The Experts below are selected from a list of 513 Experts worldwide ranked by ideXlab platform
Paul D. Crowe - One of the best experts on this subject based on the ideXlab platform.
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Scintillation Proximity Assay as a High-Throughput Method ating Nonpeptide Ligand nRH Receptor
2016Co-Authors: Paul D. CroweAbstract:Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as Kon and Koff are time and labor intensive, prompting the development of a screening Assay to identify slowly dissociating com-pounds for follow-up rate constant determination. The authors measured inhibition binding constants (Ki) for GnRH recep-tor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased Ki values over time (Ki30 min/Ki10 h> 6). They used Scintillation Proximity Assay technology to perform these binding experiments because this homogeneous Assay does not have a fixed termination end point as does filtration binding, permitting successive read-ings to be taken from the same Assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound’s Ki value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the Ki ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants. (Journal of Biomolecular Screening 2007:235-239
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Scintillation Proximity Assay as a High-Throughput Method to Identify Slowly Dissociating Nonpeptide Ligand Binding to the GnRH Receptor
2016Co-Authors: Christopher E Heise, Susan K Sullivan, Paul D. CroweAbstract:Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as Kon and Koff are time and labor intensive, prompting the development of a screening Assay to identify slowly dissociating com-pounds for follow-up rate constant determination. The authors measured inhibition binding constants (Ki) for GnRH recep-tor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased Ki values over time (Ki30 min/Ki10 h> 6). They used Scintillation Proximity Assay technology to perform these binding experiments because this homogeneous Assay does not have a fixed termination end point as does filtration binding, permitting successive read-ings to be taken from the same Assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound’s Ki value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the Ki ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants. (Journal of Biomolecular Screening 2007:1-5
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Scintillation Proximity Assay as a high throughput method to identify slowly dissociating nonpeptide ligand binding to the gnrh receptor
Journal of Biomolecular Screening, 2007Co-Authors: Christopher E Heise, Susan K Sullivan, Paul D. CroweAbstract:Many nonpeptide antagonists of the gonadotropin-releasing hormone (GnRH) receptor, as well as other drug targets, possess a broad range of dissociation kinetic rate constants. Current methods to accurately define kinetic rate parameters such as K(on) and K(off) are time and labor intensive, prompting the development of a screening Assay to identify slowly dissociating compounds for follow-up rate constant determination. The authors measured inhibition binding constants (K(i)) for GnRH receptor antagonists after 30 min and 10 h of incubation and observed several compounds with markedly decreased K(i) values over time (Ki(30 min)/Ki(10 h) > 6). They used Scintillation Proximity Assay technology to perform these binding experiments because this homogeneous Assay does not have a fixed termination end point as does filtration binding, permitting successive readings to be taken from the same Assay plate over an extended period of time. They also used a quantitative method of kinetic rate analysis to confirm that a large disparity between a compound's K(i) value at 30 min and 10 h could identify compounds that dissociate slowly. Thus, the K(i) ratio can be used to screen for and select compounds to test using more quantitative, albeit lower throughput methods to accurately define kinetic rate constants.
Emilio Diez - One of the best experts on this subject based on the ideXlab platform.
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a homogeneous method to measure aminoacyl trna synthetase aminoacylation activity using Scintillation Proximity Assay technology
Analytical Biochemistry, 2000Co-Authors: Ricardo Macarron, Lucy Mensah, Carmen Carranza, Neil Benson, Andrew J Pope, Emilio DiezAbstract:Abstract A new method to measure the aminoacylation of tRNA based upon the use of the Scintillation Proximity Assay (SPA) technology has been developed. The Assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based Assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid Scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA Assays was at least equivalent to the separation-based methods. The Assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.
James A Landro - One of the best experts on this subject based on the ideXlab platform.
