lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCCCn1c(NC(=O)c2cccs2)c(C#N)c2nc3ccccc3nc21
InChIKey: PZHBPVKRZHDXME-UHFFFAOYSA-N
Target: Dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WIS7
Assay: Inhibition Assay: Mtb PDH (Lpd+DlaT+AceE) is provided by Dr. Bryk Ruslana. The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing PDH (Lpd=15 μM; DlaT=30 μM; AceE=15 μM) was diluted 2-fold in 100 mM of potassium phosphate buffer (pH 7.0) for the assay. Another reaction solution containing 50 mM potassium phosphate buffer (pH 7.0), 200 μM TPP, 2 mM Pyruvate, 200 μM CoA, 1 mM MgCl2, 1 mM NAD+ was prepared. 5 μL/well of PDH solution was dispensed into 1536-well black plates. Next, 50 nL of each test compound (1 mM dissolved in DMSO) was added into each well. After 30 minutes of incubation, 5 μL/well of reaction solution was added. At the 30th minutes of the reaction, the florescence signal (excitation 360 nm and emission 460 nm) was obtained by Viewlux reader (PerkinElmer).
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 375.46, ALogP 4.57, HBA 6, HBD 1, PSA 83.6, QED 0.55
Compound (SMILES): CN1C(=N)N[C@](C)(c2sc(-c3nnc(-c4ccccc4)o3)cc2Cl)CS1(=O)=O
InChIKey: ZKKCLIHWNQRYQN-KRWDZBQOSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 μs delay on a RUBYstar HTRF plate reader (BMG Labtechnologies).
Measurement: Ki = 7.762 nM (pChEMBL 8.109999656677246)
Descriptors: MW 437.93, ALogP 3.13, HBA 7, HBD 2, PSA 112.18, QED 0.65
Compound (SMILES): CCN(CC)CCNC(=O)c1ccc(Nc2ncc(C)c(Nc3cc(C4CC4)n[nH]3)n2)cc1
InChIKey: BQKTYXIEHCEMMR-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 448.58, ALogP 3.94, HBA 7, HBD 4, PSA 110.86, QED 0.35
Compound (SMILES): Cc1cc2ccc1[C@@H](C)COC(=O)Nc1ccc(C3(c4nnn[nH]4)CC3)c(c1)CN(C)C(=O)[C@@H]2Nc1ccc2c(N)ncc(F)c2c1
InChIKey: FHJDRBBZAGYRKH-ADXZGYQBSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Inhibition of factor 10a (unknown origin) at 25 degC by chromogenic substrate assay
Measurement: Ki = 1050 nM (pChEMBL 5.980000019073486)
Descriptors: MW 635.7, ALogP 5.33, HBA 9, HBD 4, PSA 164.04, QED 0.2
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1ccncc1
InChIKey: ZTJXMMXXYIGJGZ-UHFFFAOYSA-N
Target: Histone deacetylase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O15379
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 1404 nM (pChEMBL 5.849999904632568)
Descriptors: MW 326.4, ALogP 2.59, HBA 4, HBD 3, PSA 97.11, QED 0.51
Compound (SMILES): Cc1ccc2nc(N3CC=CC3)nc(NCc3ccc(NC(=O)C4CCN(Cc5ccccc5)CC4)cc3)c2c1
InChIKey: WNJZFQIJHPAMLL-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf22C11 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 761 nM (pChEMBL 6.119999885559082)
Descriptors: MW 532.69, ALogP 5.78, HBA 6, HBD 2, PSA 73.39, QED 0.28
Compound (SMILES): N[C@H](C(=O)O)C(NS(=O)(=O)c1cccc2ccccc12)C(=O)O
InChIKey: QTKQWWVIXJJZGN-PXYINDEMSA-N
Target: Excitatory amino acid transporter 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P43005
Assay: Inhibition of [3H]-D-Asp uptake at human EAAT3 expressed in HEK293 cells measured after 3 mins by TopCount method
Measurement: IC50 = 63000 nM (pChEMBL 4.199999809265137)
Descriptors: MW 338.34, ALogP -0.02, HBA 5, HBD 4, PSA 146.79, QED 0.58
Compound (SMILES): CCCCCCCC#CC1(C(F)(F)F)CC(c2ccc(C)cc2)=C(c2cn[nH]n2)C(=O)N1
InChIKey: AKHWHZCWDGAKGK-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in CORNING Falcon 96-well Polypropylene plates, in a total volume of 60 uL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 uM 2-oleoylglycerol, 15 uM oleoyl-Coenzyme A and 0.0013 ug/uL Human or Mouse MGAT-2 or 0.0026 ug/uL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 uL of ice cold methanol containing 1 ug/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50x2.1 mm column, was used for the chromatography of enzyme products.
