RÉSUMÉ
Little is known about the mechanism of HCV polymerase-catalyzed nucleotide incorporation and the individual steps employed by this enzyme during a catalytic cycle. In this paper, we applied various biochemical tools and examined the mechanism of polymerase catalysis. We found that formation of a productive RNA-enzyme complex is the slowest step followed by RNA dissociation and initiation of primer strand synthesis. Various groups have reported several classes of small molecule inhibitors of hepatitis C virus NS5B polymerase; however, the mechanism of inhibition for many of these inhibitors is not clear. We undertook a series of detailed mechanistic studies to characterize the mechanisms of inhibition for these HCV polymerase inhibitors. We found that the diketoacid derivatives competitively bind to the elongation NTP pocket in the active site and inhibit both the initiation and elongation steps of polymerization. While both benzimidazoles and benzothiadiazines are noncompetitive with respect to the active site elongation NTP pocket, benzothiadiazine compounds competitively bind to the initiation pocket in the active site and inhibit only the initiation step of de novo RNA polymerization. The benzimidazoles bind to the thumb allosteric pocket and inhibit the conformational changes during RNA synthesis. We also observed a cross interaction between the thumb allosteric pocket and the initiation pocket using inhibitor-inhibitor cross competition studies. This information will be very important in designing combination therapies using two small molecule drugs to treat hepatitis C virus.
Sujet(s)
Benzimidazoles/pharmacologie , Benzothiadiazines/pharmacologie , Polymères/métabolisme , Protéines virales non structurales/antagonistes et inhibiteurs , Protéines virales non structurales/métabolisme , Benzimidazoles/composition chimique , Benzothiadiazines/composition chimique , Électrophorèse , Héparine/métabolisme , Humains , Cinétique , Liaison aux protéines , Structure secondaire des protéines , ARN viral/métabolisme , Spécificité du substrat , Matrices (génétique) , Facteurs temps , Protéines virales non structurales/composition chimiqueRÉSUMÉ
As part of our efforts to identify potent HIV-1 protease inhibitors that are active against resistant viral strains, structural modification of the azacyclic urea (I) was undertaken by incorporating acyl groups as P(1)' ligands. The extensive SAR study has yielded a series of N-acyl azacyclic ureas (II), which are highly potent against both wild-type and multiple PI-resistant viral strains.
Sujet(s)
Inhibiteurs de protéase du VIH/synthèse chimique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , Composés aza/synthèse chimique , Composés aza/pharmacologie , Conception de médicament , Multirésistance aux médicaments , Inhibiteurs de protéase du VIH/usage thérapeutique , Ligands , Tests de sensibilité microbienne , Modèles moléculaires , Ritonavir/synthèse chimique , Ritonavir/usage thérapeutique , Relation structure-activité , Urée/analogues et dérivés , Urée/synthèse chimique , Urée/pharmacologieRÉSUMÉ
A series of novel azacyclic urea HIV protease inhibitors bearing a benzenesulfonamide group at P1' were synthesized utilizing a parallel synthesis method. Structural studies of early analogs bound in the enzyme active site were used to design more potent inhibitors. The effects of substituting the P1' benzenesulfonyl group on antiviral activity and protein binding are described.
Sujet(s)
Inhibiteurs de protéase du VIH/synthèse chimique , Sulfonamides/synthèse chimique , Urée/analogues et dérivés , Urée/synthèse chimique , Inhibiteurs de protéase du VIH/composition chimique , Inhibiteurs de protéase du VIH/pharmacologie , Humains , Modèles moléculaires , Conformation moléculaire , Relation structure-activité , Sulfonamides/composition chimique , Sulfonamides/pharmacologie , Urée/composition chimique , Urée/pharmacologieRÉSUMÉ
Using measured free fraction and 50% inhibitory concentration (IC50) values for the human immunodeficiency virus protease inhibitors lopinavir (LPV) and ritonavir (RTV) in tissue culture media with various protein concentrations ranging from 5 to 50%, we estimated serum-free IC50 values for each drug. The range of serum-free IC50 values (0.64 to 0.77 ng/ml for LPV and 3.0 to 5.0 ng/ml for RTV) did not exhibit a trend with increasing protein concentrations, despite a 10-fold difference in the free fraction value (0.006 to 0.063) for LPV and a 5-fold difference in the free fraction value (0.013 to 0.057) for RTV. The mean serum-free IC50 by the MTT-MT4 assay (0.69 ng/ml for LPV and 4.0 ng/ml for RTV) may be the most accurate parameter for the estimation of the inhibitory quotient (IQ), a relative measure of in vivo potency defined as the ratio of the minimal free drug concentration in plasma (C(trough,free)) for a specific patient population and the serum-free IC50. Using this approach, we calculated the average IQs for protease inhibitor-naïve patients for LPV and RTV to be 67 and 5.6, respectively.
