lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCN(CCN1CCC(c2noc3cc(F)ccc23)CC1)C(=O)C1CNCCC1O
InChIKey: ZWSDZWGYYIXLJO-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: Binding Activity Assay: Human 5-HT2A Receptor, Human 5-HT7 Receptor, and Human D2 Receptor: Binding affinity of the present compound for human 5-HT2A receptor, human 5-HT7 receptor, and human D2 receptor was measured by the following procedures.CHO cell membrane fraction in which human 5-HT2A receptor, human 5-HT7 receptor, or human D2 receptor was expressed was purchased from PerkinElmer, Inc. In a test for evaluating binding affinity, a test compound dissolved in dimethylsulfoxide (DMSO) and each receptor membrane sample diluted in buffer were mixed with [3H]Ketanserin, [3H]SB-269970, or [3H]Spiperone (all purchased from PerkinElmer, Inc.) for 5-HT2A receptor, 5-HT7 receptor, or D2 receptor, respectively. Each mixture was incubated at room temperature for 60 minutes. Non specific binding to receptors was obtained from a competitive binding test in the presence of 10 μmol/L 8-OH-DPAT, 10 μmol/L Mianserin, or 10 μmol/L Spiperone, respectively. Radioactivity caused by binding to receptors was measured with a liquid scintillation counter (PerkinElmer, Inc.), and 50% inhibition concentration was calculated.
Measurement: Ki = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 418.51, ALogP 1.97, HBA 6, HBD 2, PSA 81.84, QED 0.75
Compound (SMILES): Cn1cnc2c(ncn2[C@@H]2C[C@@H]3OP(=O)(O)O[C@H]4[C@@H](F)[C@H](n5cnc6c(=O)n(C)cnc65)O[C@@H]4COP(=O)(S)O[C@@H]2[C@H]3O)c1=O
InChIKey: HNISUHKYGHZBOY-NCEIAANDSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: Surface Plasmon Resonance (SPR) Binding: Surface plasmon resonance (SPR) STING agonist binding studies were carried out using a Biacore T200 instrument (GE Healthcare) at 4° C. in a 150 mM KCl, 25 mM Hepes (pH 7.5), 1 mM TCEP, 2.5 mM MgCl2, 5% (v/v) glycerol, 0.005% (v/v) P20, 1% (v/v) DMSO running buffer. The recombinant protein immobilized on the streptavidin chip was either human WT or H232R STING. A truncated construct of STING was used in all studies. The STING constructs were comprised of residues 155-341 with both N- and C-terminal truncations; the N-terminal transmembrane domains were removed (1-154), as well as the C-terminal tail (342-379). A highly specific N-terminal biotinylation was achieved enzymatically with the E. coli biotin ligase (BirA) and inclusion of the high-affinity biotinylation peptide AviTag. A Carboxymethylated dextran pre-immobilized with streptavidin (series S Streptavidin CM5 Sensor Chip) was used to capture the biotinylated STING protein. Test compound injections were made at a flow rate of 100 μl per minute with a 60 second association time and variable dissociation time. A three-fold dilution series from a 10 μM starting concentration was used for all test compounds. Data analysis was performed using the BiacoreT200 data evaluation software package (GE Healthcare).
Measurement: Kd = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 690.5, ALogP 0.14, HBA 18, HBD 3, PSA 226.17, QED 0.19
Compound (SMILES): Cc1cc2nccn2c(N[C@H](c2ccc(C(F)(F)F)c(F)c2)C(C)(C)O)n1
InChIKey: HEBDUPRUMNBGAV-OAHLLOKOSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Inhibitory Activity Assay (Lab B): In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.). Cells were harvested 48 hours post-transfection and lysed by sonication (setting 3, 10×5 sec pulses) in a buffer containing 20 mM HEPES pH 7.4, 1 mM EDTA and Complete Protease Inhibitor Cocktail Tablets (Roche, Indianapolis, Ind.). Lysate was collected by centrifugation at 75,000×g for 20 minutes at 4° C. and supernatant utilized for evaluation of PDE2 activity.