Measurement: IC50 = 826 nM (pChEMBL 6.079999923706055)
Descriptors: MW 444.5, ALogP 5.21, HBA 3, HBD 2, PSA 70.67, QED 0.46
Compound (SMILES): COCCOCCOCCOCCOCCOCCOCCOCCOCc1cn(CCCC[C@H](NC(=O)N[C@@H](CCC(=O)O)C(=O)O)C(=O)O)nn1
InChIKey: JPAWLDOHKLYPRL-NSOVKSMOSA-N
Target: Diacylglycerol O-acyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75907
Assay: PMSA Inhibition Assay: For IC50 measurements, inhibitors were dissolved in Reaction Buffer containing 40 μM NAAG to a final volume of 100 μL. Then, 25 μL of this solution was transferred to each of three wells in a microtiter plate, and 5 μL aliquots were serially diluted into 20 μL of solution containing 40 μM NAAG over 10 wells (5-fold dilutions). Inhibitor concentration therefore ranged over 6 orders of magnitude in these experiments. rhPSMA (20 ng/mL in Reaction Buffer, 20 μL, R&D Research) was then added to each well. The plate was incubated at room temperature for 15 min, and then heated to 95 °C. for 3 minutes. The plate was allowed to cool to room temperature, and glutamic acid levels were measured using a commercially available Amplex® − Red Glutamic Acid/Glutamate Oxidase Assay Kit (Invitrogen). Fluorescence intensities were measured using a Synergy 2 multiwell plate reader (Biotek), fitted with excitation and emission filters of 545 nm and 590 nm, respectively. Ki values were obtained from IC50 values using the Cheng-Prusoff equation, a substrate concentration of 20 μM, and a Km value of 0.925 μM.
Measurement: IC50 = 131 nM (pChEMBL 6.880000114440918)
Descriptors: MW 767.83, ALogP -0.2, HBA 16, HBD 5, PSA 266.81, QED 0.05
Compound (SMILES): C/C(=C\c1ccccc1C(F)(F)F)c1ccnc2[nH]c3ccc(-c4ccc(N5CCN(C)CC5)cc4)cc3c12
InChIKey: HTDVWRWILSZWNP-XUTLUUPISA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 6400 nM (pChEMBL 5.190000057220459)
Descriptors: MW 526.61, ALogP 7.71, HBA 3, HBD 1, PSA 35.16, QED 0.26
Compound (SMILES): O=C(O)c1ccc(-c2c(C#Cc3ccc(C(F)(F)F)cn3)nc3c(N4CCOCC4)ccnn23)cc1
InChIKey: PIJYIBOPYRLKTG-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 493.45, ALogP 3.74, HBA 7, HBD 1, PSA 92.85, QED 0.43
Compound (SMILES): CC1(C)Cc2ccccc2N2CCNCC21
InChIKey: ZRWIQBUHNPOOGK-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2B (SINGLE PROTEIN), Homo sapiens
UniProt: P41595
Assay: Agonist activity at 5-HT2B receptor (unknown origin) expressed in HEK293 cells assessed as [3H]inositol phosphate accumulation after 2 hrs by scintillation counting
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 216.33, ALogP 2.05, HBA 2, HBD 1, PSA 15.27, QED 0.71
Compound (SMILES): COc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4cc(C)c5[nH]ncc5c4)n3)c3ccccc23)cc1
InChIKey: GISQWMVPDCAGDF-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 653.75, ALogP 8.49, HBA 9, HBD 4, PSA 143.9, QED 0.13
Compound (SMILES): O=C(c1ccccc1-n1nccn1)N1CCOC[C@H]1Cc1cccc(-c2nccs2)c1
InChIKey: FYEWZQAALXXWSM-LJQANCHMSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: OX 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 ug/mL G418, 100 U/mL penicillin, 100 ug/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.
