lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc([C@H](NC(=O)c2ccc3cnc(NC4CCOCC4)nc3c2)c2cnn(C)c2)cc1Cl
InChIKey: LZHGVCWCMKIINY-DEOSSOPVSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an N-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 1 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 506.99, ALogP 4.14, HBA 8, HBD 2, PSA 103.19, QED 0.39
Compound (SMILES): Cc1noc(C)c1S(=O)(=O)NCCN1CC2CN(CCCOc3ccc(C#N)cc3)CC(C1)O2
InChIKey: RCOLTNQLHZQCOG-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG expressed in CHO cells incubated for 10 mins after 3 hrs incubation with Rb+-load buffer by atomic absorption spectrometry based Rb+ efflux assay
Measurement: IC50 < 31622.78 nM (pChEMBL 4.5)
Descriptors: MW 489.6, ALogP 1.3, HBA 9, HBD 1, PSA 120.93, QED 0.49
Compound (SMILES): O=C(C1CCS(=O)(=O)CC1)N1CCN(c2ncc(F)cn2)CC1c1cccc(F)c1
InChIKey: DZMBGQZHTTYNNU-UHFFFAOYSA-N
Target: Sodium- and chloride-dependent glycine transporter 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48067
Assay: Cytostar-T Assay: In detail, SK-N-MC cells are seeded into 96-well Cytostar-T assay plates at a density of 200,000 cells/well and grown for 16-18 hours to confluence in growth medium as recommended by ATCC. Before starting the assay, cells are washed once with HBSS (Hank's buffered salt solution; Sigma, H8264) cont. 5 mM alanine (referred in here as HBSS/Ala) and afterwards the following reagents are added: 1.80 ul/well HBSS/A1a 2. 20 ul/well of HBSS/A1a containing 6x the concentration of compound in 6% DMSO 3. approx. 5-10 min incubation 4. 20 ul/well 3 uM glycine (3H-glycine (Perkin Elmer, NET004001MC, specific activity: 52 Ci/mmol; diluted 1:1 with unlabelled glycine) in HBSS/A1a. In the final assay, glycine concentration is 500 nM (250 nM derived from the 3H-glycine Perkin Elmer, 250 nM unlabelled glycine), DMSO concentration is 1%. The assay plate is immediately after addition of the 3H-glycine placed into a Micro-Beta Counter (Perkin Elmer) and the signal is recorded over 60 min.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 436.48, ALogP 1.97, HBA 6, HBD 0, PSA 83.47, QED 0.73
Compound (SMILES): CN(C)CCOc1ccc(/C(=C(/CCCO)c2cccnc2)c2ccc(Br)cc2)cc1.Cl
InChIKey: UUDDNYNDVOQDSG-OQKDUQJOSA-N
Target: Estrogen-related receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P62508
Assay: Inhibition of fluorescien-conjugated coactivator PGC1a binding to GST-tagged human ERRgamma LBD after 1 hr by TR-FRET assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 481.43, ALogP 5.52, HBA 4, HBD 1, PSA 45.59, QED 0.41
Compound (SMILES): O=C1c2cnc3[nH]ccc3c2N([C@H]2CC[C@H](CNCC(F)(F)F)CC2)CN1CC1CC1
InChIKey: PHECSRDWGGZDIV-KOMQPUFPSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 435.49, ALogP 3.9, HBA 4, HBD 2, PSA 64.26, QED 0.72
Compound (SMILES): O=C(NCCCN1CCN(C(=O)OCc2cc(Cl)cc(Cl)c2)CC1)c1c[nH]nn1
InChIKey: CDQQOQKRIAQNSQ-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Inhibition Assay: ATX activity was determined by measurement of released choline in reactions containing ATX (10 nM), choline oxidase (0.1 U/ml), HRP (100 U/ml), amplex red (50 μM) and LPC 18:1 (10 μM). Compounds of the invention were prepared as 10 point serial dilutions from 1 μM in duplicate and pre-incubated with ATX at 37° C. for 20 minutes prior to the addition of remaining reagents. The liberated choline was measured from changes in fluorescence intensity (λex 530 nm, λem 590 nm) of the product resurofin at 37° C. every 2 minutes over a 40-minute period. ATX activity was measured as a slope of the linear portion of the progress curve, typically between 14 to 24 minutes.
