title: Molecular basis for increased susceptibility of isolates with atazanavir resistance-conferring substitution I50L to other protease inhibitors.
abstract: Protease inhibitors (PIs) are highly effective drugs against the human immunodeficiency virus (HIV), yet long-term therapeutic use is limited by emergence of HIV type 1 (HIV-1) protease substitutions that confer cross-resistance to multiple protease inhibitor drugs. Atazanavir is a highly potent HIV protease inhibitor with a distinct resistance profile that includes effectiveness against most HIV-1 isolates resistant to one or two PIs. The signature resistance substitution for atazanavir is I50L, and it is frequently (53%) accompanied by a compensatory A71V substitution that helps restore viability and increases atazanavir resistance levels. We measured the binding affinities of wild-type (WT) and I50L/A71V HIV-1 proteases to atazanavir and other currently approved PIs (ritonavir, lopinavir, saquinavir, nelfinavir, indinavir, and amprenavir) by isothermal titration calorimetry. Remarkably, we find that all of the PIs have 2- to 10-fold increased affinities for I50L/A71V protease, except for atazanavir. The results are also manifested by thermal stability measures of affinity for WT and I50L/A71V proteases. Additional biophysical and enzyme kinetics experiments show I50L/A71V protease is a stable enzyme with catalytic activity that is slightly reduced (34%) relative to the WT. Computational modeling reveals that the unique resistance phenotype of I50L/A71V protease likely originates from bulky tert-butyl groups at P2 and P2' (specific to atazanavir) that sterically clash with methyl groups on residue L50. The results of this study provide a molecular understanding of the novel hypersusceptibility of atazanavir-resistant I50L/A71V-containing clinical isolates to other currently approved PIs.
authors.affiliation: Gene Expression and Protein Biochemistry Department, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543-4000, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Yanchunas
authors.forename: David R
authors.initials: DR
authors.lastname: Langley
authors.forename: Li
authors.initials: L
authors.lastname: Tao
authors.forename: Ronald E
authors.initials: RE
authors.lastname: Rose
authors.forename: Jacques
authors.initials: J
authors.lastname: Friborg
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Colonno
authors.forename: Michael L
authors.initials: ML
authors.lastname: Doyle
mesh_terms: D019943:Amino Acid Substitution; D000069446:Atazanavir Sulfate; D001667:Binding, Competitive / Q000187:drug effects; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D024882:Drug Resistance, Viral; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics*; D017320:HIV Protease Inhibitors / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006358:Hot Temperature; D008958:Models, Molecular; D008961:Models, Structural; D009842:Oligopeptides / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D013696:Temperature
chemical_list: D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D011725:Pyridines; D000069446:Atazanavir Sulfate; D016333:HIV Protease