lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1sc2c(c1CC(=O)Nc1nc(-c3ccc(Cl)c(Cl)c3)cs1)c(=O)n(C)c(=O)n2C
InChIKey: QSBZGESBJYBOBP-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 = 33.61 nM (pChEMBL 7.46999979019165)
Descriptors: MW 495.41, ALogP 4.22, HBA 8, HBD 1, PSA 85.99, QED 0.46
Compound (SMILES): CC(C)Nc1nnc(-c2ccc3[nH]cc(-c4cccc(C(=O)N(C)C)n4)c3c2)o1
InChIKey: MTDOMZCSLNXWQL-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 496 nM (pChEMBL 6.300000190734863)
Descriptors: MW 390.45, ALogP 3.8, HBA 6, HBD 2, PSA 99.94, QED 0.54
Compound (SMILES): CC(C)Oc1ccc(/N=c2\[nH]c(=O)n(Cc3ccccc3C(=O)O)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: DVUTVMVNMJUMHZ-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37 C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 538.96, ALogP 3.95, HBA 7, HBD 2, PSA 118.68, QED 0.35
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2CCN(c3cc(F)c(F)c(OCC(=O)O)c3)CC2)cn1
InChIKey: NSQBOWALYQMKIM-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Antagonist activity at PGD2 receptor in human platelets assessed as inhibition of PGD2-mediated cAMP accumulation preincubated for 5 mins followed by PGD2 addition measured after 2 mins by HTRF assay
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 471.48, ALogP 2.12, HBA 7, HBD 1, PSA 109.27, QED 0.62
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4cnc(OCC5(F)CCOCC5)c(F)c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: NVAGDJMXUPSQQO-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 uM (2x starting concentration; 10% DMSO) and 10 uL were transferred into a 384-well plate. Then 10 uL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. Protein:GST-Bcl-2, Probe: F-Bak Peptide Probe Acetyl-GQVGRQLAIIGDK(6-FAM)INR-amide(SEQ ID NO: 1), Protein(nM): 1, Probe (nM): 100, Antibody: Tb-anit-GST, Antibody (nm): 1.  The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 0.029 nM (pChEMBL 10.539999961853027)
Descriptors: MW 861.41, ALogP 8.34, HBA 10, HBD 2, PSA 138.98, QED 0.13
Compound (SMILES): CCN(Cc1ccc(OCCN2C(=O)CCC2=O)c(C)c1)C(CC(=O)O)c1ccc(Cl)cc1
InChIKey: ODRQJWBNWQXXBT-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 424 nM (pChEMBL 6.369999885559082)
Descriptors: MW 472.97, ALogP 4.21, HBA 5, HBD 1, PSA 87.15, QED 0.49
Compound (SMILES): Cc1nc2cc(C(=O)N3CCN(C(=O)c4cc(Cc5n[nH]c(=O)c6c5NCCC6)ccc4F)CC3)ccc2[nH]1
InChIKey: GHUMXEOTCZEDMV-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 529.58, ALogP 2.64, HBA 6, HBD 3, PSA 127.08, QED 0.37
Compound (SMILES): O=C(OC1CNC1)[C@@H]1CC[C@@H]2CN1C(=O)N2OS(=O)(=O)O
InChIKey: SZCUDVBJZPPYDP-SVRRBLITSA-N
Target: Beta-lactamase (SINGLE PROTEIN), Pseudomonas aeruginosa
UniProt: Q8RL90
Assay: Spectrophotometric Assay: Enzyme activities were measured in the presence of the test inhibitor in spectrophotometric assay.
