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.
My research will focus on determining the mechanism of action of C-terminal Cys peptide triazoles in the virolytic process of HIV-1 Env, to accomplish this I will analyze the binding affinity, antiviral potency, and the gp120 shedding of serially truncated peptide triazole thiols. We expect the decrease to be discontinuous in p24 leakage due to the need for spatial distance between peptide SH and Env gp120 disulfides to cause gp120 thiol exchange and membrane rupture. This will allow for a better understanding of the compelling, yet elusive HIV-1 entry process while developing novel agents that may be affective therapeutics.