lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(CCC#N)C1CCC(=CCn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)CC1
InChIKey: MXSSPXRDFQWUOU-GRSHGNNSSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.57 nM (pChEMBL 9.239999771118164)
Descriptors: MW 595.05, ALogP 5.81, HBA 9, HBD 1, PSA 113.37, QED 0.23
Compound (SMILES): Cc1cc(-c2n[nH]c3ccc(C(=O)N[C@H]4CCC[C@@](O)(Cn5cc6cc(F)ccc6n5)C4)cc23)ccn1
InChIKey: IWKTZWHPKLAQNY-DWACAAAGSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Mus musculus
UniProt: P63085
Assay: Active ERK2 (aERK) Assay: Activated ERK2 activity was also determined in the IMAP assay format using the procedure outlined above. 1 μl of 25× compound was added to 14 μl of kinase buffer containing 0.25 ng fully phosphorylated, active Mouse ERK2 protein. Following a 30 minute incubation, the reactions were initiated by addition of 10 μl of kinase buffer containing 1 μM ERK2 IMAP substrate peptide (0.9 μM unlabeled IPTTPITTTYFFFK-CONH2 and 100 nM IPTTPITTTYFFFK(5-carboxyfluorescein)-CONH2) and 30 μM ATP. Reactions proceeded for 30 minutes before termination by addition of 60 μl IMAP detection beads in binding buffer. Plates were read as above after 30 minute binding equilibration. Active compounds were reconfirmed in an independent assay.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 498.56, ALogP 4.53, HBA 6, HBD 3, PSA 108.72, QED 0.33
Compound (SMILES): NS(=O)(=O)c1ccc(-n2cc(CN(Cc3cn(-c4ccc(S(N)(=O)=O)cc4)nn3)c3ccccc3)nn2)cc1
InChIKey: LUROKFGCKDYVGL-UHFFFAOYSA-N
Target: Carbonic anhydrase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00915
Assay: Inhibition of human carbonic anhydrase 1 pre-incubated for 15 mins by stopped flow carbon dioxide hydrase assay
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 565.64, ALogP 1.35, HBA 11, HBD 2, PSA 184.98, QED 0.27
Compound (SMILES): CC(C)c1nc2c(c(Nc3ccc(Cl)c(Cl)c3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: ZZSJKJSXOFQQTO-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 496.36, ALogP 6.67, HBA 5, HBD 1, PSA 53.94, QED 0.44
Compound (SMILES): CC1(OC(=O)N2CCC(N(C(=O)c3ccc(-c4cnco4)cc3)C3CC3)CC2)CC1
InChIKey: RUKMTRNCJAWVKD-UHFFFAOYSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] are stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP_848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM β-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37° C. 5% CO2. For the assay, the cells are seeded in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37° C./5% CO2. After the medium is aspirated from the wells completely, 10 ul of the test compound are added, the compounds are diluted using stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes incubation at room temperature (approx. 20° C.), the cAMP concentrations are determined using the AlphaScreen cAMP Assay Kit (Cat. No. #6760625R from PerkinElmer). 10 ul of Biotin-cAMP (final concentration 1 U/well in lysing buffer (5 mM Hepes (pH 7.4), 0.1% BSA, 0.5% Tween) and 10 uL Bead solution (final concentration 1 U/well in lysing buffer) are added. The plates are incubated for another 2 hours at room temperature. The cAMP concentrations are calculated using a cAMP standard curve from the Alpha Screen Counts. The data analysis is carried out by calculating the EC50 value and the maximum value based on a positive control, using suitable software (Graphpad Prism).
Measurement: EC50 = 52 nM (pChEMBL 7.28000020980835)
Descriptors: MW 409.49, ALogP 4.1, HBA 5, HBD 0, PSA 75.88, QED 0.74
Compound (SMILES): Cc1ncoc1-c1ccc(C(=O)N(C2CC2)C2CCN(C(=O)OC(C)(C)C)CC2)cc1
InChIKey: XRLAZSGKPFLWOU-UHFFFAOYSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] are stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP_848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM β-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37° C. 5% CO2. For the assay, the cells are seeded in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37° C./5% CO2. After the medium is aspirated from the wells completely, 10 ul of the test compound are added, the compounds are diluted using stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes incubation at room temperature (approx. 20° C.), the cAMP concentrations are determined using the AlphaScreen cAMP Assay Kit (Cat. No. #6760625R from PerkinElmer). 10 ul of Biotin-cAMP (final concentration 1 U/well in lysing buffer (5 mM Hepes (pH 7.4), 0.1% BSA, 0.5% Tween) and 10 uL Bead solution (final concentration 1 U/well in lysing buffer) are added. The plates are incubated for another 2 hours at room temperature. The cAMP concentrations are calculated using a cAMP standard curve from the Alpha Screen Counts. The data analysis is carried out by calculating the EC50 value and the maximum value based on a positive control, using suitable software (Graphpad Prism).
