lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=CC(=O)N1C[C@H](Nc2nc(Nc3cnn(CC(F)(F)F)c3)nc3[nH]cc(Cl)c23)CC[C@@H]1C
InChIKey: IYHWPGRNZRXDSS-NWDGAFQWSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Measurement of Inhibitory Activity Against JAK3 and BTK Enzymes: JAK3 and BTK kinases inhibitory activities were measured for the compounds prepared in the Examples through in vitro analysis on the ADP Glow (Glo) platform.Specifically, the inhibitory activities against JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1× kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to 96 well plates (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds prepared in the previous Examples were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 ug/uL of Poly(Glu4, Tyr1) peptide (JAK3 and BTK final concentration) in the total 25 uL reactants was added to 96-well plates to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (25 uL per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (50 uL per reaction) was added and incubated (30° C.) for 30 minutes at room temperature. The kinase activity was measured by chemiluminescence according to the instructions of ADP Glo kinase assay kit, and the inhibitory activity of the compounds according to the present invention was calculated. For the analysis of the results of each compound, Microsoft Excel was used, and IC50 values were calculated by SigmaPlot software.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 482.9, ALogP 4.09, HBA 7, HBD 3, PSA 103.76, QED 0.46
Compound (SMILES): C[C@@H]1C[C@H](N)C[C@H](c2ccncc2NC(=O)c2ccc(F)c3cc(N4CCC[C@@H](F)C4)cnc23)C1
InChIKey: LTZAZXMPSNMTBX-MANSERQUSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Assay: Pim-2 kinase assay20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 479.58, ALogP 5.19, HBA 5, HBD 2, PSA 84.14, QED 0.54
Compound (SMILES): COc1cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(NS(C)(=O)=O)c4OC)c4ccccc34)ccn2)ccc1C(=O)NCCCN1CCOCC1
InChIKey: HMBIOLBRLKAILX-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: p38 MAPKα Enzyme Inhibition: The inhibitory activities of compounds of the invention against p38MAPKγ (MAPK12: Invitrogen), are evaluated in a similar fashion to that described hereinabove. The enzyme (800 ng/mL, 2.5 μL) is incubated with the test compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solution (2.5 μL, 400 μM) are then added to the enzymes/compound mixtures and the whole is incubated for 1 hr. Development reagent (protease, 5 μL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, Thermo Scientific).
Measurement: IC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 825.99, ALogP 7.55, HBA 11, HBD 5, PSA 181.48, QED 0.07
Compound (SMILES): COc1ccc(CC(=O)Nc2sccc2-c2nnn(C)n2)cc1
InChIKey: CYZHBOBJYJSPNB-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: P45983
Assay: HTRF Assay: (1) Assay buffer #1 with 0.0025% Tween and 1 mM DTT was prepared using JNK buffer stock solution.(2) Assay buffer #2 with 0.025% BSA was prepared using the assay buffer #1.(3) Test compound solution preparation: 5× compound solution was prepared using assay buffer #1 with 5% DMSO. The compound solution (10 μl/well) was added to a 384-well plate (Corning, Cat No. 3654).(4) aJNK1, 2 or 3 preparation: aJNK stock (100 μg/ml) at −80° C. was thawed on ice and an aJNK (1.6 ng/ml) solution was prepared using assay buffer #2. The aJNK solution (20 μl/well) was added to the plate and the plate was shaken. The enzyme was incubated with the compound at RT for 10 min.(5) ATP/substrate solution preparation: ATP and substrate stocks were thawed on ice. 2.5×ATP/substrate (e.g., 25 μM or 2.5 mM ATP/50 nM EPIW-1) was prepared using assay buffer #1. The ATP/substrate (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 15 min. Note: In an exemplary assay, the final ATP concentration was about 1 mM.(6) EDTA preparation: 30 mM EDTA was prepared using 0.5M EDTA stock and assay buffer #1. The EDTA (10 μl/well) was added to the plate to quench the enzyme reaction and the plate was shaken well.(7) Detection reagent preparation: Eu-63 and SA-XL stocks were thawed on ice. 4× Eu-63/SA-XL (2 nM Eu-63/9.4 nM SA-XL) was prepared using JNK detection buffer. The Eu-63/SA-XL solution (20 μl/well) was added to the plate and the plate was shaken. The plate was incubated at RT for 1 hr before reading the plate on LJL using ratiometric method named HTRF.