Measurement: IC50 = 457 nM (pChEMBL 6.340000152587891)
Descriptors: MW 431.52, ALogP 3.47, HBA 7, HBD 0, PSA 73.14, QED 0.48
Compound (SMILES): CO[C@H]1CC[C@@H](C(=O)Nc2ccc(C3CNCCO3)cc2)CC1
InChIKey: RDXXYXXWUBKOSU-JGHJKYEYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 725.5 nM (pChEMBL 6.139999866485596)
Descriptors: MW 318.42, ALogP 2.49, HBA 4, HBD 2, PSA 59.59, QED 0.9
Compound (SMILES): CS(=O)(=O)NC(=O)c1ccc(-c2c(/C=C/c3ccc4ccccc4n3)nc3c(N4CCOCC4)ccnn23)cc1
InChIKey: UWZHXTUUEVMRGJ-OUKQBFOZSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 554.63, ALogP 3.64, HBA 9, HBD 1, PSA 118.79, QED 0.34
Compound (SMILES): O=C(O)CCN1C(=O)/C(=C/c2ccccc2)SC1=S
InChIKey: MGSYHFSSOOVWOY-NTMALXAHSA-N
Target: CDGSH iron-sulfur domain-containing protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZ45
Assay: Displacement of [3H]rosiglitazone from recombinant human C-terminal His-tagged MitoNEET cytosolic domain (32 to 108 residues) expressed in Escherichia coli BL21 by Cheng-Prusoff analysis
Measurement: Ki = 412 nM (pChEMBL 6.380000114440918)
Descriptors: MW 293.37, ALogP 2.36, HBA 4, HBD 1, PSA 57.61, QED 0.68
Compound (SMILES): COC(=O)CCC/C=C\C[C@@H]1[C@@H](c2ccc(C(C)(C)C)cc2)[C@H](O)C[C@H]1Cl
InChIKey: JVJSZVIWJDEHTQ-CQJJLBMOSA-N
Target: Prostaglandin E2 receptor EP3 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43115
Assay: Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 392.97, ALogP 5.35, HBA 3, HBD 1, PSA 46.53, QED 0.29
Compound (SMILES): CCCN(Cc1ccc(OCCN2C(=O)CCC2=O)c(OC)c1)C(CC(=O)O)c1ccc(Cl)cc1
InChIKey: DWUJCNUMUUIVJW-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Radioligand Binding Assay: The composition of the binding assay buffer is determined in a course of detailed optimization procedure. This resulted in a binding assay buffer constituted by the following components: 25 mM Hepes (pH=7.4), 5 mM MgCl2, 1 mM CaCl2, 100 mM NaCl, supplemented with 0.1% of protease free BSA (as a final concentration). Competition binding assay is performed using 125I-CXCL10 (PerkinElmer, NEX348, specific activity 2200 Ci/mmol) radioligand in a final concentration of 50-70 pM. The nonspecific binding is defined by 150 pM of hr-CXCL10 (R&D Systems, Cat No 266-IP). The total assay volume is equal to 150 ul and contained 1% of DMSO (final concentration). Binding reaction is initiated by adding of membranes (10-20 ug proteins, approximately 5x105 cell equivalents) to the reaction mixture. After 60 minutes of incubation at 25° C. the reaction is terminated by rapid filtration over GF/B glass fibre filters that are pre-soaked with 0.5% polyethyleneimine (Fluka Analytical, P3143) for 1 hour, using a Skatron cell harvester device. Filters then are washed with 8 ml of ice-cold wash buffer (modified binding buffer in which BSA is omitted and the concentration of NaCl is adjusted to 500 mM concentration). The radioactivity retained on the filters is measured by a Wizard 1470 Automatic Gamma counter.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 503, ALogP 4.3, HBA 6, HBD 1, PSA 96.38, QED 0.41
Compound (SMILES): O=C(COc1ccccc1-c1ncc[nH]1)NCC(O)CN1CCc2ccccc2C1
InChIKey: DVNRPGHNGLFEEC-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 406.49, ALogP 1.99, HBA 5, HBD 3, PSA 90.48, QED 0.53
Compound (SMILES): Cc1cn(-c2ccc3n(c2=O)CCN(CC2(C)CCC(c4ccc(C(F)(F)F)cc4)O2)C3=O)cn1
InChIKey: QDUVMXKXDXAPQY-UHFFFAOYSA-N
Target: Presenilin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P49768
Assay: Cell-Based Assay: The ability of compounds to modulate production of amyloid beta protein Abeta(1-42) was determined using human WT-APP overexpressing CHO cells. Cells were plated at 22,000 cells/100 uL well in 96 well tissue culture treated, clear plates (Falcon) in DMEM/F12 based medium and incubated for 24 hours at 37° C. Compounds for testing were diluted in 100% DMSO to achieve an eleven points, half log, dose response for IC50 determinations. Compounds were added in fresh medium to achieve 1% final DMSO. Appropriate vehicle or inhibitor controls were added into control wells individually to obtain minimum or maximum inhibition values, respectively, for the assay signal window before the plates were incubated for 24 hours at 37° C. This procedure produces conditioned media in each well which is tested for Abeta(1-42) levels in the ELISA detection step described next. The remaining cell cultures in each well are also tested for cell toxicity as described below.
