ABSTRACT
Passage of human immunodeficiency virus type-1 (HIV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in sequential accumulation of mutations in HIV-1 protease. We have characterized recombinant HIV-1 proteases that contain these mutations either individually (L10F, M46I, I47V, I50V) or in combination (the double mutant L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic properties and affinities for sulfonamide inhibitors and other classes of inhibitors were determined. For the I50V mutant, the efficiency (kcat/Km) of processing peptides designed to mimic cleavage junctions in the HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple mutant had a 2-fold higher processing efficiency than the I50V single mutant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory. The effects of mutation on processing efficiency were used in conjunction with the inhibition constant (Ki) to evaluate the advantage of the mutation for viral replication in the presence of drug. These analyses support the virological observation that the addition of M46I and I47V mutations on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478. Crystal structures and molecular models of the active site of the HIV-1 protease mutants suggest that changes in the active site can selectively affect the binding energy of inhibitors with little corresponding change in substrate binding.
Subject(s)
HIV Protease/genetics , HIV-1/enzymology , HIV-1/genetics , Mutation , Amino Acid Sequence , Binding Sites , Carbamates , Furans , Genetic Variation , HIV Protease/drug effects , HIV Protease Inhibitors/pharmacology , Hydrolysis , Indinavir , Isoquinolines/pharmacology , Kinetics , Models, Molecular , Molecular Sequence Data , Oligopeptides/metabolism , Pyridines/pharmacology , Quinolines/pharmacology , Saquinavir , Selection, Genetic , Substrate Specificity , Sulfonamides/pharmacologyABSTRACT
The objective of this work was to contribute to the understanding of mechanisms for IMPDH inhibition. We over-expressed hamster type II IMPDH in Escherichia coli, purified the protein to apparent homogeneity, and used capillary electrophoresis to quantify enzyme turnover events accompanying inhibition by mycophenolic acid (MPA). We dissected two convergent pathways leading to MPA-inhibition; a rapid "forward" pathway beginning with substrates and linked to enzyme catalysis, and a slower "reverse" pathway apparently not involving catalysis. MPA-inhibition occurred rapidly in the forward direction by interrupting the enzyme turnover cycle, after IMP and NAD+ binding, after hydride transfer, and after NADH release. Slow inhibition, without substrate turnover, was achieved by incubating free enzyme with excess XMP and MPA. We propose that mycophenolic acid inhibits IMPDH by trapping a transient covalent product of the hydride transfer reaction (IMPDH approximately XMP*) before a final hydrolysis step that precedes XMP and enzyme release in the forward reaction pathway. Understanding the ligand occupancy of the protein has also proven important for producing homogeneous, chemically defined complexes for structural studies. IMPDH samples inhibited by MPA in the forward and reverse pathways yielded similar, high-quality crystals that are currently undergoing X-ray diffraction analyses.
Subject(s)
Enzyme Inhibitors/pharmacology , IMP Dehydrogenase/antagonists & inhibitors , Mycophenolic Acid/pharmacology , Animals , Base Sequence , Cricetinae , Crystallization , DNA Primers/chemistry , Electrophoresis, Capillary , Escherichia coli/genetics , IMP Dehydrogenase/chemistry , IMP Dehydrogenase/metabolism , Inosine Monophosphate/metabolism , Kinetics , Models, Chemical , Molecular Sequence Data , NAD/metabolism , Protein Binding , Recombinant Proteins/metabolismABSTRACT
Enzyme-based assays are commonly employed in clinical and pharmaceutical laboratories to aid in quantitation of organic substances. Many enzyme assays are tedious, requiring the addition of reagents at multiple time intervals. The HPLC-based analysis of reaction products requires an additional step of vialing the samples and placing them in the autosampler. Such time-consuming, repetitive procedures are ideally suited for automation. We automated an HIV protease assay for the purposes of screening compounds as inhibitors of HIV protease and determining inhibition constants. Automation was accomplished by interfacing a robotic sample processor from a Gilson Model 232/401 biocompatible automatic sample processor and injector, with a Hewlett Packard HPLC. We used this configuration to automate the following steps: (a) preparation of serial dilutions of inhibitor, (b) enzyme assay setup, and (c) quantitation of products of enzyme assays. The resulting automated method produced inhibition constants that were of comparable accuracy and precision to those determined by manual methods.