Measurement: IC50 = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 329.39, ALogP 2.13, HBA 7, HBD 1, PSA 81.93, QED 0.78
Compound (SMILES): COc1cccc(C(=O)N2C3CCC2CN(Cc2c(-c4ccc(Cl)cc4)nc4ncccn24)C3)n1
InChIKey: YJADTYIFXPYAMV-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: Inhibition of Recombinant TASK-1 and TASK-3 In Vitro: The investigations on the inhibition of the recombinant TASK-1 and TASK-3 channels were conducted using stably transfected CHO cells. The compounds according to the invention were tested in this case by application of 40 mM potassium chloride in the presence of a voltage-sensitive dye according to the method described in detail in the following references [Whiteaker et al., Validation of FLIPR membrane potential dye for high-throughput screening of potassium channel modulators, J. Biomol. Screen. 6 (5), 305-312 (2001); Molecular Devices FLIPR Application Note: Measuring membrane potential using the FLIPR® membrane potential assay kit on Fluorometric Imaging Plate Reader (FLIPR®) systems, http://www.moleculardevices.com/reagents-supplies/assay-kits/ion-channels/flipr-membrane-potential-assay-kits]. The activity of the test substances was determined as their ability to inhibit a depolarization induced in the recombinant cells by 40 mM potassium chloride. The concentration which can block half of this depolarization is referred to as IC50.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 488.98, ALogP 3.94, HBA 7, HBD 0, PSA 75.86, QED 0.42
Compound (SMILES): CC1CCCC(CC(O)c2c(C(F)(F)F)sc3cncn23)C1
InChIKey: BVKJQZXJHPGUBA-UHFFFAOYSA-N
Target: Tryptophan 2,3-dioxygenase (SINGLE PROTEIN), Homo sapiens
UniProt: P48775
Assay: Enzymatic Assay: TDO2: Recombinant human TDO comprising amino acids 19-407 with a N-terminal hexahistidine tag expressed in E. coli and purified to homogeneity is incubated at a final concentration of 15 nM in assay buffer consisting of 75 mM phosphate buffer at pH7 supplemented with 100 μM ascorbic acid, 50 U/ml Catalase, 0.01% BSA, and 0.01% Tween 20 (protocol modified from Seegers et al, JBS 2014). Example compounds are serially diluted in DMSO, further diluted in phosphate buffer, and added to the reaction mixture at final concentrations ranging from 10 μM to 0.5 nM. The final DMSO concentration is 0.6%. Following a pre-incubation of 30 minutes at RT, the reaction is started by the addition of L-tryptophan at a final concentration of 200 μM in assay buffer. After 30 minutes of incubation at RT, 3 μL of the reaction mixture are transferred to a 384 deep well plate containing 25 μL of deionized water. 100 μl of 200 nM L-Tryptophan-(indole-d5) in cold 100% methanol are added followed by a 10 minutes centrifugation at 4,000 rpm at 4° C. An additional 75 μL of deionized water are then added and followed by a 10 minutes centrifugation at 4,000 rpm at 4° C. The product of the reaction N′-Formylkynurenine (NFK) is quantified by LCMS and normalized to the L-Tryptophan-(indole-d5) signal. Samples with 0.6% DMSO (0% effect) and a TDO/IDO inhibitor (100% effect) are used as control samples to set the parameters for the non-linear regression necessary for the determination of the half-maximal inhibitory concentration (IC50) for each compound. For each compound concentration the percentage of activity compared to 0% and 100% effect is calculated as average±STDEV (each concentration measured in duplicate). IC50 values and curves are generated with XLfit software (IDBS) using Dose-Response One Site model 203 (four parameter logistic curve model). When compounds are measured multiple times, mean values are given.
