The mechanism of HIV-1 virolytic inactivation by Env-targeting peptide triazoles

Env靶向肽三唑灭活HIV-1病毒的机制

• 批准号: 8603546
• 负责人: Lauren Danielle Bailey
• 金额: $ 4.07万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8603546
• 关键词: Acquired Immunodeficiency Syndrome; Affective; Affinity; Amino Acid Sequence; Amino Acids; Antigens; Antiviral Agents; Binding; Binding Sites; Biological Assay; C-terminal; CD4 Antigens; Capsid Proteins; Cell fusion; Cells; Cysteine; Disulfides; Enhancers; Event; Exhibits; Extravasation; Fingerprint; Glycoproteins; Goals; Guidelines; HIV; HIV Envelope Protein gp120; HIV vaccine; HIV-1; Immunoblotting; Infection; Integral Membrane Protein; Left; Length; Ligands; Maps; Measures; Mediating; Membrane; Membrane Proteins; Molecular Conformation; Mutate; Patients; Peptides; Pharmaceutical Preparations; Predisposition; Process; Proline; Proteins; Receptor Cell; Research; Residual state; Role; Rupture; Site-Directed Mutagenesis; Structure; Structure-Activity Relationship; Sulfhydryl Compounds; Surface; Therapeutic; Therapeutic Uses; Triazoles; V3 Loop; Variant; Viral; Viral Proteins; Virion; Virus; Virus Shedding; base; cycloaddition; disulfide bond; env Glycoproteins; improved; inhibitor/antagonist; insight; mutant; novel; particle; pharmacophore; prevent; public health relevance; receptor; receptor binding; spatial relationship; transmission process; virucide

DESCRIPTION (provided by applicant): Interfering with the viral envelope glycoproteins which are necessary for HIV-1 entry is an ideal target for inhibiting interactions with target cells. To his end, we have identified a class of peptide triazole inhibitors, which allosterically bind gp120 and alter its conformation, thereby preventing it from engaging CD4 receptors and co-receptor surrogate mAb 17b. Peptide triazoles potently inhibit cell infection and cause gp120 shedding from the virus. Our lab has shown through site-directed mutagenesis studies of HIV-1 gp120 that peptide triazoles specifically binds to gp120 in the CD4 binding pocket as evidenced by a reduced antiviral and virolytic potency of peptide triazoles. Because these inhibitors simultaneously inhibit gp120-CD4 and gp120 co-receptor interactions they have an improved potential for being developed into entry inhibitor drugs. Moreover, peptide triazoles containing C-terminal cysteines were found to cause cell-free virolysis. Therefore, we investigated a 16- mer peptide triazole with a C-terminal Cys-containing extension (KR-13, R-I-N-N-I-X-W-S-E-A-M-M-¿A-Q-¿A-C-CONH2, where X= ferrocenyltriazole-Pro (FtP)). This variant exhibited dual-antagonistic and antiviral cell infection (EC50 = 25 nM) activities, and in addition caused cell-fre virolytic breakdown of HIV- 1. Virolysis of HIV-1 results in the leakage of the p24 capsid protein, which is necessary for infection. To determine the mechanistic role of the sulfhydryl in virolysis, KR-13b (R-I-N-N-I-X-W-S-E-A-M-M-¿A-Q-¿A-C-ACM-CONH2, X=FtP, ACM=Acetomidomethyl) was developed. In this variant, the sulfhydryl group was capped. KR-13b displayed antiviral activity in cell infection (EC50 = 66 nM), but did not cause p24 leakage. Thus, blocking the sulfhydryl decreases the potency in antiviral assays and prevented virolysis. We hypothesize that the sulfhydryl is necessary for virolysis, possibly by interfering with conserved disulfides i HIV-1 Env causing "thiol exchange", which perturbs the viral membrane stability resulting in p24 leakage. We have initiated the synthesis of truncated and extended peptides derived from KR-13 in order to investigate the structure- activity relationship between the sulfhydryl and virolysi. Serially truncated C-terminal Cys variants display reduced antiviral and virolytic potency. This suggests that there is an optimal length needed to contact pertinent disulfides of the viral envelope, which are necessary for engaging CD4 and aiding HIV-1 entry. These results, taken with the current understanding of the peptide binding, provide a spacial guideline for mutating conserved disulfides near the CD4 binding pocket of HIV-1 Env, which are necessary for infection to determine the mechanism of action of C-terminal Cys peptide triazoles in the virolytic process. The gp120 shedding and virucidal action of peptide triazoles has potential therapeutic use by preventing AIDS transmission because of its' ability to inactivate virus before it engages cells and because the viral protein gp41 retained on the residual particle retains its antigenicity. Thus, understanding the mechanism of virolysis induced by peptide triazole thiols is pertinent to developing a HIV vaccine.

