抑制剂作为阴道杀微生物剂的发展。(a)确定并纳入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)开发和优化筛选融合抑制肽的大规模细菌表达和纯化的稳健程序。(c)研究对肽抑制剂的抗性机制，以避免引起抗性。2. 研究优化后的肽融合抑制剂的特异性、效力和毒性，以及它们与CCR5抑制剂CMPD167和进入抑制剂BMS-378806的体外协同作用。(a)确定优化后的融合抑制肽对多种原代HIV-1分离株的杀病毒活性。(b)在兔模型上评价其毒性、免疫原性和药物稳定性。(c)研究体外抗病毒协同作用，对铅抑制剂组合进行合理预测，进行体内疗效试验。3. 目的：检测融合抑制肽对粘膜HIV-1感染的保护作用。(a)在人类宫颈和阴道组织感染HIV-1的体外模型中确定有效肽抑制剂的特异性和效力。(b)利用NOD/SCID-hu BLT小鼠阴道传播模型，评估高效肽抑制剂单独使用和与小分子CCR5抑制剂CMPD167和小分子进入抑制剂BMS-378806联合使用的体内效价和活性广度。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 PUBLIC HEALTH RELEVANCE: In the absence of an effective vaccine against HIV-1, there is an urgent public health need to find alternative approaches, such as vaginally applied microbicide gels, to prevent heterosexual HIV-1 transmission. Since blocking HIV-1 entry is the first line of defense against viral infection, the HIV-1 envelope glycoprotein is a favored target for microbicide development. Several HIV-1 fusion and entry inhibitors have been shown to be effective in blocking SHIV transmission in a rhesus macaque model. The results of this project will lead to preclinical proof-of-concept for optimized fusion-inhibitory peptides as a practical microbicide.