lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCN1CCN(c2nc3ccc(-c4c(C)noc4C)c4c3n2[C@@H](c2ccccn2)CO4)CC1
InChIKey: QATGQQDJALGVIA-OAQYLSRUSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 444.54, ALogP 3.83, HBA 8, HBD 0, PSA 72.45, QED 0.47
Compound (SMILES): CC(C)(C)n1ncc(OCc2ccc(C(=O)OCCCF)cc2)c(Cl)c1=O
InChIKey: KKHAMXRWWGGPAA-UHFFFAOYSA-N
Target: NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial (SINGLE PROTEIN), Bos taurus
UniProt: P25708
Assay: Submitochondrial Particle Assay: The procedure for determining catalytic activity of submitochondrial particles was adapted from Satoh et al. (e.g., see Satoh T, Miyoshi H, Sakamoto K, Iwamura H. Comparison of the inhibitory action of synthetic capsaicin analogues with various NADH-ubiquinone oxidoreductases. Biochim Biophys Acta. 1996, 1273, 21-30). NADH-DB reductase activity was measured using a stirred cuvette in a spectrophotometer (Hewlett-Packard, Houston Tex.) at 37° C., as the rate of NADH oxidation at 340 nm (ε=5.4 mM−1×cm−1) for 120 seconds. The final volume of the reaction was 2.5 mL, containing 50 mM K2HPO4 (pH 7.4), 0.4 μM Antimycin A, and 2 mM potassium cyanide. The final SMP concentration was 45 μg/mL. The enzyme reaction was initiated by the addition of 100 μM decyl ubiquinone and 50 μM NADH. Inhibitors at varying concentrations were pre-incubated with the reaction mixture containing SMPs for 4 min prior to initiation of the reaction. The IC50 value was determined as the concentration of the inhibitor required for 50% inhibition of the NADH oxidation. The IC50 value was calculated using GraphPad Prism Version 4 (GraphPad, San Diego, Calif.).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 396.85, ALogP 3.75, HBA 6, HBD 0, PSA 70.42, QED 0.53
Compound (SMILES): C[C@H](O)Cn1cc(-c2cc3c(N4CCN(c5ncc(Sc6ccccc6F)cn5)CC4)ncnn3c2)cn1
InChIKey: SZUWOLFQYJESDM-SFHVURJKSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: KIT D816V assay at Km: In each well of a 384-well plate, 0.04 ng/ul (0.5 nM) of D816V KIT (Carna Bioscience 08-156) was incubated in a total of 12.5 ul of buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 10 mM MgCl2, 1 mM DTT) with 1 uM Srctide (5-FAM-GEEPLYWSFPAKKK-NH2) and 15 uM ATP at 25 C for 90 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 ul of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.9 psi, upstream voltage −700, downstream voltage −3000, post sample sip 35s). Data was normalized to 0% and 100% inhibition controls and the IC50 or EC50 calculated using a 4-parameter fit using GraphPad Prism.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 531.62, ALogP 3.38, HBA 11, HBD 1, PSA 100.5, QED 0.34
Compound (SMILES): O=C(c1ccc(F)cc1)N1CCn2c(nnc2-c2csc(-c3ccc(N4CCCCC4)cc3)n2)C1
InChIKey: MVGLHOSJPPPKDO-UHFFFAOYSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: 3H-SB222200 Binding Competition Assay with Human NK-3 Receptor: The ability of the compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells which express the human NK3 receptor. The membranes were incubated with 5 nM 3H-SB222200_(ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 Mm/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of the compounds of the invention. The amount of tritiated SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard). Competition curves were obtained for compounds of the invention and the concentrations of compounds which displaced 50% of bound radioligand (IC50) were determined and then apparent inhibition constant Ki values were calculated by the following equation: Ki=IC50/(1+[L]/KD) where [L] is the concentration of free radioligand and KD is its dissociation constant at the receptor, derived from saturation binding experiments (Cheng and Prusoff, 1973).
