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Biomedical subjects

Atul Agarwal

Publications and source records attributed to Atul Agarwal.

13 recordsLinked to original sources
PubMed2006

Molecular mechanism of a thumb domain hepatitis C virus nonnucleoside RNA-dependent RNA polymerase inhibitor.

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 beta-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 microM. 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.

Amino Acid Substitution↗
PubMed2006

Structure-activity relationship of 6-methylidene penems bearing 6,5 bicyclic heterocycles as broad-spectrum beta-lactamase inhibitors: evidence for 1,4-thiazepine intermediates with C7 R stereochemistry by computational methods.

The design and synthesis of a series of 6-methylidene penems containing [6,5]-fused bicycles (thiophene, imidazole, or pyrazle-fused system) as novel class A, B, and C beta-lactamase inhibitors is described. These penems proved to be potent inhibitors of the TEM-1 (class A) and AmpC (class C) beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. Their in vitro and in vivo activities in combination with piperacillin are discussed. On the basis of the crystallographic structures of a serine-bound reaction intermediate of 2 with SHV-1 (class A) and GC1 (class C) enzymes, compounds 14a-l were designed and synthesized. Penems are proposed to form a seven-membered 1,4 thiazepine ring in both class A and C beta-lactamases. The interaction energy calculation for the enzyme-bound intermediates favor the formation of the C7 R enantiomer over the S enantiomer of the 1,4-thiazepine in both beta-lactamases, which is consistent with those obtained from the crystal structure of 2 with SHV-1 and GC1.

Aldehydes↗
PubMed2006

Discovery of proline sulfonamides as potent and selective hepatitis C virus NS5b polymerase inhibitors. Evidence for a new NS5b polymerase binding site.

Through high throughput screening, substituted proline sulfonamide 6 was identified as HCV NS5b RNA-dependent RNA polymerase inhibitor. Optimization of various regions of the lead molecule resulted in compounds that displayed good potency and selectivity. The crystal structure of 6 and NS5b polymerase complex confirmed the binding near the active site region. The optimization approach and SAR are discussed in detail.

Antiviral Agents↗
PubMed2006

Focal Wallerian degeneration of the corpus callosum in large middle cerebral artery stroke: serial diffusion tensor imaging.

PURPOSE: To detect lesion-related focal Wallerian degeneration (WD) changes in different segments of the corpus callosum (CC) in patients with large middle cerebral arterial (MCA) territory stroke using diffusion tensor imaging (DTI). MATERIALS AND METHODS: Eight patients underwent DTI scans at three different time points: six to eight weeks, 10-12 weeks, and beyond six months of stroke onset. Eight healthy age-matched controls were also scanned using the same protocol at three different time points. Region-of-interest (ROI) analysis was performed on seven segments of the CC to determine the fractional anisotropy (FA), mean diffusivity (MD), and corresponding callosal cross-sectional areas. RESULTS: On repeated-measures analysis of variance (ANOVA), a significant reduction in the FA values was observed from the first to the third study compared to controls, reflecting temporal degeneration in the rostrum, genu, rostral body, anterior midbody, and splenium of the CC. However, a significant temporal elevation in MD values was observed in only the rostral body and anterior midbody of the CC. This was associated with a significant region-specific reduction in the cross-sectional areas at time points beyond six months, and appears to be consistent with the loss of callosal structural components due to interruption of the cortico-callosal fibers secondary to WD. CONCLUSION: These results indicate that cortico-callosal topographical changes exhibit a significant temporal decline in observed FA values that is suggestive of cortico-callosal WD in patients with large MCA territory stroke.

Adult↗
PubMed2005

Diffusion tensor imaging in late posttraumatic epilepsy.

PURPOSE: The main objective of this study was to use diffusion tensor imaging (DTI) to search and quantify the extent of abnormality beyond the obvious lesions seen on the T2 and fluid-attenuation inversion recovery (FLAIR) magnetic resonance images in patients with chronic traumatic brain injury (TBI) with and without epilepsy. METHODS: DTI was performed on 23 chronic TBI patients (with late posttraumatic epilepsy, n=14; without epilepsy, n=9) and 11 age-matched controls. The ratios of fractional anisotropy (FA) and mean diffusivity (MD) between the regions of interest beyond the T2/FLAIR-visualized abnormality and the corresponding contralateral normal-appearing region were calculated. FA and MD ratios were compared for relative changes in these parameters among the TBI subjects with and without epilepsy and controls. Tissue volumes exhibiting abnormalities on DTI also were measured in these patients. RESULTS: The mean regional FA ratio was significantly lower, whereas the mean regional MD value was higher in patients with TBI compared with controls. The mean regional FA ratio was significantly lower in TBI patients with epilepsy (0.57+/-0.059) than in those without epilepsy (0.68+/-0.039). Although the regional MD ratio was higher in TBI patients with epilepsy (1.15+/-0.140) relative to those without epilepsy (1.09+/-0.141), the difference did not reach statistical significance. The tissue volume with low FA value also was found to be higher in TBI patients with epilepsy than without. CONCLUSIONS: Severity of injury as predicted by the DTI-derived increased volume of microstructure damage is associated with delayed posttraumatic epilepsy in TBI patients. These findings could be valuable in predicting epileptogenesis in patients with chronic TBI.

