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J. Biol. Chem., Vol. 281, Issue 35, 25813-25821, September 1, 2006

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Kinetic Dependence to HIV-1 Entry Inhibition^*

From the Department of Biochemistry and Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107

Infection by human immunodeficiency virus type 1 (HIV-1) involves the fusion of viral and cellular membranes mediated by formation of the gp41 trimer-of-hairpins. A designed protein, 5-Helix, targets the C-terminal region of the gp41 ectodomain, disrupting trimer-of-hairpins formation and blocking viral entry. Here we show that the nanomolar inhibitory potency of 5-Helix (IC₅₀ 6 nM) is 4 orders of magnitude larger than its subpicomolar binding affinity (K_D 0.6 pM). This discrepancy results from the transient exposure of the 5-Helix binding site on gp41. As a consequence, inhibitory potency is determined by the association rate, not by binding affinity. For a series of 5-Helix variants with mutations in their gp41 binding sites, the IC₅₀ and K_D values poorly correlate. By contrast, an inverse relationship between IC₅₀ values and association rate constants (k_on) extends for over 2 orders of magnitude. The kinetic dependence to inhibition places temporal restrictions on an intermediate state of HIV-1 membrane fusion and suggests that access to the C-terminal region of the gp41 ectodomain is largely free from steric hindrance. Our results support the importance of association kinetics in the development of improved HIV-1 fusion inhibitors.

Received for publication, February 14, 2006 , and in revised form, June 19, 2006.

^* This work was supported by National Institutes of Health Grant RO1GM66682. 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 Materials and Methods, Figs. S1-S3, formula derivations S1-S15, and Refs. 1-5.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Thomas Jefferson University, 233 South 10th St., Rm. 802, Philadelphia, PA 19107. Tel.: 215-503-4564; Fax: 215-923-2117; E-mail: mroot{at}mail.jci.tju.edu.

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