Measurement: IC50 = 3810 nM (pChEMBL 5.420000076293945)
Descriptors: MW 332.39, ALogP 4.66, HBA 4, HBD 1, PSA 37.53, QED 0.89
Compound (SMILES): Cc1c(Cl)cccc1-c1cccc2c1N[C@H](C)CC(=O)N2
InChIKey: AMGYKKLABABLCG-SNVBAGLBSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 7770 nM (pChEMBL 5.110000133514404)
Descriptors: MW 300.79, ALogP 4.46, HBA 2, HBD 2, PSA 41.13, QED 0.82
Compound (SMILES): CC(C)(C)Oc1ccc(-c2cc3c(N4CC(NC(=O)Cc5ccc(Cl)cc5Cl)C4)ncnn3c2)cc1
InChIKey: UPSFROZEAXWWJP-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kit D816V Assay: In each well of a 384-well plate, 0.04 ng/ul (0.5 nM) of D816V Kit (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 15 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 524.45, ALogP 5.43, HBA 6, HBD 1, PSA 71.76, QED 0.36
Compound (SMILES): COc1cc2nccc(Oc3ccc(NC(=O)c4c[nH]c(=O)n(-c5ccc(F)cc5)c4=O)cc3F)c2cc1OC
InChIKey: OMGRFVAGZMFWJX-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Kinase Assay: The cMET kinase assay was performed in 384-well Fluotrac 200 HiBase microplates using the HTRF KinEASE assay described above for AXL except that the assay volume was reduced to half. Enzyme concentration was 8 ng/mL of recombinant human baculovirus-expressed cMET while the substrate concentrations were 0.1 μM and 0.02 μM for the biotinylated peptide and ATP, respectively. Instead of the Multidrop Combi, the BioRAPTR® FRD microfluidic workstation (Beckman Coulter, Brea, Calif.) was utilized for reagent additions.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 546.49, ALogP 4.41, HBA 8, HBD 2, PSA 124.54, QED 0.31
Compound (SMILES): Cn1nnc(Cc2ccccc2-c2sc3cc(O)ccc3c2Oc2ccc(/C=C/C(=O)O)cc2)n1
InChIKey: NZTOUEOBTSJJHI-MDWZMJQESA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: ERalpha Degradation (MCF7 Cells): Plate MCF7 cells at 0.3 million cells/mL (100 l/well) in black, clear-bottom 96-well plates (Greiner, catalog number 655090) in DMEM/F12 media (Gibco, catalog number 11330) supplemented with 10% charcoal-stripped serum (Gemini Bio-Products, catalog number 100-119), and incubate them at 37° C., 5% CO2 for 24-36 hours. Next day, make 10× solution of ligands in DMSO and add the solution to the cells to achieve a final concentration of 10 uM. A DMSO control is required for relative calculations, and fulvestrant is used as a positive control for ER degradation. The cells are subjected to the in-cell Western assay after incubating cells with ligand for 18-24 hours.Media is removed from the plates by decanting, and cells are immediately fixed with 100 μl of 3.7% formaldehyde in PBS using a multi-channel pipettor. Add formaldehyde to the sides of the wells to avoid cell disruption. Plates are incubated at room temperature for 20 minutes without shaking. The fix solution is then removed and cells are permeabilized with 100 μL/well of 0.1% Triton X-100 in PBS. The lysate is then blocked by adding 50 uL/well of blocking solution (3% goat serum, 1% BSA, 0.1% cold fish skin gelatin and 0.1% Triton X-100 in PBS, pH 7.4) and allowed to shake at room temperature for 2 hr, or alternatively, at 4° C. overnight.After blocking, 40 μL/well of the primary antibody against ERα (HC-20) (Santa cruz, catalog number 543) diluted at 1:3000 in blocking buffer diluted 1:3 with PBS is added to each well, except the negative control wells (which are used for background subtraction) and the plate is sealed and incubated overnight at 4° C. Next day, the primary antibody solution is removed and the wells are washed three times with 0.1% TWEEN in PBS, with each wash lasting 5 minutes. 