Measurement: EC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 425.53, ALogP 4.65, HBA 5, HBD 0, PSA 75.88, QED 0.71
Compound (SMILES): CN1Cc2c(C3=CC4CN(CC(=O)N5CC(O)C5)CC4C3)[nH]c3nccc(c23)-c2cc(F)ccc21
InChIKey: PBKTVFZFCMOJNZ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay : CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were diluted in 100% DMSO then diluted with 1:10 in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. Equal volume of the compound dilutions was added to a final reaction mixture containing LANCE detection buffer (PerkinElmer CR97-100), 100 nM ULight MBP (PerkinElmer TRF0109M), 1000 μM ATP, and CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 1 hour before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer. The reaction was incubated for 1 hour and the signal was read in Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 94 nM (pChEMBL 7.03000020980835)
Descriptors: MW 473.55, ALogP 2.86, HBA 5, HBD 2, PSA 75.7, QED 0.61
Compound (SMILES): O=C1c2cnc3[nH]ccc3c2N([C@H]2CC[C@H](CNCC(F)F)CC2)CN1C1CC1
InChIKey: BJCQGGRBYJQHAY-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 7.1 nM (pChEMBL 8.149999618530273)
Descriptors: MW 403.48, ALogP 3.36, HBA 4, HBD 2, PSA 64.26, QED 0.78
Compound (SMILES): NC1CCN(c2cc(-c3n[nH]c4cnc(-c5cccnc5)cc34)ccn2)CC1
InChIKey: YOEKUPVJOQMNFT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 2.78 nM (pChEMBL 8.5600004196167)
Descriptors: MW 371.45, ALogP 3.01, HBA 6, HBD 2, PSA 96.61, QED 0.57
Compound (SMILES): C[C@@H](NC(=O)/C=C/c1cnc2[nH]cc(-c3ccccc3)c2c1)[C@@H](O)c1ccccc1
InChIKey: LHOQGWQNUGSEBJ-ZQINLGPMSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: Inhibition of human GSK3beta using YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE as substrate in presence of gamma-33P-ATP
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 397.48, ALogP 4.48, HBA 3, HBD 3, PSA 78.01, QED 0.42
Compound (SMILES): NCC(c1ccc(Cl)nc1)N1CCN(c2ncnc(N)c2-c2ccc(F)cc2)CC1
InChIKey: HRDNQTRURDVWED-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: Enzyme Assay: P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction.
Measurement: IC50 = 8.5 nM (pChEMBL 8.069999694824219)
Descriptors: MW 427.92, ALogP 2.74, HBA 7, HBD 2, PSA 97.19, QED 0.6
Compound (SMILES): NC(=O)OC(CNC(=O)Cn1nc(-c2ccc(Cl)cc2)n(C[C@H](O)C(F)(F)F)c1=O)c1ccccc1C(F)(F)F
InChIKey: GCPCDABXSOXIND-DJNXLDHESA-N
Target: Vasopressin V1a receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P37288
Assay: Cellular in Vitro Assay: On the day before the assay, the cells are plated out in culture medium (DMEM, 10% FCS, 2 mM glutamine, 10 mM HEPES) in 384-well microtitre plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, the culture medium is replaced by a Tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM glucose, 20 mM HEPES), which additionally contains the cofactor coelenterazine (50 μM), and the microtitre plate is then incubated for a further 3-4 hours. The test substances in various concentrations are placed for 10 to 20 minutes in the wells of the microtitre plate before the agonist [Arg8]-vasopressin is added, and the resulting light signal is measured immediately in the luminometer.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 595.88, ALogP 3.26, HBA 8, HBD 3, PSA 141.47, QED 0.32
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)CN(Cc3cccc(C(=O)O)c3)C2=O)cn1
InChIKey: VIWBWOCSCNUOFX-UHFFFAOYSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 409.4, ALogP 2.2, HBA 7, HBD 1, PSA 121.77, QED 0.62
Compound (SMILES): O=C(O)COc1ccc(Cl)cc1C1c2scnc2CCN1C(=O)NCc1ccc(F)cc1
