BDBM50359629 TROLOX
BDBM50373877 THIAMINE (VIT B1) Betaxin ThOH CA inhibitor, 3 Vitamin B 1 Thiamine
- Chan, AHY; Ho, TCS; Fathoni, I; Pope, R; Saliba, KJ; Leeper, FJ Inhibition of Thiamine Diphosphate-Dependent Enzymes by Triazole-Based Thiamine Analogues. ACS Med Chem Lett 14: 621-628 (2023)
- Chan, AHY; Fathoni, I; Ho, TCS; Saliba, KJ; Leeper, FJ Thiamine analogues as inhibitors of pyruvate dehydrogenase and discovery of a thiamine analogue with non-thiamine related antiplasmodial activity. RSC Med Chem 13: 817-821 (2022)
- Özdemir, Zÿ; Sentürk, M; Ekinci, D Inhibition of mammalian carbonic anhydrase isoforms I, II and VI with thiamine and thiamine-like molecules. J Enzyme Inhib Med Chem 28: 316-9 (2013)
- Ahn, SM; Rho, HS; Baek, HS; Joo, YH; Hong, YD; Shin, SS; Park, YH; Park, SN Inhibitory activity of novel kojic acid derivative containing trolox moiety on melanogenesis. Bioorg Med Chem Lett 21: 7466-9 (2011)
- Alspach, JD; Ingraham, LL Inhibition of acetylcholinesterase by thiamine. A structure-function study. J Med Chem 20: 161-4 (1977)
- Thomas, AA; De Meese, J; Le Huerou, Y; Boyd, SA; Romoff, TT; Gonzales, SS; Gunawardana, I; Kaplan, T; Sullivan, F; Condroski, K; Lyssikatos, JP; Aicher, TD; Ballard, J; Bernat, B; DeWolf, W; Han, M; Lemieux, C; Smith, D; Weiler, S; Wright, SK; Vigers, G; Brandhuber, B Non-charged thiamine analogs as inhibitors of enzyme transketolase. Bioorg Med Chem Lett 18: 509-12 (2008)
- Le Huerou, Y; Gunawardana, I; Thomas, AA; Boyd, SA; de Meese, J; Dewolf, W; Gonzales, SS; Han, M; Hayter, L; Kaplan, T; Lemieux, C; Lee, P; Pheneger, J; Poch, G; Romoff, TT; Sullivan, F; Weiler, S; Wright, SK; Lin, J Prodrug thiamine analogs as inhibitors of the enzyme transketolase. Bioorg Med Chem Lett 18: 505-8 (2008)
- Thomas, AA; Le Huerou, Y; De Meese, J; Gunawardana, I; Kaplan, T; Romoff, TT; Gonzales, SS; Condroski, K; Boyd, SA; Ballard, J; Bernat, B; DeWolf, W; Han, M; Lee, P; Lemieux, C; Pedersen, R; Pheneger, J; Poch, G; Smith, D; Sullivan, F; Weiler, S; Wright, SK; Lin, J; Brandhuber, B; Vigers, G Synthesis, in vitro and in vivo activity of thiamine antagonist transketolase inhibitors. Bioorg Med Chem Lett 18: 2206-10 (2008)
- Chan, AHY; Ho, TCS; Leeper, FJ Thiamine analogues featuring amino-oxetanes as potent and selective inhibitors of pyruvate dehydrogenase. Bioorg Med Chem Lett 98: (2024)
- Xie, SS; Lan, JS; Wang, XB; Jiang, N; Dong, G; Li, ZR; Wang, KD; Guo, PP; Kong, LY Multifunctional tacrine-trolox hybrids for the treatment of Alzheimer's disease with cholinergic, antioxidant, neuroprotective and hepatoprotective properties. Eur J Med Chem 93: 42-50 (2015)
- Swier, LJ; Monjas, L; Guskov, A; de Voogd, AR; Erkens, GB; Slotboom, DJ; Hirsch, AK Structure-based design of potent small-molecule binders to the S-component of the ECF transporter for thiamine. Chembiochem 16: 819-26 (2015)
- Nepovimova, E; Korabecny, J; Dolezal, R; Babkova, K; Ondrejicek, A; Jun, D; Sepsova, V; Horova, A; Hrabinova, M; Soukup, O; Bukum, N; Jost, P; Muckova, L; Kassa, J; Malinak, D; Andrs, M; Kuca, K Tacrine-Trolox Hybrids: A Novel Class of Centrally Active, Nonhepatotoxic Multi-Target-Directed Ligands Exerting Anticholinesterase and Antioxidant Activities with Low In Vivo Toxicity. J Med Chem 58: 8985-9003 (2015)
- Evaluation of ketoclomazone and its analogues as inhibitors of 1-deoxy-d-xylulose 5-phosphate synthases and other thiamine diphosphate (ThDP)-dependent enzymes.
