Steger H Kirby, Root Michael J
Department of Biochemistry and Molecular Biology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
J Biol Chem. 2006 Sep 1;281(35):25813-21. doi: 10.1074/jbc.M601457200. Epub 2006 Jun 27.
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 approximately 6 nm) is 4 orders of magnitude larger than its subpicomolar binding affinity (K(D) approximately 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.
1型人类免疫缺陷病毒（HIV-1）感染涉及由gp41发夹三聚体形成介导的病毒膜与细胞膜的融合。一种设计蛋白5-Helix靶向gp41胞外结构域的C末端区域，破坏发夹三聚体的形成并阻断病毒进入。我们在此表明，5-Helix的纳摩尔抑制效力（IC50约为6 nM）比其亚皮摩尔结合亲和力（K(D)约为0.6 pM）大4个数量级。这种差异源于gp41上5-Helix结合位点的短暂暴露。因此，抑制效力由缔合速率决定，而非结合亲和力。对于一系列在其gp41结合位点发生突变的5-Helix变体，IC50和K(D)值相关性较差。相比之下，IC50值与缔合速率常数（k(on)）之间的反比关系延伸超过2个数量级。抑制的动力学依赖性对HIV-1膜融合的中间状态施加了时间限制，并表明接近gp41胞外结构域的C末端区域在很大程度上不受空间位阻的影响。我们的结果支持缔合动力学在改进HIV-1融合抑制剂开发中的重要性。
