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: 8514470
• 负责人: ALEXANDER MICHAEL COLE
• 金额: $ 43.6万
• 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 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8514470
• 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; cervicovaginal; 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一直是 HIV-1 在全世界传播的最常见途径，尽管 HIV-1 疫苗的开发受到了很多关注，但进展缓慢且紧迫。需要寻找替代方法，例如局部杀微生物剂，以针对 HIV-1 的传播。 β-防御素有助于抵抗微生物和病毒感染的先天免疫防御；然而，这些产生的内源性因子在预防 HIV 传播方面效果甚微。某些非人灵长类动物也具有 β-防御素 - 18 残基环肽，可作为 HIV-1 进入抑制剂。存在多种人类β-防御素基因，但它们含有一个阻止翻译的过早终止密码子，人类基因组内编码的β-防御素（在人类中称为逆转录环素）是。基于原本完整的逆环素基因中包含的遗传密码，我们合成地重新创建了逆环素，并发现它们在体内产生时对来自多种HIV-1分支的R5和X4分离株具有笨拙的活性。恒河猴中的β-防御素涉及两个九残基肽的翻译后连接，这一独特过程的机制及其在人类细胞中的存在都不为人所知 - 直到现在，我们提议通过使用廉价且广泛使用的氨基糖苷类抗生素来增强阴道对 HIV-1 的先天免疫力，从而改变杀菌剂预防 HIV-1 传播的方式。在真核生物中，氨基糖苷类抗生素可以抑制过早终止密码子。在初步研究中，我们通过在其位置掺入氨基酸，使用抑制氨基糖苷类的药物恢复了人类早幼粒细胞和 HeLa 细胞的逆转录环素肽的表达。根据我们的研究，我们发现，氨基糖苷类药物可以恢复宫颈阴道粘膜中逆环素的自然产生，并且可以配制为快速释放膜以赋予其抗HIV-1活性。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) 评估氨基糖苷类诱导持续表达的能力。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的逆环素，以及2)评估局部应用的氨基糖苷类在阴道粘膜中诱导体外和离体毒性和促炎细胞因子的能力，同时表现出最大的活性。对阴道粘膜表现出最小的不良影响，将进入 R33 阶段，在此阶段我们将： 3) 将氨基糖苷配制成快速释放膜，以诱导天然抗 HIV-1 逆转录酶表达，4) 评估薄膜配制的氨基糖苷在猪尾猕猴中的阴道安全性，氨基糖苷介导的逆转录酶翻译可能会产生一类新的杀微生物剂，以预防 HIV-1 传播。