Publications by authors named "Kaveri Das"

7 Publications

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A physical phantom for amine chemical exchange saturation transfer (CEST) MRI.

MAGMA 2021 Jan 23. Epub 2021 Jan 23.

UCLA Brain Tumor Imaging Laboratory (BTIL), Center for Computer Vision and Imaging Biomarkers, University of California, Los Angeles, Los Angeles, CA, USA.

Objective: To develop a robust amine chemical exchange saturation transfer (CEST) physical phantom, validate the temporal stability, and create a supporting software for automatic image processing and quality assurance.

Materials And Methods: The phantom was designed as an assembled laser-cut acrylic rack and 18 vials of phantom solutions, prepared with different pHs, glycine concentrations, and gadolinium concentrations. We evaluated glycine concentrations using ultraviolet absorbance for 70 days and measured the pH, relaxation rates, and CEST contrast for 94 days after preparation. We used Spearman's correlation to determine if glycine degraded over time. Linear regression and Bland-Altman analysis were performed between baseline and follow-up measurements of pH and MRI properties.

Results: No degradation of glycine was observed (p > 0.05). The pH and MRI measurements stayed stable for 3 months and showed high consistency across time points (R = 1.00 for pH, R, R, and CEST contrast), which was further validated by the Bland-Altman plots. Examples of automatically generated reports are provided.

Discussion: We designed a physical phantom for amine CEST-MRI, which is easy to assemble and transfer, holds 18 different solutions, and has excellent short-term chemical and MRI stability. We believe this robust phantom will facilitate the development of novel sequences and cross-scanners validations.
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http://dx.doi.org/10.1007/s10334-020-00902-zDOI Listing
January 2021

Assessment of Mycobacterium tuberculosis pantothenate kinase vulnerability through target knockdown and mechanistically diverse inhibitors.

Antimicrob Agents Chemother 2014 Jun 31;58(6):3312-26. Epub 2014 Mar 31.

Department of Bioscience, iMED infection, AstraZeneca India Pvt. Ltd., Bangalore, India

Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis.
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http://dx.doi.org/10.1128/AAC.00140-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4068421PMC
June 2014

Assay development for identifying inhibitors of the mycobacterial FadD32 activity.

J Biomol Screen 2013 Jun 30;18(5):576-87. Epub 2013 Jan 30.

Institut de Pharmacologie et de Biologie Structurale (IPBS), Centre National de la Recherche Scientifique (CNRS), Département "Mécanismes Moléculaires des Infections Mycobactériennes," Toulouse, France.

FadD32, a fatty acyl-AMP ligase (FAAL32) involved in the biosynthesis of mycolic acids, major and specific lipid components of the mycobacterial cell envelope, is essential for the survival of Mycobacterium tuberculosis, the causative agent of tuberculosis. The protein catalyzes the conversion of fatty acid to acyl-adenylate (acyl-AMP) in the presence of adenosine triphosphate and is conserved in all the mycobacterial species sequenced so far, thus representing a promising target for the development of novel antituberculous drugs. Here, we describe the optimization of the protein purification procedure and the development of a high-throughput screening assay for FadD32 activity. This spectrophotometric assay measuring the release of inorganic phosphate was optimized using the Mycobacterium smegmatis FadD32 as a surrogate enzyme. We describe the use of T m (melting temperature) shift assay, which measures the modulation of FadD32 thermal stability, as a tool for the identification of potential ligands and for validation of compounds as inhibitors. Screening of a selected library of compounds led to the identification of five novel classes of inhibitors.
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http://dx.doi.org/10.1177/1087057112474691DOI Listing
June 2013

Kinetic modelling of GlmU reactions - prioritization of reaction for therapeutic application.

PLoS One 2012 27;7(8):e43969. Epub 2012 Aug 27.

Innovative Medicine for Infection (iMed Infection), AstraZeneca India Pvt. Ltd., Bangalore, Karnataka, India.

Mycobacterium tuberculosis(Mtu), a successful pathogen, has developed resistance against the existing anti-tubercular drugs necessitating discovery of drugs with novel action. Enzymes involved in peptidoglycan biosynthesis are attractive targets for antibacterial drug discovery. The bifunctional enzyme mycobacterial GlmU (Glucosamine 1-phosphate N-acetyltransferase/ N-acetylglucosamine-1-phosphate uridyltransferase) has been a target enzyme for drug discovery. Its C- and N- terminal domains catalyze acetyltransferase (rxn-1) and uridyltransferase (rxn-2) activities respectively and the final product is involved in peptidoglycan synthesis. However, the bifunctional nature of GlmU poses difficulty in deciding which function to be intervened for therapeutic advantage. Genetic analysis showed this as an essential gene but it is still unclear whether any one or both of the activities are critical for cell survival. Often enzymatic activity with suitable high-throughput assay is chosen for random screening, which may not be the appropriate biological function inhibited for maximal effect. Prediction of rate-limiting function by dynamic network analysis of reactions could be an option to identify the appropriate function. With a view to provide insights into biochemical assays with appropriate activity for inhibitor screening, kinetic modelling studies on GlmU were undertaken. Kinetic model of Mtu GlmU-catalyzed reactions was built based on the available kinetic data on Mtu and deduction from Escherichia coli data. Several model variants were constructed including coupled/decoupled, varying metabolite concentrations and presence/absence of product inhibitions. This study demonstrates that in coupled model at low metabolite concentrations, inhibition of either of the GlmU reactions cause significant decrement in the overall GlmU rate. However at higher metabolite concentrations, rxn-2 showed higher decrement. Moreover, with available intracellular concentration of the metabolites and in vivo variant of model, uncompetitive inhibition of rxn-2 caused highest decrement. Thus, at physiologically relevant metabolite concentrations, targeting uridyltranferase activity of Mtu GlmU would be a better choice for therapeutic intervention.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3428340PMC
February 2013

An assay for exogenous sources of purified MurG, enabled by the complementation of Escherichia coli murG(Ts) by the Mycobacterium tuberculosis homologue.