Measurement: IC50 = 1040 nM (pChEMBL 5.980000019073486)
Descriptors: MW 486.49, ALogP 4.13, HBA 6, HBD 0, PSA 69.36, QED 0.56
Compound (SMILES): COc1cc(C(=O)c2ccccc2)cc(O)c1-c1cccc(C)c1
InChIKey: XGIFDISIJIJHKW-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: CB2 Receptor Binding Protocol: 15.3 ug of membranes (20 ul of 1:20 dilution in incubation buffer) was incubated with 5 ul of drug solution (10E-4M to 10E-12M) and 5ul of 5.4 nM [3H]CP55,940 in a total volume of 200 ul for 90 mins at 30 C. Non-specific binding was determined using 10 uM WIN55,212-2 (Ki=4.4 nM). The membranes were filtered and the filters washed 7x with 0.2 ml ice-cold incubation buffer and allowed to air dry under vacuum.
Measurement: Ki = 224.98 nM (pChEMBL 6.650000095367432)
Descriptors: MW 318.37, ALogP 4.61, HBA 3, HBD 1, PSA 46.53, QED 0.71
Compound (SMILES): C/C(=C\c1cc(F)c(Oc2ccc(S(=O)(=O)NCCOCCOCCOCCNC(=O)CN(CCN(CC(=O)O)CC(=O)NCCOCCOCCOCCNS(=O)(=O)c3ccc(Oc4c(F)cc(/C=C(\C)C(=O)N=C(N)N)cc4F)cc3)CC(=O)O)cc2)c(F)c1)C(=O)N=C(N)N
InChIKey: FGTVAAJSIAMUST-NXRKLTDKSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human 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 = 3.981 nM (pChEMBL 8.399999618530273)
Descriptors: MW 1427.47
Compound (SMILES): CCN1CCC(CCOc2ccc(-c3nc(C#N)nc4c3ccn4CCO)cc2C(F)(F)F)CC1
InChIKey: MDNJDINYGFVFHJ-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Mus musculus
UniProt: O70370
Assay: Inhibition Assay: First, spleen cells collected from C57BL/6 mice were seeded in a 96-well plate so as to be a 1× 105 cells/well, then, 5-fold dilution of 10 mM DMSO solution of the test compound was serially performed with RPMI 1640 medium (including 10% fetal bovine serum (FCS), 5×10^−5 M 2-mercaptoethanol, 50 IU/mL penicillin and 50 μg/mL streptomycin) nine times such that the final concentration becomes 0.026 nM to 10 μM, and the resultant products were added thereto (the final DMSO concentration was 0.1%). At the same time, LPS (Sigma L4005) was added to the wells such that the final concentration becomes 2 μg/mL, and culture was performed at 37° C. for 48 hours in 5% CO2. After culturing, the cells were stained at 4° C. for 20 minutes with PE-labeled streptavidin (BD BIOSCIENCE 554061) and biotin-labeled YAe antibody (EBIOSCIENCE 13-5741-85) together with FITC-labeled anti-mouse B220 antibody (BD BIOSCIENCE 553088), and an expression level (fluorescence intensity of YAe-bioti
Measurement: IC50 = 0.91 nM (pChEMBL 9.039999961853027)
Descriptors: MW 487.53, ALogP 4.48, HBA 7, HBD 1, PSA 87.2, QED 0.5
Compound (SMILES): O=C(CCc1cc2c(cc1CN1CCC[C@H]1c1nc(C(=O)NCCCCC3CCCCC3)co1)OCO2)NS(=O)(=O)C(F)(F)F
