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（由申请人提供）：通过粘膜表面的性传播是HIV-1在全世界传播的最常见途径。尽管人们对HIV-1疫苗的开发给予了很大关注，但进展缓慢，迫切需要找到替代方法，如局部杀微生物剂，以遏制HIV-1的传播。人类的一个-和- 防御素有助于对微生物和病毒感染的先天免疫防御;然而，这些内源性因素在预防HIV传播方面的效果最低。某些非人类的灵长类动物也会产生？防御素-作为HIV-1进入抑制剂的18个残基的环肽。多个人类？-存在防御素基因，但它们具有阻止翻译的提前终止密码子。因此，？在人类基因组中编码的防御素（在人类中称为逆转录细胞周期蛋白）不表达为肽。基于包含在其他完整的retrocyclin基因的遗传密码，我们重新合成retrocyclins，并揭示了它们对R5和X4分离株从各种各样的HIV-1分支显着的活性。当体内产生？-恒河猴中的防御素涉及两个9个残基肽的翻译后连接，直到现在，这种独特过程的机制及其在人类细胞中的存在都是未知的。 我们正在提出一种模式转变，即通过使用廉价且广泛可用的氨基糖苷类抗生素来增强阴道对HIV-1的先天免疫力，从而使杀微生物剂能够起到预防HIV-1传播的作用。在真核生物中，氨基糖苷类可以通过在其位置掺入氨基酸来抑制提前终止密码子。在初步研究中，我们恢复了人早幼粒细胞和HeLa细胞的retrocyclin肽的表达，使用氨基糖苷类抑制retrocyclin的提前终止密码子，并揭示了这些天然的retrocyclins对HIV-1的活性。根据我们的研究，我们假设氨基糖苷类可以恢复宫颈阴道粘膜中retrocyclins的自然产生，并可以配制成快速释放膜，以增强对性传播HIV-1的保护。在多学科团队的协助下，我们将在R21阶段通过以下方式测试这些假设：1）评估氨基糖苷类药物诱导人阴道上皮细胞、器官型宫颈阴道组织和宫颈器官培养物中retrocyclins持续表达的能力，2）评估局部应用氨基糖苷类药物诱导阴道粘膜中体外和离体毒性和促炎细胞因子的能力。一种先导氨基糖苷类药物，表现出最大的活性，同时表现出对阴道粘膜的最小的副作用，将进入R33阶段，在该阶段我们将：3）将氨基糖苷类药物配制成快速释放的膜剂，以诱导天然的抗HIV-1逆转录细胞周期蛋白的表达，和4）评价膜剂配制的氨基糖苷类药物在猪尾猕猴中的阴道安全性。氨基糖苷类药物介导的逆转录细胞周期蛋白的翻译可能导致一类新的杀微生物剂，以防止HIV-1的传播。