Measurement: IC50 = 282 nM (pChEMBL 6.550000190734863)
Descriptors: MW 441.32, ALogP 2.19, HBA 6, HBD 2, PSA 103.45, QED 0.64
Compound (SMILES): Brc1cnc(NCC(c2ccccc2)c2ccccc2)nc1Nc1cc(C2CC2)[nH]n1
InChIKey: XJSSNBCOWSWUMM-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) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 475.39, ALogP 5.83, HBA 5, HBD 3, PSA 78.52, QED 0.3
Compound (SMILES): C[C@@]1(c2cc(Nc3nc4ccc(Cl)cc4o3)ccc2F)C[C@@H](C(F)(F)F)OC(N)=N1
InChIKey: UPLXDXPJVCXTNE-YJBOKZPZSA-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 > 40000 nM (pChEMBL 4.3979997634887695)
Descriptors: MW 442.8, ALogP 5.25, HBA 6, HBD 2, PSA 85.67, QED 0.54
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2c2ccncc2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: UNSUYJXQOBXBNF-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 1659.59 nM (pChEMBL 5.78000020980835)
Descriptors: MW 492.58, ALogP 5.38, HBA 8, HBD 0, PSA 78.19, QED 0.29
Compound (SMILES): N#Cc1ccc(C2C3=C(CCC3=O)N(c3cccc(C(F)(F)F)c3)C(=O)N2CC(=O)NCC2CCOCC2)cc1
InChIKey: ZMXKTFBBLHSKDJ-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available. The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL of these compound dilutions were mixed with 10 ul Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 = 9.7 nM (pChEMBL 8.010000228881836)
Descriptors: MW 552.55, ALogP 4.72, HBA 5, HBD 1, PSA 102.74, QED 0.56
Compound (SMILES): CCC(C(=O)Nc1ccc(-c2nnc(C(F)(F)C(F)(F)C(=O)O)[nH]2)cc1)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: RBCDTSHNKBJBHR-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 632.96, ALogP 5.24, HBA 8, HBD 3, PSA 162.99, QED 0.2
Compound (SMILES): CN1CCN(C(=O)CN2C(=O)N(c3cccc(C(F)(F)F)c3)C3=C(C(=O)CC3)C2c2ccc(C#N)cc2)CC1
InChIKey: ASRKFPCXPUJFLX-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available. The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 uL of these compound dilutions were mixed with 10 ul Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 uL substrate solution in assay buffer were added (250 uM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 537.54, ALogP 3.95, HBA 5, HBD 0, PSA 87.96, QED 0.59
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(C2CCOCC2)c(OCC2CC2)n1)C(=O)NC
InChIKey: QGNMTFHVDPFUHG-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
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 = 186.2 nM (pChEMBL 6.730000019073486)
Descriptors: MW 403.52, ALogP 2.8, HBA 5, HBD 2, PSA 89.55, QED 0.66
Compound (SMILES): O=C(CC1CC1)N1CCC(Oc2ccc(NC(=O)N3Cc4ccncc4C3)cc2)CC1
InChIKey: SKKBHNUQSAPEFR-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 = 19.2 nM (pChEMBL 7.71999979019165)
Descriptors: MW 420.51, ALogP 3.8, HBA 4, HBD 1, PSA 74.77, QED 0.8
Compound (SMILES): CN(C)c1ncc(S(=O)(=O)N[C@H]2CCN([C@H](Cc3cc4ccnc(N)c4cc3Cl)C(=O)O)C2=O)cc1Cl
InChIKey: KLFSLDKLJLSVFR-RBUKOAKNSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (4.9 nM to 5 µM final) and deposited in a proportion of 25 µl per well. After the deposition of 50 µl of Spectrozyme 229 substrate (American Diagnostica), at the final concentration of 625 µM, the reaction is triggered by the addition of 25 µl of FIXa (human factor IXa supplied by Enzyme Research Laboratories (ERL)), at the final concentration of 2.5 U/ml. Reading is carried out at 405 nm for 15 min at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 567.46, ALogP 2.16, HBA 8, HBD 3, PSA 158.82, QED 0.37