Measurement: Ki > 2955 nM (pChEMBL 5.5289998054504395)
Descriptors: MW 382.36, ALogP 4.12, HBA 5, HBD 2, PSA 62.45, QED 0.67
Compound (SMILES): Cc1cnc(-c2cc(OCC3CCOCC3)cc(C(=O)NC(C)c3ncc(Cl)cc3F)c2)s1
InChIKey: WLBXKEKEIMKGRV-UHFFFAOYSA-N
Target: P2X2/P2X3 heterotrimeric receptor (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9UBL9
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity : A fluorescent imaging plate reader (FLEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Fluo-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C. The final assay DMSO concentration is 1%.The agonist, α,β-Me-ATP, was added in a volume of 20 μl at a concentration representing the EC80 value. The fluorescence was measured for an interval of 90 sec at 2 sec intervals and analyzed based on the increase in peak relative fluorescence units (RFU) compared to the basal fluorescence. Peak fluorescence was used to determine the response to agonist obtained at each concentration of test compound by the following equation: % Response=100*(RFU(test compound)−RFU(control))/(RFU(DMSO)−RFU(control)).
Measurement: IC50 = 129 nM (pChEMBL 6.889999866485596)
Descriptors: MW 490, ALogP 5.6, HBA 6, HBD 1, PSA 73.34, QED 0.47
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2ncc(Cc3cccc(C#N)c3)s2)COc2ccccc21
InChIKey: LNCGJECMHCFQDK-KRWDZBQOSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding (FP Binding) assay: Assay was performed in polystyrene low volume 384-well black plate, at Room Temperature (RT) in a final volume of 10.1 μl/well using 10 nM of GST-hRIPK1 (8-327) enzyme and 5 nM of fluorescent labeled ligand (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino) propyl]amino}-2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo[2″,3″]indolizino[8″,7″:5′,6]pyrano[3′,2′:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate.Test Compounds were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final). In each well of a 384-well Plate were dispensed 0.1 μL of compound solution (or DMSO for controls) followed by 5 μL of GST-hRIPK1 (8-327) at twice the final concentrations in assay buffer (50 mM HEPES pH 7.5, 10 mM NaCl, 50 mM MgCl2, 0.02% CHAPS, 0.5 mM DTT and 0.01% Pluronic F127). For negative control the enzyme addition was replaced by assay buffer only.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 418.48, ALogP 2.76, HBA 6, HBD 1, PSA 95.32, QED 0.7
Compound (SMILES): N#Cc1cccc(F)c1-c1cc(-n2ccc(N3CCC[C@H](CO)C3)n2)c2c(n1)CNC2=O
InChIKey: RUHRJFNUOYWIBG-AWEZNQCLSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 432.46, ALogP 2.4, HBA 7, HBD 2, PSA 107.07, QED 0.66
Compound (SMILES): CCCc1ccc(S(=O)(=O)Nc2ccc(F)c(C(=O)c3c[nH]c4ncnc(Cl)c34)c2F)cc1
InChIKey: WLPFZVLMFBVNJD-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Biochemical Assay: Assays for biochemical and cell based activity are known in the art, for example, as described in PCT publication WO 2007/002433, the disclosure of which is hereby incorporated by reference as it relates to such assays. For example, the biochemical activity IC50 values are determined with respect to inhibition of B-Raf kinase activity, c-Raf-1 kinase activity, or B-Raf V600E kinase activity, where inhibition of phosphorylation of a peptide substrate is measured as a function of compound concentration. Compounds to be tested are diluted in dimethyl sulfoxide to a concentration of 0.1 mM. These are serially diluted 15 μL into 30 μL of dimethyl sulfoxide seven times in 96 well plates for a total of 8 dilution points, and for each dilution point 1 μL is added to a well of an assay plate. Plates are prepared such that each well in a 384 well plate contains 1 μL of compound in 10 μL volume with 0.1 ng Raf enzyme (i.e. any of B-Raf, c-Raf-1 or B-Raf V600E, Upstate Biotechnology or prepared by methods known to one of skill in the art), 50 mM HEPES, pH 7.0, 50 mM NaCl, 2 mM MgCl2, 1 mM MnCl2, 0.01% Tween-20, 1 mM DTT, and 100 nM biotin-MEK1 as substrate. The reaction is started with addition of 10 μL of 200 μM ATP (i.e. final 100 μM ATP). After incubation of the kinase reaction for 45 minutes at room temperature, 5 μL/well of Stop Solution