Adolescent↗
PubMed2004

Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum beta-lactamase inhibitors: crystallographic structures show unexpected binding of 1,4-thiazepine intermediates.

The design and synthesis of a series of seven tricyclic 6-methylidene penems as novel class A and C serine beta-lactamase inhibitors is described. These compounds proved to be very potent inhibitors of the TEM-1 and AmpC beta-lactamases and less so against the class B metallo-beta-lactamase CcrA. In combination with piperacillin, their in vitro activities enhanced susceptibility of all class C resistant strains from various bacteria. Crystallographic structures of a serine-bound reaction intermediate of 17 with the class A SHV-1 and class C GC1 enzymes have been established to resolutions of 2.0 and 1.4 A, respectively, and refined to R-factors equal 0.163 and 0.145. In both beta-lactamases, a seven-membered 1,4-thiazepine ring has formed. The stereogenic C7 atom in the ring has the R configuration in the SHV-1 intermediate and has both R and S configurations in the GC1 intermediate. Hydrophobic stacking interactions between the tricyclic C7 substituent and a tyrosine side chain, rather than electrostatic or hydrogen bonding by the C3 carboxylic acid group, dominate in both complexes. The formation of the 1,4- thiazepine ring structures is proposed based on a 7-endo-trig cyclization.

Binding Sites↗
PubMed2004

Novel imidazole substituted 6-methylidene-penems as broad-spectrum beta-lactamase inhibitors.

Beta-lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against beta-lactam antibiotics. Production of class-A, class-B, and class-C enzymes by the bacteria make the use of beta-lactam antibiotics ineffective in certain cases. To overcome resistance to beta-lactam antibiotics, several beta-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with beta-lactam antibiotics. However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved beta-lactamase inhibitors. Although the commercially available beta-lactamase inhibitor/beta-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum beta-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various beta-lactamase producing isolates. The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.

Imidazoles↗
PubMed2004

Peritoneal calcification: causes and distinguishing features on CT.

OBJECTIVE: We undertook this study to determine the causes of peritoneal calcification seen on CT and to investigate which CT features distinguish benign from malignant peritoneal calcification. MATERIALS AND METHODS: Seventeen patients with peritoneal calcification were identified through retrospective review of reports from 74765 abdominopelvic CT examinations performed during a 7-year period. We determined the cause of peritoneal calcification by examining medical and histopathologic records. Calcification morphology was classified as nodular or sheetlike on the basis of the consensus interpretation by two independent radiologists. The radiologists also recorded the presence or absence of associated soft-tissue components or lymph node calcification. The association between the CT findings and the cause of calcification was assessed using chi-square analysis. RESULTS: Peritoneal calcification was due to peritoneal dialysis (n = 4), prior peritonitis (n = 3), cryptogenic origin (n = 1), or peritoneal spread of ovarian carcinoma (n = 9). Sheet-like calcification was more common in patients with benign calcification (seven of eight patients) than in those with malignant calcification (two of nine patients, p < 0.05). Nodal calcification was seen only in patients with malignant calcification (five of nine patients vs none of eight, p < 0.05). CONCLUSION: Common causes of peritoneal calcification are dialysis, prior peritonitis, or ovarian cancer; sheetlike calcification indicates a benign cause, whereas associated lymph node calcification strongly suggests malignancy.

Adult↗
PubMed2004

Epilepsy with focal cerebral calcification: role of magnetization transfer MR imaging.