Compound (SMILES): O=C1CCC2N1CCCN2C(=O)c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1
InChIKey: BKRMBLUWRKKSDZ-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 12200 nM (pChEMBL 4.909999847412109)
Descriptors: MW 456.42, ALogP 4.24, HBA 5, HBD 0, PSA 75.63, QED 0.59
Compound (SMILES): COc1ccc(Nc2nc(-c3sc(C)nc3C)cs2)nc1
InChIKey: ZPYHZOHERCFPAF-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Antagonist activity at human recombinant TRPV4 expressed in CHO-K1 cells assessed as blockade of 1000 nM 4alphaPDD-induced calcium mobilization by FLIPR assay
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 318.43, ALogP 4.03, HBA 7, HBD 1, PSA 59.93, QED 0.79
Compound (SMILES): Nc1ccc(S(=O)(=O)N(Cc2ccc(F)c(C(F)(F)F)c2)c2sc3ccccc3c2Br)cc1
InChIKey: GGTHLFHSVCAQPO-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 559.4, ALogP 6.8, HBA 4, HBD 1, PSA 63.4, QED 0.22
Compound (SMILES): O=C(NCC1CC1)c1ccc(NC(=O)N2Cc3ccncc3C2)cc1
InChIKey: YPUUEPDIVJYYTQ-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 = 44.5 nM (pChEMBL 7.349999904632568)
Descriptors: MW 336.4, ALogP 2.77, HBA 3, HBD 2, PSA 74.33, QED 0.9
Compound (SMILES): CN1C2COCC1CC(OC(=O)c1cn(CC(F)(F)F)c3ncccc13)C2
InChIKey: KRGNCEFMJYXXEC-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: In Vitro Inhibition of Ca Flux Assay: The 5HT3 antagonist activity of the compounds of the invention was determined by measuring the ability of the compounds to inhibit the calcium flux activity of 3HT3a receptor expressed in HEK-293T cells. HEK-293T cells were transfected with the 5HT3a expression construct using Xtreme Gene 9 (Roche) in 150 mm tissue culture treated plates and incubated for 24 hours at 37 °C. Cells were then split and plated at a density of 60K cells/well in poly-lysine coated, black 96-well plates with clear bottoms (BD BioSciences) and incubated overnight at 37 °C. Growth media was removed and cells loaded with 200 uL calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 5 Assay kit, Molecular Devices) and incubated at 37 °C. for 1 hour. While cells were incubating, the 10× antagonist and agonist/antagonist addition plates were made. For 10× antagonist plate: half log serial dilutions (final concentrations range from 10−7 through 10−10 with the bottom well a negative, no ligand control) were made from test compounds in DMSO at a 1000× concentration and then diluted to 10× in HBSS/20 mM HEPES. For addition plate: 5HT was diluted to 100× in HBSS/20 mM HEPES (final concentration in the assay−216 nM) and 15 uL was added to each well of the addition plate, 15 uL of 10× compound was also added to the addition plate, and finally 120 uL of HBSS/20 mM HEPES (for a total of 150 uL). Cells were then removed from the incubator and equilibrated to room temperature for 10 minutes, then 22.5 uL of 10× test compounds were added in triplicate to the plates and incubated at room temperature for 10 minutes (Tropisetron was used as a positive control in every assay). Test plate and addition plate were loaded into the FlexStation III (Molecular Devices), and using the fluidics, 22.5 uL compound additions were made (at t=~17 seconds), and fluorescence was measured for 90 seconds, reading every 2.2 seconds.