Sujet(s)
Inhibiteurs de protéase du VIH/pharmacologie , Inhibiteurs de protéase du VIH/pharmacocinétique , Pyrimidinones/pharmacologie , Pyrimidinones/pharmacocinétique , Ritonavir/pharmacologie , Ritonavir/pharmacocinétique , Algorithmes , Animaux , Dosage biologique , Bovins , Chromatographie en phase liquide à haute performance , Protéase du VIH/métabolisme , Humains , Cinétique , Lopinavir , Liaison aux protéinesRÉSUMÉ
The HIV protease inhibitor ABT-378 (lopinavir) has a six-member cyclic urea in the P-2 position. A series of analogues in which the six-member cyclic urea is replaced by various heterocycles was synthesized and the structure-activity relationships explored.
Sujet(s)
Inhibiteurs de protéase du VIH/synthèse chimique , Pyrimidinones/pharmacologie , Lignée cellulaire tumorale , Antienzymes/synthèse chimique , Antienzymes/pharmacologie , Inhibiteurs de protéase du VIH/pharmacologie , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , Composés hétérocycliques/composition chimique , Humains , Lopinavir , Tests de sensibilité microbienne , Pyrimidinones/synthèse chimique , Relation structure-activité , Urée/composition chimiqueRÉSUMÉ
Isopropyl substituted 4-thioazolyl valine side chains are highly optimized P(2)-P(3) ligands for C2 symmetry-based HIV protease inhibitors, as exemplified by the drug ritonavir. Replacement of the side chain with the conformationally constrained hexahydrofurofuranyloxy P(2) ligand in combination with a dimethylphenoxyacetate on the other end of the ritonavir core diamine yielded highly potent HIV protease inhibitors. The in vitro antiviral activity in MT4 cells increased by 10- and 20-fold, respectively, in the absence and presence of 50% human serum compared to ritonavir. The structure-activity relationships of inhibitor series with this combination of ligands were investigated. Preliminary pharmacokinetic studies in rats indicated rapid elimination of the inhibitors from the blood, and the plasma levels were not significantly enhanced by coadministration with ritonavir. However, the novel structural features and the high intrinsic antiviral potency of this series provides potential for the future exploration of prodrug strategies.
Sujet(s)
Inhibiteurs de protéase du VIH/synthèse chimique , Inhibiteurs de protéase du VIH/pharmacologie , Phénoxy-acétates/synthèse chimique , Phénoxy-acétates/pharmacologie , Animaux , Biodisponibilité , Lignée cellulaire , Inhibiteurs de protéase du VIH/pharmacocinétique , Période , Humains , Rats , Ritonavir/pharmacologie , Relation structure-activitéRÉSUMÉ
The HIV protease inhibitor Lopinavir has a pseudosymmetric core unit incorporating benzyl groups at both P-1, P-1' positions. A series of analogues incorporating non-aromatic side chains at the P-1 position were synthesized and the structure-activity relationships explored.
Sujet(s)
Inhibiteurs de protéase du VIH/synthèse chimique , Inhibiteurs de protéase du VIH/pharmacologie , Pyrimidinones/synthèse chimique , Pyrimidinones/pharmacologie , Lignée cellulaire , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/effets des médicaments et des substances chimiques , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/enzymologie , Humains , Lopinavir , Tests de sensibilité microbienne , Relation structure-activitéRÉSUMÉ
The in vitro inhibition of wild-type human immunodeficiency virus (HIV) by combinations of lopinavir and six other protease inhibitors over a range of two-drug combination ratios was evaluated. Combinations of lopinavir with indinavir, nelfinavir, amprenavir, tipranavir, and BMS-232632 generally displayed an additive relationship. In contrast, a consistent, statistically significant synergistic inhibition of HIV type 1 replication with combinations of lopinavir and saquinavir was observed. Analysis of the combination indices indicated that lopinavir with saquinavir was synergistic over the entire range of drug combination ratios tested and at all levels of inhibition in excess of 40%. Cellular toxicity was not observed at the highest drug concentrations tested. These results suggest that administration of combinations of the appropriate dose of lopinavir with other protease inhibitors in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity. More importantly, the observed in vitro synergy between lopinavir and saquinavir provides a theoretical basis for the clinical exploration of a novel regimen of lopinavir-ritonavir and saquinavir.
Sujet(s)
Agents antiVIH/pharmacologie , Inhibiteurs de protéase du VIH/pharmacologie , VIH (Virus de l'Immunodéficience Humaine)/effets des médicaments et des substances chimiques , Pyrimidinones/pharmacologie , Association de médicaments , Infections à VIH/traitement médicamenteux , Lopinavir , Tests de sensibilité microbienneRÉSUMÉ
The HIV protease inhibitor ABT-378 (Lopinavir) has a 2,6-dimethylphenoxyacetyl group in the P-2' position. Analogues in which this group is replaced with various substituted phenyl or heteroaryl groups were synthesized and the structure-activity relationships explored.