BETAINE BDBM50357226
BDBM50557959 CHEBI:15710 BETAINE ALDEHYDE
- Barrett, TN; Taylor, JA; Barker, D; Procopiou, PA; Thompson, JDF; Barrett, J; Le, J; Lynn, SM; Pogany, P; Pratley, C; Pritchard, JM; Roper, JA; Rowedder, JE; Slack, RJ; Vitulli, G; Macdonald, SJF; Kerr, WJ Profile of a Highly Selective Quaternized Pyrrolidine Betaine α J Med Chem 62: 7543-7556 (2019)
- Jiracek, J; Collinsova, M; Rosenberg, I; Budesinsky, M; Protivinska, E; Netusilova, H; Garrow, TA S-alkylated homocysteine derivatives: new inhibitors of human betaine-homocysteine S-methyltransferase. J Med Chem 49: 3982-9 (2006)
- Vanek, V; Budesínský, M; Kabeleová, P; Sanda, M; Kozísek, M; Hanclová, I; Mládková, J; Brynda, J; Rosenberg, I; Koutmos, M; Garrow, TA; Jirácek, J Structure-activity study of new inhibitors of human betaine-homocysteine S-methyltransferase. J Med Chem 52: 3652-65 (2009)
- Pícha, J; Vanek, V; Budešínský, M; Mládková, J; Garrow, TA; Jirácek, J The development of a new class of inhibitors for betaine-homocysteine S-methyltransferase. Eur J Med Chem 65: 256-75 (2013)
- Mládková, J; Vanek, V; Budešínský, M; Elbert, T; Demianová, Z; Garrow, TA; Jirácek, J Double-headed sulfur-linked amino acids as first inhibitors for betaine-homocysteine S-methyltransferase 2. J Med Chem 55: 6822-31 (2012)
- Jørgensen, L; Al-Khawaja, A; Kickinger, S; Vogensen, SB; Skovgaard-Petersen, J; Rosenthal, E; Borkar, N; Löffler, R; Madsen, KK; Bräuner-Osborne, H; Schousboe, A; Ecker, GF; Wellendorph, P; Clausen, RP Structure-Activity Relationship, Pharmacological Characterization, and Molecular Modeling of Noncompetitive Inhibitors of the Betaine/γ-Aminobutyric Acid Transporter 1 (BGT1). J Med Chem 60: 8834-8846 (2017)
- ChEMBL_520157 (CHEMBL947845) Binding affinity to human recombinant BHMT expressed in Escherichia coli using variable levels of betaine substrate by Dixon plot
- ChEMBL_520155 (CHEMBL947843) Inhibition of human recombinant BHMT expressed in Escherichia coli using 2 mM betaine and 1 mM DL-homocysteine as substrate
- ChEMBL_970672 (CHEMBL2405889) Inhibition of human recombinant BHMT using N-methyl-[14C]-betaine/D,L-homocysteine as substrate after 30 mins by scintiillation counting analysis
- Fluorescence Based Assay The fluorescence based assay to monitor the activity of human nSMase2 in the presence or absence of potential inhibitors has been described recently. Figuera-Losada, et al. Lysate of cells expressing recombinant nSMase2 is used to catalyze the hydrolysis of sphingomyelin (SM) to ceramide and phosphorylcholine. Phosphorylcholine undergoes dephosphorylation in a reaction catalyzed by alkaline phosphatase (4 U/mL) to produce choline which in turn is oxidized by choline oxidase (0.1 U/mL) to betaine and hydrogen peroxide (H2O2). Hydrogen peroxide is made to react with Amplex red (50 μM) in the presence of horseradish peroxidase (HRP, 1 U/mL) to generate the fluorescent molecule resorufin. Generation of fluorescence is monitored by measuring relative florescence units (RFU) with excitation at 530 nm and emission at 590 nm. Extent of fluorescence is directly proportional to the extent of SM hydrolysis. Substrate stock solution is prepared in 2% Triton X-100 and sonicated for 1 min. Reactions are carried out for 1 h at 37° C. in 100 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Triton X-100. This assay has been optimized in 384-well format (50 μL total volume per well) under conditions where nSMase2-catalyzed hydrolysis of SM is linear with respect to nSMase2 concentration, time of incubation and SM concentration (FIGS. 3A-3C). The assay has high reliability (Z′=0.8-0.9). It is used for compound screening, IC50 determinations and mode of inhibition studies
- Choline Release Assasy The purpose of this assay is to detect autotaxin inhibition using a choline release assay.Test compound (10 mM stocks in 100% DMSO) is serially diluted in 100% DMSO resulting in 10 concentrations of 100× inhibitor in half area 96 well plates (Corning 3992). Each of these 10 wells in 100% DMSO is diluted 1:33.33 in assay buffer in round bottom 96 well plates (Fisher 12565502) resulting in 3× concentrations in well containing 3% DMSO. The assay buffer is 50 mM Tris pH8.0, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.01% TRITON X-100 (Sigma T9284) and 0.01% fatty acid free bovine serum albumin (Sigma A8806). A 20 μl aliquot of each 3× test compound is then added to black flat bottom 96 well plates (Corning 3991) in singlicate. A 20 μl aliquot per well of 3× recombinant human autotaxin, (Echelon, E-4000) (full length human autotaxin with a C-terminal His tag transfected into 293E cells and purified via nickel chelate and size exclusion chromatography) is then added to every well except for the no enzyme control wells. A 20 μl aliquot per well of assay buffer is added to the no enzyme control wells. A 20 μl aliquot of a 3× cocktail containing choline oxidase (Sigma C5896), horseradish peroxidase (Sigma P8125), amplex ultrared (Invitrogen A36006) and the autotaxin substrate lysophosphatidylcholine (LPC) 16:0 (Avanti Polar Lipids 855675P) is added to each well while avoiding exposure to light. The final concentrations in the well of choline oxidase, horseradish peroxidase, amplex ultrared and LPC 16:0 are 0.4 units/ml, 4 units/ml, 40 μM and 30 μM respectively. The plate is then sealed with aluminum foil seals and incubated at 37° C. for 1 hour in a Labline Imperial III incubator. During this incubation, LPC is cleaved by autotaxin resulting in Lysophosphatidic Acid (LPA) 16:0 and choline. The choline that is released is oxidized by choline oxidase resulting in betaine and hydrogen peroxide. The hydrogen peroxide reacts with the horseradish peroxide and amplex ultrared to form the fluorescent molecule resorufin.
