Design of Peptide-based Inhibitors for Human Immunodeficiency Virus Type 1 Strains Resistant to T-20

Abstract

Enfuvirtide (T-20) is a fusion inhibitor that suppresses replication of human immunodeficiency virus (HIV) variants withmulti-drug resistance to reverse transcriptase and protease inhibitors. It is a peptide derived from the C-terminal heptad repeat (C-HR) of HIV-1 gp41, and it prevents interactions between the C-HR and the N-terminal HR (N-HR) of gp41, thus interfering with conformational changes that are required for viral fusion. However, prolonged therapies with T-20 result in the emergence of T-20-resistant strains that contain primary mutations such as N43D in the N-HR of gp41 (where T-20 and C-HR bind) that help the virus escape at a fitness cost. Such variants often go on to acquire a secondary mutation, S138A, in the C-HR of gp41 region that corresponds to the sequence of T-20. We demonstrate here that the role of S138A is to compensate for the impaired fusion kinetics of HIV-1s carrying primary mutations that abrogate binding of T-20. To preempt this escape strategy, we designed a modified T-20 variant containing the S138A substitution and showed that it is a potent inhibitor of both T-20-sensitive and T-20-resistant viruses. Circular dichroism analysis revealed that the S138A provided increased stability of the 6-helix bundle. We validated our approach on another fusion inhibitor, C34. In this case, we designed a variant of C34 with the secondary escape mutation N126K and showed that it can effectively inhibit replication of C34-resistant HIV-1. These results prove that it is possible to design improved peptide-based fusion inhibitors that are efficient against a major mechanism of drug resistance.

Resistance Profiles of Novel Electrostatically Constrained HIV-1 Fusion Inhibitors

“…X-ray crystallographic and circular dichroism (CD) analyses revealed that the S138A substitution contributed to stability of the N-HR⅐C-HR complex (19). Similar results were observed for C34 with N126K (C34 N126K ), also secondary mutations for T-20-resistant variants in vivo (20,21) and C34-resistant variants in vitro (18). Therefore, the novel strategy to design inhibitor peptides utilizing resistance mutations has resulted in antivirals that can suppress variants resistant to the parental peptides.…”

“…An HIV-1 molecular clone, pNL4-3 (23), was employed as a wild-type HIV-1 (HIV-1 NL4-3 ), and used for the construction of various gp41-recombinant viruses as described previously (15,18,24,25). The viruses were harvested from the supernatant of transfected 293T cells and stored at Ϫ80°C.…”

“…Circular Dichroism (CD) Spectroscopic Analysis-CD analysis was performed as described previously (15,18,27), with some modifications. In brief, spectra of a complex of N-HR and C-HR peptides (each 10 M) or an N-HR peptide alone (10 M) in 5 mM HEPES buffer (pH 7.2) were collected using a J-710 CD spectrometer (Jasco, Tokyo, Japan) equipped with a thermoelectric temperature controller.…”

“…Inhibitory Activity of Modified Fusion Inhibitors-We have previously demonstrated that introduction of resistance mutations to T-20 restores anti-HIV-1 activity against T-20-resistant variants (18), suggesting that this strategy can result in the design of peptides with improved potential for the treatment of resistant HIV-1. We examined whether this strategy was also applicable to SC34-and SC34EK-selected mutations.…”

“…These new changes enhance the antiviral potency against resistant variants. Hence, T-20 with a S138A substitution (T-20 S138A ), one of the secondary resistance mutations observed in patients that fail to respond to T-20, regained anti-HIV-1 activity against T-20-resistant variants (18). X-ray crystallographic and circular dichroism (CD) analyses revealed that the S138A substitution contributed to stability of the N-HR⅐C-HR complex (19).…”