Measurement: IC50 = 1.82 nM (pChEMBL 8.739999771118164)
Descriptors: MW 383.37, ALogP 2.62, HBA 6, HBD 0, PSA 56.59, QED 0.76
Compound (SMILES): O=C(OC(c1ccccc1)C1CNC1)c1ccccc1
InChIKey: IIYIEZQVFYKYOZ-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Binding Assay: 5-HT transporter binding assay for evaluating binding of the compound to the serotonin transporter was carried out using human recombinant serotonin transporter membrane (PerkinElmer Life and Analytical Sciences, USA) expressed in HEK293 cells and radioisotope [3H]Imipramine (PerkinElmer).That is, the test drug, 2 nM [3H]Imipramine, serotonin transporter membrane (9 ug/well), 120 mM NaCl and 50 mM Tris-HCl buffer (pH 7.4) containing 5 mM KCl were added to obtain a reaction mixture with a final volume of 0.25 ml. After incubation for 30 minutes at 27° C., the mixture was quickly passed through a Filtermat A glass fiber filter (PerkinElmer) pre-soaked with 0.5% (w/v) PEI (polyethyleneimine) using Inotech Harvester (Inotech) to terminate the reaction. After washing with cold washing buffer (50 mM Tris-HCl, pH 7.4, 154 mM NaCl) solution, the filter was covered with MeltiLex and sealed in a sample bag. After drying in an oven, radioactivity was counted using MicroBeta Plus (Wallac).
Measurement: IC50 = 402 nM (pChEMBL 6.400000095367432)
Descriptors: MW 267.33, ALogP 2.8, HBA 3, HBD 1, PSA 38.33, QED 0.87
Compound (SMILES): CCCn1cc(-c2cc3c(-c4cccc(N5CCNCC5)n4)n[nH]c3cn2)cn1
InChIKey: XKPHITFYRRNWMQ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 0.039 nM (pChEMBL 10.40999984741211)
Descriptors: MW 388.48, ALogP 2.7, HBA 7, HBD 2, PSA 87.55, QED 0.55
Compound (SMILES): O=C(O)CNC(=O)C1=C(O)CCN(Cc2ccc(Oc3ccccc3F)nc2)C1=O
InChIKey: XUPHUYVPEDVZQC-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Fluorescence Polarization Assay: The enzyme and the substrate were each diluted with a 50 mM tris-hydrochloric acid buffer (pH 7.5) containing 12.5 mM KCl, 3.75 mM MgCl2, 25 μM iron sulfate, 5 mM ascorbic acid, and 2.5 mM DTT, whereas each test compound was diluted with dimethyl sulfoxide (DMSO).A test compound and the substrate solution were preliminarily added onto 384-well plates and reaction was initiated by adding a human PHD2 enzyme solution (40 ng/well). After 20-min incubation at 30° C., an EDTA-containing quench solution was added and the amount of the proline residues hydroxylated via binding to an added HIF-OH antibody solution was quantified by fluorescence polarization.
Measurement: IC50 = 108 nM (pChEMBL 6.96999979019165)
Descriptors: MW 415.38, ALogP 1.76, HBA 6, HBD 3, PSA 129.06, QED 0.59
Compound (SMILES): Cc1c(Cc2ccccn2)nnc(N2CCN(c3cnc(C(C)(C)O)cn3)[C@H](C)C2)c1C
InChIKey: GQWJKCWJQXXCSQ-MRXNPFEDSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 433.56, ALogP 2.81, HBA 8, HBD 1, PSA 91.16, QED 0.66
Compound (SMILES): CC(C)(C)OC(=O)N1CC(CNc2cc(=O)[nH]c3ccc(C(c4ccc(Cl)cc4)c4nccs4)cc23)C1
InChIKey: MNIPANZRLRIDDG-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 2330 nM (pChEMBL 5.630000114440918)
Descriptors: MW 537.09, ALogP 6.1, HBA 6, HBD 2, PSA 87.32, QED 0.31
Compound (SMILES): CC(C)(C)Nc1c(-c2cccc(C#N)c2)nc2ccc(Cl)cn12
InChIKey: AAOJDMVYFBSKFT-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: Dose Response Assay: Harvested cells with 0.025% trypsin/EDTA. Resuspended cells in growth media without selection antibiotics. Measured cell density and diluted to 2.4×10^5 cells/ml in media containing 1 μg/ml Doxycycline Plate 25 μl/well into 384 well black/clear tissue culture-treated plates. Incubated overnight at 37° C. Growth media was removed from the cell plates (20 μl) and 20 μl of the Replacement Buffer was added followed by addition of 25 μl of diluted dye. All three steps were performed using a Plate Washer BioTek 407. The plates were then incubated for 30' at RT. After incubation, both the cell and compound plates were brought to the FLIPR and 20 μl of the diluted compounds/antagonist/bk were transferred to the cell plates by the FLIPR. Plates were then incubated for 30' at room temperature. After 30' incubation, plates were returned to the FLIPR and 20 ul of 4.5× Cinnamaldehyde was added to the cell plates. During the compound addition as well as agonist addition, fluorescence readings were taken simultaneously from all 384 wells of the cell plate every 1.5 seconds. Five readings were taken to establish a stable baseline, then 20 μl of sample was rapidly (30 μl/sec) and simultaneously added to each well of the cell plate. The fluorescence was continuously monitored before, during and after sample/agonist addition for a total elapsed time of 100 seconds (compound addition) and 120 seconds (agonist addition).