InChIKey: OYIWYIIDTHEYIY-DEOSSOPVSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Cell Based Assay: Ca2+ signaling studies were performed using a FLIPR TETRA system (Molecular Devices, Sunnyvale, Calif., USA) in the 384-format. This is a high-throughput instrument for cell-based assays to monitor Ca2+ signaling associated with GPCRs and ion channels. Cells were seeded at a density of 5×104 cells/well in BioCoat poly-D-lysine coated, black wall, clear bottom 384-well plates (BD Biosciences, Franklin lakes, NJ, USA) and allowed to attach overnight in an incubator at 37° C. The cells were then washed twice with HBSS-HEPES buffer (Hanks' balanced salt solution without bicarbonate and phenol red, 20 mM HEPES, pH 7.4) using an ELx405 Select CW Microplate Washer (BioTek, Winooski, Vt., USA). After 60 min of dye-loading in the dark using the Ca2+-sensitive dye Fluo-4AM (Invitrogen, Carlsbad, Calif., USA), at a final concentration of 2×10^−6M, the plates were washed 4 times with HBSS-HEPES buffer to remove excess dye and leaving 50 μl of buffer in each well. The plates were then placed in the FLIPR TETRA instrument and allowed to equilibrate at 37° C. AGN-211377 was added in a 25 μl volume to each well to give final concentrations of 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 30 μM; or 0.067 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.67 μM, and 1 μM for cells over-expressing TP receptors. After 4.5 minutes, a 7-point serial dilution of the standard agonist for the corresponding receptor, in a 25 μl volume was injected at the final concentrations from 10^−11M to 10^−5M in 10-fold serial dilution increments for cells expressing human recombinant DP1, EP1, EP2, EP3, EP4, FP, and IP receptors. The dose range for the standard agonist for human recombinant TP receptors was from 10^−12M to 10^−6M. HBSS-HEPES buffer was used as the negative control for the standard agonists. Cells were excited with LED (light emitting diode) excitation at 470-495 nm and emission was measured through an emission filter at 515-575 nm. Assay plates were read for 3.5 minutes using the FLIPRTETRA.
Measurement: EC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 656.72, ALogP 5.12, HBA 9, HBD 2, PSA 140.07, QED 0.28
Compound (SMILES): CCCCN(CCCC)C(=O)c1cc(C)n(-c2ccc(N[S+]([O-])c3cc(Cl)ccc3F)cc2C(=O)N2Cc3ccccc3C[C@H]2CO)n1
InChIKey: CHBZCZLKVLQQQK-AOPCHTBDSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Surface plasmon resonance (SPR): Methods for Determining IC50s.The present method includes utility of a Surface plasmon resonance (SPR)- based biosensor (Biacore GE Healthcare, Uppsala, Sweden) to characterize BCL-2 inhibitors.Biacore utilizes the phenomenon of surface plasmon resonance (SPR) to detect and measure binding interactions. In a typical Biacore experiment, one of the interacting molecules (ligand) is immobilized on a flexible dextran matrix while the interacting partner (analyte) is allowed to flow across that surface. A binding interaction results in an increase in mass on the sensor surface and a corresponding direct change in the refractive index of the medium in the vicinity of the sensor surface. Changes in refractive index or signal are recorded in resonance units (R.U.) Signal changes due to association and dissociation of complexes are monitored in a non-invasive manner, continuously and in real-time, the results of which are reported in the form of a sensorgram.