- ChEMBL_210409 (CHEMBL814138) Antiparasitic activity against thiamine transporter of chicken intestine
- ChEMBL_210410 (CHEMBL814139) Antiparasitic activity against thiamine transporter of Eimeria tenella
- ChEMBL_2346878 Inhibition of porcine PDHc E1-subunit incubated for 30 mins followed by pyruvate addition in presence of thiamine pyrophosphate by microplate reader analysis
- ChEMBL_2472799 Inhibition of human recombinant PDHK2 assessed as residual PDH activity in presence of ATP using sodium pyruvate/Coenzyme A/thiamine pyrophosphate as substrates incubated for 90 mins by UV-transparent microplate analysis
- AChE Inhibition Bioassay AChE enzymatic activity was measured using an adaptation of the method previously described [Ingkaninan et al., J. Ethnopharmacol., 89:261-264]; 98 μl of 50 mM Tris-HCl buffer (pH 8), 30 μl of a solution sample of the inhibitor, at different concentrations in methanol, and 7.5 μl of AChE solution containing 0.26 U/ml were mixed in a microplate and left to incubate for 15 min. Subsequently, 22.5 μl of 0.023 mg/ml AChI and 142 μl of 3 mM DTNB were added. The initial rate of the enzymatic reaction was followed by reading the absorbance at 405 nm during the first 5 min of reaction. Samples were prepared in a range of concentrations of the compounds in water (choline caffeate, choline trolox, choline cinnamate) or in an aqueous solution of 50% methanol (choline 3,4-dimethoxicinnamate, choline rosmarinate). A control reaction was carried out using the sample solvent instead of sample and it was considered 100% activity.
- Biochemical Activity Assay The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 μl ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements.
- Biochemical Activity Assay The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 μl ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements. As full value for the PDHK2 assay the inhibitor-free PDHK2 reaction is used.
- Biochemical Activity Assay The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 l ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements. As full value for the PDHK2 assay the inhibitor-free PDHK2 reaction is used. The pharmacological zero value used is DCA (Sigma-Aldrich) in a final concentration of 3 mM. The inhibitory values (IC50) were determined using either the program Symyx Assay Explorer or Condosseo from GeneData.
- Biochemical Activity Assay The biochemical activity assay for PDHK2 is based on the inactivation of PDC through phosphorylation by PDHK2. The assay is run in two steps: the enzymatic PDHK2 reaction in which isolated PDC is phosphorylated by PDHK2 with ATP as co-substrate and the PDC activity assay in which pyruvate and NAD are converted to acetyl-CoA and NADH. The PDC activity correlates to the increase in NADH and thereby is detectable directly via the increasing fluorescence signal (Exc 340 nm, Em 450 nm). Inhibition of PDHK2 results in a lower phosphorylation status and thereby a less decrease in activity of PDC and a stronger increase in NADH fluorescence signal.The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 μl ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements.
- Biochemical Activity Testing of PDHK2 The biochemical activity assay for PDHK2 is based on the inactivation of PDC through phosphorylation by PDHK2. The assay is run in two steps: the enzymatic PDHK2 reaction in which isolated PDC is phosphorylated by PDHK2 with ATP as co-substrate and the PDC activity assay in which pyruvate and NAD are converted to acetyl-CoA and NADH. The PDC activity correlates to the increase in NADH and thereby is detectable directly via the increasing fluorescence signal (Exc 340 nm, Em 450 nm). Inhibition of PDHK2 results in a lower phosphorylation status and thereby a less decrease in activity of PDC and a stronger increase in NADH fluorescence signal.The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 μl ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements. As full value for the PDHK2 assay the inhibitor-free PDHK2 reaction is used. The pharmacological zero value used is DCA (Sigma-Aldrich) in a final concentration of 3 mM. The inhibitory values (1050) were determined using either the program Symyx Assay Explorer or Condosseo from GeneData.
