lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccnc(-n2nnc3c2CCN(C(=O)c2cccc(C(F)(F)F)c2Cl)[C@H]3C)c1
InChIKey: YBKVGRQSYAWVKA-LBPRGKRZSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 = 9036.5 nM (pChEMBL 5.039999961853027)
Descriptors: MW 435.84, ALogP 4.4, HBA 5, HBD 0, PSA 63.91, QED 0.6
Compound (SMILES): CC(C)(C)C[C@@H]1N[C@@H](C(=O)NC2CCC(O)CC2)[C@H](c2cccc(Cl)c2)[C@]12C(=O)Nc1cc(Cl)ccc12
InChIKey: PSABHJAMVJLCQH-JVUXWEBQSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Fluorescence-polarization Binding Assay: The binding affinity of the MDM2 inhibitors was optionally determined using a fluorescence polarization-based (FP-based) binding assay using a recombinant human MDM2 protein (residues 5-109) and PMDM6-F as follows: MDM2 protein was serially diluted with a step of 1.8 in a Costar 96-well, black, non binding surface reference 3686 plate, and the PMDM6-F peptide was added at 5 nM concentration. The assay was performed in the buffer: 100 mM potassium phosphate, pH 7.5; 100 μg/mL bovine gamma globulin, 0.01% Triton X-100) and the anisotropy values were measured at equilibrium using a Fusion reader (Packard).
Measurement: IC50 = 289.62 nM (pChEMBL 6.539999961853027)
Descriptors: MW 544.52, ALogP 5.16, HBA 4, HBD 4, PSA 90.46, QED 0.43
Compound (SMILES): COc1ccc2[nH]c3c(c2c1)C[C@H]1c2cc4c(cc2CCN1C3)OCO4
InChIKey: HOTTYHVEFWTDND-IBGZPJMESA-N
Target: Alpha-1A adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35348
Assay: Antagonist activity at human full-length N-terminal SNAP-tagged alpha1A adrenoceptor expressed in HEK293 cells assessed as inhibition of agonist-induced calcium mobilization preincubated for 10 mins followed by agonist addition by fluo-4 AM dye-based assay
Measurement: IC50 = 12.8 nM (pChEMBL 7.889999866485596)
Descriptors: MW 348.4, ALogP 3.56, HBA 4, HBD 1, PSA 46.72, QED 0.73
Compound (SMILES): CC1(C)c2cc(N3CCC(N)CC3)c(Br)cc2C(=O)c2c1[nH]c1cc(C#N)ccc21
InChIKey: UBSJOCKVYNOYPO-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 0.168 nM (pChEMBL 9.779999732971191)
Descriptors: MW 463.38, ALogP 4.6, HBA 4, HBD 2, PSA 85.91, QED 0.55
Compound (SMILES): CS(=N)(=O)Cc1cccc(Nc2ncc(F)c(-c3ccc(F)cc3OCc3ccc(F)cc3)n2)c1
InChIKey: ZVYXVPZGGHYOJL-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: CDK9/CycT1 Kinase Assay: Recombinant full-length His-tagged human CDK9 and CycT1, expressed in insect cells and purified by Ni-NTA affinity chromatography, were purchased from Invitrogen (Cat. No PV4131). As substrate for the kinase reaction biotinylated peptide biotin-Ttds-YISPLKSPYKISEG (C-terminus in amid form) was used which can be purchased e.g. form the company JERINI Peptide Technologies (Berlin, Germany) For the assay 50 nl of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 ul of a solution of CDK9/CycT1 in aqueous assay buffer [50 mM Tris/HCl pH 8.0, 10 mM MgCl2, 1.0 mM dithiothreitol, 0.1 mM sodium ortho-vanadate, 0.01% (v/v) Nonidet-P40 (Sigma)] were added and the mixture was incubated for 15 min at 22 C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 498.53, ALogP 6.06, HBA 6, HBD 2, PSA 87.96, QED 0.31
Compound (SMILES): CN1C(=O)N(c2ccc(C#Cc3cnn(C)c3)cn2)CC1(C)C
InChIKey: FGFZTQXFKXNZHI-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Intracellular Ca2+ Mobilization Assay: A monoclonal HEK-293 cell line stably transfected with a cDNA encoding for the human mGlu5a receptor was generated; for the work with mGlu5 Positive Allosteric Modulators (PAMs), a cell line with low receptor expression levels and low constitutive receptor activity was selected to allow the differentiation of agonistic versus PAM activity. Cells were cultured according to standard protocols (Freshney, 2000) in Dulbecco's Modified Eagle Medium with high glucose supplemented with 1 mM glutamine, 10% (vol/vol) heat-inactivated bovine calf serum, Penicillin/Streptomycin, 50 μg/ml hygromycin and 15 μg/ml blasticidin (all cell culture reagents and antibiotics