Measurement: IC50 = 849.56 nM (pChEMBL 6.070000171661377)
Descriptors: MW 694.27, ALogP 6.71, HBA 7, HBD 2, PSA 113.76, QED 0.15
Compound (SMILES): CCc1cc(-c2ccnc3[nH]cc(C#N)c23)ccc1CN1CCOCC1
InChIKey: PHGSTYLIFXUHNG-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Homogeneous Time Resolved-fluorescence Resonance Energy Transfer (TR-FRET) Assay: LRRK2 kinase activity is measured using a LanthaScreen kinase activity assay available from Invitrogen (Life Technologies Corporation). The assay is a homogeneous time resolved-fluorescence resonance energy transfer (TR-FRET) assay that measures phosphorylation of a fluorescein-labelled peptide substrate (fluorescein-LRRKtide, fluorescein-GAGRLGRDKYKTLRQIRQ) (SEQ ID NO 1) as a result of LRRK2 kinase activity. The phosphorylated peptide is recognized by a terbium-labelled phospho-specific anti-LRRKtide antibody and, subsequently, the phosphorylated LRRKtide can be quantified by the extent of TR-FRET between the terbium donor and fluorescein acceptor.The LRRK2 kinase is obtained from Invitrogen (Life Technologies Corporation) and comprises residues 970 to 2527 of the full length human wild-type LRRK2 kinase or a similar sequence with the G2019S mutation. As discussed above, this mutation increases the kinase activity relative to the wild-type. The kinase reactions are performed in a 20 uL volume in 384-well plates. The kinase reaction buffer consists of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT.Â  In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase is incubated with the test compound (typically at 0 to 30 uM) for 30 minutes before the kinase reaction is initiated by addition of 1.3 mM ATP and 0.4 uM fluorescein-LRRKtide. The reaction mixture (20 ul total volume) is incubated for 2 hours at 30° C. before the reaction is terminated by addition of 10 mM EDTA and 1 nM terbium-labelled anti-phospho-LRRKtide antibody (final volume 20 ul). The mixture is further incubated for 30 minutes at RT. TR-FRET is measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 us) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 us. The measurement is repeated 10 times for fluorescein and 10 times for terbium emission with a 2000 us time window between repeats. TR-FRET measurements are performed on a Biomek Synergy plate. The TR-FRET signal is calculated as the emission-ratio at 520 nm over 495 nm.
Measurement: IC50 = 149 nM (pChEMBL 6.829999923706055)
Descriptors: MW 346.43, ALogP 3.5, HBA 4, HBD 1, PSA 64.94, QED 0.79
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(C2CC2)c(OCC2CC2)n1)C(=O)NCCOC
InChIKey: DSJZBTQJBGKLNQ-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 403.52, ALogP 2.8, HBA 5, HBD 2, PSA 89.55, QED 0.52
Compound (SMILES): O=C(Nc1ccc(C(=O)C2CCNC2)cc1)c1ccc(Cl)cc1
InChIKey: OJDNZFIZSBZKHT-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 328.8, ALogP 3.38, HBA 3, HBD 2, PSA 58.2, QED 0.85
Compound (SMILES): CC1(C)CN(c2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(Cc5ccccc5-c5ccc(Cl)cc5)CC4)cc3Oc3ccccc3)cc2)C1=O
InChIKey: VWIURBKQCUIQRJ-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 735.31, ALogP 7.61, HBA 7, HBD 1, PSA 99.26, QED 0.15
Compound (SMILES): NC(=O)c1cc(N2CCOCC2C(N)=O)nc(-c2ccc(Oc3ccc(F)cc3)cc2)n1
InChIKey: RAXSCIQUOWLHGV-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: FLIPR Assay: Assay Buffer: The assay buffer was formulated by removing 120 mL from a 1 L bottle of fresh, sterile dH2O (Mediatech, Herndon, Va.) and adding 100 mL of 10×HBSS that does not contain Ca++ or Mg++ (Gibco, Invitrogen, Grand Island, N.Y.) followed by 20 mL of 1.0 M Hepes, pH 7.3 (Fisher Scientific, BP299-100). The final buffer consisted of 20 mM Hepes, pH 7.3, 1.261 mM CaCl2, 0.493 mM MgCl2, 0.407 mM Mg(SO)4, 5.33 mM KCl, 0.441 mM KH2PO4, 137 mM NaCl, 0.336 mM Na2HPO4 and 0.556 mM D-glucose (Hanks et al., Proc. Soc. Exp. Biol. Med. 71:196 (1949)), and the simple formulation was typically the basic buffer throughout the assay (i.e., all wash and addition steps).