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 321.31, ALogP -1.5, HBA 7, HBD 2, PSA 125.48, QED 0.47
Compound (SMILES): CC(C)(C(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(C(N)=O)c1)C2)CC1CCC1)S(C)(=O)=O.O=C(O)C(F)(F)F
InChIKey: NRIPQSUIOFHGEC-JCNHLJMTSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Cavia porcellus
UniProt: P41144
Assay: Displacement of [3H]-diprenorphine from guinea pig kappa opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 489.68, ALogP 2.95, HBA 5, HBD 1, PSA 100.78, QED 0.57
Compound (SMILES): C[C@@H]1/C=C\C(=O)[C@@H](O)[C@@H](O)C/C=C/c2cc(NCC3CCNCC3)cc(O)c2C(=O)O[C@H]1C
InChIKey: VIMRATAOFMWODG-RJUOWCSDSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: Inhibition of MEK1 (unknown origin) using ERK2 as substrate after 40 mins by ELISA
Measurement: IC50 = 356 nM (pChEMBL 6.449999809265137)
Descriptors: MW 458.56, ALogP 2.25, HBA 8, HBD 5, PSA 128.12, QED 0.44
Compound (SMILES): O=C(O)c1ccc2[nH]c(-c3ccc(-c4ccc(Cl)cc4)cc3)nc2c1
InChIKey: LEXFBVDJGJMLED-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk2 (SINGLE PROTEIN), Homo sapiens
UniProt: O96017
Assay: Inhibition of recombinant human CDS1 using N-terminal biotin-labeled SGLYRSPSMPENLNRPR as substrate in presence of [gamma-33P]-ATP after 3 hrs by scintillation counting method
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 348.79, ALogP 5.25, HBA 2, HBD 2, PSA 65.98, QED 0.53
Compound (SMILES): O=C(NCCCCC1CCN(C(=O)C2CCS(=O)(=O)C2)CC1)N1Cc2ccncc2C1
InChIKey: ATQSSCJFPDWMIN-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 18.4 nM (pChEMBL 7.739999771118164)
Descriptors: MW 448.59, ALogP 1.95, HBA 5, HBD 1, PSA 99.68, QED 0.67
Compound (SMILES): CCC#CC(=O)Nc1cc(-c2cnc3[nH]cc(Cl)c3c2)ccc1F
InChIKey: CNKBQTIZCNURMG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 341.77, ALogP 4.37, HBA 2, HBD 2, PSA 57.78, QED 0.68
Compound (SMILES): Cc1cccc(C)c1N1C[C@@](CN(C)C(C)C)(C(=O)Nc2cc(C(F)(F)F)cc(C(F)(F)F)c2)[C@H](C)C1=O
InChIKey: QCGNFCVZGFVZGC-IQGLISFBSA-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 <= 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 543.55, ALogP 6.29, HBA 3, HBD 1, PSA 52.65, QED 0.44
Compound (SMILES): O=C(Nc1ccc(C2CNCCO2)cc1)c1ccc(C(F)(F)F)nc1
InChIKey: UVEZVCKZYWIVDM-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse 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 = 21.4 nM (pChEMBL 7.670000076293945)
Descriptors: MW 351.33, ALogP 3.01, HBA 4, HBD 2, PSA 63.25, QED 0.89
Compound (SMILES): COc1ncc(-c2cc(NC(=O)c3cccc(C(F)(F)F)c3)ccc2C)cc1N1CCOCC1
InChIKey: BHICALKRKYZBTH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 1.07 nM (pChEMBL 8.970000267028809)
Descriptors: MW 471.48, ALogP 5.17, HBA 5, HBD 1, PSA 63.69, QED 0.56
Compound (SMILES): CCN(C(=O)Oc1ccc(F)cc1)[C@@H]1CN(C(=O)C2CCN(C(=O)c3ccc(F)cn3)CC2)C[C@H]1c1ccc(Cl)c(F)c1
InChIKey: TXSPZUXEGDDBOU-RBJSKKJNSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: [3H]SR142801 Competition Binding Assay: hNK3 receptor binding experiment were performed using [3H]SR142801 (Catalog No. TRK1035, specific activity: 74.0 Ci/mmol, Amersham, GE Healthcare UK limited, Buckinghamshire, UK) and membrane isolated from HEK293 cells transiently expressing recombinant human NK3 receptor. After thawing, the membrane homogenates were centrifuged at 48,000×g for 10 mM at 4° C., the pellets were resuspended in the 50 mM Tris-HCl, 4 mM MnCl2, 1 μM phosphoramidon, 0.1% BSA binding buffer at pH 7.4 to a final assay concentration of 5 μg protein/well. For inhibition experiments, membranes were incubated with [3H]SR142801 at a concentration equal to KD value of radioligand and 10 concentrations of the inhibitory compound (0.0003-10 μM) (in a total reaction volume of 500 μl) for 75 min at room temperature (RT). At the end of the incubation, membranes were filtered onto unitfilter (96-well white microplate with bonded GF/C filter preincubated 1 h in 0.3% PEI+0.3% BSA, Packard BioScience, Meriden, Conn.) with a Filtermate 196 harvester (Packard BioScience) and washed 4 times with ice-cold 50 mM Tris-HCl, pH 7.4 buffer. Nonspecific binding was measured in the presence of 10 μM SB222200 for both radioligands. The radioactivity on the filter was counted (5 min) on a Packard Top-count microplate scintillation counter with quenching correction after addition of 45 μl of microscint 40 (Canberra Packard S.A., Zürich, Switzerland) and shaking for 1 h.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 615.05, ALogP 5.52, HBA 5, HBD 0, PSA 83.05, QED 0.37
