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

作者：Park Joon H; Sayer Jane M; Aniana Annie; Yu Xiaxia; Weber Irene T; Harrison Robert W; Louis John M^*

来源：Biochemistry, 2016, 55(16): 2390-2400.

DOI：10.1021/acs.biochem.6b00012

• Autoprocessing
• Dimer formation
• Drug resistance
• High resistance
• Multidrug resistants
• Natural variation
• Orders of magnitude
• Protease inhibitor

We have systematically validated the activity and inhibition of a HIV-1 protease (PR) variant bearing 17 mutations (PRS17), selected to represent high resistance by machine learning on genotype phenotype data. Three of five mutations in PRS17 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. PRS17, which forms a stable dimer (<10 nM), is similar to 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 PRS17 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-PRS17 and mature PRS17. Resistance of PRS17 to eight protease inhibitors (PIs) relative to PR (K-i) increases by 1.5-5 orders of magnitude from 0.01 to 8.4 mu M. 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 similar to 7.5 to 60 mu M. Thus, this process, crucial for stable dimer formation, shows inhibition similar to 200-800-fold weaker than that of the mature PRS17. TFP/p6pol cleavage, which occurs faster, is inhibited even more weakly by all PIs except darunavir (IC50 = 15 mu M); amprenavir shows a 2-fold increase in IC50 (similar to 15 mu M), and atazanavir and lopinavir show increased IC50 values of >42 and >70 mu M, respectively.

• 出版日期2016-4-26