Peptide Triazole Inhibitors of HIV-1: Hijackers of Env Metastability

Abstract

With 1.5 million new infections and 690,000 AIDS-related deaths globally each year, HIV- 1 remains a pathogen of significant public health concern. Although a wide array of effective antiretroviral drugs have been discovered, these largely target intracellular stages of the viral infectious cycle, and inhibitors that act at or before the point of viral entry still require further advancement. A unique class of HIV-1 entry inhibitors, called peptide triazoles (PTs), has been developed, which irreversibly inactivates Env trimers by exploiting the protein structure's innate metastable nature. PTs, and a related group of inhibitors called peptide triazole thiols (PTTs), are peptide compounds that dually engage the CD4 receptor and coreceptor binding sites of Env's gp120 subunit. This triggers dramatic conformational rearrangements of Env, including the shedding of gp120 (PTs and PTTs) and lytic transformation of the gp41 subunit to a post-fusion-like arrangement (PTTs). Due to the nature of their dual receptor site engagement, PT/PTT-induced conformational changes may elucidate mechanisms behind the native fusion program of Env trimers following receptor and coreceptor engagement, including the role of thiols in fusion. In addition to inactivating Env, PTT-induced structural transformation enhances the exposure of important and conserved neutralizable regions of gp41, such as the membrane proximal external region (MPER). PTT-transformed Env could present an intriguing potential vaccine immunogen prototype. In this review, we discuss the origins of the PT class of peptide inhibitors, our current understanding of PT/PTT-induced structural perturbations and viral inhibition, and prospects for using these antagonists for investigating Env structural mechanisms and for vaccine development.

Keywords: HIV-1 Env; conformational dynamics; disulfide exchange; peptide triazoles; protein metastability; therapy; vaccine; virus inactivators.

Fig. (1).

Path of peptide triazole development. The HIV-1 entry inhibitor peptide, 12p1, was first identified in a phage display library [31]. Potency-enhancing modifications were made by the addition of azidoproline (HNG-105) [32] and ferrocene moiety (HNG-156) [33]. The addition of a linker and cysteine residue to the HNG-156 template resulted in novel virolytic properties (KR13) [34]. Peptide minimization to the active pharmacophore (UM-15) [35] and subsequent cyclization (AAR029b) [36] resulted in a more drug-like molecule with greater stability and comparable potency.

Fig. (2).

Peptide triazole interactions with HIV-1 Env receptor binding pocket. (A) HIV-1 envelope trimer (PDB: 5VN8) with a macrocyclic peptide triazole antagonist, AAR029N2 (an optimization on AAR029b shown in Fig. 1), bound to the receptor/coreceptor binding site of gp120. The isoleucine, benzo [b]thiophene-triazole-Pro, and tryptophan pharmacophore residues embed deeply within the receptor-binding pocket, while the polar amino acids of the macrocycle remain solvent-exposed. (B) Zoomed-in view of the hydrophobic I-X-W pharmacophore (where X=Benzo [b]thiophene-Triazole-Pro) of AAR029N2, which binds with high affinity to hydrophobic patches within the Env receptor/coreceptor binding pocket, inhibiting HIV-1 cell entry. Unpublished docking simulation of AAR029N2 with 5VN8 Env performed by Dr. Adel Rashad.

Fig. (3).

Effects of peptide triazole and peptide triazole thiol antagonists on metastable HIV-1 Env. (A) Experimental observations of PT and PTT effect on gp120 monomers versus whole Env trimers on viral or cell surface; (B) Diagram of an energy landscape of hypothesized Env conformational changes in response to peptide triazole (PT) or peptide triazole thiol (PTT) inhibitors. Intact Envelope trimer exists in a metastable state. Perturbation by treatment with a PT inhibitor (left direction) results in the shedding of gp120 subunits, leaving a gp41 trimer. Perturbation by treatment with a PTT inhibitor (right direction) results in a fusion-like sequence of conformational events, resulting in a hypothetical structure denoted a “Sunken Trimer”.

Fig. (4).

Hypothetical model of peptide triazole (PT) and peptide triazole thiol (PTT) binding and structural perturbation of HIV-1 Env. The non-thiolated PT, HNG-156, binds the dual receptor-binding pocket of gp120, triggering the shedding of gp120 subunits and revealing an intact gp41 trimer core, thereby inactivating the fusion capacity of Env [33]. The thiolated PTT, KR13, similarly binds the receptor-binding pocket of gp120, but the sulfhydryl tail engages with a gp120-associated disulfide bond(s), triggering the fusion-related program of the gp41 trimer, inactivating Env and disrupting the viral membrane to cause p24 leakage [34, 45, 68]. The stoichiometry of PT/PTT interaction with Env protomers remains an area of active research. Transparent versions of HNG-156 and KR13 represent the possibility that the engagement of multiple protomers is necessary for transformation and virolysis. Model inspired by Ang et al. [30].

Fig. (5).

Epitope exposure profile in pre- and post-KR13-treated Env. (A) The sunken trimer has been experimentally demonstrated to exhibit reduced binding of 35O22 (indicating loss of gp120), increased binding of NC-1 (indicating the presence of the six-helix bundle), decreased binding of 50–69 and VRC34.01 (potentially pointing to the ‘burying’ of the immunodominant loops and fusion peptides within the residual trimer stump), and increased binding of 10E8 (indicating enhanced exposure of MPER). Antibody binding results from Ang et al. [30] (B) Composite artistic renderings of HIV-1 Env trimer and the hypothesized post-KR13 “sunken trimer” derived and modified from PDB structures 6VFL [75] and 6UJV [76]. The model illustrates the treatment of intact Env trimer with KR13, resulting in the shedding of gp120 subunits, the triggering of the gp41 fusion mechanism, and the resulting “pseudo-post-fusion” sunken trimer structure.

Fig. (6).

Broadly neutralizing antibody recognition of Env MPER epitopes. (A) Schematic representation of gp41 protein sequence showing fusion peptide (FP), N-heptad repeat (NHR), immunodominant loop (Loop), C-heptad repeat (CHR), membrane proximal external region (MPER), transmembrane domain (TMD), and C-terminal domain (CTD). (B-E) The MPER binding epitopes of four MPER-specific antibodies are delineated, showing antibody V_L and V_H domain co-crystalized with the respective cognate peptide epitopes. (B) 2F5 antibody (PDB: 2P8L) [109]. (C) 4E10 antibody (PDB: 4XC3) [105]. (D) 10E8 antibody (PDB: 5IQ7) [108]. (E) Z13e1 antibody (PDB: 3FN0) [110].