is added (25 mM Hepes pH 7.5, 100 mM EDTA, 0.01% BSA with donor beads (Streptavidin coated beads, Perkin Elmer), acceptor beads (Protein A coated, Perkin Elmer), and anti phosphor MEK1/2 antibody (CellSignal), each at final concentration 10 μg/mL). The plates are incubated for 3 hours at room temperature and read on Envision reader (Perkin Elmer). Phosphorylation of Mek1 results in binding of the anti-phosphor-MEK1/2 antibody and association of the donor and acceptor beads such that signal correlates with kinase activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 490.92, ALogP 4.87, HBA 5, HBD 2, PSA 104.81, QED 0.28
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)CCCC[C@H](c1ccc(-c3cc(Cl)ccc3-n3cc(C)cn3)c[n+]1[O-])c1nc-2c(C)[nH]1
InChIKey: BMLGENRLCJEYSQ-RUZDIDTESA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: Kallikrein Assay: The effectiveness of a compound of the present invention as an inhibitor of Kallikrein can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at rt or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Kallikrein determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; Fisher Scientific). Determinations were made using purified Human plasma Kallikrein at a final concentration of 0.5 nM (Enzyme Research Laboratories) and the synthetic substrate, Acetyl-K-P-R-AFC (Sigma # C6608) at a concentration of 100 mM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.2 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. The reactions were performed under linear progress curve conditions and fluorescence increase measured at 405 Ex/510 Em nm. Values were converted to percent inhibition of the control reaction (after subtracting 100% Inhibition value). IC50 was determined by inflection point from a four parameter logistic curve fit. Ki was calculated using the Cheng Prusoff equation, Ki=IC50/(1+([S]/Km)).
Measurement: Ki = 82.8 nM (pChEMBL 7.079999923706055)
Descriptors: MW 626.12, ALogP 6.66, HBA 7, HBD 3, PSA 140.87, QED 0.15
Compound (SMILES): O=C(NNS(=O)(=O)CC1CCCCC1)c1cc(-c2cn(Cc3ncco3)cn2)cc(Cl)c1F
InChIKey: OYZMUCDEJUEFAI-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 495.96, ALogP 3.52, HBA 7, HBD 2, PSA 119.12, QED 0.46
Compound (SMILES): COc1ccc(C)c(C(=O)N2CCN(Cc3c(-c4ccc(C#N)cc4)nc4ccccn34)CC2)n1
InChIKey: XLCSMRVTVQNPBL-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: Inhibition In Vito Assay: The investigations on the inhibition of the recombinant TASK-1 and TASK-3 channels were carried out using stably transfected CHO cells. Here, the compounds according to the invention were tested with administration of 40 mM of potassium chloride in the presence of a voltage-sensitive dye for high-throughput screening of potassium channel from Molecular Devices FLIPR Application Note: Measuring membrane potential using the FLIPR membrane potential assay kit on Fluorometric Imaging Plate Reader (FLIPR) systems. The activity of the test substances was determined as their ability to inhibit a depolarization induced in the recombinant cells by 40 mM potassium chloride.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 466.55, ALogP 3.54, HBA 7, HBD 0, PSA 86.76, QED 0.45
Compound (SMILES): COCCOc1cc2ncnc(Oc3cccc(NC(=O)Nc4cc(C5CCOCC5)no4)c3)c2cc1OC
InChIKey: JKENTMLQHVTGQZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Competition Binding Assay: Competition binding assays used herein were developed, validated and performed as described in Fabian et al., Nature Biotechnology 2005, 23, 329-336. Kinases were produced as fusions to T7 phage (See, Fabian et al. or WO04/015142) or alternatively, the kinases were expressed in HEK-293 cells and subsequently tagged with DNA for PCR detection (See, WO08/005310). For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 μL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 μL. The assay plates were incubated at room temperature with shaking for 1 hour, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1×PBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1×PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. KdS were determined using eleven serial threefold dilutions.
