ABSTRACT
The identification of a low-permeability scavenger receptor BI (SR-BI) inhibitor starting from the ITX-5061 template is described. Structure-activity and structure-permeability relationships were assessed for analogs leading to the identification of compound 8 as a potent and nonabsorbable SR-BI inhibitor.
Subject(s)
Phenylenediamines/pharmacology , Scavenger Receptors, Class B/antagonists & inhibitors , Sulfonamides/pharmacology , Animals , Dogs , Dose-Response Relationship, Drug , Humans , Madin Darby Canine Kidney Cells , Molecular Structure , Organ Specificity , Phenylenediamines/administration & dosage , Phenylenediamines/chemistry , Rats , Structure-Activity Relationship , Sulfonamides/administration & dosage , Sulfonamides/chemistryABSTRACT
Mutations in the YMDD motif of the hepatitis B virus (HBV) DNA polymerase result in reduced susceptibility of HBV to inhibition by lamivudine, at a cost in replication fitness. The mechanisms underlying the effects of YMDD mutations on replication fitness were investigated using both a cell-based viral replication system and an in vitro enzyme assay to examine wild-type (wt) and YMDD-mutant polymerases. We calculated the affinities of wt and YMDD-mutant polymerases for each natural deoxyribonucleoside triphosphate (dNTP) and determined the intracellular concentrations of each dNTP in HepG2 cells under conditions that support HBV replication. In addition, inhibition constants for lamivudine triphosphate were determined for wt and YMDD-mutant polymerases. Relative to wt HBV polymerase, each of the YMDD-mutant polymerases showed increased apparent K(m) values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased K(i) values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent K(m) values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 microM). However, assays using dNTP concentrations equivalent to those measured in HepG2 cells under physiological conditions showed decreased enzymatic activity of YMDD-mutant polymerases relative to wt polymerase. Therefore, the decrease in replication fitness of YMDD-mutant HBV strains results from the lower affinities (increased K(m) values) of the YMDD-mutant polymerases for the natural dNTP substrates and physiological intracellular concentrations of dNTPs that are limiting for the replication of YMDD-mutant HBV strains.
Subject(s)
DNA-Directed DNA Polymerase/metabolism , Deoxyribonucleotides/pharmacology , Hepatitis B virus/enzymology , Baculoviridae/drug effects , Baculoviridae/genetics , Catalysis , Cell Line , DNA-Directed DNA Polymerase/genetics , Hepatitis B virus/genetics , Hepatitis B virus/immunology , Humans , Kinetics , Mutation/genetics , Nucleic Acid Hybridization , Plasmids/genetics , Reverse Transcriptase Polymerase Chain Reaction , Transduction, Genetic , Transfection , Virus Replication/drug effectsABSTRACT
A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in biotechnology.