from Invitrogen, Basel, Switzerland). bout 24 hrs before an experiment, 5×104 cells/well were seeded in poly-D-lysine coated, black/clear-bottomed 96-well plates. The cells were loaded with 2.5 μM Fluo-4AM in loading buffer (1×HBSS, 20 mM HEPES) for 1 hr at 37 °C. and washed five times with loading buffer. The cells were transferred into a Functional Drug Screening System 7000 (Hamamatsu, Paris, France), and 11 half logarithmic serial dilutions of test compound at 37 °C. were added and the cells were incubated for 10-30 min. with on-line recording of fluorescence. Following this pre-incubation step, the agonist L-glutamate was added to the cells at a concentration corresponding to EC20 (typically around 80 μM) with on-line recording of fluorescence; in order to account for day-to-day variations in the responsiveness of cells, the EC20 of glutamate was determined immediately ahead of each experiment by recording of a full dose-response curve of glutamate. Responses were measured as peak increase in fluorescence minus basal (i.e. fluorescence without addition of L-glutamate), normalized to the maximal stimulatory effect obtained with saturating concentrations of L-glutamate.
Measurement: EC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 309.37, ALogP 1.87, HBA 4, HBD 0, PSA 54.26, QED 0.76
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1Cl)c1nc2cccc(Cl)c2c(=O)n1-c1cccnc1
InChIKey: BTUCPZBBCNQCSB-VIFPVBQESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 443.3, ALogP 3.22, HBA 9, HBD 3, PSA 137.63, QED 0.44
Compound (SMILES): Cc1cncc(-c2cc3c(cn2)cnn3-c2ccc(CO)c(N3CCC[C@H](N)C3)n2)n1
InChIKey: BZGCHCBKEVJNKB-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase Binding Assay: PIM-1, -2, and -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.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.06 nM (pChEMBL 10.220000267028809)
Descriptors: MW 416.49, ALogP 2, HBA 9, HBD 2, PSA 118.87, QED 0.52
Compound (SMILES): Cn1ccc2cc(N(C(=O)c3cc(-c4cc(OC(F)(F)F)ccc4C(=O)N4Cc5ccccc5C[C@H]4CN4CCOCC4)n4c3CCCC4)c3ccc(O)cc3)ccc21
InChIKey: ZMEFLWOEVJYYCG-DHUJRADRSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Fluorescence Polarisation Assay: The fluorescence polarisation tests were carried out on microplates (384 wells). The Bcl-2 protein, at a final concentration of 2.50×10−8 M, is mixed with a fluorescent peptide (Fluorescein-REIGAQLRRMADDLNAQY), at a final concentration of 1.00×10−8 M in a buffer solution (Hepes 10 mM, NaCl 150 mM, Tween20 0.05%, pH 7.4), in the presence or in the absence of increasing concentrations of test compounds. After incubation for 2 hours, the fluorescence polarisation is measured.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 803.88, ALogP 8.47, HBA 8, HBD 1, PSA 92.41, QED 0.17
Compound (SMILES): COCc1c(-c2cc3cc(C)cc(OC)c3s2)c2c(N)ncnn2c1CN1CCNCC1
InChIKey: KBUPLPBZRGPNCS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: High ATP Kinase Assay: For the assay, 50 nl of each stock solution of the test compound in DMSO was pipetted into a black, low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany) 2 Î¼l of a solution of the above FGFR-1 fusion protein in aqueous assay buffer [8 mM MOPS pH 7.0, 10 mM magnesium acetate, 1.0 mM dithiothreitol, 0.05% (w/v) bovine serum albumin (BSA), 0.07% (v/v) Tween-20, 0.2 mM EDTA] was added, and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme. Then, the kinase reaction was started by the addition of 3 ul of a solution of adenosine triphosphate (ATP, 3.3 mM; final concentration in the 5 ul assay volume=2 mM) and substrate (0.16 uM; final concentration in the 5 ul assay volume=0.1 uM) in assay buffer, and the resulting mixture was incubated for a reaction time of 15 min at 22° C. The concentration of FGFR-1 fusion protein was adjusted depending on the activity of the enzyme lot.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 452.58, ALogP 3.06, HBA 9, HBD 2, PSA 89.94, QED 0.46