Measurement: IC50 = 360 nM (pChEMBL 6.440000057220459)
Descriptors: MW 437.43, ALogP 1.86, HBA 7, HBD 2, PSA 133.66, QED 0.6
Compound (SMILES): N#Cc1cnc2[nH]cc(C(=O)c3c(F)ccc(NS(=O)(=O)c4cccnc4)c3F)c2c1
InChIKey: VJGBPYCHRVNHQC-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf V600E mutant (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 439.4, ALogP 3.14, HBA 6, HBD 2, PSA 128.6, QED 0.46
Compound (SMILES): FC(F)(F)c1ccc2nc(C3C4Oc5ccc(Oc6ccnc(-c7nc(C(F)(F)F)c[nH]7)c6)cc5C43)[nH]c2c1
InChIKey: GGPZUHATGVDJQP-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 543.43, ALogP 6.82, HBA 5, HBD 2, PSA 88.71, QED 0.24
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCN(Cc2cccc(Cl)c2)CC1
InChIKey: KWOPWTXIXUJBFZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF 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 uL of 1.11x enzyme and 1.11x substrate in 1x 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 uL of 10xATP in 1x 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 uL of 2x 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.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 467.94, ALogP 3.82, HBA 7, HBD 2, PSA 112.86, QED 0.51
Compound (SMILES): COc1cccc(N2CCOc3cc(S(=O)(=O)Nc4nccs4)ccc32)c1
InChIKey: GAOVCEUYHFSWJK-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 2070 nM (pChEMBL 5.679999828338623)
Descriptors: MW 403.49, ALogP 3.48, HBA 7, HBD 1, PSA 80.76, QED 0.7
Compound (SMILES): Cc1ccccc1S(=O)(=O)N1CCC(Nc2ncc(C(=O)NO)cn2)(c2ccccc2)CC1
InChIKey: QPBLZOUYURAUJO-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: HDAC Enzyme Assay: The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 uM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 uM (HDAC1), 10 uM (HDAC2), 17 uM (HDAC3) and 14 uM (HDAC6). Five ul of compounds and 20 ul of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five u1 of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated.
Measurement: IC50 = 423 nM (pChEMBL 6.369999885559082)
Descriptors: MW 467.55, ALogP 2.7, HBA 7, HBD 3, PSA 124.52, QED 0.38
Compound (SMILES): Cn1cc2c(Nc3nc(N[C@@H]4CCCC[C@@H]4N)nnc3C(N)=O)cccc2n1
InChIKey: GZYSRRCMPCBHEP-SMDDNHRTSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Inhibition of human SYK using 5-Fluo-Ahx-GAPDYENLQELNKK-Amid as substrate in presence of ATP by microfluidic mobility shift assay
Measurement: IC50 = 0.8 nM (pChEMBL 9.100000381469727)
Descriptors: MW 381.44, ALogP 1.28, HBA 9, HBD 4, PSA 149.66, QED 0.52
Compound (SMILES): C=CC(=O)Nc1ccccc1Nc1cc(N(Cc2ccccc2)C(=O)Nc2c(Cl)c(OC)cc(OC)c2Cl)ncn1
InChIKey: XPADWPKAVAOLCN-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Kinase HotSpotâ¿¢ Assay: Compounds were profiled for FGFR inhibition activity at Reaction Biology Corporation (Malvern, Pa.) with their Kinase HotSpot assay. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.Recombinant FGFR1 (2.5 nM), FGFR2 (1 nM), FGFR3 (5 nM), or FGFR4 (12 nM) (Invitrogen) was prepared as a mixture with substrate KKKSPGEYVNIEFG (SEQ ID NO:1) (20 uM, FGFR1 substrate); and Poly [E,Y]4:1 (0.2 mg/ml, FGFR2,3,4 substrate)] in kinase reaction buffer (20 mM HEPES-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.1 mM Na3VO4, 0.02 mg/ml BSA, 2 mM DTT, and 1% DMSO). Compound was added to the enzyme/substrate mixture using acoustic technology (Labcyte Echo 550, Sunnyvale, Calif.) (see, Olechno et al., 2006, Improving IC50 results with acoustic droplet ejection. JALA 11, 240-246) and pre-incubated for 0, 15, or 60 minutes at room temperature. After compound pre-incubation, a mixture of ATP (Sigma-Aldrich) and 33P-γ-ATP (PerkinElmer) was added to a final concentration of 10 uM to initiate kinase reactions. Reactions were incubated for 120 minutes at room temperature and then spotted onto Whatman P81 ion exchange filter paper. Unbound phosphate was removed by extensively washing filters in 0.75% phosphoric acid. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 593.47, ALogP 6.91, HBA 7, HBD 3, PSA 117.71, QED 0.17