BACKGROUND: Some patients with focal cerebral calcification (FCC) have no seizure or a benign course of epilepsy, whilst others with a similar lesion have uncontrolled epilepsy. AIMS: To look for perilesional hyperintensity, presumed to be indicative of gliosis, around FCC on magnetization transfer (MT) MRI and to correlate seizure outcome with its presence. SETTING AND DESIGN: Case control study. MATERIAL AND METHODS: Fifty-one patients with epilepsy and 30 controls with single calcified cerebral lesion on CT were studied. Clinical and treatment details were noted. EEG and T1, T2, MT and contrast enhanced MRI were done. STATISTICAL ANALYSIS USED: Student's t test. RESULTS: On MT MRI, perilesional gliosis was seen around the focal calcified lesion in 17 (33.3%) patients. None of the controls had perilesional gliosis. The mean monthly seizure frequency was significantly higher in the 17 patients having perilesional gliosis (2.63+1.15) as compared to the 34 patients without it (0.59+0.63; P= 0.0014). Perilesional gliosis was seen in 8 out of 11 (72.7%) patients who were on 2 AEDs and in all 5 (100%) patients who were on 3 or more AEDs. It was present only in 4 (11.4%) out of 35 patients who were on one AED. CONCLUSION: Gliosis around a cerebral calcified lesion as seen on T1 weighted MT MRI indicates poor seizure control.

Adolescent↗
PubMed2003

Short course of prednisolone in Indian patients with solitary cysticercus granuloma and new-onset seizures.

PURPOSE: To evaluate the role of a short course of oral corticosteroids in Indian patients with solitary cysticercus granuloma with seizures. METHODS: In this open-label, randomized, prospective follow-up study, 97 patients with new-onset seizures and a single enhancing computed tomography (CT)-detected lesion of cysticercosis were randomly divided in two groups to receive either antiepileptic monotherapy alone (n = 48) or antiepileptic monotherapy with prednisolone (n = 49). The patients in the latter group received prednisolone, 1 mg/kg/day for 10 days, followed by tapering over next 4 days. The patients were followed up for 6 months. Repeated CT scans were performed after 1 and 6 months. RESULTS: The majority of patients were young. Simple partial seizure, with or without secondary generalization, was the commonest seizure type encountered. Follow-up CT scans at 1 and 6 months demonstrated a significantly better response for prednisolone as far as complete resolution of CT lesion was concerned. Kaplan-Meier analysis suggested significantly less probability of seizure recurrence for prednisolone-treated patients. At 6 months, Kaplan-Meier estimated risk of seizure after first seizure was 2% in prednisolone-treated patients in comparison to 13% for those who were not given prednisolone. CONCLUSIONS: Short-term prednisolone therapy helps in rapid resolution of solitary cysticercus granuloma in Indian patients with new-onset seizures. Resolution of lesions is associated with improved seizure-related prognosis.

Adolescent↗
PubMed2002

Modulation of hepatitis C virus RNA-dependent RNA polymerase activity by structure-based site-directed mutagenesis.

The hepatitis C virus (HCV) encodes an RNA-dependent RNA polymerase (NS5B), which is indispensable for the viral genome replication. Although structural comparison among HCV NS5B, poliovirus 3D-pol, and human immunodeficiency virus-reverse transcriptase RNA-dependent polymerase reveals the canonical palm, fingers, and thumb domains, the crystal structure of HCV NS5B highlights the presence of a unique A1-loop, which extends from the fingers to the thumb domain (amino acids 12-46), providing many contact points for the proposed "closed" conformation of the enzyme. The polymerase also possesses a tunnel, which starts at the active site and terminates on the back surface of the enzyme. This tunnel of 19 A contains five basic amino acids, which may be engaged in NTP trafficking. In the present study, we exploited the crystal structure of the enzyme to elucidate the involvement of these two structural motifs in enzyme activity by site-directed mutagenesis. As predicted, the replacement of leucine 30 located in the Lambda 1-loop is detrimental to the NS5B activity. Heparin-Sepharose column chromatography and analytical ultracentrifugation experiments strongly suggest a local alteration in the structure of the Leu-30 mutant. An analysis of amino acid substitutions in Arg-222 and Lys-151 within the putative NTP tunnel indicates that Arg-222 was critical in delivering NTPs to the active site, whereas Lys-151 was dispensable. Interestingly, the substitution of lysine 151 for a glutamic acid resulted in an enzyme that was consistently more active in de novo synthesis as well as by "copy-back" mechanism of a self-primed substrate when compared with the wild type NS5B enzyme. Burst kinetic analyses indicate that the gain in function of K151E enzyme was primarily the result of the formation of more productive pre-initiation complexes that were used for the elongation reaction. In contrast to the recent observations, both the wild type and mutant enzymes were monomeric in solution, whereas molecules of higher order were apparent in the presence of RNA template.

Base Sequence↗