Compound (SMILES): O=C1NCC2(CCCCC2)n2c1cc1cnc(Nc3ccc(N4CCC(N5CCC(C(F)(F)F)CC5)CC4)cn3)nc12
InChIKey: JKTDPJGOPWGJLD-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 11600 nM (pChEMBL 4.940000057220459)
Descriptors: MW 582.68, ALogP 5.22, HBA 8, HBD 2, PSA 91.21, QED 0.44
Compound (SMILES): CNC(=O)c1cc(F)cc(Nc2ncnc(N3CCC(c4nc(-c5ccccc5)cs4)CC3)n2)c1
InChIKey: OQQDCZCPECONEK-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 = 1560 nM (pChEMBL 5.809999942779541)
Descriptors: MW 489.58, ALogP 4.62, HBA 8, HBD 2, PSA 95.93, QED 0.41
Compound (SMILES): CN1CC(N2CC(C(=O)N[C@H]3CC[C@@H](c4ccccc4)CC3)=C(O)C2=O)CC1=O
InChIKey: SFUCWHPQMUEFBE-SJPCQFCGSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Enzymatic Assay: Endothelial lipase activity was measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 285 uL of 1 mMDMPG in a 1:1 mixture of MeOH and CHCl3 with 15 uL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen and resuspended in 150 uL of 50 mM HEPES pH 8.0 buffer containing 100 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at rt for 15 min and then was sonicated 3x4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.05 for the FRET substrate.The enzymatic assay was measured using white, opaque 96-well half area plates. Each well contained 60 uL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 2 ul of a DMSO solution containing compound of interest. Conditioned media obtained from HT-1080 cells, which were transformed by RAGE technology (Athersys) to overexpress endogenous EL, was added and the reaction was allowed to incubate for 20 min at 37deg; C. with gentle agitation. The reaction was started by the addition of 20 uL of a 1:4 dilution of vesicles. The final total reaction volume was 100 uL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 488 nm and a emission of 530 nm. Readings were taken every 20 seconds for 10 min with agitation between each reading. The slope of the linear portion of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 8254 nM (pChEMBL 5.079999923706055)
Descriptors: MW 397.48, ALogP 1.71, HBA 4, HBD 2, PSA 89.95, QED 0.81
Compound (SMILES): Cc1cccc(Nc2nn(C3(CC#N)CCN(C(=O)OC(C)(C)C)CC3)cc2C(N)=O)n1
InChIKey: BJCRIWFJHIWJQZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 439.52, ALogP 3.07, HBA 8, HBD 2, PSA 139.16, QED 0.73
Compound (SMILES): COc1cc(N2CCNCC2)ccc1Nc1ncc(C(F)(F)F)c(Nc2ccc(N3CCNCC3)cc2OC)n1
InChIKey: JEKOHYYOZUSNGX-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 558.61, ALogP 3.82, HBA 10, HBD 4, PSA 98.84, QED 0.33
Compound (SMILES): CN1Cc2c(C3=CCN(C4CCC(C(=O)O)CC4)CC3)[nH]c3nccc(c23)-c2ccccc21
InChIKey: HKHSLKKZKCVNJT-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay : CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were diluted in 100% DMSO then diluted with 1:10 in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. Equal volume of the compound dilutions was added to a final reaction mixture containing LANCE detection buffer (PerkinElmer CR97-100), 100 nM ULight MBP (PerkinElmer TRF0109M), 1000 μM ATP, and CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 1 hour before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer. The reaction was incubated for 1 hour and the signal was read in Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 442.56, ALogP 4.91, HBA 4, HBD 2, PSA 72.46, QED 0.6
Compound (SMILES): CCn1c(=O)c2cn[nH]c2c2ccc(-c3ccccc3N(C)C)cc21
InChIKey: SPQXHLWSWJVURN-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzymatic Screening Assay: The enzymatic test is a test in two steps.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 332.41, ALogP 3.63, HBA 4, HBD 1, PSA 53.92, QED 0.62
Compound (SMILES): CN(C)c1nc2c(c(Nc3ccc(C(F)(F)F)c(Cl)c3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: UVMVELIEPPDUAU-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 530.9, ALogP 5.98, HBA 6, HBD 1, PSA 57.18, QED 0.42
Compound (SMILES): CC(C)(C)c1ccc(Nc2nc(CN3CCCCC3)nc3c2CCN(c2ncccc2C(F)(F)F)CC3)cc1