Compound (SMILES): CCCCOC(=O)N1CCc2c(F)ccc(-c3cc(CC(=O)O)ccc3OCC)c2C1
InChIKey: MELGDABUWKVEBC-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 ul. First, 25 ul 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 ul Binding-Buffer, 50 ul 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 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/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 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 429.49, ALogP 4.81, HBA 4, HBD 1, PSA 76.07, QED 0.61
Compound (SMILES): CCCN(CC1CC1)c1cc(C(=O)Nc2ccc3c(cnn3CC(=O)O)c2)ncn1
InChIKey: KQHOYQIJZWBWKF-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: 35S-GTPgammaS Binding: Measurements of 35S-GTPgammaS Binding: Membranes (1 to 10 ug protein) prepared as described above, were incubated in 96-well Scintiplates (PerkinElmer) with test compounds diluted in DMSO, in 180 ul of 20 mM HEPES, pH 7.4, 10 mM MgCl2, 2 ug/well Saponin, 0.2% fatty acid free BSA (Assay buffer), 140 mM NaCl and 1.7 uM GDP. The assay was initiated with the addition of 20 ul of 1.5 nM [35S]-GTPgammaS (1100 Ci/mmol; GE Healthcare) in assay buffer. After 60 min incubation at 30° C. on a shaker, plates were centrifuged for 10 min at 2000 RPM. Supernatant was discarded and membrane bound radioactivity was measured on a PerkinElmer 1450 MicroBeta counter. Triplicate samples were averaged and expressed as % response relative to S1P activation in absence of compound (n=2).
Measurement: EC50 = 3265 nM (pChEMBL 5.489999771118164)
Descriptors: MW 408.46, ALogP 2.79, HBA 7, HBD 2, PSA 113.24, QED 0.56
Compound (SMILES): NC(=O)c1c(-c2ccccc2)c(C2CC2)n2c1CN(C(=O)NCC13CC4CC(CC(C4)C1)C3)CC2
InChIKey: VLDWWEORHAWBEQ-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: In Vitro ATP-33P Casein Assay: "U"-bottomed Falcon plates are prepared by placing 5 uL of solutions of the compounds according to the invention at concentrations of 10, 1, 0.1, 0.01 or 0.001 uM in different wells. The solutions of the compounds according to the invention at these various concentrations are prepared by diluting in a test buffer (Tris 50 mM pH 7.5, MgCl2 10 M, DTT 2 mM and EGTA 1 mM) a stock solution in DMSO at a concentration of 10 mM. Next, 5 uL of dephosphorylated casein are added to a final concentration of 0.2 ug/uL, 20 uL of CK1 epsilon to a final concentration of 3 ng/uL, and 20 uL of ATP-33P to a final concentration of 0.02 uCi/uL mixed with cold ATP (10 uM final approximately 2x106 CPM per well). The final total test volume per well is equal to 50 uL. The "U"-bottomed Falcon test plate mentioned above is vortexed, and then incubated at room temperature for 2 hours. After 2 hours the reaction is stopped by adding an ice-cold solution of 65 uL of cold ATP (2 mM) prepared in test buffer. 100 uL of the reaction mixture are then transferred from the "U"-bottomed Falcon plate into Millipore MAPH filter plates, preimpregnated with 25 uL of ice-cold 100% TCA. The Millipore MAPH filter plates are agitated gently and are left to stand at room temperature for at least 30 minutes to precipitate the proteins. After 30 minutes, the filter plates are sequentially washed and filtered with 2x150 uL of 20% TCA, 2x150 uL of 10% TCA and 2x150 uL of 5% TCA (6 washes in total per plate/900 uL per well). The plates are left to dry overnight at room temperature. Next, 40 uL of Microscint-20 Packard scintillation liquid are added per well and the plates are closed in a leaktight manner. The radiation emitted by each well is then measured for 2 minutes in a TopCount NXT Packard scintillation counter, in which the values of CPM/well are measured.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 472.63, ALogP 4.87, HBA 3, HBD 2, PSA 80.36, QED 0.65