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high throughput screening by mass spectrometry comparison with the Scintillation Proximity Assay with a focused file screen of akt1 pkbα
Journal of Biomolecular Screening, 2007Co-Authors: Andrea K Quercia, Jayhyuk Myung, James A Landro, William A Lamarr, Can C Ozbal, Kevin J LumbAbstract:Mass spectrometry is an emerging format for label-free high-throughput screening. The main limitation of mass spectrometry is throughput, due to the requirement to purify samples prior to ionization. Here the authors compare an automated high-throughput mass spectrometry (HTMS) system (RapidFire) with the Scintillation Proximity Assay (SPA). The cancer therapy target AKT1/PKBalpha was screened against a focused library of kinase inhibitors and IC50 values determined for all compounds that exhibit > 50% inhibition. A selection of additional compounds that exhibited
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a high throughput competitive Scintillation Proximity aminoacyl trna synthetase charging Assay to measure amino acid concentration
Analytical Biochemistry, 2007Co-Authors: Chris D Forbes, Jayhyuk Myung, James A LandroAbstract:Abstract An enhanced method to measure the concentration of individual naturally occurring free amino acids in solution is described. This relatively simple but robust method combines two previously reported procedures: the use of Scintillation Proximity Assay (SPA) technology to measure aminoacyl–tRNA synthetase (aaRS) activity and the use of aaRS activity to measure amino acid concentration using the enzymatic isotope dilution technique. The format described is called an aaRS competitive Scintillation Proximity Assay (cSPA). This cSPA takes advantage of competition between a fixed concentration of radiolabeled amino acid and an unknown concentration of the same nonradiolabeled amino acid for its cognate tRNA catalyzed by the aaRS specific for that amino acid. Under equilibrium conditions, in the case of limiting tRNA, the rate of the enzyme-catalyzed reaction relative to substrate concentration becomes irrelevant and the enzymatic isotopic dilution technique becomes the simple isotopic dilution technique. Due to the exquisite specificity of the reaction, a crude mixture of tRNAs and aaRSs can be used to detect the concentration of a particular amino acid without interference from noncognate amino acids. When used to monitor aminopeptidase M activity, this Assay produced similar results in time course and inhibition experiments as compared with a traditional fluorescent Assay. High-throughput compatibility was demonstrated by screening 12,000 compounds against aminopeptidase M in 384-well microtiter plates with Z factors ranging from 0.53 to 0.70. This competitive Assay can be used as a general method to detect amino acids at concentrations less than 100 nM and to monitor enzyme activity in biological samples, and it is amenable to high-throughput screening.
Eric Gouaux - One of the best experts on this subject based on the ideXlab platform.
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thermostabilization and purification of the human dopamine transporter hdat in an inhibitor and allosteric ligand bound conformation
PLOS ONE, 2018Co-Authors: Vikas Navratna, Dilip K Tosh, Kenneth A Jacobson, Eric GouauxAbstract:The human dopamine transporter (hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space using secondary active transport. Dopamine is an essential monoamine chemical messenger that regulates reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants such as cocaine primarily target the central binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of dopamine reuptake leads to accumulation of dopamine in the synapse causing heightened signaling. In addition, hDAT is implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the structural studies of hDAT have proven difficult. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, we identified ligands that stabilize hDAT in detergent micelles. We then screened ~200 single residue mutants of hDAT using a high-throughput Scintillation Proximity Assay and identified a thermostable variant (I248Y). Here we report a robust strategy to overexpress and successfully purify a thermostable variant of hDAT in an inhibitor and allosteric ligand bound conformation.
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thermostabilization and purification of the human dopamine transporter hdat in an inhibitor and allosteric ligand bound conformation
bioRxiv, 2018Co-Authors: Vikas Navratna, Dilip K Tosh, Kenneth A Jacobson, Eric GouauxAbstract:The human dopamine transporter (hDAT) plays a major role in dopamine homeostasis and regulation of neurotransmission by clearing dopamine from the extracellular space using secondary active transport. Dopamine is an essential monoamine chemical messenger that regulates reward seeking behavior, motor control, hormonal release, and emotional response in humans. Psychostimulants such as cocaine primarily target the central binding site of hDAT and lock the transporter in an outward-facing conformation, thereby inhibiting dopamine reuptake. The inhibition of dopamine reuptake leads to accumulation of dopamine in the synapse causing heightened signaling. In addition, hDAT is implicated in various neurological disorders and disease-associated neurodegeneration. Despite its significance, the molecular architecture of hDAT and its various conformational states are poorly understood. Instability of hDAT in detergent micelles has been a limiting factor in its successful biochemical, biophysical, and structural characterization. To overcome this hurdle, first we identified ligands that stabilize hDAT in detergent micelles. Then, we screened ~200 single residue mutants of hDAT using high-throughput Scintillation Proximity Assay, and identified a thermostable variant (I248Y). Here we report a robust strategy to overexpress and successfully purify a thermostable variant of hDAT in an inhibitor and allosteric ligand bound conformation.
