Aminoglycoside microbicides restore natural expression of anti-HIV-1 retrocyclins

氨基糖苷类杀菌剂恢复抗 HIV-1 逆转录环素的自然表达

• 批准号: 8318565
• 负责人: ALEXANDER MICHAEL COLE
• 金额: $ 47.1万
• 依托单位: UNIVERSITY OF CENTRAL FLORIDA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-25 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8318565
• 关键词: Adverse effects; Affect; Affinity; Amino Acids; Aminoglycoside Antibiotics; Aminoglycosides; Antibiotics; Antiviral Agents; Attention; Binding; Biological Assay; Cells; Cervical; Clinical Trials; Complex; Cyclic Peptides; Defensins; Endogenous Factors; Epithelial Cells; Eukaryota; Exhibits; Film; Genes; Genetic Code; Goals; HIV; HIV-1; Hela Cells; Human; Human Genome; Immune; Immunity; In Vitro; Indigenous; Infection; Lactobacillus; Lead; Ligation; Local Microbicides; Macaca mulatta; Macaca nemestrina; Measures; Mediating; Mucous Membrane; Natural Immunity; Nonsense Codon; Organ Culture Techniques; Peptides; Pharmaceutical Preparations; Phase; Pilot Projects; Process; Production; Progranulocytes; Proteins; Route; Safety; Seminal Plasma; Sexual Partners; Sexual Transmission; Site; Surface; Terminator Codon; Testing; Time; Tissues; Topical application; Toxic effect; Translations; Vagina; Virus Diseases; Woman; Work; base; cytokine; empowered; human tissue; in vivo; inhibitor/antagonist; microbial; microbicide; multidisciplinary; nonhuman primate; preclinical study; prevent; prophylactic; rectal; retrocyclin; transmission process; vaccine development; vaginal fluid

DESCRIPTION (provided by applicant): Sexual transmission through mucosal surfaces has been the most common route of HIV-1 spread throughout the world. Although much attention has been focused on vaccine development for HIV-1, progress has been slow and there is an urgent need to find alternative approaches, such as topical microbicides, to target the spread of HIV-1. Human a- and ¿-defensins contribute to innate immune defenses against microbial and viral infections; however, these endogenous factors are minimally effective in preventing the spread of HIV. Certain nonhuman primates also produce ?-defensins - 18 residue cyclic peptides that act as HIV-1 entry inhibitors. Multiple human ?-defensin genes exist, but they harbor a premature termination codon that blocks translation. Consequently, the ?-defensins encoded within the human genome (called retrocyclins in humans) are not expressed as peptides. Based on the genetic code contained within the otherwise intact retrocyclin genes, we recreated retrocyclins synthetically, and revealed them to be remarkably active against R5 and X4 isolates from a wide variety of HIV-1 clades. While in vivo production of ?-defensins in rhesus macaques involves the post-translational ligation of two nine-residue peptides, neither the mechanism of this unique process nor its existence in human cells was known - until now. We are proposing a paradigm shift in how a microbicide could act to prevent HIV-1 transmission, through the use of inexpensive and widely available aminoglycoside antibiotics to enhance vaginal innate immunity against HIV-1. In eukaryotes, aminoglycosides can suppress premature termination codons through the incorporation of an amino acid in its place. In preliminary studies, we restored the expression of retrocyclin peptides by human promyelocytic and HeLa cells using aminoglycosides that suppressed retrocyclin's premature termination codon, and revealed that these natural retrocyclins were active against HIV-1. Based on our studies, we hypothesize that aminoglycosides can restore the natural production of retrocyclins in the cervicovaginal mucosa, and can be formulated as quick-release films to confer increased protection against sexually transmitted HIV-1. With assistance from a multidisciplinary team, we will test these hypotheses in the R21 phase by: 1) assessing the ability of aminoglycosides to induce sustained expression of retrocyclins in human vaginal epithelial cells, organotypic cervicovaginal tissues and cervical organ cultures, and 2) evaluating the ability of topically applied aminoglycosides to induce in vitro and ex vivo toxicity and proinflammatory cytokines in the vaginal mucosa. A lead aminoglycoside, exhibiting the greatest activity while exhibiting minimal adverse effects to the vaginal mucosa, will proceed to the R33 phase in which we will: 3) formulate the aminoglycoside as a quick-release film to induce natural anti-HIV-1 retrocyclin expression, and 4) evaluate the vaginal safety of the film-formulated aminoglycoside in pigtailed macaques. Aminoglycoside-mediated translation of retrocyclins could result in a new class of microbicide to prevent HIV-1 transmission.

描述（通过应用程序提供）：整个粘膜表面的性传播一直是HIV-1的最常见途径。尽管已经关注HIV-1的疫苗开发，但进步却很慢，迫切需要寻找替代方法，例如局部菌心剂，以靶向HIV-1的传播。人类A-和�-防御素有助于对微生物和病毒感染的先天免疫防御措施；但是，这些内源性因素在防止艾滋病毒的传播方面至少有效。某些非人类私人也会产生吗？ - 防御素-18个残留的循环宠物，充当HIV-1进入抑制剂。存在多个人类？ - 防御素基因，但它们具有阻止翻译的过早终止密码子。因此，在人类基因组中编码的 - 二防御素（人类中称为retrocyclens）未表达为宠物。基于原本完整的折叠基因基因中包含的遗传密码，我们通过合成重新创建了折叠蛋白，并揭示了它们对来自各种HIV-1进化枝的R5和X4分离株的活性非常活跃。当恒河猕猴中的体内产生？ - 二弱素涉及两种九个二氧化胡椒的翻译后连接，但直到现在，这种独特过程的机制也不是其在人类细胞中的存在的机制 - 到目前为止。我们提出，通过使用廉价且广泛可用的氨基糖苷抗生素来增强对HIV-1的阴道先天性免疫力，杀菌剂如何起作用以防止HIV-1传播的范式转移。在真核生物中，氨基糖苷可以通过将氨基酸掺入其位置来抑制过早的终止密码子。在初步研究中，我们使用氨基糖苷抑制了逆转录蛋白的过早终止密码子的氨基糖苷恢复了人类寄生虫细胞和HeLa细胞的折叠蛋白胡椒体的表达，并揭示了这些天然折叠蛋白对HIV-1具有活性。基于我们的研究，我们假设氨基糖苷可以恢复子宫颈粘膜中折返蛋白的自然产生，并且可以作为快速释放膜形成，以增加对性传播HIV-1的保护。在多学科团队的协助下，我们将通过以下方面在R21阶段测试这些假设，评估氨基糖苷在人类阴道上皮细胞，有机宫颈组织和宫颈培养物中诱导返回蛋白的持续表达的能力，以及评估对氨基化剂量的氨基化剂量的能力，并诱导氨基化剂的能力，并赋予氨基化剂的能力。阴道粘膜中的细胞因子。铅氨基糖苷在对阴道粘膜中表现出最小的不良反应时表现出最大的活性，将继续进入R33阶段，我们将在其中：3）制定氨基糖苷作为快速释放膜，以诱导天然抗HIV-HIV-1逆转录蛋白表达，并评估膜型AmInogcos的阴道安全性。氨基糖苷介导的折叠剂的翻译可能会导致一类新的杀菌剂，以防止HIV-1传播。