Measurement: Kd > 1000 nM (pChEMBL 6)
Descriptors: MW 535.56, ALogP 4.98, HBA 10, HBD 2, PSA 139.09, QED 0.27
Compound (SMILES): Cc1c(C#N)nn(C)c1C(=O)Nc1ccc2[nH]nc(-c3cnoc3)c2c1
InChIKey: ZIBKDPZGSFXEKZ-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen Kinase Activity Assay: The assay was carried out under the following protocol conditions: 1 mM compound in DMSO was serially diluted 1:3, 11 points in DMSO with a Biomek FX and 0.1 μL of the diluted compound was subsequently stamped into the assay plate (384-well format Lumitrac 200, Greiner, 781075) with an Echo Labcyte such that the final compound concentration in the assay was 10 μM to 169 μM. Subsequently, 5 μL of 2× kinase solution (2.9 nM final concentration) was added to the assay plate in assay buffer composed of 50 mM Tris pH 8.5 (Sigma, T6791), 5 mM MgCl2 (Fluka, 63020), 1 mM EGTA (Sigma, E3889), 0.01% BRIJ-35 (Sigma, P1254) and 2 mM DTT. The reaction was started by addition of 2×ATP/LRRKtide solution in assay buffer such that the final concentration was 400 nM LRRKtide and 25 μM ATP. After 60 min incubation at room temperature, the reaction was stopped by addition of 10 μL of 2× stop solution containing a final concentration of 2 nM anti-pERM antibody and 10 mM EDTA. After a 30 min incubation at RT, the TR-FRET signal was measured on a Wallac 2104 EnVision multilabel reader at an excitation wavelength of 340 nm and reading emission at 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 347.34, ALogP 2.38, HBA 7, HBD 2, PSA 125.42, QED 0.59
Compound (SMILES): COc1cn(C(Cc2ccn(C)n2)C(=O)Nc2ccc(C(N)=O)nc2)c(=O)cc1-c1cc(F)ccc1C#N
InChIKey: GQVNMLVJSIXAMF-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: The factor XIa inhibition of the substances according to the invention is determined using a biochemical test system which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.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). 1 μl of the diluted substance solutions is placed into each of 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; 5 mM of calcium chloride; 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 mM 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). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 515.51, ALogP 2.18, HBA 9, HBD 2, PSA 157.92, QED 0.36
Compound (SMILES): CC(=O)Nc1cn(C)c2c1C(=O)N(c1cc(C)c3nnn(C)c3n1)C2c1ccc(Cl)cc1
InChIKey: DGXUFGAFFVEFEU-UHFFFAOYSA-N
Target: Bromodomain-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25440
Assay: TR-FRET In-Vitro Binding Assay: All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 μL per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5 μL per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 μL per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (1050 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 449.9, ALogP 3.37, HBA 7, HBD 1, PSA 97.94, QED 0.52
Compound (SMILES): C[C@H](c1ccccc1)N1CCC(Oc2cc(F)c(S(=O)(=O)Nc3cscn3)cc2Cl)CC1
InChIKey: PNFLMUWDTCSMFS-OAHLLOKOSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 27908 nM (pChEMBL 4.550000190734863)