Sunita M De Sousa - One of the best experts on this subject based on the ideXlab platform.
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Microplate Assay for Inhibitors of the Transpeptidase Activity of PBP1b of Escherichia coli
2016Co-Authors: Ramesh K Jha, Sunita M De SousaAbstract:The transpeptidase (TP) activity of penicillin-binding proteins (PBPs), target of the β-lactam antibiotics, is a well-validated antibacterial drug target. The TP activity of PBP1b converts un–cross-linked peptidoglycan to the cross-linked form. Directly measuring TP activity is difficult because cross-linked and un–cross-linked peptidoglycan have very similar chromatographic properties. The authors report a microdilution plate method to directly measure the TP enzyme activity, uncoupled from the transglycosylase (TG), for detection of TP inhibitors. Escherichia coli membranes were incubated with 100 mM ampicillin, fol-lowed by removal of unbound ampicillin. The substrate for the TP, un–cross-linked peptidoglycan, was prepared by incubating these membranes with peptidoglycan sugar precursors, 1 of which was radiolabeled. Subsequently, solubilized PBP1b was added and TP activity Assayed. The cross-linked peptidoglycan formed was monitored by addition of wheat germ agglutinin Scintillation Proximity Assay beads plus N-laurylsarcosine, which selectively captures cross-linked peptidoglycan. The PBP1b-catalyzed activity was inhibited by penicillin G but not by cephalexin or cephradine, which have higher affinity for PBP1a. Moenomycin, a TG inhibitor, also inhibited TP activity. Because this is a true enzyme Assay, it has the potential to detect novel, non-β-lactam TP inhibitors and could lead to the discovery of new antibacterial agents. (Journal of Biomolecular Screenin
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a microplate Assay for the coupled transglycosylase transpeptidase activity of the penicillin binding proteins a vancomycin neutralizing tripeptide combination prevents penicillin inhibition of peptidoglycan synthesis
Biochemical and Biophysical Research Communications, 2014Co-Authors: Vidya Prasanna Kumar, Chandrakala Basavannacharya, Sunita M De SousaAbstract:Abstract A microplate, Scintillation Proximity Assay to measure the coupled transglycosylase–transpeptidase activity of the penicillin binding proteins in Escherichia coli membranes was developed. Membranes were incubated with the two peptidoglycan sugar precursors UDP-N-acetyl muramylpentapeptide (UDP-MurNAc(pp)) and UDP-[3H]N-acetylglucosamine in the presence of 40 μM vancomycin to allow in situ accumulation of lipid II. In a second step, vancomycin inhibition was relieved by addition of a tripeptide (Lys- d -ala- d -ala) or UDP-MurNAc(pp), resulting in conversion of lipid II to cross-linked peptidoglycan. Inhibitors of the transglycosylase or transpeptidase were added at step 2. Moenomycin, a transglycosylase inhibitor, had an IC50 of 8 nM. Vancomycin and nisin also inhibited the Assay. Surprisingly, the transpeptidase inhibitors penicillin and ampicillin showed no inhibition. In a pathway Assay of peptidoglycan synthesis, starting from the UDP linked sugar precursors, inhibition by penicillin was reversed by a ‘neutral’ combination of vancomycin plus tripeptide, suggesting an interaction thus far unreported.
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an Assay for exogenous sources of purified murg enabled by the complementation of escherichia coli murg ts by the mycobacterium tuberculosis homologue
Fems Microbiology Letters, 2012Co-Authors: Ramesh K Jha, Nainesh Katagihallimath, Swetansu K Hota, Kaveri Das, Sunita M De SousaAbstract: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.