Chemical optimization of macrocyclic HIV-1 inactivators for improving potency and increasing the structural diversity at the triazole ring.

HIV-1 entry inhibition remains an urgent need for AIDS drug discovery and development. We previously reported the discovery of cyclic peptide triazoles (cPTs) that retain the HIV-1 irreversible inactivation functions of the parent linear peptides (PTs) and have massively increased proteolytic resistance. Here, in an initial structure-activity relationship investigation, we evaluated the effects of variations in key structural and functional components of the cPT scaffold in order to produce a platform for developing next-generation cPTs. Some structural elements, including stereochemistry around the cyclization residues and Ile and Trp side chains in the gp120-binding pharmacophore, exhibited relatively low tolerance for change, reflecting the importance of these components for function. In contrast, in the pharmacophore-central triazole position, the ferrocene moiety could be successfully replaced with smaller aromatic rings, where a p-methyl-phenyl methylene moiety gave cPT 24 with an IC50 value of 180 nM. Based on the observed activity of the biphenyl moiety when installed on the triazole ring (cPT 23, IC50 ∼ 269 nM), we further developed a new on-resin synthetic method to easily access the bi-aryl system during cPT synthesis, in good yields. A thiophene-containing cPT AAR029N2 (36) showed enhanced entropically favored binding to Env gp120 and improved antiviral activity (IC50 ∼ 100 nM) compared to the ferrocene-containing analogue. This study thus provides a crucial expansion of chemical space in the pharmacophore to use as a starting point, along with other allowable structural changes, to guide future optimization and minimization for this important class of HIV-1 killing agents.

Peptide Triazole Thiol Irreversibly Inactivates Metastable HIV-1 Env by Accessing Conformational Triggers Intrinsic to Virus-Cell Entry.

KR13, a peptide triazole thiol previously established to inhibit HIV-1 infection and cause virus lysis, was evaluated by flow cytometry against JRFL Env-presenting cells to characterize induced Env and membrane transformations leading to irreversible inactivation. Transiently transfected HEK293T cells were preloaded with calcein dye, treated with KR13 or its thiol-blocked analogue KR13b, fixed, and stained for gp120 (35O22), MPER (10E8), 6-helix-bundle (NC-1), immunodominant loop (50-69), and fusion peptide (VRC34.01). KR13 induced dose-dependent transformations of Env and membrane characterized by transient poration, MPER exposure, and 6-helix-bundle formation (analogous to native fusion events), but also reduced immunodominant loop and fusion peptide exposure. Using a fusion peptide mutant (V504E), we found that KR13 transformation does not require functional fusion peptide for poration. In contrast, simultaneous treatment with fusion inhibitor T20 alongside KR13 prevented membrane poration and MPER exposure, showing that these events require 6-helix-bundle formation. Based on these results, we formulated a model for PTT-induced Env transformation portraying how, in the absence of CD4/co-receptor signaling, PTT may provide alternate means of perturbing the metastable Env-membrane complex, and inducing fusion-like transformation. In turn, the results show that such transformations are intrinsic to Env and can be diverted for irreversible inactivation of the protein complex.