Descriptors: MW 496.03, ALogP 5.34, HBA 6, HBD 1, PSA 71.53, QED 0.48
Compound (SMILES): CN1Cc2cc(Cl)ccc2-n2c(nnc2C2CC3(C2)CN(c2ccccn2)C3)C1
InChIKey: MIAGFSZQJXZDTB-UHFFFAOYSA-N
Target: Vasopressin V1b receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P47901
Assay: Vasopressin V1B Receptor Antagonist Assay: The purpose of the assay is to determine the inhibitory effect of synthesized compounds on the Vasopressin V1b receptor. The assay was performed in Chinese Hamster Ovary (CHO) cells expressing the human Arginine Vasopressin Receptor 1b (AVPR1b). Arginine Vasopressin (AVP) evokes an increase in intracellular calcium in CHO-AVPR1b cells which is measured in a fluorescence assay on the FLIPRTETRA using calcium sensitive dyes. Test compounds were assessed for their ability to affect the magnitude of the response to AVP, with antagonists showing a concentration-dependent reduction in the AVP-mediated fluorescence.CHO-AVPR1b cells were maintained in routine culture in T175 Flasks at 37° C., 5% CO2. The growth medium consisted of Ham's F12 media supplemented with 10% v/v fetal bovine serum, 1× non-essential amino acids, and 0.4 mg/ml Geneticin G418.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 406.92, ALogP 3.65, HBA 6, HBD 0, PSA 50.08, QED 0.65
Compound (SMILES): CC(=O)Nc1cc(Nc2nc(-c3ccccn3)nn3ccc(CN4CCC(F)(F)CC4)c23)ccn1
InChIKey: CHAIHARSRAHKQQ-UHFFFAOYSA-N
Target: TGF-beta receptor type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P37173
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1.) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 478.51, ALogP 4.12, HBA 8, HBD 2, PSA 100.34, QED 0.43
Compound (SMILES): CC(=O)N[C@H]1CC[C@H](CS(=O)(=O)NNC(=O)c2cc(-c3cn(C)cn3)cc(Cl)c2F)CC1
InChIKey: AOERMBVQGQVBDX-CTYIDZIISA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 485.97, ALogP 2.14, HBA 6, HBD 3, PSA 122.19, QED 0.52
Compound (SMILES): O=C(c1cc(F)ccc1Cl)N1CC2CN(Cc3c(-c4ccc(Br)cc4)nc4ccccn34)CC2C1
InChIKey: VXCUZFYDDABRKE-UHFFFAOYSA-N
Target: Potassium channel subfamily K member 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NPC2
Assay: In Vitro Electrophysiological Assay: In Vitro Electrophysiological Analysis of the Human TASK-1 and TASK-3 Channels Via Two-Electrode Voltage Clamp Technique in Xenopus laevis OocytesSubsequently, the oocytes were injected with 0.5-5 ng of a cRNA solution coding for TASK-1 or TASK-3. For the electrophysiological analysis of the channel proteins expressed in the oocytes, the two-electrode voltage clamp technique was used. The measurements were conducted at room temperature (21-22° C.) using a Turbo TEC 10CD amplifier (NPI), recorded at 2 kHz and filtered with 0.4 kHz. Substance administration was performed using a gravitation-driven perfusion system. Here, the oocyte is located in a measuring chamber and exposed to the solution stream of 10 ml/min. The level in the measuring chamber is monitored and regulated by sucking off the solution using a peristaltic pump.
