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

doi:10.1074/jbc.M807169200

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

^‡Institute for Virus Research, Kyoto University, 53 Kawaramachi, Shogoin, Kyoto 606-8507, Japan, the ^§Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida, Shimoadachi-cho, Kyoto 606-8501, Japan, and the ^¶Christopher S. Bond Life Sciences Center and Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, Missouri 65211

1 To whom correspondence should be addressed. Tel. and Fax: 81-75-751-3986; E-mail: ekodama{at}virus.kyoto-u.ac.jp.

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.

Footnotes

↵2 The abbreviations used are: HIV, human immunodeficiency virus; T-20, enfuvirtide; HR, heptad repeat; MAGI, multinuclear activation of galactosidase indicator; EC₅₀, 50% effective concentration; T_m, melting temperature; CD, circular dichroism; shRNA, short hairpin RNA; WT, wild-type.
↵* This work was supported, in part, by National Institutes of Health Grants AI076119, AI079801, and AI074389 (to S. G. S.). This work was also supported in part by grants from the Ministry of Health and Welfare and the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to E. K., S. O., and N. F.), the Japan Health Sciences Foundation (to E. K., S. O., N. F., and M. M.), the 21st Century COE program (to K. I., S. I., and H. N.), and a Japan Society for the Promotion of Science research fellowship (to H. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and Tables 1 and 2.
- Received September 16, 2008.
- Revision received December 3, 2008.
The American Society for Biochemistry and Molecular Biology, Inc.