Resistance and synergism in the antiviral functions of peptide triazole inactivators of the HIV-1 Env

Shiyu Zhang

doi:10.17918/00000525

In the first aim of my thesis, we investigated potential mechanisms of viral escape from this promising class of HIV-1 entry inhibitors, developed in Chaiken lab. They are drug-like peptide triazoles (PTs) inducing HIV-1 Envelope gp120 shedding, altering gp41 formation and irreversibly inactivating virions. HIV-1 resistance to cyclic (AAR029b) and linear (KR13) PTs was obtained by dose escalation in viral passaging experiments. High-level resistance for both inhibitors developed slowly (relative to escape from gp41-targeted C-peptide inhibitor C37) by acquiring mutations in gp120 both within (Val255) and distant to (Ser143) the putative PT binding site. The similarity in the resistance profiles for AAR029b and KR13 suggests that the shared IXW pharmacophore provided the primary pressure for HIV-1 escape. In single-round infectivity studies employing recombinant virus, V255I/S143N double escape mutants reduced PT antiviral potency by 150- to 3900-fold. Curiously, the combined mutations had a much smaller impact on PT binding affinity for monomeric gp120 (4- to 9-fold). This binding disruption was entirely due to the V255I mutation, which generated few steric clashes with PT in molecular docking simulations. However, this minor effect on PT affinity belied large, offsetting changes to association enthalpy and entropy. The escape mutations had negligible effect on CD4 binding and utilization during entry, but significantly altered both binding thermodynamics and inhibitory potency of the conformationally-specific, anti-CD4i antibody 17b. Moreover, the escape mutations substantially decreased gp120 shedding induced by either soluble CD4 or AAR029b. Together, the data suggest that the escape mutations significantly modified the energetic landscape of Env's prefusogenic state, altering conformational dynamics to hinder PT-induced irreversible inactivation of Env. The second aim of my thesis has focused on novel HIV-1 antagonists development considering the urgent needs for patient treatment due to the threat of drug resistance. To help address this need, we previously reported a highly potent synthetic chimeric HIV-1 entry inhibitor, LJC240-L4-UM15, consisting of a CCR5 coreceptor inhibitor (CoRI, LJC240) covalently conjugated to an HIV-1 Env inactivator (EnvI, UM15) through a flexible linker. Here, we used linker length variation to evaluate linker dependence for the simultaneous engagement of the chimera inhibitor components with Env and coreceptor in chimera inhibition. We synthesized and evaluated chimeras with three different linker lengths (LJC240-Ln-UM15, n=0/2/4), along with their constituent components. In single-round HIV-1 neutralization assays, antiviral potency with the LJC240-Ln component alone decreased as the linker length increased from n=0 to n=4, suggesting functional interference with increasing linker size. Furthermore, while adding EnvI to CoRI-Ln in noncovalent mixtures enhanced function, enhancement decreased as linker length increased, again reflecting interference by the linker. In contrast, the covalently-linked chimera LJC240-Ln-UM15 increased in potency with increasing linker length, showing that the presence of both inhibitor components covalently attached in the same molecule overcame linker interference. Taken together, the findings argue that increasing linker length provides sufficient spatial distance to enable simultaneous engagement of both EnvI and CoRI components of the chimera to the Env and CoR proteins at the virus-cell interface. Overall, our work reinforces the promising strategy to design dual-function HIV entry chimeric inhibitors with strong potency and, due to the presence of components with two different modes of action, potentially delayed onset of resistance. The third aim of my thesis was continuously focused on the high-potency chimeric HIV-1 entry inhibitor. We derived a high-potency chimeric molecule by covalently linking a small molecule (CoRI, LJC240) targeting host-cell CCR5 coreceptor and a cyclic peptide triazole (cPT, AAR029b) targeting the HIV-1 envelope protein (Env). Denoted LJC240-L4-AAR029b (Compound 2), this CoRI-linker-cPT chimera exhibited broad-spectrum, subnanomolar antiviral potency against Envs from diverse HIV-1 strains. The enhanced potency of the chimera vs noncovalent LJC240-L4 and cPT mixtures was demonstrated in combinatorial neutralization assays. Owing to the macrocyclic nature of the cPT, the LJC240-L4-AAR029b chimera (Compound 2) was refractory to degradation by a serum protease cocktail for over 30 hours. This work advances a promising strategy to develop potent bifunctional HIV-1 entry inhibitors that both block virus-cell interaction and irreversibly inactivate Env. Overall, my work demonstrated the mechanisms of resistance arising from the drug-like peptide triazoles treatment, and of synergy between the two classes of HIV-1 Entry inhibitors targeting both the viral surface glycoproteins and the cell coreceptors. With these gained knowledges with PTs and dual-action chimeras, we confirmed the appealing strategy to develop novel HIV-1 entry inhibitors with designed property and expanded our understanding of the intrinsic metastability of Env as well as the viral Env- cell coreceptor interactions.

Resistance and synergism in the antiviral functions of peptide triazole inactivators of the HIV-1 Env

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Resistance and synergism in the antiviral functions of peptide triazole inactivators of the HIV-1 Env

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