title: 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.
abstract: 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 (Ki) 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.
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services , Bethesda, Maryland 20892, United States.
authors.forename: Joon H
authors.initials: JH
authors.lastname: Park
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services , Bethesda, Maryland 20892, United States.
authors.forename: Jane M
authors.initials: JM
authors.lastname: Sayer
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services , Bethesda, Maryland 20892, United States.
authors.forename: Annie
authors.initials: A
authors.lastname: Aniana
authors.forename: Xiaxia
authors.initials: X
authors.lastname: Yu
authors.forename: Irene T
authors.initials: IT
authors.lastname: Weber
authors.forename: Robert W
authors.initials: RW
authors.lastname: Harrison
authors.affiliation: Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Department of Health and Human Services , Bethesda, Maryland 20892, United States.
authors.forename: John M
authors.initials: JM
authors.lastname: Louis
mesh_terms: D024882:Drug Resistance, Viral; D015658:HIV Infections / Q000188:drug therapy* / Q000821:virology*; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000737:chemistry / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D006801:Humans; D017354:Point Mutation; D055503:Protein Multimerization; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism
chemical_list: D017320:HIV Protease Inhibitors; D054301:gag Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease; C514803:p16 protease, Human immunodeficiency virus 1