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: 8900736
• 负责人: Lauren Danielle Bailey
• 金额: $ 4.11万
• 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/8900736
• 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(由申请人提供)：干扰HIV-1进入所必需的病毒包膜糖蛋白是抑制与目标细胞相互作用的理想靶点。为此，我们已经确定了一类多肽三氮唑抑制剂，它能变构结合gp120和gp120。 改变其构象，从而阻止其与CD4受体和共受体替代物mAb17b结合。多肽三唑能有效地抑制细胞感染，并导致gp120从病毒中脱落。我们的实验室已经通过对HIV-1 gp120的定点突变研究表明，多肽三唑与CD4结合口袋中的gp120特异性结合，证明多肽三唑的抗病毒和裂解病毒的效力降低。因为这些抑制剂同时抑制gp120-CD4和gp120共同受体的相互作用，所以它们有更大的潜力被开发成进入抑制药物。此外，含有C-末端半胱氨酸的多肽三氮唑还能引起无细胞的病毒裂解。因此，我们研究了一个C端含半胱氨酸的16肽三唑化合物(KR-13，R-I-N-N-I-X-W-S-E-A-M-M-？A-Q-？A-C-CONH2，式中X=二茂铁基三氮唑-Pro(Ftp))。该变异体具有双重拮抗和抗病毒细胞感染活性(EC50=25 nM)，并能引起HIV-1的细胞裂解。HIV-1的裂解导致p24衣壳蛋白的泄漏， 这是感染所必需的。为了确定巯基在病毒裂解中的机械作用， KR-13b(R-I-N-N-I-X-W-S-E-A-M-M-A-Q-A-C-ACM-CONH2，X=ftp，acm=乙酰咪甲基)。在这个变体中，巯基是封端的。KR-13b在细胞感染中表现出抗病毒活性(EC50=66 nM)，但不引起p24泄漏。因此，封闭巯基会降低抗病毒检测的效力，并防止病毒裂解。我们推测，巯基是病毒裂解所必需的，可能是通过干扰保守的二硫键I HIV-1 Env引起“硫醇交换”，从而扰乱病毒膜的稳定性，导致p24泄漏。我们已经开始了从KR-13衍生的截短肽和延伸肽的合成，以研究巯基和病毒素之间的构效关系。连续截短的C-末端半胱氨酸变异体表现出抗病毒和裂解病毒的效力降低。这表明，有一个最佳的长度需要接触病毒被膜的相关二硫化物，这是结合CD4和帮助HIV-1进入所必需的。这些结果结合目前对肽结合的理解，为突变HIV-1 Env的CD4结合口袋附近的保守二硫化物提供了空间指南，这是感染所必需的，以确定C末端半胱氨酸肽三氮唑在病毒裂解过程中的作用机制。三氮唑多肽的gp120脱落和杀灭病毒的作用具有潜在的治疗用途，可以防止艾滋病的传播，因为它能够在病毒与细胞接触之前灭活病毒，而且保留在残留颗粒上的病毒蛋白gp41保持其抗原性。因此，了解三唑硫醇诱导病毒裂解的机制对于HIV疫苗的研制具有重要意义。