Query String: CoCl2
- ChEMBL_422673 (CHEMBL913341) Inhibition of CSK measured as poly-E4Y phosphorylation by acid precipitation assay in presence of 0.2 mM CoCl2
- ChEMBL_576753 (CHEMBL1032020) Inhibition of CoCl2-induced HIF1alpha expression in mouse 4T1 cells transfected with oxygen-dependent-degradation domain of HIFalpha by luciferase reporter gene assay
- ChEMBL_1923162 (CHEMBL4426118) Inhibition of carbonic anhydrase 9 in human HT-29 cells assessed as reduction in cell viability in presence of CoCl2-induced hypoxic conditions after 72 hrs by cell counter method
- ChEMBL_1793107 (CHEMBL4265026) Inhibition of Streptococcus pneumoniae Pgda expressed in Escherichia coli BL21 (DE3) cells transformed with pET28bSpPgdA232-431 plasmid using acetoxymethyl-4-methylumbelliferone as substrate measured at 5 min intervals over 60 mins in presence of CoCl2 by fluorescence assay
- ChEMBL_1793106 (CHEMBL4265025) Inhibition of Escherichia coli PgaB expressed in Escherichia coli BL21 (DE3) cells transformed with pET28 plasmid coding for PgaB42-655 using acetoxymethyl-4-methylumbelliferone as substrate measured at 2 min intervals over 10 mins in presence of CoCl2 by fluorescence assay
- ChEMBL_1636403 (CHEMBL3879301) Inhibition of recombinant full length human His-tagged methionine aminopeptidase 1 expressed in Escherichia coli BL21(DE3) using methionylprolyl-p-nitroanilide as substrate preincubated for 20 mins followed by substrate addition measured for 20 mins in presence of CoCl2 by proline aminopeptidase coupled enzyme assay
- ChEMBL_1793119 (CHEMBL4265038) Inhibition of Streptococcus pneumoniae Pgda expressed in Escherichia coli BL21 (DE3) cells transformed with pET28bSpPgdA232-431 plasmid using compound pre-incubated with 1Eq DTT and using acetoxymethyl-4-methylumbelliferone as substrate measured at 5 min intervals over 60 mins in presence of CoCl2 by fluorescence assay
- ChEMBL_1793117 (CHEMBL4265036) Inhibition of Escherichia coli PgaB expressed in Escherichia coli BL21 (DE3) cells transformed with pET28 plasmid coding for PgaB42-655 using compound pre-incubated with 1Eq DTT and using acetoxymethyl-4-methylumbelliferone as substrate measured at 2 min intervals over 10 mins in presence of CoCl2 by fluorescence assay
- ChEMBL_1793109 (CHEMBL4265028) Inhibition of His6-tagged Streptococcus pneumoniae Pgda C-terminal de-N-acetylase domain (232 to 431 residues) expressed in Escherichia coli BL21 (DE3) cells transformed with pET28b-SpPgdA232-431 using chitotriose as substrate measured at 75 or 100 sec intervals up to 5 mins in presence of CoCl2 by fluorescence assay
- GCPII Activity Assay Inhibition potencies against GCPII (IC50 values) were determined using previously described methods with minor modification. Rojas et al., 2002. Briefly, reactions were carried out in the presence of NAA-[3H]-G and human recombinant GCPII enzyme in Tris-HCl and CoCl2 at 37° C. for 20 minutes. Reactions were stopped with ice-cold sodium phosphate buffer containing 1 mM EDTA. Aliquots were then transferred to 96-well spin columns containing AG1X8 ion-exchange resin and centrifuged. NAA-[3H]-G was bound to the resin and [3H]-G eluted in the flow through. Columns were washed with formate to ensure complete elution of [3H]-G. The flow-through and the washes were collected, aliquots transferred and dried to completion in a solid scintillator-coated 96-well plate. The radioactivity corresponding to [3H]-G was determined with a scintillation counter. Subsequently, IC50 curves were generated from CPM results.
- Biochemical Assays Stock solutions for all assays were prepared in DMSO (10 mM) and stored at −20 °C. Assays were performed in 96 well plates (Greiner) at a total reaction volume of 100 μL. First, enzyme (final concentration: 50 nM) was incubated with inhibitor (25 μM) or positive control (DMSO, 0.25%) in a reaction buffer (finalconcentration: 100 μM CoCl2 6H2O, 100 mM NaCl, 0.075% bovine serum albumine, 50 mM Tris pH 7.5) for 20 min at 37 °C. Then, the substrate Met-Gly-Met-Met was added to a final concentration of 400 μM. After an incubation period of 15 min (HsMetAP1), 20 min (EcMetAP), or 60 min (HsMetAP2) at 37 °C, the enzyme reactionwas stopped by adding 10 μL 4% trifluoroacetic acid (TFA), and the plate was centrifuged at 1000g for 10 min. Detection of the product Gly-Met-Met was performed by HPLC: C18 column (Dr. Maisch, ReproSil-Pur 120 ODS-3, 3 μm, 50 × 2 mm). An 80 μL injection volume was eluted with mobile phases A (H2O and 0.1% TFA, filtered, degasse
- Enzymatic Activity Assay of MetAP2 The compounds exemplified herein are tested essentially as described below and exhibit an IC50 for the human and mouse MetAP2 assay as shown in Table 1.Full length MetAP2 (human and mouse) proteins are generated from Sf9 cells using procedure similar to that described in Biochemistry 2003, 42, 5035-5042. MetAP2 is purified in the presence of 5 mM MnCl2 and 2 mM CoCl2 respectively, and stored at −78° C. before use.Inhibition of the catalytic activity of human and mouse MetAP2 by compounds in the present invention is measured by monitoring the formation of the product peptide (Gly-Lys-Val-Lys-Val-Gly-Val-Asn-Gly) from the substrate peptide (Met-Gly-Lys-Val-Lys-Val-Gly-Val-Asn-Gly) via LC/MS. The reaction is typically conducted by incubating the enzyme, test compound and substrate (150 μM) in an assay buffer (100 μl) (50 mM HEPES, 100 mM NaCl, 50 mg/mL BSA, 0.17 mM Triton X-100 at pH 7.5) for 40 minutes. After the reaction is stopped by the addition of ACN (200 μl), the levels of product and remaining substrate are quantified with a mass spectrometer. The IC50 value is calculated typically from a 10-point dose titration curve using a 4-parameter equation.