Measurement: IC50 = 4300 nM (pChEMBL 5.369999885559082)
Descriptors: MW 553.86, ALogP 5.76, HBA 4, HBD 0, PSA 40.85, QED 0.32
Compound (SMILES): CC12CC(NC(=O)c3c(O)c4cccnc4n(Cc4ccc(F)cc4)c3=O)(C1)C2
InChIKey: KQHRWAYCWXVUJT-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: CB1 and CB2 Receptor Binding Assay: CB1R binding protocol involves the use of the same solution buffer used for both incubation and washing reaction (Tris-HCl, 50 mM; EDTA, 2.5 mM; MgCl2, 2.5 mM; BSA, 0.5 mg/mL at pH 7.4), 0.4 nM for [3H]CP-55,940, test compounds (concentrations from 0.001 to 10 μM), and finally 8 μg/sample membrane in a total volume of 200 μL. CB2R binding assays were carried out with two different buffers: incubation buffer (Tris-HCl, 50 mM; MgCl2, 5 mM; CaCl2 1 mM; BSA, 0.2% at pH 7.4) and washing buffer (Tris-HCl, 50 mM; NaCl 500 mM; BSA, 0.1% at pH 7.4). The assay mixture contained incubation buffer, 0.4 nM [3H]CP-55,940, test substances (concentrations from 0.001 to 10 μM), and 4 μg/sample membrane in a total assay volume of 200 μL. Assays were performed in duplicate and incubated for 120 mM at 37° C. After the incubation, the assay mixture is filtered through 96 GF/C filter plates (Perkin Elmer #6005174) using Perkin Elmer Filtermate Harvester, and then washed four times with ice-cold washing buffer.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 393.42, ALogP 2.96, HBA 5, HBD 2, PSA 84.22, QED 0.71
Compound (SMILES): Cn1cc(-c2c(-c3ccc(F)cc3)nc(N)n3nc(Cc4ncccc4OC(F)F)nc23)ccc1=O
InChIKey: IVLZUGWDYRIRPP-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Binding Affinities to Different Adenosine Receptors: The compounds at different concentrations were incubate with hA1 membrane (from PerkinElmer) and [3H]-8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) for 50 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 L ULTIMA GOLD was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA2a membrane (from PerkinElmer) and [3H]-CGS21680 for 90 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 L ULTIMA GOLD was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA2b membrane (from PerkinElmer) and [3H]-DPCPX for 60 min at 27° C., and the binding reactions were stopped by rapid filtration through 0.5% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 L of scintillation cocktail was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA3 membrane (from PerkinElmer) and [1211]-AB-MECA for 60 min at 27° C., the binding reactions were stopped by rapid filtration through 0.50% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 L of scintillation cocktail was added into each well, and CPM was read by TopCount.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 493.45, ALogP 3.47, HBA 9, HBD 1, PSA 113.22, QED 0.39
Compound (SMILES): CNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2nccc(C(F)(F)F)c2F)CC1
InChIKey: XCYMFAKBKBNHID-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Enzyme Assay: MER: Compounds of Formula I were screened for their ability to inhibit MER kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human MER cytoplasmic domain (5 nM) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 489.48, ALogP 4.99, HBA 8, HBD 1, PSA 82.68, QED 0.4
Compound (SMILES): Cc1cc(C)c(CN2CCc3c(Br)c4c(c(C)c3C2=O)OC(C)(C23CCC(N(C)C)(CC2)CC3)O4)c(=O)[nH]1
InChIKey: YUGYQZBNPLWZEK-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibitory Activity Assay: The inhibitory activity of the synthesized compounds above against EZH1 or EZH2 methyltransferase was measured. The experiments were consigned to Reaction Biology, and EZH1 or EZH2 activity was measured using a radiometric scintillation proximity assay. To measure the IC50 of the synthesized compounds for EZH1 or EZH2, 2.3 nmol/L EZH1 or EZH2, 1 μmol/L histone H3 (21-44)-lys(biotin), 1.5 μmol/L S-adenyl methionine (SAM), and 500 nmol/L 3H-SAM were added to the compounds or DMSO with a buffer solution and reacted for 90 minutes at room temperature. The buffer solution was comprised of 50 mmol/L Tris-HCl pH 8.0, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmoL/L DTT, 1 mmol/L PMSF and 1% DMSO. Trichloroacetic acid was added to end the reaction, and SPA beads coated with PVT streptavidin were added followed by 1 hour reaction at room temperature. A TopCount NXT plate reader was used to measure the methylation value of the substrate peptide. The measured values were converted to percent activity, setting the mean value for wells treated with DMSO as 100% and the background mean value as 0%.