Measurement: Ki = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 488.59, ALogP 4.85, HBA 7, HBD 0, PSA 67.15, QED 0.41
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(-c3cccc(CN=S(C)(C)=O)c3)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)O1
InChIKey: MBEYTHIZYADQNY-DGPALRBDSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 638.74, ALogP 5.48, HBA 8, HBD 2, PSA 110.08, QED 0.24
Compound (SMILES): COc1cc(-c2cnc(N3CCC(C)(NC(=O)c4ncc(F)cc4Cl)CC3)cn2)c2c(C#N)cnn2c1
InChIKey: QYNFBOVCEQNTPT-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 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 520.96, ALogP 3.65, HBA 9, HBD 1, PSA 121.33, QED 0.42
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCN([C@H]4CC[C@H](CC(=O)NS(=O)(=O)C(C)C)CC4)CC3)cc12
InChIKey: MVMUDRKGVOKWBI-JKIUYZKVSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), 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 directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes 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 (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 568.72, ALogP 5.27, HBA 6, HBD 2, PSA 104.39, QED 0.38
Compound (SMILES): Cn1nc(C(=O)N[C@H](Cc2ccc(-c3ccccc3)cc2)C[C@@H](O)C(=O)O)cc1C(=O)O
InChIKey: XTPSJUFNDWCPHA-YLJYHZDGSA-N
Target: Neprilysin (SINGLE PROTEIN), Homo sapiens
UniProt: P08473
Assay: Enzyme Inhibition Assay: The assays were performed in 384-well white opaque plates at 37° C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO4; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO4). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37° C.Test compounds were assayed over the range of concentrations from 10 μM to pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 437.45, ALogP 1.96, HBA 6, HBD 4, PSA 141.75, QED 0.4
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2n[nH]c(Cc3ccccc3)n2)COc2ccc(N3CCC4(CCCN(Cc5ccccc5)C4=O)CC3)cc21
InChIKey: DDWBYAKSADZWND-NDEPHWFRSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Biochemical Assay: A 500 ml stock volume of 5× Reaction Kinase Buffer was made by mixing 1000 μl of 1M MgCl2, 500 μl of 1M Tris-HCL pH7.4, 0.5 mg/ml (25 mg) of BSA, and 3475 μl of distilled H2O. A 3 ml 2× working stock volume of Reaction Kinase Buffer was made containing a final concentration of 100 M DTT and 4 mM MnCl2.Components of RIPK1 enzyme (Rigel Pharmaceuticals, South San Francisco, Calif., USA) were thawed on ice. Diluted RIPK1 was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer) to 31 ng/well. A 166 μM working stock ATP assay solution was prepared in 1× Kinase Reaction Buffer (diluted from 2× buffer).Compounds were serially diluted in DMSO from 250 uM in 4-fold dilutions then diluted 1:5 in 2× Reaction Buffer in a 96 well plate. 1.0 ul of diluted compound was added to a 384 well plate in duplicate. 2 μl of diluted Active RIPK1 was added to 384 well plate (do not add to column1) add 2×r×n buffer to column 1. AKT (Anaspec, Fremont, Calif., USA) at 150 nM was combined with ATP working stock at equal volume and 2 ul/well were added to the 384 well plate. The final reaction volume was 5.0 μl.