Compound (SMILES): O=C(O)CCC/C=C\C[C@H]1C(=O)C[C@@H](O)[C@@H]1c1ccc2c(c1)CCC2O
InChIKey: KARPJTLGVFQYJV-MMJXDPMESA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Displacement of [3H]PGE2 from from human EP2 receptor expressed in HEK293 cells at pH 7.3 membranes incubated for 60 mins
Measurement: IC50 = 2281 nM (pChEMBL 5.639999866485596)
Descriptors: MW 358.43, ALogP 2.9, HBA 4, HBD 3, PSA 94.83, QED 0.51
Compound (SMILES): O=C1CCc2c(Oc3ccc4c(c3)C3C(O4)C3c3nc4cc(Cl)c(C(F)(F)F)cc4[nH]3)ccnc2N1
InChIKey: CBEGWCPFZLRDSX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
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 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 512.88, ALogP 5.95, HBA 5, HBD 2, PSA 89.13, QED 0.35
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(N(C(=O)c2ccc(-c3cnco3)cc2F)C2CC2)CC1
InChIKey: JZEACRPAEFOIBH-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 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 429.49, ALogP 4.48, HBA 5, HBD 0, PSA 75.88, QED 0.72
Compound (SMILES): Fc1ccc2c(c1Cc1ccccc1)CCC1CNCCN21
InChIKey: SYDFROBMNFAPBV-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2C (SINGLE PROTEIN), Homo sapiens
UniProt: P28335
Assay: Agonist activity at unedited 5-HT2C receptor (unknown origin) expressed in HEK293 cells assessed as [3H]inositol phosphate accumulation after 2 hrs by scintillation counting
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 296.39, ALogP 3.14, HBA 2, HBD 1, PSA 15.27, QED 0.92
Compound (SMILES): CS(=O)(=O)NCCNc1nonc1/C(=N/O)Nc1cccc(C(F)(F)F)c1
InChIKey: VFEHBKRICPOHDX-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: Human indoleamine 2,3-dioxygenasae (IDO) with an N-terminal His tag was expressed in E. coli and purified to homogeneity. IDO catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N′-formylkynurenine. The assays were performed at room temperature as described in the literature using 95 nM IDO and 2 mM D-Trp in the presence of 20 mM ascorbate, 5 μM methylene blue and 0.2 mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5). The initial reaction rates were recorded by continuously following the absorbance increase at 321 nm due to the formation of N′-formlylkynurenine (See: Sono, M., et al., 1980, J. Biol. Chem. 255, 1339-1345).
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 408.36, ALogP 1.3, HBA 8, HBD 4, PSA 141.74, QED 0.18
Compound (SMILES): CNC(=O)c1cccc(Nc2ncnc(N3CCN(c4cccc(C)c4)CC3)n2)c1
InChIKey: HZQUVBGOTRETTJ-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Cell-Based Assay: Compounds were tested on human Nav1.7 (hNav1.7) expressed stably in 293 cells, which have no endogenous sodium channels. Two forms of whole-cell patch-clamp assays were used. In both, peak current through Nav1.7 was evoked by a depolarizing voltage pulse, and currents were monitored as a function of time in response to exposure to several different concentrations of drug. Automated electrophysiology uses the PatchXpress 7000A system. Up to sixteen cells at a time were voltage-clamped in parallel, and the steady-state voltage dependence of inactivation was determined experimentally for each cell. For each cell, fractional inactivation was set at 20%, and peak current through Nav1.7 as a function of time was measured in response to increasing concentrations of test compound. Manual electrophysiology refers to conventional whole-cell patch-clamping (Hamill et al., 1981). After formation of the whole-cell clamp, cells were lifted off the bottom of the dish with the patch pipette and positioned directly in front of an array of glass microperfusion tubes each with internal diameter about 250 microns, with individual compound concentrations flowing from each tube. Current as a function of time in response to increasing drug concentrations was measured with Nav1.7 fully in the resting state (holding voltage set to −140 mV) and on the same cells with the holding voltage set to produce about 20% inactivation. Compounds were not applied for a prespecified time, but for as long as required to achieve a stable level of block.