InChIKey: JODQOHBWOLGAHR-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 μl. First, 25 μl of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 μl Binding-Buffer, 50 μl of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 μl CRTH2 membrane fragments, reaching a final concentration of 20 μg/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 μl of Microscint-40 (Packard) was added to each well and the re
Measurement: IC50 = 124 nM (pChEMBL 6.909999847412109)
Descriptors: MW 475.93, ALogP 4.26, HBA 5, HBD 2, PSA 91.76, QED 0.56
Compound (SMILES): Cc1c(C2COC2)sc(NC(=O)C2=C(C(=O)O)CCC2)c1-c1nc(C2CC2)no1
InChIKey: PCMZGTDLLGBMMV-UHFFFAOYSA-N
Target: Fatty acid-binding protein, adipocyte (SINGLE PROTEIN), Homo sapiens
UniProt: P15090
Assay: Time Resolved-Fluorescence Energy Transfer (TR-FRET) Assay: Stock DMSO solutions (1.8 mM) of compounds were serially diluted 3-fold for ten concentrations with 100% DMSO (50 μM to 0.003 μM final compound concentration). 1 μl of these compound dilutions and 1 μl of Bodipy labeled fatty acid 4.5 μM in 100% DMSO (Bodipy FL C11, cat. no. D3862, Invitrogen) were sequentially pipetted in wells of 384-well black polypropylene plates (Thermo Matrix cat. no. 4344). FABP4 or FABP5 protein was then added (28 μl of 64 nM protein in 25 mM Tris pH 7.5, 0.4 mg/ml α-globulin, 1 mM DTT, 0.012% NP40, final protein concentration: 50 nM). Assay blanks contained ligand, but no protein. Neutral controls contained ligand, but no compound. After adding the detection reagent (Tb antiHis6 antibody, Columbia Biosciences, TB-110, 6 μl of a 24 nM Ab solution in 25 mM Tris pH 7.5, 0.4 mg/ml γ-globulin, final Tb antiHis6 Ab concentration: 4 nM), plates were spun one minute at 1000 rpm.
Measurement: IC50 = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 415.47, ALogP 3.6, HBA 7, HBD 2, PSA 114.55, QED 0.74
Compound (SMILES): O=S(=O)(c1ccccc1)N(CCN1CCOCC1)c1sc2ccccc2c1Br
InChIKey: MQZNJXAROISMSF-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 24.8 nM (pChEMBL 7.610000133514404)
Descriptors: MW 481.44, ALogP 4.19, HBA 5, HBD 0, PSA 49.85, QED 0.53
Compound (SMILES): Cc1cnc(NS(=O)(=O)c2ccc3c(c2)OCCN3c2ccc(C(F)(F)F)cc2Cl)s1
InChIKey: QAXMZLLPTWVHQQ-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 489.93, ALogP 5.46, HBA 6, HBD 1, PSA 71.53, QED 0.53
Compound (SMILES): COc1cc(F)c(C2COC(=O)N2c2ccc3[nH]cnc3c2)c(F)c1
InChIKey: VJQHZPJDDALUIV-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometic Assay: Spectrophotometic Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R.C.J. 1989 J Neurosci Methods 30, 23-28).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 345.31, ALogP 3.55, HBA 4, HBD 1, PSA 67.45, QED 0.79
Compound (SMILES): COc1ccncc1-c1ccc2[nH]nc(-c3cc(N4CCC(F)(F)CC4)ncn3)c2c1
InChIKey: FKAKVEBQFNPPRE-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 6.66 nM (pChEMBL 8.180000305175781)
Descriptors: MW 422.44, ALogP 4.33, HBA 6, HBD 1, PSA 79.82, QED 0.53
Compound (SMILES): COC(=O)[C@@H](NC(=O)Nc1cc2[nH]nc(OC)c2c(CO)n1)c1ccccn1
InChIKey: PGIOAGFEIOETAT-AWEZNQCLSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 105.4 nM (pChEMBL 6.980000019073486)
Descriptors: MW 386.37, ALogP 0.89, HBA 8, HBD 4, PSA 151.35, QED 0.46
Compound (SMILES): CCOC(Oc1ncccc1Nc1ncnc2cc(N=S(C)(C)=O)cc(C)c12)C(F)(F)F
InChIKey: RIOULVCMYYGLQB-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In vitro Kinase Assay: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 469.49, ALogP 4.74, HBA 8, HBD 1, PSA 98.59, QED 0.5
Compound (SMILES): CN(c1ccc(C(=O)NCc2ccccc2)cc1F)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: PUNOHMZQRDFAEB-LJQANCHMSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z'-LYTE™ Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE™ Tyr 6 peptide or Z-LYTE™ Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100M, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer™) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 = 0.055 nM (pChEMBL 10.260000228881836)