- BCA Protein Assay Escherichia coli BL21*DE3 pET30a-Ec yeaWX #1 (Ec YeaWX) strain was generated as described below. The contiguous Escherichia coli coding sequence yeaW (equivalent to uniprot ID P0ABR7.1 (YeaW) (SEQ ID NO: 2)) and yeaX (equivalent to uniprot ID P76254.1 (YeaX) (SEQ ID NO: 3)) were PCR amplified from Escherichia coli strain K-12 substr. BW25113 genomic DNA. PCR primers (YeaW_Nde I_fwd2-SEQ ID NO: 4; YeaX_rev2-SEQ ID NO: 5) were designed to create a 5′ NdeI restriction site including the ATG start codon of yeaW and create a PstI restriction site just 3′ of the yeaX TAG stop codon.The bacteria were grown aerobically in 50 mL LB broth (Difco #244620; 10 g/L Tryptone, 5 g/L yeast extract, 10 g/L NaCl, 50 μg/mL kanamycin), in a 500 mL Erlenmeyer flask. The cultures were inoculated from glycerol stock of BL21*DE3 pET30a-Ec yeaWX #1 strain. Strains were cultured all day at 37° C. with 250 rpm shaking. Two 300 mL Minimal M9 Medium (6 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 0.1 mM CaCl2, 1 mM MgSO4, 0.2% Dextrose, 1 mg/L Thiamine, 50 μg/mL kanamycin), in 1 L Erlenmeyer flasks, were inoculated with 5 mL of the LB broth day culture and cultured overnight at 37° C. with 250 rpm shaking. The overnight cultures were used to inoculate twelve 1 L cultures of Minimal M9 media in 2.8 L fluted Erlenmeyer flasks to an OD 600 nm of 0.05 (typically approximately 28 mLs), which were grown at 37° C. with 250 rpm shaking until an OD600 of approximately 0.4 was reached. Expression of YeaWX was induced with 1 mM IPTG and the induced cultures were further grown overnight at 37° C. with 250 rpm shaking. The biomass was pelleted by centrifugation at 6000×g for 12 minutes at 4° C. The cell pellet was suspended in 240 mL of ice-cold 1× Phosphate Buffered Saline (Ca2+ and Mg2+ free). Ninety micrograms of Lysozyme (Sigma #L6876 Lot #SLBG8654V; Sigma-Aldrich Corp., St. Louis, Mo.) was added and incubated with 320 rpm shaking for 30 minutes at 4° C. Lysis was achieved via French press with a 4° C. prechilled 1″ diameter chamber at 1000 psi (high ratio; internal PSI equivalent 16000). The lysate was centrifuged at 6,000×g for 12 minutes at 4° C. to pellet extra debris. Glycerol was added to the centrifuged lysate supernatant at a final concentration of 15% A protein concentration of the centrifuged lysate supernatant was determined by a BCA Protein Assay Kit (Pierce #23225), typically in the 2.5 to 4.5 mg/ml range. The centrifuged Ec YeaWX lysate supernatant was aliquoted into 20 mL volumes and stored frozen at −80° C.Ec YeaWX lysate was diluted to 2.0 mg/mL protein with 1× Dulbecco's phosphate buffered saline (DPBS) plus 15% glycerol. Nicotinamide adenine dinucleotide phosphate (NADPH) was added to 250 μM. One hundred and fifty microliters of Ec YeaWX lysate was dispensed into a deep-well plate (polypropylene, 2 mL volume, Corning Axygen catalogue #P-DW-20-C). Candidate IC50 compounds from TABLE 1 and vehicle control (respective vehicle control of DMSO or water), or control compounds (IC50 control, 8-Quinolinol hemisulfate salt (Sigma Catalog #55100)) were added at a 1:100 dilution (e.g., 1.5 μL per well). The plates were agitated on a plate shaker for 1 minute. d9-carnitine chloride (1.5 μL of 5 mM) was added to all wells to reach a final d9-carnitine chloride concentration of 50 μM.