Measurement: IC50 = 1830 nM (pChEMBL 5.739999771118164)
Descriptors: MW 403.49, ALogP 2.61, HBA 7, HBD 2, PSA 86.28, QED 0.68
Compound (SMILES): CCCc1cc(-c2n[nH]cc2-c2ccc3c(c2)OCCO3)c(O)cc1O
InChIKey: OLPMQMKSKFCDRC-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Protein was prepared by dialysis for 24 hours against 1 liter of 30 mM Hepes pH 7.5, 150mM NaCl and 0.5mM TCEP. The final protein concentration was determined by Bradford Assay. The final protein concentration in the sample cell was 50 uM. Ligand stock solutions of 10 mM in DMSO were diluted to 5uM concentrations with ITC buffer and adjusted to 2% (v/v) DMSO. Both the titrate and titrant solutions were degassed prior to loading the calorimeter cell and injection syringe.The integrated heat data were fit with a one-site binding model using the Origin-7 software provided with the MicroCal VP-ITC. Protein concentration was corrected by titration of a reference compound and normalized for the concentration of each ligand. All ligands bound to N-HSP90 have a stoichiometry of approximately unity.
Measurement: Kd = 35.07 nM (pChEMBL 7.460000038146973)
Descriptors: MW 352.39, ALogP 3.88, HBA 5, HBD 3, PSA 87.6, QED 0.66
Compound (SMILES): COC1CCC2(CC1)C(C)(C)c1ccc(Br)cc1C21N=C(N)N(C)C1=O
InChIKey: XVHUQEBLDZZFQJ-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 22780 nM (pChEMBL 4.639999866485596)
Descriptors: MW 420.35, ALogP 3.3, HBA 4, HBD 1, PSA 67.92, QED 0.76
Compound (SMILES): NC1=N[C@](CF)(c2cc(Oc3ncccc3F)ccc2F)C[C@@H](C(F)(F)F)O1
InChIKey: PZOIBDWDOFHTCP-XJKSGUPXSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 398 nM (pChEMBL 6.400000095367432)
Descriptors: MW 405.3, ALogP 3.98, HBA 5, HBD 1, PSA 69.73, QED 0.78
Compound (SMILES): COc1cc(N2CCN3CCOC[C@@H]3C2)ccc1Nc1nccc(-c2c(-c3ccc(OC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: CRAVHEUWLSRUKN-SANMLTNESA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 718.76, ALogP 6.27, HBA 11, HBD 2, PSA 118.38, QED 0.18
Compound (SMILES): COCCOc1n[nH]c2cc(NC(=O)NC[C@H](C)c3ccccc3)ncc12
InChIKey: APNLHOVEQFBCKI-ZDUSSCGKSA-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 = 14.52 nM (pChEMBL 7.840000152587891)
Descriptors: MW 369.43, ALogP 2.91, HBA 5, HBD 3, PSA 101.16, QED 0.53
Compound (SMILES): C[C@@H]1CN(C(=O)C2(C#N)CC2)C[C@@H]1Nc1c(C(N)=O)cnn2cc(-c3cnn(C4CC4)c3)nc12
InChIKey: GIZRTWZXZFTZPD-DYVFJYSZSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Kinase Assay: The assay reactions were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (100 mM HEPES pH 7.4, 10 mM MgCl2, 25 mM beta-glycerol phosphate 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of GST-JAK3 enzyme with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA to each sample. Each reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product Inhibition data were calculated by comparison with no enzyme control reactions for 100% inhibition, and vehicle-only treated reactions for 0% inhibition. The final concentration of reagents in the assay was: ATP, 8 μM; fluoresceinated peptide, 1.5 μM; GST-JAK3, 4.5 nM; and DMSO, 1.6%.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 459.51, ALogP 1.59, HBA 9, HBD 2, PSA 147.23, QED 0.57
Compound (SMILES): Cc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4cc(C)c5[nH]ncc5c4)n3)c3ccccc23)cc1
InChIKey: ZCIMSJOHBOGKMX-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 = 263 nM (pChEMBL 6.579999923706055)
Descriptors: MW 637.75, ALogP 8.79, HBA 8, HBD 4, PSA 134.67, QED 0.14
Compound (SMILES): Cn1nc(N2CCCC2)nc1CCc1nc2ccc(Cl)cn2n1
InChIKey: JWWFKKRFLPBIRO-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Scintillation Proximity Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 μl contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 μl of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate solution (stop reagent). After 1 h under shaking, the plate was centrifuged one minute at 170 g to allow beads to settle. Afterwards, radioactive counts were measured on a Perkin Elmer Top-Count Scintillation plate reader.