40 μL/well of secondary antibody (Biotium CF770 goat anti-rabbit 1:2000, catalog number 20078) and DRAQ5 (DNA stain, 5 mM, Thermo Scientific, catalog number 62251) diluted at 1:10000 in blocking buffer diluted 1:3 with PBS is then added to all the well, including the negative control wells, and the plate is allowed to incubate on shaker at room temperature for 2 hr. The secondary antibody solution is then removed and the plates are washed three times as described above. The plate is then washed one final time with PBS alone to minimize auto-fluorescence. The plate is then cleaned and read on LiCor Odyssey imager.For % response calculations, first divide integrated intensities for 700 channel (ER) by integrated intensities for 800 channel (DNA normalization); 700 (ER)/800 (DNA). This will be referred to as the normalized value. Then subtract average of negative control wells (no primary antibody) from all normalized values. This corresponds to negative subtraction. % response=(Valueunknown/ValueDMSO control)*100.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 484.54, ALogP 5.28, HBA 8, HBD 2, PSA 110.36, QED 0.3
Compound (SMILES): CC(F)(F)COc1ccc([C@H]2COC(=O)N2c2ccc3[nH]cnc3c2)cc1F
InChIKey: IQQIFBUARJSBEK-MRXNPFEDSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Fluorometric Assays: All measurements were performed with a BioAssay Reader HTS-7000Plus for microplates (Perkin Elmer) at 30° C. QC activity was evaluated fluorometrically using H-Gln-βNA. The samples consisted of 0.2 mM fluorogenic substrate, 0.25 U pyroglutamyl aminopeptidase (Unizyme, Horsholm, Denmark) in 0.2 M Tris/HCl, pH 8.0 containing 20 mM EDTA and an appropriately diluted aliquot of QC in a final volume of 250 ul. Excitation/emission wavelengths were 320/410 nm. The assay reactions were initiated by addition of glutaminyl cyclase. QC activity was determined from a standard curve of β-naphthylamine under assay conditions. One unit is defined as the amount of QC catalyzing the formation of 1 umol pGlu-βNA from H-Gln-βNA per minute under the described conditions.In a second fluorometric assay, QC was activity determined using H-Gln-AMC as substrate. Reactions were carried out at 30° C. utilizing the NOVOStar reader for microplates (BMG labtechnologies). The samples consisted of varying concentrations of the fluorogenic substrate, 0.1 U pyroglutamyl aminopeptidase (Qiagen) in 0.05 M Tris/HCl, pH 8.0 containing 5 mM EDTA and an appropriately diluted aliquot of QC in a final volume of 250 ul. Excitation/emission wavelengths were 380/460 nm. The assay reactions were initiated by addition of glutaminyl cyclase. QC activity was determined from a standard curve of 7-amino-4-methylcoumarin under assay conditions. The kinetic data were evaluated using GraFit software.
Measurement: Ki = 19.19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 391.35, ALogP 4.43, HBA 4, HBD 1, PSA 67.45, QED 0.7
Compound (SMILES): C=C1CN(C(=O)c2nc(N(C)C)sc2-c2ccc(C)c(C)c2)C(C)(c2nc3cc(OC)ccc3[nH]2)C1
InChIKey: MDOFWYOTVXOOFK-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 501.66, ALogP 5.7, HBA 6, HBD 1, PSA 74.35, QED 0.36
Compound (SMILES): CCCCOc1nc(N)c2nc(O)n(Cc3ccc4c(c3)Sc3cc(CNC)ccc3N4C)c2n1
InChIKey: IDFWOLDHCSUASK-UHFFFAOYSA-N
Target: Interleukin-6 (SINGLE PROTEIN), Homo sapiens
UniProt: P05231
Assay: Interleukin 6 Induction Assay: Compounds diluted in DMSO were transferred to individual wells of a Matrix Technologies clear, V-bottom 384-well plate using ECHO acoustic liquid handling technology (25 nL per well). Human whole-blood samples (25 uL) were added to each well using a CyBio FeliX liquid handling instrument. The plate was shaken on a plate shaker for three min before incu-bating the reaction mixtures at 37° C. for 20 h. Basel RPMI 1640 medium (supplemented with L-glutamine) was then added to each well (25 uL per well) prior to liberating plasma from each sample by centrifugation (450×g, 5 min, ambient temperature). Treated plasma samples (3 uL) were subsequently transferred to individual wells of a white, shallow, 384-well ProxiPlate (Perkin Elmer) using the FeliX liquid handling instrument and their interleukin 6 levels were measured using AlphaLISA technology as described by the manufacturer, PerkinElmer. Data analyses software was used to determine compound EC50 values where the baseline was established using average DMSO values and 100% induction established using reference compound values at the highest concentration tested. EC50's can be determined with software such as Graphpad Prism.
