Inhibition of Wild-Type and Mutant Human Immunodeficiency Virus Type 1 Proteases by GW0385 and Other Arylsulfonamides

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• 作者：Mary H. Hanlon ; David J. T. Porter ; Eric S. Furfine ; Andrew Spaltenstein ; H. Luke Carter ; Dana Danger ; Arthur Y. L. Shu ; Istvan W. Kaldor ; John F. Miller ; and Vicente A. Samano
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：November 16, 2004
• 年：2004
• 卷：43
• 期：45
• 页码：14500 - 14507
• 全文大小：116K
• 年卷期：v.43,no.45(November 16, 2004)
• ISSN：1520-4995

文摘

The arylsulfonamide derivatives described herein were such potent inhibitors of humanimmunodeficiency virus type 1 (HIV-1) protease (enzyme, E) that values for the inhibition constants (K_i)could not be determined by conventional steady-state kinetic techniques (i.e., the minimal enzymeconcentration usable for the activity assay was much greater than the value of the dissociation constant).Consequently, two alternative methods were developed for estimation of K_i values. The first methodemployed kinetic determinations of values for k₁ and k_-1, from which K_i was determined (k_-1/k₁). Thesecond method was a competitive displacement assay used to determine binding affinities of other inhibitorsrelative to that of GW0385. In these assays, the inhibitor of unknown affinity was used to displace [³H]GW0385 from E·[³H]GW0385. From the concentration of E·[³H]GW0385 at equilibrium, the concentrationsof enzyme-bound and free inhibitors were calculated, and the ratio of the K_i value of the unknown to thatof GW0385 was determined (K_i,unknown/K_i,GW0385). The values of k₁ were calculated from data in whichchanges in the intrinsic protein fluorescence of the enzyme associated with inhibitor binding were directlyor indirectly monitored. In the case of saquinavir, the fluorescence changes associated with complexformation were large enough to monitor directly. The value of k₁ for saquinavir was 62 ± 2 M^-1 s^-1.In the case of GW0385, the fluorescence changes associated with complex formation were too small tomonitor directly. Consequently, the value of k₁ was estimated from a competition experiment in whichthe effect of GW0385 on the binding of E to saquinavir was determined. The value of k₁ for GW0385was estimated from these experiments to be 137 ± 4 M^-1 s^-1. Because E·[³H]GW0385 was stable inthe standard buffer at room temperature for greater than 33 days, the value of the first-order rate constantfor dissociation of E·[³H]GW0385 (k_-1) could be estimated from the time-course for exchange of E·[³H]GW0385 with excess unlabeled GW0385. The value of k_-1 calculated from these data was (2.1 ± 0.1) ×10^-6 s^-1 (t_1/2 = 91 h). The K_i value of wild-type HIV-1 protease for GW0385, calculated from thesevalues for k₁ and k_-1, was 15 ± 1 fM. Three multidrug resistant enzymes had K_i values for GW0385 thatwere less than 5 pM.