InChIKey: NEWBGYVANRXSCX-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: Functional Assay: HEK293 cells were transfected with human TRPV1 cloned in pcDNA3.1zeo(+) using the Effectene non-liposomal lipid based transfection kit (Qiagen) (hTRPV1/HEK293). hTRPV1/HEK293 cells were routinely grown as monolayers under selection in zeocin (200 μg/mL; Invitrogen) in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, and penicillin/streptomycin (50 units/mL) in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 420 nM (pChEMBL 6.380000114440918)
Descriptors: MW 538.66, ALogP 6.52, HBA 6, HBD 1, PSA 57.18, QED 0.4
Compound (SMILES): CC1Cc2nc(NCC(F)F)c(N3CCC(Oc4ccc(F)cc4F)CC3)nc2CN1S(C)(=O)=O
InChIKey: BNECLYPVBLBWJE-UHFFFAOYSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.1): This cell based assay measures the ability of compounds to inhibit the constitutive cAMP activity of GPR6 receptor expressed in CHO-K1 cells. CHO cells were stably expressed with GPR6 receptor, whose expression is controlled by a tetracycline inducable element. The cells were cultured in medium containing F12K, 10% FBS, 1% PennStrep, 200 ug/mL Hygromycin. GPR6 receptor expression was induced for 20 hrs with 1 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 250-500 cells per well in half-volume black clear bottom plates (Costar) and place in an incubator (37° C, 5% C(O)2) for 20 hours prior to cAMP assays.Culture media was removed from cells and they were washed with 50 uL of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHC(O)3 1.26 mg/mL, D-glucose 1.8 mg/mL, NaCl 7 mg/mL; pH=7.4). Compounds suspended in DMSO were diluted in Ringer's Buffer.
Measurement: IC50 = 0.0104 nM (pChEMBL 10.979999542236328)
Descriptors: MW 517.55, ALogP 3.19, HBA 7, HBD 1, PSA 87.66, QED 0.56
Compound (SMILES): CCC(O)(c1cn(Cc2ccc3c(-c4ccc(OC)cc4)cc(C(N)=O)nc3c2)cn1)C(F)(F)F
InChIKey: NXCMSLDZYALNKL-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Biological Assay: The utility of the compounds in accordance with the present invention as antagonists of metabotropic glutamate receptor activity, in particular mGluR2 activity, may be demonstrated by methodology known in the art. Antagonist constants are determined as follows. The compounds of the present invention were tested in a fluorescence laser imaging plate reader based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr- cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with various concentrations of antagonists of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 484.48, ALogP 4.41, HBA 6, HBD 2, PSA 103.26, QED 0.4
Compound (SMILES): COCCS(=O)(=O)Nc1noc2cc(COc3ccc(OCC(C)C)c(Cl)c3)c(Cl)cc12
InChIKey: FIIKAIDVEXDROV-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay: Sodium currents were measured using the patch clamp technique in the whole-cell configuration using either a PatchXpress automated voltage clamp or manually using an Axopatch 200B (Axon Instruments) or Model 2400 (A-M systems) amplifier. The manual voltage clamp protocol was as follows: Borosilicate glass micropipettes were fire-polished to a tip diameter yielding a resistance of 2-4 Mohms in the working solutions. The pipette was filled with a solution comprised of 5 mM NaCl, 10 mM CsCl, 120 mM CsF, 0.1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA; and adjusted to pH 7.2 with CsOH. The external solution had the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES; and adjusted to pH 7.4 with NaOH. In some studies, the external sodium was reduced by equimolar replacement with choline.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 503.4, ALogP 5.14, HBA 7, HBD 1, PSA 99.89, QED 0.39
Compound (SMILES): NC[C@@H](c1ccc(C(F)(F)F)cc1)N1CCN(c2ncnc(N)c2-c2cn[nH]c2)CC1
InChIKey: LRVNLSGZICUQLS-INIZCTEOSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: Enzyme Assay: P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction.
