Binding of Clinical Inhibitors to a Model Precursor of a Rationally Selected Multidrug Resistant HIV-1 Protease Is Significantly Weaker Than That to the Released Mature Enzyme

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• 作者：Joon H. Park ; Jane M. Sayer ; Annie Aniana ; Xiaxia Yu ; Irene T. Weber ; Robert W. Harrison ; John M. Louis
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：April 26, 2016
• 年：2016
• 卷：55
• 期：16
• 页码：2390-2400
• 全文大小：577K
• 年卷期：0
• ISSN：1520-4995

文摘

We have systematically validated the activity and inhibition of a HIV-1 protease (PR) variant bearing 17 mutations (PR^S17), selected to represent high resistance by machine learning on genotype–phenotype data. Three of five mutations in PR^S17 correlating with major drug resistance, M46L, G48V, and V82S, and five of 11 natural variations differ from the mutations in two clinically derived extreme mutants, PR20 and PR22 bearing 19 and 22 mutations, respectively. PR^S17, which forms a stable dimer (<10 nM), is ∼10- and 2-fold less efficient in processing the Gag polyprotein than the wild type and PR20, respectively, but maintains the same cleavage order. Isolation of a model precursor of PR^S17 flanked by the 56-amino acid transframe region (TFP-p6pol) at its N-terminus, which is impossible upon expression of an analogous PR20 precursor, allowed systematic comparison of inhibition of TFP-p6pol-PR^S17 and mature PR^S17. Resistance of PR^S17 to eight protease inhibitors (PIs) relative to PR (K_i) increases by 1.5–5 orders of magnitude from 0.01 to 8.4 μM. Amprenavir, darunavir, atazanavir, and lopinavir, the most effective of the eight PIs, inhibit precursor autoprocessing at the p6pol/PR site with IC₅₀ values ranging from ∼7.5 to 60 μM. Thus, this process, crucial for stable dimer formation, shows inhibition ∼200–800-fold weaker than that of the mature PR^S17. TFP/p6pol cleavage, which occurs faster, is inhibited even more weakly by all PIs except darunavir (IC₅₀ = 15 μM); amprenavir shows a 2-fold increase in IC₅₀ (∼15 μM), and atazanavir and lopinavir show increased IC₅₀ values of >42 and >70 μM, respectively.