Measurement: EC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 491.62, ALogP 4.29, HBA 10, HBD 3, PSA 114.35, QED 0.31
Compound (SMILES): COC1CCN(c2cccc3c2CCN(C(=O)c2cnn(-c4cccnc4)c2)C3C(=O)Nc2ccc(C(=O)O)cc2)CC1
InChIKey: CXGWBRHGBOMOMU-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Enzyme Inhibition Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 6000 nM (pChEMBL 5.21999979019165)
Descriptors: MW 580.65, ALogP 3.96, HBA 8, HBD 2, PSA 129.89, QED 0.34
Compound (SMILES): CC1(N2Cc3cc(Nc4nn([C@H]5CCCC[C@@H]5C#N)c5cc[nH]c(=O)c45)ccc3C2=O)CCOCC1
InChIKey: JZCKKPDNCNCXLJ-UTKZUKDTSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 486.58, ALogP 4.25, HBA 7, HBD 2, PSA 116.04, QED 0.57
Compound (SMILES): CC1(C)CN(c2ccccc2NC(=O)Nc2ccc(OC(F)(F)F)cc2)c2c(O)cc(F)c(-c3ccc(C(F)(F)F)cc3)c21
InChIKey: LSRDDBSNRVLFDR-UHFFFAOYSA-N
Target: P2Y purinoceptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P47900
Assay: Scintillation Proximity Assay (SPA): A SPA membrane binding assay was used to identify inhibitors of [33P] 2MeS-ADP binding to cloned human P2Y1 receptors (The P2Y1 receptor membranes were provided by Biology and the cloning of the receptor and P2Y1 receptor membrane preparation is same as described by Biology). Binding reactions were performed in 384-well OptiPlates (PerkinElmer Life Sciences, Cat #6007299) in a volume of 50 μL containing 15 fmol of P2Y1 receptor (1.7 μg of total protein), 0.3 nM [33P] 2MeS-ADP (PerkinElmer; 2,000 Ci/mmol), various concentrations of the test compound (usually between 10 μM and 160 pM) in Buffer B containing 1% DMSO in assay buffer (15 mM, HEPES, 145 mM potassium chloride, 5 mM sodium chloride, 5 mM EDTA, 0.1 mM MgCl2, pH 7.4) and 100 μg of SPA bead (WGA polystyrene Image beads, #RPNQ 0260V, Amersham). Reactions were allowed to proceed to completion at room temperature for 1 hour followed by centrifugation of the plate for 5 min. About 40 μL of the aqueous solution was aspirated. Plates were sealed and the [33P] 2MeS-ADP bound to the P2Y1 receptor membranes that were bound to the SPA bead were determined in a Gen 4 LEADSEEKER (Amersham) Image Reader.
Measurement: Ki = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 619.54, ALogP 9.19, HBA 4, HBD 3, PSA 73.83, QED 0.19
Compound (SMILES): Cc1cnc(-c2cc(OC3CCNC(C(F)(F)F)C3)cc(C(=O)N[C@H](C)c3cnc(C(F)(F)F)nc3)c2)s1
InChIKey: RLIMWYRKEDYMPQ-UYCDIGJJSA-N
Target: P2X purinoceptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBL9
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity at Human P2X3 and Human P2X2/3 Receptors: A fluorescent imaging plate reader (FLEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Fluo-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C. The final assay DMSO concentration is 1%. The agonist, α,β-Me-ATP, was added in a volume of 20 μl at a concentration representing the EC80 value. The fluorescence was measured for an interval of 90 sec at 2 sec intervals and analyzed based on the increase in peak relative fluorescence units (RFU) compared to the basal fluorescence. Peak fluorescence was used to determine the response to agonist obtained at each concentration of test compound by the following equation: % Response=100*(RFU(test compound)−RFU(control))/(RFU(DMSO)−RFU(control))The Examples were tested in triplicates per plate and mean values were plotted in Excel XLFit to determine IC50 values at the human P2X3 and human P2X2/3 receptors, percentage of maximal inhibition and the Hill coefficients.