Measurement: IC50 = 5.7 nM (pChEMBL 8.239999771118164)
Descriptors: MW 432.45, ALogP 2.29, HBA 7, HBD 3, PSA 112.98, QED 0.57
Compound (SMILES): CC(C)Nc1nc2c(nc1N1CCN(Cc3ccc(F)cc3F)CC1)CCN(C(=O)C(C)C)C2
InChIKey: IWFKEJLLLIHOQD-UHFFFAOYSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.2): This cell based assay measures the ability of compounds to inhibit the constitutive cAMP activity of GPR6 receptor expressed in CHO-K1 cells. CHO cells were stably expressed with GPR6 receptor, whose expression is controlled by a tetracycline inducable element. The cells were cultured in medium containing F12K, 10% FBS, 1% PennStrep, 200 ug/mL Hygromycin. GPR6 receptor expression was induced for 20 hrs with 2 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 450-750 cells per well in 96-well half-volume black tissue culture plates (Costar) and placed in an incubator (37°, 5% CO2) for 20 hours prior to cAMP assays. Culture media was removed from cells and they were washed with 50 uL/well of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHCO3 1.26 mg/mL, D-glucose 1.8 mg/mL, NaCl 7 mg/mL; pH=7.4). Compounds suspended in DMSO were diluted in Ringer's Buffer.
Measurement: IC50 = 0.029 nM (pChEMBL 10.539999961853027)
Descriptors: MW 472.58, ALogP 3.44, HBA 6, HBD 1, PSA 64.6, QED 0.7
Compound (SMILES): NC(=O)C1CSCN1C(=O)c1ccc(C2CC2)c(OCC2CC2)n1
InChIKey: PACWXBITGZWEJG-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×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 °C. for 30 min. Compounds were added to a final assay volume of 100 μl 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 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM 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 as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 103.4 nM (pChEMBL 6.989999771118164)
Descriptors: MW 347.44, ALogP 1.75, HBA 5, HBD 1, PSA 85.52, QED 0.85
Compound (SMILES): N#Cc1c(-c2ccccc2)nc2[nH]nc(-c3ccccc3)c2c1C1CC1
InChIKey: IBKHRKCVFDSEOT-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Positive allosteric modulation of mGlu5 receptor (unknown origin) assessed as increase in L-glutamate-induced calcium release after 60 mins by cell based FLIPR assay
Measurement: EC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 336.4, ALogP 5.04, HBA 3, HBD 1, PSA 65.36, QED 0.57
Compound (SMILES): COc1ccc(CNC(=O)c2nc(N)nc3c(OC)cccc23)nc1
InChIKey: MDOJLXHRFIJLOH-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Displacement of [3H]5-amino-7-[2- phenethyl]-2-(furan-2-yl)-7H-pyrazolo[4,3-e][l,2,4]triazolo[l,5-c]pyrimidine from human adenosine A2A receptor expressed in HEK293 cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: Ki = 7.8 nM (pChEMBL 8.109999656677246)
Descriptors: MW 339.36, ALogP 1.55, HBA 7, HBD 2, PSA 112.25, QED 0.72
Compound (SMILES): O=C1COc2cc(S(=O)(=O)Nc3nccs3)ccc2N1c1ccc(C(F)(F)F)cc1CO
InChIKey: COWSPFLQNHBOFG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to −10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 3350 nM (pChEMBL 5.46999979019165)
Descriptors: MW 485.46, ALogP 3.51, HBA 7, HBD 2, PSA 108.83, QED 0.57
Compound (SMILES): O=C(NCc1ccc(S(=O)(=O)c2cncc(C(F)(F)F)c2)cn1)c1cc2ccncc2o1
InChIKey: ZPIPRSGXAFONKA-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT Enzymatic Reaction. The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 pM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 49.5 nM (pChEMBL 7.300000190734863)