Descriptors: MW 444.51, ALogP 3.74, HBA 5, HBD 2, PSA 77.15, QED 0.47
Compound (SMILES): O=C(O[C@H]1C[N+]2(CCCc3cccs3)CCC1CC2)C1(O)c2ccccc2Oc2ccccc21
InChIKey: LULZMULPNLKOML-KDMOYCQDSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: [3H]-NMS Binding Assay: The binding of [3H]-NMS to human muscarinic receptors was performed according to Waelbroek et al (1990) (1). Assays were carried out at 25° C. Membrane preparations from stably transfected chinese hamster ovary-K1 cells (CHO) expressing the genes for the human muscarinic receptors Hm3 were used.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 476.62, ALogP 4.87, HBA 5, HBD 1, PSA 55.76, QED 0.41
Compound (SMILES): CC(C)C[C@@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)N)CSCc2cc3cc(c2)CSC[C@H](NC(=O)[C@@H]2CCCN2C(=O)[C@@H]2CCCN2C(=O)[C@H](CC(C)C)NC1=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)NCC(N)=O)CSC3
InChIKey: JIAPWZJNDOSBJY-YCKSDESNSA-N
Target: Sortase A (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q9S446
Assay: Inhibition of Staphylococcus aureus His6-biotinylated SrtA expressed in Escherichia coli XL1-blue using Dabcyl-LPETG-Edans as substrate assessed as residual activity by fluorescence assay
Measurement: Ki = 19300 nM (pChEMBL 4.710000038146973)
Descriptors: MW 1475.83
Compound (SMILES): NCc1ccc(Cl)cc1CNC(=O)[C@@H]1CCN1C(=O)C1NCc2ccccc21
InChIKey: SKZVYVLIAHIJQN-OYKVQYDMSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: In Vitro Assay: Relevant in vitro assays are referenced in Morrissette, et al., Bioorg. Med. Chem. Lett. 2004, 14, 4161-4164 and described in Lewis, et al. Thromb. Res. 1993, 70, 173 (assays of human α-thrombin and human trypsin), and Lewis, et al. Thromb. Haemostasis 1995, 74, 1107-1112. The assays were carried out at 25° C. in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1% PEG. Trypsin assays also contained 1 mM CaC12. In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate were determined, a Thermomax 96-well plate reader was used was used to measure (at 405 nm) the time dependent appearance of p-nitroaniline. sar-PR-pna was used to assay human α-thrombin (Km=125 μM) and bovine trypsin (Km=125 μM). p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm−1M−1.
Measurement: Ki = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 398.89, ALogP 1.86, HBA 4, HBD 3, PSA 87.46, QED 0.72
Compound (SMILES): CS(=O)(=O)c1ccc(-n2nc(C(N)=O)c3c2-c2cc(NC(=O)c4cccc(F)c4F)ccc2CC3)cc1
InChIKey: JIJWGZYNYGNTJB-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus using biotinylated IkappaBalpha peptide as substrate assessed as decrease in [gamma-33P]-ATP incorporation in substrate after 30 mins by scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 522.53, ALogP 3.67, HBA 6, HBD 2, PSA 124.15, QED 0.41
Compound (SMILES): Cc1ccc(-c2nc(C(=O)NCCC(=O)Nc3ccc(C4CCC(CC(=O)O)CC4)cc3)c(C(F)(F)F)o2)cc1
InChIKey: VEXSZMTUQHGIFR-UHFFFAOYSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: Enzyme Inhibition Assay: Human DGAT-1 activity was determined as follows: Assay buffer [20 mM HEPES (pH 7.4), 100 mM MgCl2, 0.04% BSA] containing 500 μM of enzyme substrate (didecanoyl glycerol) and 7.5 μM radiolabeled acyl-CoA substrate ([14C]decanoyl-CoA) was added to each well of a phospholipid FlashPlate (PerkinElmer Life Sciences). A small aliquot of lysate (5 μg/well) or mouse liver microsome (0.1 mg/well) was added to start the reaction, which was allowed to proceed in a 20° C. bath for 1 hour. and maintained overnight at room temperature. Next day, measurement was performed by Wallac 1450 Microbeta Trilux Liquid Scintillation Counter and Luminometer (PerkinElmer Life Science). In the control group, 0.1% DMSO was only added, and for the background value, 0.1% DMSO was only added without the cell fragment having the over-expressed human DGAT1 (5 μg/well) or the liver microsome of mice (0.1 mg/well), which was the same as the control group.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 557.57, ALogP 6.18, HBA 5, HBD 3, PSA 121.53, QED 0.29