- beta-lactamase assay Expression and Purification of Beta-Lactamases. For his tag KPC-2 beta-lactamase, bacteria were grown overnight at 30 C with shaking in 50 mL LB broth supplemented with 50 μg/mL kanamycin. Two liters of LB broth supplemented with 50 μg/mL kanamycin, 200 mM sorbitol, and 5 mM betaine were each inoculated with 10 mL of overnight bacterial culture. Cultures were then grown at 37 C until an optical density at 600 nm (OD600) of 0.6-0.7. Protein expression was then initiated by the addition of IPTG (final concentration 0.5 mM), followed by growth for 16 hr at 20 C. Cells were pelleted by centrifugation and stored at −80 C until further use. The his tag KPC-2 beta-lactamase was purified by nickel affinity chromatography and gel filtration. Briefly, the cell pellets were thawed and re-suspended in 40 mL of buffer A (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole) with one complete protease inhibitor cocktail tablet (Roche) and disrupted by sonication, followed by ultracentrifugation to clarify the lysate. After ultracentrifugation, the supernatant was passed through a 0.22 μm filter before loading onto a 5 mL HisTrap HP affinity column (GE Healthcare Life Sciences, USA) pre-equilibrated with buffer A. His tag KPC-2 was eluted by a linear imidazole gradient (20 mM to 500 mM). Fractions were analyzed by SDS-PAGE. Fractions containing his tag KPC-2 were concentrated using a 10 k NMWL Amicon Ultra-15 Centrifugal Filter Unit. Concentrated his tag KPC-2 was then loaded onto a superdex 75 gel filtration column (GE Healthcare Life Sciences) pre-equilibrated with 20 mM Tris-HCl pH 8.0, 300 mM NaCl. Protein concentration was determined by absorbance at 280 using an extinction coefficient of 39,545. SDS-PAGE analysis indicated that the eluted protein was more than 95% pure.For sumo tag NDM-1 metallo-beta-lactamase, bacteria were grown overnight at 30 C with shaking in 50 mL LB broth supplemented with 100 μg/mL ampicillin. Two liters of LB broth supplemented with 100 μg/mL ampicillin were each inoculated with 10 mL of overnight bacterial culture. Cultures were then grown at 37 C until an optical density at 600 nm (OD600) of 0.6-0.7. Protein expression was then initiated by the addition of IPTG (final concentration 0.5 mM), followed by growth for 16 hr at 20 C. Cells were pelleted by centrifugation and stored at −80 C until further use. The sumo tag NDM-1 beta-lactamase was purified by nickel affinity chromatography and gel filtration. Briefly, the cell pellets were thawed and re-suspended in 40 mL of buffer A (20 mM HEPES pH 7.4, 0.5 M NaCl, 20 mM imidazole) with one complete protease inhibitor cocktail tablet (Roche) and disrupted by sonication, followed by ultracentrifugation to clarify the lysate. After ultracentrifugation, the supernatant was passed through a 0.22 μm filter before loading onto a 5 mL HisTrap HP affinity column (GE Healthcare Life Sciences, USA) pre-equilibrated with buffer A. Sumo tag NDM-1 was eluted by a linear imidazole gradient (20 mM to 500 mM). Fractions were analyzed by SDS-PAGE. Fractions containing sumo tag NDM-1 were buffer exchanged into 20 mM HEPES pH 7.0, 100 mM NaCl. Cleavage of the sumo tag was then carried out with ULP1 protease overnight at room temperature and then concentrated using a 10 k NMWL Amicon Ultra-15 Centrifugal Filter Unit. The sample was then loaded back onto a nickel affinity column and the flow through was collected, containing the untag NDM-1. NDM-1 was concentrated and loaded onto a gel filtration column (GE Healthcare Life Sciences) pre-equilibrated with 20 mM HEPES pH 7.0, 100 mM NaCl. Protein concentration was determined by absorbance at 280 using an extinction coefficient of 27,960. SDS-PAGE analysis indicated that the eluted protein was more than 95% pure.Steady-State Kinetic Analysis. Steady-state kinetic parameters were determined by using a Biotek Cytation Multi-Mode Reader. For KPC-2, each assay was performed in 100 mM Tris-HCl pH 7.0,