Measurement: IC50 = 74.5 nM (pChEMBL 7.130000114440918)
Descriptors: MW 633.75, ALogP 3.96, HBA 7, HBD 2, PSA 123.76, QED 0.32
Compound (SMILES): C=CC(=O)N[C@@H]1CCC[C@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1ccc(Oc2ccccc2)cc1C
InChIKey: MGHYGYDMVWSPBS-NHCUHLMSSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 7.76 nM (pChEMBL 8.109999656677246)
Descriptors: MW 553.64, ALogP 6.03, HBA 6, HBD 3, PSA 112.66, QED 0.24
Compound (SMILES): O/N=C\c1ccccc1-c1ccccc1-c1nnnn1-c1ccccc1F
InChIKey: RAYPYMSGHQSSCY-XKZIYDEJSA-N
Target: Intermediate conductance calcium-activated potassium channel protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15554
Assay: Inhibition Assay: Human HEK293 cells over-expressing human IK are grown in culture medium (DMEM supplemented with 10% foetal bovine serum), in polystyrene culture flasks (175 mm2) in a humidified atmosphere of 5% CO2 in air, at 37° C. Cell confluence should be 80-90% on day of plating. Cells are rinsed with 4 mL of PBS (phosphate buffered saline) and incubated 2 min with 1 mL of Trypsin-EDTA. After addition of 25 mL of culture medium cells are re-suspended by trituration with a 25 mL pipette.The cells are seeded at a density of 3×106 cells/mL (25 μL/well) in black-walled, clear bottom, 384-well plates pre-treated with 0.01 g/L poly-D-lysin (20 μL/well for ≧30 min). Plated cells were allowed to proliferate for 24 h before loading with dye.BTC-AM (50 mg, Invitrogen) is added 25.5 μl DMSO. The BTC-AM stock solution (2 mM) is diluted to a final concentration of 2 μM in Cl+ free assay buffer (in mM: 140 Na+-gluconate, 2.5 K+-gluconate, 6 Ca2+-gluconate, 1 Mg2+ gluconate, 5 glucose, 10 HEPES, pH 7.3) containing 2 μM ouabain, 2 mM amaranth and 1 mM tartrazine.The culture medium is aspirated from the wells, and 25 μl of the BTC-AM loading solution are added to each well. The cells are incubated at 37° C. for 60 min.After the loading period, the Tl+-sensitive BTC fluorescence signal is measured over time using a FLIPR.
Measurement: IC50 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 359.36, ALogP 3.94, HBA 6, HBD 1, PSA 76.19, QED 0.34
Compound (SMILES): Cc1ccc(C(=O)N(C(C)C)C2CCN(C(=O)[C@@](C)(O)C(F)(F)F)CC2)c(F)c1
InChIKey: DGPPISQVIGMVBR-LJQANCHMSA-N
Target: [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q15119
Assay: Biochemical Activity Assay: The PDC inactivation assay is performed in Greiner 384-well microtiter plates and is used for high throughput screen. 4 μl of PDHK2 (human, rec, Carna Bioscience, 10 ng/μl-137 nM final concentration) and PDC (isolated from porcine heart, Sigma-Aldrich, 20 mU/ml final concentration) are incubated in the absence or presence of the test compound (10 dilution concentrations) for 30 min at room temperature in kinase buffer (15 mM potassium phosphate buffer, pH 7.0, 60 mM KCl, 1.5 mM DTT, 2.5 mM MgCl2, 0.0125% (w/v) BSA, 0.125% Pluronic F-68). The kinase reaction is started by the addition of 4 μl ATP substrate solution (fc 5 μM in kinase buffer). After 30 min incubation at 37° C. 40 μl of PDC reaction solution (100 mM Tris/HCl, pH 7.8, 0.5 mM EDTA, 1 mM MgCl2, 50 mM NaF, 0.25 mM Coenzyme A, 5 mM pyruvate, 1 mM NAD, 5 mM DTT, 1 mM thiamine pyrophosphate) is added. The first fluorescence measurement is performed on a Perkin Elmer Envision (Exc 340 nm, Em 450 nm). The reaction is incubated for 45 min at room temperature. Afterwards a second fluorescence measurement is performed and the PDC activity is calculated by the difference between both measurements. As full value for the PDHK2 assay the inhibitor-free PDHK2 reaction is used.