Measurement: IC50 = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 559.54, ALogP 5.48, HBA 7, HBD 2, PSA 89.03, QED 0.39
Compound (SMILES): CC(F)(F)[C@H]1CN(C2CCN(c3nnc(N)[nH]3)CC2)[C@@H](Cc2ccc(Cl)cc2)CO1
InChIKey: QAZJYDLZLLBKGO-DLBZAZTESA-N
Target: Acidic mammalian chitinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BZP6
Assay: CHIT1 Activity Assay: An enzymatic assay with recombinant human CHIT1 was used in order to establish inhibitory activity of the compounds (Boot et al., 2001, JBC:276). The assay was run in the 96-well plate format, each reaction in the total volume of 100 μL. 4-methylumbelliferyl β-D-N,N′,N″-triacetylchitotriose was used as a substrate for the enzyme. Upon hydrolysis by CHIT1, the substrate releases 4-methylumbelliferyl (4MU) that, when ionized in basic pH, emits fluorescence at 460 nm. Briefly, 40 μL of a substrate was added to each well, followed by 10 μL of compound dilution and 50 μL of CHIT1 recombinant enzyme solution. The reaction was carried out in citrate buffer, pH 5.2, in the dark, at 37° C. for 60 minutes with shaking. After that time the reaction was stopped by adding 195 μL of Stop Solution (pH 10.5) to each well. The fluorescence of the reaction product was measured in Tecan Spark multimode plate reader at an excitation wavelength of 355 nm. The IC50 values were calculated using GraphPad Prism.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 440.93, ALogP 2.98, HBA 6, HBD 2, PSA 83.3, QED 0.74
Compound (SMILES): CCN1Cc2ccc(NC(=O)[C@@H]3C[C@@H](F)CN3C(=O)Cn3cc(C(C)=O)c4cc(-c5ccnnc5)ccc43)cc2C1=O
InChIKey: COEXMOUYCYGEKN-DFHRPNOPSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Factor D Assay: Human Factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration was incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. Absorbance at 405 nm (A405) was recorded at 30 second intervals for 30 minutes using a microplate spectrophotometer. IC50 values are calculated by nonlinear regression of complement Factor D reaction rates as a function of test compound concentration.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 568.61, ALogP 3.85, HBA 7, HBD 1, PSA 117.5, QED 0.34
Compound (SMILES): Cc1nc(-c2cccnc2)sc1C(=O)Nc1cn2nc(-c3c(-c4ccc(F)cc4)ncn3C3CN(C)C3)ccc2n1
InChIKey: NRVYFAQGTJFUMF-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 0.915 nM (pChEMBL 9.039999961853027)
Descriptors: MW 565.64, ALogP 4.96, HBA 10, HBD 1, PSA 106.13, QED 0.3
Compound (SMILES): COc1cc(CCc2cnc3[nH]c(-c4ccc(C5CCN(C)CC5)cc4)cc3n2)cc(OC)c1
InChIKey: VTZHEKKAPSQDPL-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Enzymatic Assay: The inhibitor potency of compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; added fresh 30 mM EDTA and Perkin Elmer Lance Reagents for HTRF at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader.FGFR1 and FGFR2 were measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 μM, respectively and FGFR2, 0.01 nM and 100 μM, respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 456.59, ALogP 5.24, HBA 5, HBD 1, PSA 63.27, QED 0.41
Compound (SMILES): COC(=O)c1ccccc1CN1C(=O)N(Cc2ccc(OC)cc2)C[C@]12CC[C@](c1ccccc1)(N(C)C)CC2
InChIKey: UYRRUFWQQVZIOQ-CZFRINIUSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl. 10 mM MgCl2. 1 mM EDTA (pH 7.4). The final assay volume (250 ul/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 1 uM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 60 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux beta-counter.