Compound (SMILES): O=C(NCc1ccco1)Nc1cc(-n2cccn2)cc(-n2cnc3cc(-n4cccn4)ccc32)c1
InChIKey: CAKIDSSBYAPZGC-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: TR-FRET Assay: Compounds were screened in the TR-FRET assay with FGFR1 kinase. 5 ng of FGFR1 [Upstate, USA] kinase was used for assay. The compound was incubated with the kinase for 30 minutes at RT. After the incubation, substrate mix [40 nM Ultra light poly GT (Perkin Elmer, USA) and 13 μM ATP (Sigma)] was added. The above reaction was stopped by the addition of 40 mM EDTA after the 30 mM kinase reaction. The Eu-labelled antiphospho-tyrosine antibody [Perkin Elmer, USA] was added at 0.5 nM and the fluorescence emission at 615 nm/665 nm [excitation at 340 nm] was measured. The compounds were initially screened at 100 nM and 1 μM concentrations. The compounds with >50% inhibition at 100 nM of FGFR1 were taken for the full dose response studies. The final DMSO concentration in the assay was 1%.
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 464.49, ALogP 4.31, HBA 8, HBD 2, PSA 107.73, QED 0.38
Compound (SMILES): OC(COCC1CC1)CN1CCc2ccccc2C1
InChIKey: OVKWNDBUOLGGTR-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 261.36, ALogP 1.83, HBA 3, HBD 1, PSA 32.7, QED 0.85
Compound (SMILES): CCOC(=O)N1CCC(S(=O)(=O)c2c(Cl)ccc(NC(=O)Nc3cccc(F)c3C)c2O)CC1
InChIKey: XIWDXADXPBRSJR-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P25024
Assay: Displacement of [125I]IL8 from recombinant human CXCR1 expressed in CHO cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: IC50 < 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 513.98, ALogP 4.53, HBA 6, HBD 3, PSA 125.04, QED 0.5
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5c(c4)OCCO5)n3)c3ccccc23)n(-c2ccc(OCCN3CCOCC3)cc2)n1
InChIKey: VTQGLZJCCQHUJG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: p38 MAPKα Enzyme Inhibition Assay: The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen), are evaluated indirectly by determining the level of activation/phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPK&dlpha; protein (80 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL of either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL or 0.004 ug/mL) for 2 hr at RT. The mix solution (2.5 uL) of the p38&dlpha; inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 uM; a phosphorylation target for MAPKAP-K2) is then added and the kinase reaction is initiated by adding ATP (40 uM, 2.5 uL). The mixture is incubated for 1 hr at RT. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 756.86, ALogP 7.78, HBA 12, HBD 3, PSA 146.15, QED 0.12
Compound (SMILES): C#Cc1cccc(NC(=O)c2ccccc2C(=O)c2ccc3nc(NC(=O)OC)[nH]c3c2)c1
InChIKey: MUPACCSCNPRESR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 438.44, ALogP 4.21, HBA 5, HBD 3, PSA 113.18, QED 0.32
Compound (SMILES): Cc1cc(Oc2cnc(NC(=O)C(O[C@@H]3CCOC3)c3ccc(S(=O)(=O)C4CC4)cc3)s2)ccc1C(=O)O
InChIKey: VKFAKECKYCKINR-FKSKYRLFSA-N
Target: Hexokinase-4 (SINGLE PROTEIN), Homo sapiens
UniProt: P35557
Assay: Coupled Enzymatic Assay: The assay is carried out according to the protocol outlined in Hariharan et al (1997), Diabetes 46: 11-16. Briefly, the test compounds are incubated in a reaction mix containing 25 mM HEPES (pH 7.2), 10 mM MgCl2, 100 mM KCl, 5 mM ATP, 2 mM DTT, 0.5 mM NAD, 1 U/mL Leuconostoc mesenteroides G6PD, 0.3 U/mL of purified human recombinant GK, and different concentrations of glucose. Enzymatic activity is calculated from the initial reaction velocity, measured from the change in NADH absorbance as a function of time.
