Lack of synergy for inhibitors targeting a multi‐drug‐resistant HIV‐1 protease

Abstract

The three-dimensional structures of indinavir and three newly synthesized indinavir analogs in complex with a multi-drug-resistant variant (L63P, V82T, I84V) of HIV-1 protease were determined to ∼2.2 Å resolution. Two of the three analogs have only a single modification of indinavir, and their binding affinities to the variant HIV-1 protease are enhanced over that of indinavir. However, when both modifications were combined into a single compound, the binding affinity to the protease variant was reduced. On close examination, the structural rearrangements in the protease that occur in the tightest binding inhibitor complex are mutually exclusive with the structural rearrangements seen in the second tightest inhibitor complex. This occurs as adaptations in the S1 pocket of one monomer propagate through the dimer and affect the conformation of the S1 loop near P81 of the other monomer. Therefore, structural rearrangements that occur within the protease when it binds to an inhibitor with a single modification must be accounted for in the design of inhibitors with multiple modifications. This consideration is necessary to develop inhibitors that bind sufficiently tightly to drug-resistant variants of HIV-1 protease to potentially become the next generation of therapeutic agents.

Structural basis and distal effects of Gag substrate coevolution in drug resistance to HIV-1 protease

“…The gene encoding the HIV protease was subcloned into the heat-inducible pXC35 expression vector (ATCC) and transformed into E. coli TAP-106 cells. Protein expression and purification were performed as previously described (41). Protease purified from size exclusion column (equilibrated with gel filtration buffer containing 0.05 M sodium acetate at pH 5.5, 5% (vol/vol) ethylene glycol, 10% (vol/vol) glycerol, and 5 mM DTT) was concentrated to 2 mg/mL using an Amicon Ultra-15 10-kDa device (Millipore) for crystallization.…”

Structural and Thermodynamic Basis of Amprenavir/Darunavir and Atazanavir Resistance in HIV-1 Protease with Mutations at Residue 50

“…Each variant was subcloned into the heat-inducible pXC35 expression vector (American Type Culture Collection [ATCC], Manassas, VA) and transformed into Escherichia coli TAP-106 cells. Protein overexpression, purification, and refolding were carried out as previously described (22). Protein used for crystallographic studies was further purified with a Pharmacia Superdex 75 fast-performance liquid chromatography column (GE Healthcare, Chalfont St. Giles, United Kingdom) equilibrated with refolding buffer (50 mM sodium acetate, pH 5.5, 10% glycerol, 5% ethylene glycol, 5 mM dithiothreitol).…”

Context Surrounding Processing Sites Is Crucial in Determining Cleavage Rate of a Subset of Processing Sites in HIV-1 Gag and Gag-Pro-Pol Polyprotein Precursors by Viral Protease

“…Protease Expression and Purification-The HIV-1 protease was expressed in E. coli TAP 106 cells and purified from inclusion bodies as previously described (32). Briefly, the inclusion body centrifugation pellet was dissolved in 50% acetic acid followed by another round of centrifugation to remove impurities.…”