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1.  ACH-806, an NS4A Antagonist, Inhibits Hepatitis C Virus Replication by Altering the Composition of Viral Replication Complexes 
Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.
PMCID: PMC3697357  PMID: 23629709
2.  Experimental posterolateral spinal fusion with beta tricalcium phosphate ceramic and bone marrow aspirate composite graft 
Indian Journal of Orthopaedics  2010;44(4):402-408.
Beta tricalcium phosphate is commonly used in metaphyseal defects but its use in posterolateral spinal fusion remains controversial. There are very few published animal studies in which use of beta tricalcium phosphate has been evaluated in the posterolateral lumbar arthrodesis model. Hence we conducted a study to evaluate the potential of composite graft of beta tricalcium phosphate and bone marrow aspirate in comparison to autologous bone graft, when used for posterolateral spinal fusion.
Materials and Methods:
Single level posterolateral lumbar fusion was performed in 40 adult male Indian rabbits, which were assigned randomly into one of the four groups based on graft materials implanted; a) 3 gm beta tricalcium phosphate plus 3 ml bone marrow aspirate (Group I); b) 3 ml bone marrow aspirate alone (Group II); c) 3 gm beta tricalcium phosphate (Group III) and d) 3 gm autologous bone graft (Group IV). Each group had 10 rabbits. Half of the rabbits were sacrificed by injecting Phenobarbitone intraperitoneally after eight weeks and the remaining after 24 weeks, and were evaluated for fusion by X-rays, computed tomography (CT) scans, manual palpation test and histology.
Beta tricalcium phosphate used with bone marrow aspirate produced best results when compared to other groups (P =.0001). When beta tricalcium phosphate was used alone, fusion rates were better as compared to fusion achieved with autologous iliac crest bone graft though statistically not significant (P =0.07). Autologous bone graft showed signs of new bone formation. However, the rate of new bone formation was comparatively slow.
Composite graft of beta tricalcium phosphate and bone marrow aspirate can be used as an alternative to autologous iliac crest bone graft.
PMCID: PMC2947727  PMID: 20924481
Beta tricalcium phosphate; bone marrow aspirate; posterolateral spinal fusion
3.  Reliability and validity of Hindi translation of the migraine disability assessment and headache impact test-6 questionnaires 
The objective of the study was to assess the reliability and validity of the Hindi translation of the Migraine Disability Assessment (MIDAS) and Headache Impact Test-6 (HIT-6) questionnaires.
Materials and Methods:
The study was conducted on the migraine patients. For test–retest reliability, the respondents filled the MIDAS and HIT-6 questionnaires twice, at an interval of three weeks. For validity, the same population of patients filled the headache diary for three months. After three months they filled the MIDAS and HIT-6 questionnaires again. The patients were subgrouped according to their occupation and level of education. The test–retest reliability and validity were calculated by the Pearson correlation coefficient. Internal consistency was calculated using the Cronbach alpha.
A total of 236 migraine patients were screened. Seventy-nine patients fulfilled the inclusion criteria. A total of 69 patients completed the study. The HIT-6 questionnaire was applicable to all the subgroups of patients and had better comprehensibility than the MIDAS. Housewives missed out on the first two questions of the MIDAS and had lower mean MIDAS scores than HIT-6. The test–retest correlation coefficients for the total MIDAS and HIT-6 scores were 0.94 and 0.81, respectively. The correlation coefficients between the total score in the headache diary equivalent and the MIDAS and HIT-6 total score were 0.91 and 0.77, respectively. Cronbach alpha, a measure of internal consistency for the MIDAS questionnaire was > 0.90 at all the compilations. For the HIT-6 questionnaire, it ranged from 0.67 to 0.79.
The Hindi versions of MIDAS and HIT-6 questionnaires were reliable and valid, but could not be interchanged. HIT-6 had better comprehensibility.
PMCID: PMC3021931  PMID: 21264136
Headache Disability; HIT-6; migraine disability assessment; migraine; reliability; validity
4.  Selection of Replicon Variants Resistant to ACH-806, a Novel Hepatitis C Virus Inhibitor with No Cross-Resistance to NS3 Protease and NS5B Polymerase Inhibitors▿  
We have discovered a novel class of compounds active against hepatitis C virus (HCV), using a surrogate cellular system, HCV replicon cells. The leading compound in the series, ACH-806 (GS-9132), is a potent and specific inhibitor of HCV. The selection of resistance replicon variants against ACH-806 was performed to map the mutations conferring resistance to ACH-806 and to determine cross-resistance profiles with other classes of HCV inhibitors. Several clones emerged after the addition of ACH-806 to HCV replicon cells at frequencies and durations similar to that observed with NS3 protease inhibitors and NS5B polymerase inhibitors. Phenotypic analyses of these clones revealed that they are resistant to ACH-806 but remain sensitive to other classes of HCV inhibitors. Moreover, no significant change in the susceptibility to ACH-806 was found when the replicon cellular clones resistant to NS3 protease inhibitors and NS5B polymerase inhibitors were examined. Sequencing of the entire coding region of ACH-806-resistant replicon variants yielded several consensus mutations. Reverse genetics identified two single mutations in NS3, a cysteine-to-serine mutation at amino acid 16 and an alanine-to-valine mutation at amino acid 39, that are responsible for the resistance of the replicon variants to ACH-806. Both mutations are located at the N terminus of NS3 where extensive interactions with the central hydrophobic region of NS4A exist. These data provide evidence that ACH-806 inhibits HCV replication by a novel mechanism.
PMCID: PMC2415816  PMID: 18411324
5.  Molecular Mechanism of a Thumb Domain Hepatitis C Virus Nonnucleoside RNA-Dependent RNA Polymerase Inhibitor▿  
Antimicrobial Agents and Chemotherapy  2006;50(12):4103-4113.
A new pyranoindole class of small-molecule inhibitors was studied to understand viral resistance and elucidate the mechanism of inhibition in hepatitis C virus (HCV) replication. HCV replicon variants less susceptible to inhibition by the pyranoindoles were selected in Huh-7 hepatoma cells. Variant replicons contained clusters of mutations in the NS5B polymerase gene corresponding to the drug-binding pocket on the surface of the thumb domain identified by X-ray crystallography. An additional cluster of mutations present in part of a unique β-hairpin loop was also identified. The mutations were characterized by using recombinant replicon variants engineered with the corresponding amino acid substitutions. A single mutation (L419M or M423V), located at the pyranoindole-binding site, resulted in an 8- to 10-fold more resistant replicon, while a combination mutant (T19P, M71V, A338V, M423V, A442T) showed a 17-fold increase in drug resistance. The results of a competition experiment with purified NS5B enzyme with GTP showed that the inhibitory activity of the pyranoindole inhibitor was not affected by GTP at concentrations up to 250 μM. Following de novo initiation, the presence of a pyranoindole inhibitor resulted in the accumulation of a five-nucleotide oligomer, with a concomitant decrease in higher-molecular-weight products. The results of these studies have confirmed that pyranoindoles target the NS5B polymerase through interactions at the thumb domain. This inhibition is independent of GTP concentrations and is likely mediated by an allosteric blockade introduced by the inhibitor during the transition to RNA elongation after the formation of an initiation complex.
PMCID: PMC1693979  PMID: 16940072
Indian Journal of Psychiatry  1992;34(4):385-387.
A lady who had repeated spontaneous pathological laughter after cerebral infarction is described. The role of right hemispheric damage in its causation is discussed.
PMCID: PMC2982981  PMID: 21776152

Results 1-7 (7)