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

描述（由申请方提供）：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靶点。为此，我们已经鉴定了一类肽三唑抑制剂，其变构结合gp 120， 改变其构象，从而阻止其与CD 4受体和共受体替代物mAb 17 b结合。肽三唑有效地抑制细胞感染，并导致gp 120从病毒中脱落。我们的实验室已经通过HIV-1 gp 120的定点诱变研究表明，肽三唑特异性结合CD 4结合口袋中的gp 120，这通过肽三唑的抗病毒和病毒裂解效力降低来证明。由于这些抑制剂同时抑制gp 120-CD 4和gp 120共受体相互作用，因此它们具有开发成进入抑制剂药物的改进潜力。此外，发现含有C-末端半胱氨酸的肽三唑引起无细胞病毒裂解。因此，我们研究了具有C-末端含Cys延伸的16- mer肽三唑（KR-13，R-I-N-N-I-X-W-S-E-A-M-M-<$A-Q-<$A-C-CONH 2，其中X=二茂铁基三唑-Pro（FtP））。该变体表现出双重拮抗和抗病毒细胞感染（EC 50 = 25 nM）活性，此外还导致HIV- 1的细胞自由病毒裂解。HIV-1的病毒裂解导致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制作用， KR-13 b（R-I-N-N-I-X-W-S-E-A-M-M-<$A-Q-<$A-C-ACM-CONH 2，X=FtP，ACM=乙酰亚胺基甲基）。在该变体中，巯基被封端。KR-13 b在细胞感染中显示抗病毒活性（EC 50 = 66 nM），但不引起p24渗漏。因此，阻断巯基降低了抗病毒测定中的效力并防止了病毒裂解。我们假设巯基是病毒裂解所必需的，可能是通过干扰HIV-1 Env中的保守二硫化物引起“巯基交换”，这扰乱了病毒膜的稳定性，导致p24泄漏。我们已经开始了KR-13的截短和延伸肽的合成，以研究巯基和病毒裂解之间的结构-活性关系。连续截短的C-末端Cys变体显示降低的抗病毒和病毒裂解效力。这表明，有一个最佳的长度需要接触相关的二硫化物的病毒包膜，这是必要的接合CD 4和帮助HIV-1进入。这些结果与目前对肽结合的理解一起，为突变HIV-1 Env的CD 4结合口袋附近的保守二硫键提供了空间指南，这对于确定C-末端Cys肽三唑在病毒裂解过程中的作用机制是感染所必需的。肽三唑类的gp 120脱落和杀病毒作用具有潜在的治疗用途，因为它能够在病毒与细胞接触之前将其清除，并且因为保留在残留颗粒上的病毒蛋白gp 41保留了其抗原性。因此，了解由肽三唑硫醇诱导的病毒裂解的机制与开发HIV疫苗有关。

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.