Abstract Body

Background: We have identified peptide triazole thiol inhibitors which bind Env gp120, allosterically inhibit CD4 receptor and co-receptor surrogate mAb 17b binding, and cause both gp120 shedding and p24 release from the virus lumen. The prototype peptide triazole thiol KR-13 (R-I-N-N-I-X-W-S-E-A-M-M-βAQ-βA-C-CONH2, X= ferrocenyltriazole-Pro) causes p24 release, however KR-13b (C-terminal SH capped by acetomidomethyl) inhibited cell infection but did not cause p24 leakage. We are investigating the mode of action by which the C-terminal sulfhydryl group causes irreversible inactivation. From a molecular dynamics simulated model of the peptide triazole-gp120 complex we know that the IXW pharmacophore of KR13 binds in the CD4 binding site. Therefore, a working model is that the thiol interferes with conserved disulfides clustered proximal to the CD4 binding site in gp120 through “disulfide exchange”. These disulfides are located within gp120 at 296-331 (V3 loop), 385-418 (C4), 378-445 (C3), and 119-205 (V1/V2 loop). Methodology: Peptide triazoles are synthesized by solid phase Fmoc chemistry, followed by click conjugation. Infectivity assays are performed with single round pseudo-typed HIV-1 BaL.01 and a luciferase gene, which is detected by chemiluminescence. p24 release was assayed using sandwich ELISA. Results: To evaluate the importance of the spatial relationship between PT-SH and gp120 disulfide groups we synthesized truncated peptides derived from KR-13 and compared p24 release and antiviral activities. We observed a strong dependence of lysis activity on length of the linker between the IXW pharmacophore and SH group. We also examined the ability of KR13 to cause p24 release after HIV-1 treatment with Ellman’s reagent, a disulfide exchange inhibitor. These results demonstrate that release of p24 induced by KR13 is significantly inhibited by Ellman’s reagent. Additionally, we have shown that 2G12, a conformational antibody which binds to a glycosyl group near the V3 loop, competes with the p24 release of virolytic peptides in a dose dependent manner. Conclusions: We have shown that there is a discontinuous relationship between virolysis and peptide length, indicating that a minimal length is required for efficient virolysis. Our findings suggest that virolytic peptides disrupt the stable conformation of Env by interfering in disulfide exchange within gp120, thus sensitizing the viral membrane to rupture. Furthermore, our data implicates the V3 loop disulfide as a potential site that triggers this disulfide exchange cascade. We currently plan to investigate the functional cross-talk between the fusion mechanism and events leading to virolysis as well as examine the importance of specific Env disulfides that may be involved in disulfide exchange.