Measurement: EC50 = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 558.63, ALogP 4.36, HBA 9, HBD 2, PSA 141.12, QED 0.37
Compound (SMILES): C[C@H](Nc1ncc(F)c(N2C(=O)OC[C@@H]2CF)n1)c1cn(-c2ccc(C(F)F)c(F)c2)cn1
InChIKey: YEEAJLVWHWXJEY-JQWIXIFHSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 468.39, ALogP 4.35, HBA 7, HBD 1, PSA 85.17, QED 0.52
Compound (SMILES): CN1CCN(C(=O)Cn2cc(NC(=O)c3cnn4cccnc34)c(-c3cc(Cl)ccc3OC(F)F)n2)C(C)(C)C1
InChIKey: GTTCHFDBALQIQT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.56 nM (pChEMBL 9.25)
Descriptors: MW 573, ALogP 3.65, HBA 9, HBD 1, PSA 109.89, QED 0.36
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)cc(C2CCCCC2)n1CCCN1CCOCC1
InChIKey: CKUBDISBCAFOFK-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: Ki = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 477.57, ALogP 5.84, HBA 4, HBD 1, PSA 46.5, QED 0.55
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2[C@H](c2cccc(S(=O)(=O)NCCOCCOCCNC(=O)CN(CCOCCOCCOCCN)CC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)c2)C1
InChIKey: JQCYSTFMXKEZOP-WLTNIFSVSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 15.85 nM (pChEMBL 7.800000190734863)
Descriptors: MW 1277.23
Compound (SMILES): O=C(O)c1ccc(-n2nc([S+]([O-])c3c(Cl)cccc3Cl)c3ccccc32)cc1
InChIKey: MCANXLFLBQAKTG-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Inverse agonist activity at GAL4 DBD-fused RORgammat LBD (unknown origin) expressed in HG5LN cells after 18 hrs by luciferase reporter gene assay
Measurement: IC50 = 6200 nM (pChEMBL 5.210000038146973)
Descriptors: MW 431.3, ALogP 5.2, HBA 4, HBD 1, PSA 78.18, QED 0.45
Compound (SMILES): O=C(c1cc(-c2cc(Cl)ccc2C(=O)N2Cc3ccccc3C[C@H]2CN2CCOCC2)n2c1CCCC2)N(c1ccccc1)c1ccc(O)cc1
InChIKey: KDAFMWREKTUAQP-UMSFTDKQSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 701.27, ALogP 7.73, HBA 6, HBD 1, PSA 78.25, QED 0.19
Compound (SMILES): CC[C@@H]1C(=O)N(c2c(C)cccc2C)C[C@]1(C)C(=O)Nc1cc(C(F)(F)F)cc(C(F)(F)F)c1
InChIKey: KOTKRADTFVZNPE-GCJKJVERSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 486.46, ALogP 6.36, HBA 2, HBD 1, PSA 49.41, QED 0.51
Compound (SMILES): O=C(NCCc1c[nH]c2ccc(Cl)cc12)c1ccc(Cc2ccc(F)cc2)cc1
InChIKey: ZZNUHICVJZHONP-UHFFFAOYSA-N
Target: Alpha-synuclein (SINGLE PROTEIN), Homo sapiens
UniProt: P37840
Assay: In Vitro Inhibition Assay: α-Synucleinopathies are characterised by aggregation of α-synuclein in neurons. Aggregation of purified α-synuclein is performed essentially as described by Gerard et al. FASEB. 20(3):524-6 (2006). 20-100 μg purified α-synuclein (Sigma; S7820) at a concentration of about 2.5 μg/mL is incubated in the presence of spermin (250 μM) or paraquat (32 mM) or 6-hydroxydopamine (400 μM) or vehicle in a 384 well plate. Spermin, paraquat and 6-hydroxydopamine promote the α-synuclein aggregation process. Aggregation kinetics is determined by measuring turbidity at 340 nm, every 1-15 minutes for at least one hour.
Measurement: EC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 406.89, ALogP 5.52, HBA 1, HBD 2, PSA 44.89, QED 0.43
Compound (SMILES): Cc1c(Cl)cccc1NC(=O)c1cc(NC(=O)c2ccccc2C(F)(F)F)cc2c1ncn2C
InChIKey: WAAZIOTZPUBZFP-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition Assay: mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 486.88, ALogP 6.06, HBA 4, HBD 2, PSA 76.02, QED 0.37