- Inhibitory Action of PDHK2 Activity In Vitro In the case of human PDHK2 (hPDHK2, NCBI Reference Database Accession number NM_002611.4), modified hPDHK2 cDNA wherein FLAG-Tag sequence was added to the N terminus of hPDHK2 cDNA clone (pReceiver-M01/PDK2-GeneCopoeia) as the base was prepared by PCR and ligated to the NdeI/EcoRI site of pET-17b vector. The recombinant construct was transformed into Escherichia coli DH5a. The recombinant clones were identified, and plasmid DNA was isolated and subjected to the DNA sequence analysis. One clone which had the expected nucleic acid sequence was selected for expression work.For expression of hPDHK2 activity, Escherichia coli strain BL21(DE3) cells were transformed with the pET17b vector containing modified hPDHK2 cDNA. The Escherichia coli were grown to an optical density 0.6 (600 nmol/L) at 30° C. Protein expression was induced by the addition of 500 μmol/L isopropyl-β-thiogalactopyranoside. The Escherichia coli were cultured at 20° C. for 17-18 hr and harvested by centrifugation. The harvested Escherichia coli was resuspended in a suspension buffer (20 mmol/L HEPES-NaOH, 500 mmol/L sodium chloride, 1% ethylene glycol, 0.1% pluronic F-68 (pH 8.0), cOmplete, EDTA-free (pH 8.0)), and disrupted by a microfluidizer. The precipitate was removed by centrifugation and the supernatant was added to DDDDK-tagged Protein PURIFICATION GEL. DDDDK-tagged Protein PURIFICATION GEL was washed with a washing buffer (20 mmol/L HEPES-NaOH, 500 mmol/L sodium chloride, 1% ethylene glycol, 0.1% pluronic F-68 (pH 8.0)) and the bound protein was eluted with elution buffer 1 (20 mmol/L HEPES-NaOH, 100 μg/mL peptide (amino acid sequence DYKDDDDK) (SEQ ID NO: 1), 500 mmol/L sodium chloride, 1% ethylene glycol, 0.1% pluronic F-68 (pH 8.0)). The eluted fractions containing FLAG-Tagged protein were pooled, concentrated by an ultrafiltration method, added to a gel filtration column (HiLoad 26/60 Superdex 200), and eluted with elution buffer 2 (20 mmol/L HEPES-NaOH, 150 mmol/L sodium chloride, 0.5 mmol/L ethylenediaminetetraacetic acid (EDTA), 1% ethylene glycol, 0.1% pluronic F-68 (pH 8.0)). The eluted fractions were pooled and preserved at −80° C.0.025 U/mL PDH and 0.5 μg/mL hPDHK2 were mixed in an assay buffer (50 mmol/L 3-morpholinopropanesulfonic acid (pH 7.0), 20 mmol/L dipotassium hydrogen phosphate, 60 mmol/L potassium chloride, 2 mmol/L magnesium chloride, 0.4 mmol/L EDTA, 0.2% poloxamer, 2 mmol/L dithiothreitol), and the mixture was incubated at 4° C. overnight to obtain a PDH/hPDHK2 complex solution. In the assay buffer, 0.025 U/mL PDH was mixed and incubated at 4° C. overnight to prepare a PDH solution.The test compounds were diluted with DMSO. To measure an inhibitory action of the test compound on the PDHK activity in the PDH/hPDHK2 complex solution, PDH/hPDHK2 complex solution (20 μL), test compound (1.5 μL) and 1.06 μmol/L ATP (diluted with assay buffer) (8.5 μL) were added to a 384 well microplate and PDHK reaction was performed at room temperature for 45 min (test compound well). DMSO (1.5 μL) was added to control wells instead of test compound. In addition, DMSO (1.5 μL) was added to blank wells instead of the test compound, and PDH solution was added instead of the PDH/hPDHK2 complex solution. To measure an inhibitory action of the test compound on the PDHK activity inherent in the PDH solution, a test compound was added and the PDH solution instead of the PDH/hPDHK2 complex solution was added to a blank+test compound well.Then, 10 μL of substrates (5 mmol/L sodium pyruvate, 5 mmol/L Coenzyme A, 12 mmol/L NAD, 5 mmol/L thiamine pyrophosphate, diluted with assay buffer) were added. The mixture was incubated at room temperature for 90 min, and the residual PDH activity was measured.The absorbance of each well at 340 nm was measured using a microplate reader to detect NADH produced by the PDH reaction.