FEMS Microbiol Lett 2012 Jan 30;326(2):161-7. Epub 2011 Nov 30.

AstraZeneca India Pvt. Ltd, Bangalore, India.

The Mycobacterium tuberculosis murG gene, Rv2153, was expressed in Escherichia coli murG(Ts) strain OV58 on a plasmid under the control of the arabinose-inducible araBAD promoter. Mycobacterium tuberculosis murG rescued the growth of E. coli murG(Ts) at the nonpermissive temperature: transformants were only obtained in the presence of 0.2% arabinose at 42 °C, and their growth rate was dependent on arabinose concentrations. However, no MurG activity was detected in membranes from the transformant grown in arabinose at 42 °C, while MraY activity was normal. This observation led to the development of a membrane-based scintillation proximity assay for exogenous sources of MurG. Addition of purified E. coli MurG resulted in the reconstitution of MurG and peptidoglycan synthesis in these membranes. MurG is an attractive target for drug discovery, but assays to measure the activity of purified MurG are challenging. This presents an easy method to measure the activity of exogenous sources of MurG for structure-activity studies of mutant MurG proteins. It can also be used to compare the activity of, or effect of inhibitors on, MurG from other bacterial species.
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http://dx.doi.org/10.1111/j.1574-6968.2011.02446.xDOI Listing
January 2012

Screening, identification, and characterization of mechanistically diverse inhibitors of the Mycobacterium tuberculosis enzyme, pantothenate kinase (CoaA).

J Biomol Screen 2012 Mar 15;17(3):293-302. Epub 2011 Nov 15.

AstraZeneca R & D, Bangalore, India.

The authors describe the discovery of anti-mycobacterial compounds through identifying mechanistically diverse inhibitors of the essential Mycobacterium tuberculosis (Mtb) enzyme, pantothenate kinase (CoaA). Target-driven drug discovery technologies often work with purified enzymes, and inhibitors thus discovered may not optimally inhibit the form of the target enzyme predominant in the bacterial cell or may not be available at the desired concentration. Therefore, in addition to addressing entry or efflux issues, inhibitors with diverse mechanisms of inhibition (MoI) could be prioritized before hit-to-lead optimization. The authors describe a high-throughput assay based on protein thermal melting to screen large numbers of compounds for hits with diverse MoI. Following high-throughput screening for Mtb CoaA enzyme inhibitors, a concentration-dependent increase in protein thermal stability was used to identify true binders, and the degree of enhancement or reduction in thermal stability in the presence of substrate was used to classify inhibitors as competitive or non/uncompetitive. The thermal shift-based MoI assay could be adapted to screen hundreds of compounds in a single experiment as compared to traditional biochemical approaches for MoI determination. This MoI was confirmed through mechanistic studies that estimated K(ie) and K(ies) for representative compounds and through nuclear magnetic resonance-based ligand displacement assays.
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http://dx.doi.org/10.1177/1087057111423069DOI Listing
March 2012

Glycine and alanine dehydrogenase activities are catalyzed by the same protein in Mycobacterium smegmatis: upregulation of both activities under microaerophilic adaptation.

Can J Microbiol 2002 Jan;48(1):7-13

AstraZeneca India Pvt Ltd., Malleswaram, Bangalore, India.

Microaerophilic adaptation has been described as one of the in vitro dormancy models for tuberculosis. Studies on Mycobacterium tuberculosis adapted to low oxygen levels showed an enhancement of glycine dehydrogenase (deaminating) activity. We studied the physiology of the fast-growing, nonpathogenic strain of Mycobacterium smegmatis ATCC 607 under low oxygen by shifting the actively growing M. smegmatis cells to static microaerophilic growth conditions. This shifting of M. smegmatis culture resulted in a similar phenomenon as seen with M. tuberculosis, i.e., elevated glycine dehydrogenase activity. Further purification of glycine dehydrogenase from M. smegmatis demonstrated glyoxylate amination, but failed to demonstrate glycine deamination, even in the purified fraction. Moreover, the purified protein showed pyruvate amination as well as L-alanine deamination activities. By activity staining, the protein band positive for glyoxylate amination demonstrated only pyruvate amination in the presence of NAD. Absence of glycine deamination activity strongly suggested that alanine dehydrogenase of M. smegmatis was responsible for glyoxylate amination in the cell lysate. This was further confirmed by demonstrating the similar level of upregulation of both glyoxylate and pyruvate amination activities in the cell lysate of the adapted culture.
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http://dx.doi.org/10.1139/w01-126DOI Listing
January 2002
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