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 418.43, ALogP 3.29, HBA 3, HBD 1, PSA 60.85, QED 0.76
Compound (SMILES): C[C@@H]1CNc2c(sc3ccc4nc(Oc5nc(Cl)ncc5CN(C)C(C)(C)C)ccc4c23)C(=O)N1
InChIKey: BXAGSZRPCYRDDN-CYBMUJFWSA-N
Target: MAP kinase-activated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P49137
Assay: Mass Spectrometry Assay: Compounds I-1 through I-50 of the invention were assayed in a mass spectrometric assay to measure their ability to covalently modify MK2 protein. The procedure for this assay follows. Intact MK2 protein (Invitrogen, Cat. No. PR5320A) was incubated for 60 minutes at room temperature with a 10-fold excess of test compound to protein. Aliquots of the resulting mixture (6 μL each) were diluted with 0.2% trifluoroacetic acid (TFA, 10 μL) prior to micro C4 ZipTipping directly onto the MALDI target using sinapinic acid as the desorption matrix (10 mg/ml in 0.1% TFA:acetonitrile 50:50, v/v). The centroid mass of the target protein in the control sample was compared with the centroid mass of the target protein incubated with compound. A shift in the centroid mass of the treated protein compared to the untreated protein was divided by the molecular weight of the compound. This number corresponds to the percentage of modified protein (a measure of the proportion of total target protein covalently bound to the test compound) after one hour incubation. Results from this assay are reported in Table A under the column ¿Mass Modification¿.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 511.05, ALogP 5.46, HBA 8, HBD 2, PSA 92.27, QED 0.35
Compound (SMILES): CC#C[C@@H](CC(=O)O)c1ccc(OCc2ccc(CN3CCN(c4nc5ccccc5s4)CC3)cc2)cc1
InChIKey: PZZMSRISSQRTAU-SANMLTNESA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Activity Assay: The GPR40 protein activity according to the novel 3-(4-(benzyloxy)phenyl)hex-4-inoic acid derivative of the present invention was measured by investigating the changes in intracellular calcium concentration affected by the GRP40 activation. First, HEK-293 cells were transfected with human GPR40 DNA (Origene, RC218370) by using Fugene HD (Promega, E2311). The transfected HEK-293 cells were distributed in a 96-well black clear bottom floor plate (Costar, 3603), followed by culture. 24 hours later, the cell culture medium was replaced with Dulbecco's Modified Eagle Medium (DMEM, 50 μl/well) supplemented with 1% fetal bovine serum (FBS). To measure the calcium concentration, 50 μl of Fluo-4 reagent (Invitrogen, F10471) was added to each well, followed by culture in a 37° C. incubator for 2 hours. During the culture, the compounds of Examples and the compounds of Comparative Examples 1 and 2 were diluted with 1×HBSS (Hank's Buffered Salt Solution) containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, resulting in the preparation of the samples for treating the cells. 2 hours after the culture began, those prepared samples were automatically injected to the cells by using Flexstation 3 (Molecular Devices). Then, intracellular calcium concentration was measured for 120 seconds by using SoftMax® Pro software. At this time, dimethylsulfoxide (DMSO) was injected to the cells for the non-treated group, followed by measuring the calcium concentration therein.
Measurement: EC50 > 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 525.67, ALogP 5.78, HBA 6, HBD 1, PSA 65.9, QED 0.28
Compound (SMILES): CC#CC(=O)N1CC[C@@H](n2cc(-c3ccc(Oc4ccccc4Cl)cc3)c3c(N)n[nH]c(=O)c32)C1
InChIKey: BFKDSWWFOVGCHG-QGZVFWFLSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: The Btk kinase assay was performed using a ADP-Glo Btk kinase assay kit purchased from Promega (Madison, Wis.). The assay was conducted according to the protocols provided in the assay kit. In brief, the enzyme reaction was carried out in the kinase reaction buffer containing Btk (2 ng/μl), ATP (1.2 μM), poly GT peptide (0.3 μM), DTT (40 nM), MnCl2 (1.4 mM), and 1×kinase buffer (included in the kit) in the presence or absence of the tested articles at various concentrations in 384-well plate at room temperature (22±1° C.) for 60 minutes. The final reaction volume for each reaction was 10 μl. Then, 4 μl of ADP-Glo reagent (included in the kit) was added into the reaction and the plate was further incubated for another 40 minutes to terminate the reaction and deplete the remaining ATP. Finally, 10 μl of the kinase detection reagent was added into each reaction to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured by a plate-reading luminometer (Victor X5 2030 multilabel reader, PerkinElmer). IC50 value was calculated using appropriate programs in GraphPad Prism by plotting the logarithm of the concentration versus percent inhibition as compared with a vehicle (DMSO) control.