描述（由适用提供）：干扰HIV-1进入所必需的病毒包膜糖蛋白是抑制与靶细胞相互作用的理想目标。最后，我们已经确定了一类肽三唑抑制剂，它们将GP120和 更改其会议，从而阻止其吸引CD4受体和共受体代理MAB 17B。肽三唑可能会抑制细胞感染并导致病毒脱落的GP120。我们的实验室通过对HIV-1 GP120的位置定向诱变研究表明，肽三唑特异性与CD4结合口袋中的GP120结合，这证明了降低的抗病毒和毒素效力的肽三唑。由于这些抑制剂只是抑制GP120-CD4和GP120共受体相互作用，因此具有发展为进入进入抑制剂药物的潜力提高了潜力。此外，发现含有C末端半胱氨酸的肽三唑会引起无细胞的病毒溶解。因此，我们研究了一个16-Mer肽三唑，其含有C末端Cys的扩展（KR-13，R-I-N-N-N-I-n-i-X-W-S-e-A-M-M-M-M-M-M-q-C-C-Conh2，其中X = Ferrocenylylyzole-Pro（FTP））。这种变体暴露于双抗病毒细胞感染（EC50 = 25 nm）的活性，另外引起了HIV-1的细胞酸性分解。HIV-1的Violysys导致P24 CAPSID蛋白的泄漏， 这是感染所必需的。为了确定硫赖尔在病毒分析中的机械作用， 开发了KR-13b（R-I-N-N-N-I-X-W-S-E-M-M-Q-ACM-CONH2，X = FTP，ACM =乙酰甲基甲基）。在此变体中，硫酸基团被封顶。 KR-13b在细胞感染（EC50 = 66 nm）中表现出抗病毒活性，但不会引起P24泄漏。那阻断硫酰亚胺可以降低抗病毒测定的效力并防止静脉曲张。我们假设通过干扰构成的二硫化物I HIV-1 env，从而导致“硫醇交换”，这可能会导致病毒膜稳定性导致p24泄漏。为了研究硫赖尔和Virolysi之间的结构活性关系，我们启动了截短和扩展的截短和扩展的肽的合成。串行截断的C末端CYS变体显示降低的抗病毒和毒力效力。这表明与病毒包膜相关的二硫化物需要最佳的长度，这对于参与CD4和帮助HIV-1的进入是必不可少的。这些结果是在当前对肽结合的理解中取得的结果，为在HIV-1 Env的CD4结合袋附近突变成组成的二硫化物提供了一个空间指南，这对于确定CyloLytic过程中C末端CYS肽肽三唑的作用机理是必不可少的。 GP120的脱落和基于病毒的辣椒三唑作用具有潜在的治疗用途，可以防止AIDS传播，因为它的能力在其参与细胞之前会失活，并且由于残留颗粒上保留的病毒蛋白GP41保留了其抗原性。这是了解辣椒三唑硫醇引起的毒液的机制与开发HIV疫苗有关。