Measurement: IC50 = 62.68 nM (pChEMBL 7.199999809265137)
Descriptors: MW 331.81, ALogP 1.9, HBA 7, HBD 0, PSA 64.14, QED 0.73
Compound (SMILES): COc1cc(-c2cc(F)cc(Cl)c2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccc(F)cn3)ccc21
InChIKey: COVNSVBFEZSMAP-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 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 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 571.96, ALogP 5.93, HBA 6, HBD 1, PSA 90.29, QED 0.27
Compound (SMILES): CN(C)C/C=C/C(=O)Nc1ccc2ncc(C#N)c(Nc3ccc(Oc4ccccc4)cc3)c2c1
InChIKey: ZGRDDSBWZLAOAF-RMKNXTFCSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase-Glo Plus Luminescence Kinase Assay: Method B conducts the kinase assay with the following condition and procedure: The assay was performed using Kinase-Glo Plus luminescence kinase assay kit (Promega). It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The compounds were diluted in 10% DMSO and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. All of the enzymatic reactions were conducted at 30° C. for 40 minutes. The 50 μl reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT, 0.2 mg/ml substrate peptide, 10 μM ATP and BTK. After the enzymatic reaction, 50 μl of Kinase-Glo Plus Luminescence kinase assay solution (Promega) was added to each reaction and incubate the plate for 5 minutes at room temperature. Luminescence signal was measured using a BioTek Synergy 2 microplate reader. Btk activity assays were performed in duplicate at each concentration.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 463.54, ALogP 5.7, HBA 6, HBD 2, PSA 90.28, QED 0.33
Compound (SMILES): c1cc(N2CCN(CCCOc3ccc4c(c3)CNCC4)CC2)c2ccsc2c1
InChIKey: FRUDQTZRVOVULE-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P61169
Assay: Dopamine D2 Receptor Binding Assay: The assay was performed according to the method by Kohler et al. (Kohler C, Hall H, Ogren S O and Gawell L, Specific in vitro and in vivo binding of 3H-raclopride. A potent substituted benzamide drug with high affinity for dopamine D-2 receptors in the rat brain. Biochem. Pharmacol., 1985; 34: 2251-2259). The binding assay was performed using 40 ul of the membrane specimen, 20 ul of [3H]-raclopride (final concentration 1 to 2 nM), 20 ul of a test drug and 50 mM Tris-hydrochloric acid buffer (containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) so that the total amount was 200 ul (final dimethylsulfoxide concentration 1%). The reaction was performed at room temperature for 1 hour and terminated by conducting suction filtration with a cell harvester on a glass fiber filter plate. The filter plate made of glass fiber was washed with 50 mM Tris-hydrochloric acid buffer (pH 7.4), and after dried, a microplate liquid scintillation cocktail was added.
Measurement: Ki = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 407.58, ALogP 4.14, HBA 5, HBD 1, PSA 27.74, QED 0.62
Compound (SMILES): CC(C)N1CCN(C(=O)OC2CCN(c3cnc(C(N)=O)cn3)CC2)CC1
InChIKey: YCVHLRCFFFNZGB-UHFFFAOYSA-N
Target: Histamine H3 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5N1
Assay: LANCE Ultra cAMP Assay: The potency of compounds of the invention as H3 receptor antagonists can be assessed by measuring the blockade of (R)-alpha-methylhistamine-mediated cAMP production utilizing a LANCE Ultra cAMP kit (PE #TRF0263) in CHO cells expressing human H3 receptors (GenBank: BC096840; Strausberg R L et al, Proc. Natl. Acad. Sci. U.S.A. 99(26), 16899-16903; 2002).Protocol:1. Preparation of the stimulation buffer (30 ml): 29.4 ml HBSS (GIBCO #14025), 150 ul 1 M HEPES (GIBCO #15630), 30 ul 500 mM IBMX (CALBIOCHEM #410957) and 400 ul 7.5% BSA (GIBCO #10438-026).2. Preparation of assay plate: Different concentrations of the compounds of the invention (0.01-1000 nM), H3 positive controls and cAMP calibration standards; 3 mM Forskolin (CALBIOCHEM #344270); 5 uM (R)-alpha-methylhistamine (H3 receptor agonist); 1% DMSO (SIGMA #D2650); total volume: 95 nl.3. Preparation of the cell solution: Collect cells with stimulation buffer, final density: 100,000 cells/ml.