Measurement: IC50 = 0.35 nM (pChEMBL 9.460000038146973)
Descriptors: MW 584.56, ALogP 5.4, HBA 5, HBD 1, PSA 74.87, QED 0.52
Compound (SMILES): Cn1cnc2ncn(Cc3nc([C@H]4[C@@H]5CN(c6cc7ccccc7cn6)C[C@@H]54)no3)c(=O)c21
InChIKey: CGKGVIMZSQQQKI-MNZLEMJZSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (90 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 90 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 81 nM (pChEMBL 7.090000152587891)
Descriptors: MW 440.47, ALogP 1.96, HBA 10, HBD 0, PSA 107.76, QED 0.42
Compound (SMILES): CN1CCN(C(=O)Cn2cc(Nc3nc4c(N5CC6CCC(C5)N6C(=O)C5CCSCC5)cccn4n3)cn2)CC1
InChIKey: IATNNMPFTSNKQN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assays: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications. Ki values were then determined using the Morrison tight binding model (Morrison, J. F., Biochim. Biophys. Acta. 185:269-296 (1969); William, J. W. and Morrison, J. F., Meth. Enzymol., 63:437-467 (1979)) modified for ATP-competitive inhibition [Ki=Ki,app/(1+[ATP]/Km,app)].
Measurement: Ki = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 578.75, ALogP 1.77, HBA 11, HBD 1, PSA 107.14, QED 0.47
Compound (SMILES): COc1nc2ccc([C@@](O)(c3cnnn3C)c3cnc(C)n3C)cc2c(Cl)c1CC(C)C
InChIKey: ILDWWDUAUOFPRU-QHCPKHFHSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORgammat ThermoFluor Assay: For the RORγt construct used in the ThermoFluor® assay, numbering for the nucleotide sequences was based on the reference sequence for human RORγt, transcript variant 2, NCBI Accession: NM_001001523.1 (SEQ ID NO:1). Nucleotides 850-1635 (SEQ ID NO:2) coding for the wild type human RORγt ligand binding domain (RORγt LBD) were cloned into the pHIS1 vector, a modified pET E. coli expression vector (Accelagen, San Diego), containing an in-frame N-terminal His-tag and a TurboTEV protease cleavage site (ENLYFQG, SEQ ID NO:3) upstream of the cloned insert sequence. The amino acid sequence for the RORγt construct used in the Thermofluor® assay is shown as SEQ ID NO:4.ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows:0.065 mg/mL RORγt60 M 1,8-ANS100 mM Hepes, pH 7.010 mM NaCl2.5 mM GSH0.002% Tween-20Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1x=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 454.96, ALogP 3.55, HBA 8, HBD 1, PSA 90.88, QED 0.48
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(Oc4ccccn4)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: VDCSGSALVXWJLG-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 24.6 nM (pChEMBL 7.610000133514404)
Descriptors: MW 440.51, ALogP 4.11, HBA 8, HBD 0, PSA 88.57, QED 0.45
Compound (SMILES): NC(=O)c1c(-c2ccc(F)c(Cl)c2)nn2c1CN(C(=O)Nc1ccc(Cl)c(Cl)c1)CC2
InChIKey: OWZQWDOSMXBYIL-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 482.73, ALogP 4.8, HBA 4, HBD 2, PSA 93.25, QED 0.56
Compound (SMILES): C[C@H](NC(=O)[C@H](N)Cc1c[nH]c2ccccc12)C(=O)NCc1ccc(C(=N)N)cc1
InChIKey: PRLYEWZKXXESCR-SCLBCKFNSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Enzymatic Assay for Thrombin: The thrombin assay utilizes a fluorogenic peptide substrate (Boc-VPR-AMC (R&D Systems) and was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed. John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 406.49, ALogP 1.14, HBA 4, HBD 6, PSA 149.88, QED 0.25