Measurement: IC50 = 2133 nM (pChEMBL 5.670000076293945)
Descriptors: MW 324.82, ALogP 4.74, HBA 4, HBD 1, PSA 53.12, QED 0.74
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1ccc(C(F)(F)F)cn1)c1c(F)cccc1-n1nccn1
InChIKey: BQVBWAOFAJBNQP-GJZGRUSLSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 434.4, ALogP 3.58, HBA 6, HBD 2, PSA 84.73, QED 0.6
Compound (SMILES): N#Cc1cccc(-c2cnc3nnn(C[C@@H]4CN(c5ncc(-c6cnn(C7CCNCC7)c6)cn5)CCO4)c3n2)c1
InChIKey: OBUVRORYNUKALY-DEOSSOPVSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Lanthanide Fluoro-Immunoassay: The c-Met kinase inhibitory activity was analyzed using dissociation enhanced lanthanide fluoro-immunoassay (DELFIA, Perkin Elmer), which is a kind of time-resolved fluorescence (TRF). 10 mL of the compound prepared in the present invention, as a test compound, was put in a Greiner 96-well V-shaped bottom plate, and a tyrosin kinase buffer (20 mL) mixed with a c-Met enzyme was added thereto, and then the enzyme and the test compound were stirred for 15 minutes, followed by culturing. An ATP solution (10 mL) was added thereto to proceed with a kinase reaction at room temperature for 30 minutes, and then a 50 mM ethylene diamine acetic acid solution (EDTA, 40 mL) was added to stop the reaction. After that, the reaction mixture was transferred to a plate coated with Streptavidin, followed by culturing under shaking. After 2 hours, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). The europium-labeled anti-phosphotyrosine antibody was diluted to 1:2,500, and 100 mL of the diluent was added per well, followed by culturing under shaking. After 1 hour, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). 100 mL of an enhancement solution was added, followed by shaking culturing for 5 minutes, and then the reaction material was read out within a wavelength range of 615/665 nm using a Wallac Envision 2103 device.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 548.62, ALogP 2.24, HBA 13, HBD 1, PSA 148.38, QED 0.33
Compound (SMILES): Cc1cccc(C2=N[C@H](NC(=O)[C@H](CCC(F)(F)F)[C@H](CCC(F)(F)F)C(N)=O)C(=O)Nc3c(Cl)cccc32)c1
InChIKey: AHQDKFUMSUHJRJ-WJONJSRFSA-N
Target: Neurogenic locus notch homolog protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P46531
Assay: Biological Assay: The pharmacological properties of the compounds of this invention may be confirmed by a number of biological assays.