Measurement: Ki = 3.8 nM (pChEMBL 8.420000076293945)
Descriptors: MW 376.46, ALogP 0.71, HBA 7, HBD 1, PSA 104.89, QED 0.82
Compound (SMILES): O=C(O)c1ccc(CN(CCCCc2ccccc2)C(=O)c2ccc(Oc3ccccc3F)cc2)cc1
InChIKey: BKRGCMOFALKWJU-UHFFFAOYSA-N
Target: Lysophosphatidic acid receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H1C0
Assay: Receptor Assay: Engagement of the LPA5 receptor by its ligand, oleoyl-L-Î±-lysophosphatidic acid (LPA), leads to the release of intracellular stores of calcium into the cytoplasm mediated through a Gq-driven increase in inositol triphosphate (IP3) levels. Gq is a heterotrimeric G protein subunit that activates phospholipase C.The ability of test compounds to prevent LPA-driven intracellular release of stored calcium from RH7777 cells expressing human LPA5 receptors was assayed by stimulating the cells with LPA in the presence of various test compounds to measure the test compounds' antagonism, i.e. activity in vitro. Approximately 10,000 cells in normal culture medium (Minimal Essential Media, 10% Foetal Calf Serum) were dispensed per assay well in a black clear bottom poly-D-lysine coated 384 well plate (Corning). Cells were allowed to settle for thirty minutes at room temperature before being incubated overnight at 37° C., 5% CO2. Sixteen to twenty hours after dispensing, the cells were loaded with a calcium-sensitive fluorescent dye by replacing the culture medium with assay buffer (1x Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH7.4) containing 1.25 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells were incubated at 37° C. for one hour to allow for dye uptake.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 497.57, ALogP 6.98, HBA 3, HBD 1, PSA 66.84, QED 0.23
Compound (SMILES): COC(=O)C(c1ccccc1)(c1ccc(SCc2ccccc2C(=O)O)cc1)c1ncco1
InChIKey: MRHVWDNZDAVGRG-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q07869
Assay: Reporter Gene Assay: Transactivation Assay Expression vectors were prepared by inserting the ligand binding domain cDNA (complementary DNA) of human PPARalpha (amino acid 168-468) and human PPARgamma (amino acid 205-505), 3′ to and in frame with, the yeast GAL4 transcription factor DNA binding domain and the nuclear localization signal from the T-antigen of Polyoma Virus into the mammalian expression vector pSGS (Stratagene). The resulting expression vectors pSGGAL-PPARalpha and pSGGAL-PPARgamma were used in co-transfection experiments together with a modified pGL3 promoter plasmid (Promega) containing five copies of the UAS GAL4 recognition site. 2.5 μg pSGGAL-PPARalpha or pSGGAL-PPARgamma were mixed with 25 μg pGL3p 1×UAS and 22.5 μg pBluescript (Stratagene) in 0.95 mL ice cold PBS containing between 9-12 million U-2 OS (human osteosarcoma) cells. The cell/DNA mixture was incubated on ice for 5 minutes and then divided between two 0.4 cm cuvettes and electroporated at 960 μF, 2
Measurement: EC50 = 13000 nM (pChEMBL 4.889999866485596)
Descriptors: MW 459.52, ALogP 5.17, HBA 6, HBD 1, PSA 89.63, QED 0.29
Compound (SMILES): O=C(c1ccc(OCC(F)(F)F)nc1)N1CCCC(c2ccccc2)C1
InChIKey: FDVBTJLRSSCFDC-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: 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 = 1597 nM (pChEMBL 5.800000190734863)
Descriptors: MW 364.37, ALogP 4.04, HBA 3, HBD 0, PSA 42.43, QED 0.82