[Show abstract][Hide abstract] ABSTRACT: The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.
Full-text · Article · Mar 2010 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Optimization through parallel synthesis of a novel series of hepatitis C virus (HCV) NS5B polymerase inhibitors led to the identification of (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(6-methylpyridine-2-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zc and (R)-11-(4-benzyloxy-2-fluorophenyl)-6-hydroxy-3,3-dimethyl-10-(2,5-dimethyloxazol-4-carbonyl)-2,3,4,5,10,11-hexahydro-dibenzo[b,e][1,4]diazepin-1-one 11zk as potent (replicon EC(50)=400nM and 270nM, respectively) and selective (CC(50)>20muM) inhibitors of HCV replication. These data warrant further lead-optimization efforts.
No preview · Article · Mar 2009 · Bioorganic & medicinal chemistry letters
[Show abstract][Hide abstract] ABSTRACT: The exogenous control of hepatitis C virus (HCV) replication can be mediated through the inhibition of the RNA-dependent RNA
polymerase (RdRp) activity of NS5B. Small-molecule inhibitors of NS5B include nucleoside and nonnucleoside analogs. Here,
we report the discovery of a novel class of HCV polymerase nonnucleoside inhibitors, 1,5-benzodiazepines (1,5-BZDs), identified
by high-throughput screening of a library of small molecules. A fluorescence-quenching assay and X-ray crystallography revealed
that 1,5-BZD 4a bound stereospecifically to NS5B next to the catalytic site. When introduced into replicons, mutations known
to confer resistance against chemotypes that bind at this site were detrimental to inhibition by 1,5-BZD 7a. Using a panel
of enzyme isolates that covered genotypes 1 to 6, we showed that compound 4a inhibited genotype 1 only. In mechanistic studies,
4a was found to inhibit the RdRp activity of NS5B noncompetitively with GTP and to inhibit the formation of the first phosphodiester
bond during the polymerization cycle. The specificity for the HCV target was evaluated by profiling the 1,5-BZDs against other
viral and human polymerases, as well as BZD receptors.
Full-text · Article · Nov 2008 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: The use of multi-objective function optimization to superimpose a flexible source molecule onto a rigid target molecule is explored. The objective functions are the SEAL similarity score between the source and target molecules, which has to be maximized, and the conformational strain of the source molecule, which has to be minimized. The optimization algorithm used is an elitist non-dominated sorting genetic algorithm. The algorithm is tested with the superposition of two non-nucleoside HIV-reverse transcriptase inhibitors and the superposition of methotrexate onto dihydrofolate.
No preview · Article · May 2005 · Chemometrics and Intelligent Laboratory Systems
[Show abstract][Hide abstract] ABSTRACT: Objective: To investigate the important factors that determine the bioavailability and the antiviral activity of the diaryltriazine (DATA) and diarylpyrimidine (DAPY) non-nucleoside reverse transcriptase inhibitors (NNRTIs) of HIV-1 in animal species and humans using cell-based assays, physicochemical and comput ed parameters.
Methods: This naturalistic study included 15 parameters ranging from molecular mechanics calculations to phase I clinical trials. The calculated parameters were solvent-accessible surface area (SASA), polar surface area and Gibbs free energy of solvation. Physicochemical parameters comprised lipophilicity (octanol/water partition coefficient [cLogP]), ionisation constant (pKa), solubility and aggregate radius. Cell-based assays included human colonic adenocarcinoma cell (Caco-2) permeability (transepithelial transport), drug metabolism and antiviral activity (negative logarithm of the molar effective concentration inhibiting viral replica tion by 50% [pEC50]). Exposure was tested in rats, dogs and human volunteers.
Results: Of the 15 parameters, eight correlated consistently among one another. Exposure (area under the plasma concentration-time curve [AUC]) in humans correlated positively with that in rats (r = 1.00), with transepithelial transport (r = 0.83), lipophilicity (r = 0.60), ionisability (r = 0.89), hydrodynamic radius of aggregates (r = 0.66) and with antiviral activity (r = 0.61). Exposure in humans was also seen to correlate negatively with SASA (r = −0.89). No consistent correlation was found between exposure in dogs and the eight parameters. Of the 14 DATA/DAPY molecules, 11 form aggregates with radii between 34 and 100nm.
Conclusions: We observed correlations between exposure in humans with expo sure in rats, transepithelial transport (Caco-2 cells), ionisability, lipophilicity, aggregate radius and SASA in the class of DATA/DAPY NNRTI compounds. The lipophilic DATA/DAPY compounds form aggregates. It can be assumed that absorption in the intestinal tract and endocytosis in infected cells of these lipophilic compounds are governed by the common phenomenon of aggregate formation. As the lymphatic system offers a pathway for intestinal uptake of aggregates, this may offer a therapeutic advantage in the treatment of HIV-1 infection. Although it was not the objective of the study, we found that the rat was a better in vivo model than the dog for the prediction of systemic exposure in this particular set of compounds.