Measurement: Ki = 1170 nM (pChEMBL 5.929999828338623)
Descriptors: MW 541.69, ALogP 5.69, HBA 5, HBD 0, PSA 62.32, QED 0.34
Compound (SMILES): Cn1nc(Nc2cnccc2C(=O)O)c2c(F)cccc21
InChIKey: MVLYGYPENISRGG-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGL1
Assay: Enzymatic Assay: The ability of test compounds to inhibit the activity of Jarid1B was determined in 384-well plate format under the following reaction conditions: 0.8 nM Jarid1B, 300 nM H3K4me3-biotin labeled peptide (Anaspec cat #64357), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-mono- or di-methylated histone H3 lysine 4 (H3K4me1-2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 25 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K4me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO was added to each well of the plate, followed by the addition of 2 μl of 2.4 nM Jarid1B to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 50 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K4me1-2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 286.27, ALogP 2.55, HBA 5, HBD 2, PSA 80.04, QED 0.77
Compound (SMILES): COc1cc(C(OC)C(=O)Nc2nnc(N[C@@H]3CCN(c4ccc(F)nn4)C3)s2)ccc1F
InChIKey: UIPMTIIMXUDEQN-MTATWXBHSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: Enzyme Potency Assay: A Glutamate Oxidase/AmplexRed coupled assay was used to measure the ability of compounds to bind to and inhibit the activity of GLS1 in vitro. 6His tagged GLS protein (amino acids 63-669) expressed in E. Coli was purified and stored at −80° C. in aliquots. GLS1 was diluted to 2× working concentration and incubated at room temperature to allow the tetrameric/dimeric forms to reach steady state. Assay measurements were performed in buffer comprising 50 mM TRIS pH 7.8, 100 mM NaPO4, pH 7.8, 0.001% v/v Tween20. Purified recombinant GLS1 protein was diluted in assay buffer to 12 nM and pre-incubated at room temperature for 30 minutes. Test compounds were prepared by dilution in 100% DMSO to give the correct dose range for 12 point concentration response and an appropriate volume (2.5-60 nl) dispensed into 384 well micro assay plates (Greiner product code 784900) using a Labcyte Echo 555 acoustic dispenser. DMSO concentration was maintained at 2% by back filling with DMSO solution. 3 μL of diluted GLS1 protein (12 nM) was then dispensed into each well using a BioRaptr automated dispenser (Beckman-Coulter) and incubated for 15 minutes at room temperature. 3 μL of 100 mM glutamine diluted in assay buffer was then added and the reaction incubated at room temperature for 60 minutes. The reaction was then stopped by addition of 45 μM 6-(2-bromoethynyl)-2,3-dimethyl-quinazolin-4-one, 75 μM Amplex Red, 0.375 units/mL Horseradish Peroxidase, 0.12 units/mL Glutamate Oxidase in 100 mM TRIS pH7.5. After 30 minutes at room temp in the dark, plates were read on a Perkin Elmer EnVision using 535/590 nm optic filters and raw data analysed using Genedata to generate IC50 values. An artefact version of the assay where the 6His tagged GLS protein and glutamine were replaced with assay buffer was also used to rule out non specific effects on the assay components.
Measurement: IC50 = 71.3 nM (pChEMBL 7.150000095367432)
Descriptors: MW 477.5, ALogP 2.63, HBA 10, HBD 2, PSA 114.39, QED 0.51
Compound (SMILES): CCc1c(-c2cc(C)nc(C)c2)[nH]c2ccc(C3CCN(CC(=O)NC)CC3)cc12
InChIKey: SHLCZWWNBDBPQU-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 63.6 nM (pChEMBL 7.199999809265137)
Descriptors: MW 404.56, ALogP 4.33, HBA 3, HBD 2, PSA 61.02, QED 0.67
Compound (SMILES): COc1ccc(COc2ccc(Nc3ccc4ncc(N(C)C)nc4c3C#N)cc2OC)cc1C#N
InChIKey: UXQVASIWKQXCEL-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Kinase Assay: c-KIT: Inhibition of c-KIT kinase activity by a test compound disclosed herein was estimated by radiometric assay. Standard assay conditions were 20 ng of recombinant c-Kit (SignalChem) with 2 μg of substrate Poly-(Glu 4:Tyr 1) (sigma) in the assay buffer in kinase reaction buffer (10 mM MOPS pH 7.0, 0.21 mM EDTA, 0.5% glycerol, 0.001% Brij-35, 0.01 2-mercaptoehtanol, 1 mg/ml BSA, 10 mM MgCl2, 10 mM MnCl2, 10 μM ATP, 0.1 μCi per well [33P]-ATP, in the presence of test compound (diluted in final concentration of 4% DMSO) or DMSO control, with a final volume of 25 μl for 60 minutes at 30° C. The reaction was stopped by adding 5 μl of 3% phosphoric acid solution. Total reaction solution was then harvested onto a filter plate (UniFilter-96 GF/B, PerkinElmer), and washed 20 times for 5 min with dH2O. 30 μl of MicroScint-20 Cocktail (PerkinElmer) was added to dried plate. The plate was sealed and counted using a TopCount scintillation detector (PerkinElmer). The readout from control reaction (complete reaction mixture) was designated as 0% inhibition and the readout for the reaction without enzyme as 100% inhibition. The IC50 values of quinoxaline compounds of this invention against c-KIT kinase were determined using the GraphPad Prism 5 software.