Measurement: IC50 = 4.8 nM (pChEMBL 8.319999694824219)
Descriptors: MW 590.95, ALogP 5.28, HBA 4, HBD 3, PSA 113.65, QED 0.35
Compound (SMILES): CC[C@]1(C(=O)O)CC[C@H](C(=O)N2CC[C@](c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: NMDBMKMFARAOPP-ATANMQQVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 171 nM (pChEMBL 6.769999980926514)
Descriptors: MW 655.6, ALogP 6.69, HBA 4, HBD 1, PSA 91.75, QED 0.27
Compound (SMILES): Cn1cc(-c2cnc3nnn(C[C@@H]4CN(c5ncc(-c6ccc(OCCN7CCOCC7)c(F)c6)cn5)CCO4)c3n2)cn1
InChIKey: OOJTWYQBPMINTM-QHCPKHFHSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Lanthanide Fluoro-Immunoassay: The c-Met kinase inhibitory activity was analyzed using dissociation enhanced lanthanide fluoro-immunoassay (DELFIA, Perkin Elmer), which is a kind of time-resolved fluorescence (TRF). 10 mL of the compound prepared in the present invention, as a test compound, was put in a Greiner 96-well V-shaped bottom plate, and a tyrosin kinase buffer (20 mL) mixed with a c-Met enzyme was added thereto, and then the enzyme and the test compound were stirred for 15 minutes, followed by culturing. An ATP solution (10 mL) was added thereto to proceed with a kinase reaction at room temperature for 30 minutes, and then a 50 mM ethylene diamine acetic acid solution (EDTA, 40 mL) was added to stop the reaction. After that, the reaction mixture was transferred to a plate coated with Streptavidin, followed by culturing under shaking. After 2 hours, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). The europium-labeled anti-phosphotyrosine antibody was diluted to 1:2,500, and 100 mL of the diluent was added per well, followed by culturing under shaking. After 1 hour, the cultured material was washed three times with a PBS-T buffer (PBS 0.05% Tween 20). 100 mL of an enhancement solution was added, followed by shaking culturing for 5 minutes, and then the reaction material was read out within a wavelength range of 615/665 nm using a Wallac Envision 2103 device.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 601.65, ALogP 1.83, HBA 14, HBD 0, PSA 134.26, QED 0.24
Compound (SMILES): COc1cc(-c2cccc(F)c2)c(C)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: PFBPTAQNIZCENY-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 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 505.53, ALogP 4.9, HBA 7, HBD 1, PSA 103.43, QED 0.35
Compound (SMILES): Cc1c(N(Cc2cccc(OC(F)(F)F)c2)S(=O)(=O)C2CC2)sc2ccccc12
InChIKey: MQTDIWLXYDAHEM-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 7.2 nM (pChEMBL 8.140000343322754)
Descriptors: MW 441.5, ALogP 5.61, HBA 4, HBD 0, PSA 46.61, QED 0.5
Compound (SMILES): O=C1CCC(=O)N1c1cc(Cc2n[nH]c(=O)c3c2CCCC3)ccc1F
InChIKey: TZKWXSZXKCDRLK-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 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 355.37, ALogP 2.03, HBA 4, HBD 1, PSA 83.13, QED 0.85
Compound (SMILES): CCCNC(=O)c1ccc(NC(=O)N2Cc3ccc(CO)cc3C2)cc1
InChIKey: AHZLOPJUFWUDAM-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1: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-1251667.0. 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 = 197 nM (pChEMBL 6.710000038146973)
Descriptors: MW 353.42, ALogP 2.87, HBA 3, HBD 3, PSA 81.67, QED 0.77
Compound (SMILES): COc1ccc(-c2cn3c(c2C(N)=O)CN(C(=O)NC(C)(C)C)CC3)cc1
InChIKey: BKRNZNIZTXRBAP-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 < 1 nM (pChEMBL 9)
Descriptors: MW 370.45, ALogP 2.59, HBA 4, HBD 2, PSA 89.59, QED 0.87
Compound (SMILES): O=S(=O)(Nc1nccs1)c1ccc2c(c1)OCCN2c1ncc(Cl)cc1Cl
InChIKey: XJJAFLCLPIFLGE-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 397 nM (pChEMBL 6.400000095367432)
Descriptors: MW 443.34, ALogP 4.18, HBA 7, HBD 1, PSA 84.42, QED 0.65