Abstract
Enfuvirtide (T-20) is a fusion inhibitor that suppresses replication of human immunodeficiency virus (HIV) variants with multi-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.
Highlights
We demonstrate here that the role of S138A is to compensate for the impaired fusion kinetics of human immunodeficiency virus (HIV)-1s carrying primary mutations that abrogate binding of T-20
Circular Dichroism—To clarify the mechanism by which the substitutions at Ser-138 influence the antiviral activity of T-20 derivatives, we examined the binding affinities of these peptides to N-terminal HR (N-HR) using circular dichroism (CD) analysis (Fig. 2)
Antiviral Activity of Substituted C34 at Ser-138—To confirm that binding of C34 to N-HR is representative of T-20 binding to N-HR, we examined the anti-HIV-1 activities of Antiviral activity of C34N126K peptides against C34-resistant gp41 recombinant viruses
Summary
Action by a primary mutation, I37K [13]. N126K was initially identified in background of V38A, another primary mutation, for T-20 resistance in vivo [17]. Baldwin et al [17, 18] demonstrated a striking T-20-dependent replication phenotype in the V38A/N126K variant and proposed that T-20 acts as a safety pin to prevent premature formation of helical bundle, as N126K enhanced binding capacity of the introduced C-HR to N36 with V38A Taken together, these studies suggest that mutations in the C-HR serve as secondary mutations. In this study we show that the main role of secondary mutations that follow the appearance of primary mutations during treatment with peptide-based fusion inhibitors is to compensate for the impairment in replication kinetics that is caused by the primary mutations (supplemental Fig. 1) Based on this finding we hypothesized that analogs of T-20 carrying substitutions corresponding to secondary T-20 resistance mutations should be active against both wild-type and T-20-resistant viruses containing primary mutations. We demonstrate that this restoration is concomitant to improved binding of C-HRS138A to N-HRN43D, suggesting that our approach utilizing the resistance-associated mutations to design peptides may provide useful broad insights into effective peptide-based therapies
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