Descriptors: MW 462.41, ALogP 3.4, HBA 7, HBD 1, PSA 115.05, QED 0.48
Compound (SMILES): Oc1ccccc1CCCNCc1ccccc1
InChIKey: MPHXLAROGRREKY-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: Inhibition of MPO chlorination activity (unknown origin) assessed as taurine chloramine formation after 5 mins in presence of H2O2/Cl- by HTS method
Measurement: IC50 = 360 nM (pChEMBL 6.440000057220459)
Descriptors: MW 241.33, ALogP 3.11, HBA 2, HBD 2, PSA 32.26, QED 0.76
Compound (SMILES): Cc1cc(CNc2c(F)ccc(OCC(=O)O)c2F)cc(-c2cccc(F)c2)c1
InChIKey: KMAAOGSFQXFAOW-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Cellular Functional Assay: Human CHO cells expressing the recombinant human prostanoid EP2 receptor were suspended in assay medium (HBSS buffer (Invitrogen) containing 20 mM HEPES (pH7.4) and 500 μM isobutyl-methylxanthine IBMX) and plated out to yield approximately 1×104 cells/well in a 96-well. Following this, cells were incubated with agonists for 30 min in the presence of the test compounds. For stimulated control measurements, separate assay wells contain 10 μM PGE2 (control specific agonist). All incubations were carried out at 37° C. in a 5% CO2 atmosphere. Following incubation, the amount of cAMP in each well was determined by HTRF method. The cells are lysed and the fluorescence acceptor (D2-labeled cAMP) and fluorescence donor (anti-cAMP antibody labeled with europium cryptate) are added.
Measurement: EC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 401.38, ALogP 5.15, HBA 3, HBD 2, PSA 58.56, QED 0.57
Compound (SMILES): O=C(c1ccc2cc(Oc3ccc(C(F)(F)F)cn3)ccc2n1)N1CC(NCC(F)(F)F)C1
InChIKey: SUALZRSRONPDNT-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 = 2690 nM (pChEMBL 5.570000171661377)
Descriptors: MW 470.37, ALogP 4.42, HBA 5, HBD 1, PSA 67.35, QED 0.56
Compound (SMILES): CS(=O)(=O)c1ccc(S(=O)(=O)c2ccc(NC(=O)/C=C/c3cccnc3)cc2)cc1
InChIKey: FEFVYHADALRIRN-YIXHJXPBSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mix was incubated for 15 minutes at room temperature, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 uM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 minutes at room temperature, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine.
Measurement: IC50 = 11.4 nM (pChEMBL 7.940000057220459)
Descriptors: MW 442.52, ALogP 2.97, HBA 6, HBD 1, PSA 110.27, QED 0.59
Compound (SMILES): CN1c2cc(-c3cnc(N4CCOCC4)c4nc(/C=C/c5ccc6ccccc6n5)cn34)ccc2NS1(=O)=O
InChIKey: TZWJYSWIFUSSLL-MDZDMXLPSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay rPDE10: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi 3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter and results were expressed as cpm. For blank values the enz
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 539.62, ALogP 4.06, HBA 8, HBD 1, PSA 104.96, QED 0.37
Compound (SMILES): C#CCCCC(=O)Nc1nc2nn(C)cc2c2nc(-c3ccco3)nn12
InChIKey: FUMIWYQFHWMBFT-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Radioligand Binding Assays: Radioligand binding assays at hA1, hA2A, and hA3ARs were performed according to the procedures described in Gao, Z. G., et al., Biochem Pharmacol. 2003; 65: 1675-84, using the radiolabeled agonists [3H]R-PIA from Moravek Biochemicals (Brea, Calif.) or [3H]CGS21680 and [125I]I-AB-MECA from PerkinElmer (Waltham, Mass.) for the hA1, hA2AAR, and hA3AR assays, respectively.
Measurement: Ki = 199 nM (pChEMBL 6.699999809265137)
Descriptors: MW 349.35, ALogP 2.01, HBA 8, HBD 1, PSA 103.14, QED 0.44