Genetic Pathway of HIV-1 Resistance to Novel Fusion Inhibitors Targeting the Gp41 Pocket

Abstract

The peptide drug enfuvirtide (T20) is the only HIV-1 fusion inhibitor in clinical use, but it easily induces drug resistance, calling for new strategies for developing effective drugs. On the basis of the M-T hook structure, we recently developed highly potent short-peptide HIV-1 fusion inhibitors (MTSC22 and HP23), which mainly target the conserved gp41 pocket and possess high genetic barriers to resistance. Here, we focused on the selection and characterization of HIV-1 escape mutants of MTSC22, which revealed new resistance pathways and mechanisms. Two mutations (E49K and L57R) located at the inhibitor-binding site and two mutations (N126K and E136G) located at the C-terminal heptad repeat region of gp41 were identified as conferring high resistance either singly or in combination. While E49K reduced the C-terminal binding of inhibitors via an electrostatic repulsion, L57R dramatically disrupted the N-terminal binding of M-T hook structure and pocket-binding domain. Unlike E49K and N126K, which enhanced the stability of the endogenous viral six-helical bundle core (6-HB), L57R and E136G conversely destabilized the 6-HB structure. We also demonstrated that both primary and secondary mutations caused the structural changes in 6-HB and severely impaired the capability for HIV-1 entry. Collectively, our data provide novel insights into the mechanisms of short-peptide fusion inhibitors targeting the gp41 pocket site and help increase our understanding of the structure and function of gp41 and HIV-1 evolution. IMPORTANCEThe deep pocket on the N-trimer of HIV-1 gp41 has been considered an ideal drug target because of its high degree of conservation and essential role in viral entry. Short-peptide fusion inhibitors, which contain an M-T hook structure and mainly target the pocket site, show extremely high binding and inhibitory activities as well as high genetic barriers to resistance. In this study, the HIV-1 mutants resistant to MTSC22 were selected and characterized, which revealed that the E49K and L57R substitutions at the inhibitor-binding site and the N126K and E136G substitutions at the C-terminal heptad repeat region of gp41 critically determine the resistance phenotype. The data provide novel insights into the mechanisms of action of the M-T hook structurebased fusion inhibitors which will help further our understanding of the structure-function relationship of gp41 and molecular pathways of HIV-1 evolution and eventually facilitate the development of new anti-HIV drugs.

Development of potent and long-acting HIV-1 fusion inhibitors

“…So far, we have failed to induce HIV-1 mutants resistant to HP23 during in-vitro selection, different from its templates SC22EK and MTSC22 [36,37]. The L57R mutation located in the deep gp41 pocket site was selected by MTSC22, which might correlate with the M-T hook structure [37]. Although LP-11 could not efficiently inhibit the L57R mutant, we think that it has a higher genetic barrier to developing 1194 AIDS 2016, Vol 30 No 8 resistance, similar to its template HP23.…”

“…Previously, we demonstrated that HP23 is a potent fusion inhibitor with a high genetic barrier [17]. So far, we have failed to induce HIV-1 mutants resistant to HP23 during in-vitro selection, different from its templates SC22EK and MTSC22 [36,37]. The L57R mutation located in the deep gp41 pocket site was selected by MTSC22, which might correlate with the M-T hook structure [37].…”

“…For example, the I37T/N43K or V38A/N42T double mutations resulted in considerable high fold-changes of resistance to T20 and SFT, but they had little effect to the activity of HL23L and LP-11. Recently, we generated multiple SC22EK and MTSC22-resistant mutant viruses, which also displayed high resistance to HP23 [36,37]. Here we determined their susceptibility to HP23L and LP-11.…”

A Helical Short-Peptide Fusion Inhibitor with Highly Potent Activity against Human Immunodeficiency Virus Type 1 (HIV-1), HIV-2, and Simian Immunodeficiency Virus

“…We also constructed a panel of 15 HIV-1 NL4-3 -based pseudoviruses with Envs carrying T20-or HP23-resistant mutations (43,44). The inhibition data in Table 4 showed that (i) the long peptides T20 and P3 exhibited relatively higher resistance on T20-resistant mutants, but the short peptides HP23 and 2P23 could maintain their potency, and (ii) 2P23 also displayed improved inhibition over some HP23-resistant mutants (e.g., L57R and L57R/E136G).…”

A Lipopeptide HIV-1/2 Fusion Inhibitor with Highly PotentIn Vitro,Ex Vivo, andIn VivoAntiviral Activity

“…As shown in Table 1 We also constructed two panels of inhibitor-resistant pseudoviruses using NL4-3 Env as a template. The panel 1 Envs were introduced with single or double mutations that rendered resistance to T-20, while the panel 2 Envs were generated to carry HP23-resistant mutations (21,22). The data in Table 1 LP-19 has strong and long-acting ex vivo antiviral activity.…”