Measurement: Ki = 275 nM (pChEMBL 6.559999942779541)
Descriptors: MW 480.53, ALogP 4.78, HBA 9, HBD 1, PSA 116.32, QED 0.38
Compound (SMILES): CCC(Nc1cc(F)c(S(=O)(=O)Nc2nccs2)cc1Cl)c1cccnc1
InChIKey: SXFQXWOPGZNRAJ-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 7086 nM (pChEMBL 5.150000095367432)
Descriptors: MW 426.93, ALogP 4.69, HBA 6, HBD 2, PSA 83.98, QED 0.57
Compound (SMILES): Clc1ccc(NC(c2ccccc2)c2ccc(-c3ccccc3)cn2)cc1Cl
InChIKey: JVJSZSHHFYZFNB-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: TBD: TBD
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 405.33, ALogP 7.26, HBA 2, HBD 1, PSA 24.92, QED 0.38
Compound (SMILES): CC(c1ccc(Br)cc1)N1CCC(n2c(=O)[nH]c3cc(Cl)ccc32)CC1
InChIKey: NHFZJLPFUWQCRF-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: cAMP inhibition assays: he CisBio cAMP cell based assay kit that uses HTRF technology was utilized to assess MOR-mediated inhibition of cAMP. Assays were performed according to manufacturer's instructions, in the presence of 20 μM forskolin and 25 μM PDE-IV. All compounds were run in parallel with DAMGO and morphine.
Measurement: EC50 = 10 nM (pChEMBL 8)
Descriptors: MW 434.77, ALogP 5.14, HBA 3, HBD 1, PSA 41.03, QED 0.62
Compound (SMILES): NCCC1CN(c2ccc(S(=O)(=O)NC[C@H](O)CN)c(S(N)(=O)=O)c2-c2nn[nH]n2)C1
InChIKey: NOKSEJLLZNSRQL-SNVBAGLBSA-N
Target: Beta-lactamase (SINGLE PROTEIN), Pseudomonas aeruginosa
UniProt: Q79MP6
Assay: Enzyme Activity: Determination of IC50 : The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 μM to 0.00063 μM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 89.6 nM (pChEMBL 7.050000190734863)
Descriptors: MW 475.56, ALogP -3.1, HBA 11, HBD 6, PSA 236.3, QED 0.2
Compound (SMILES): COCc1ccc(N(C)C(=O)c2cn(C(C)(C)C)c3c2C(N)=NCN3)cc1
InChIKey: KVWRHCWWCSVZLW-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Measurement of Inhibitory Activity Against RET (V804L, V804M): Regarding the conditions for measurement of in vitro inhibitory activity of compounds against RET (V804L or V804M) (i.e., RET with V804L or V804M mutation) kinase activity, the website of AnaSpec states that Srctide (GEEPLYWSFPAKKK) corresponds to the substrate peptide for reaction to measure RET kinase activity. Thus, the amino acid sequence was partly modified and biotinylated to prepare biotinylated peptides (biotin-EEPLYWSFPAKKK). The purified recombinant human RET (V804L or V804M) protein used in the test was purchased from Eurofins.To measure the inhibitory activity, first, the test compounds were individually diluted with dimethyl sulfoxide (DMSO) stepwise. Subsequently, RET (V804L or V804M) protein, the substrate peptides (the final concentration: 250 nM), magnesium chloride (the final concentration: 10 mM), ATP (the final concentration: 10 μM), and a solution of a test compound in DMSO (the final concentration of DMSO: 5%) were added to a buffer for kinase reaction (13.5 mM Tris, pH of 7.5, 2 mM dithiothreitol, 0.009% Tween-20). Each of the mixtures was incubated at 25° C. for 120 minutes to perform a kinase reaction. EDTA was then added thereto to give a final concentration of 40 mM so that the reaction was terminated. A detection solution containing Eu-labeled antiphosphotyrosine antibody PT66 (PerkinElmer) and SureLight APC-SA (PerkinElmer) was added thereto, and each mixture was allowed to stand at room temperature for 2 hours or more. Finally, the intensity of fluorescence under excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at the two wavelengths of 620 nm and 665 nm. The phosphorylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which phosphorylation was inhibited by 50% was defined as the IC50 value (nM).
Measurement: IC50 = 1410 nM (pChEMBL 5.849999904632568)
Descriptors: MW 369.47, ALogP 2.75, HBA 6, HBD 2, PSA 84.88, QED 0.87