Measurement: IC50 = 505 nM (pChEMBL 6.300000190734863)
Descriptors: MW 487.95, ALogP 4.22, HBA 6, HBD 2, PSA 106.24, QED 0.42
Compound (SMILES): C=CC(=O)N1C[C@H](C)N(c2nc(=O)n(-c3c(C)ccnc3C(C)C)c3nc(-c4ccccc4F)c(F)cc23)C[C@H]1C
InChIKey: RKZKGOPAMQUMJB-UXHICEINSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: The following assay conditions were employed: Coupled Nucleotide Exchange Assay: Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated with a compound dose-response titration for 5 min in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100). Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min. To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 5 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 206 nM (pChEMBL 6.690000057220459)
Descriptors: MW 558.63, ALogP 5.16, HBA 7, HBD 0, PSA 84.22, QED 0.31
Compound (SMILES): Cc1ccc(-n2cnc(C)n2)c(C(=O)N2CCC[C@@H](C)[C@H]2CNc2nc3cc(Cl)ccc3o2)c1
InChIKey: REPWWTRXQVIIOM-OPAMFIHVSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 87 nM (pChEMBL 7.059999942779541)
Descriptors: MW 478.98, ALogP 5.03, HBA 7, HBD 1, PSA 89.08, QED 0.43
Compound (SMILES): COc1ccc([C@H]2C[C@@H]2COc2nc(C)ncc2-c2ccc(C(=O)O)nn2)nc1
InChIKey: IBQSSPZWGONAPG-OCCSQVGLSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60;
Measurement: Ki = 2.68 nM (pChEMBL 8.569999694824219)
Descriptors: MW 393.4, ALogP 2.53, HBA 8, HBD 1, PSA 120.21, QED 0.65
Compound (SMILES): CCOc1cc(-c2ccc(N3CCC(Cc4ccccn4)(NC(=O)[C@@H](N)[C@@H](C)O)CC3)nc2)c2c(C#N)cnn2c1
InChIKey: NXHMJLNCFLADJF-HRFSGMKKSA-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 = 13.7 nM (pChEMBL 7.860000133514404)
Descriptors: MW 554.66, ALogP 2.47, HBA 10, HBD 3, PSA 154.69, QED 0.28
Compound (SMILES): Cn1ccc(-c2cc(C(=O)Nc3cn4c(CO)ncc4nc3-c3ccccc3)c(F)cc2C(F)(F)F)n1
InChIKey: RRMFGAXKOOLMEP-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Biological Assay: TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK); additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA). Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 510.45, ALogP 4.7, HBA 7, HBD 2, PSA 97.34, QED 0.33
Compound (SMILES): c1ccc(Sc2cc(N3CCOCC3)nc3c(-c4ccn[nH]4)nccc23)cc1
InChIKey: OSPZQSDNXAQOQT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 542 nM (pChEMBL 6.269999980926514)
Descriptors: MW 389.48, ALogP 4.01, HBA 6, HBD 1, PSA 66.93, QED 0.57
Compound (SMILES): CC1=C(N2CCC3(CCN(C[C@H](O)c4ccc5c(c4C)CCc4nnnn4-5)CC3)C2=O)COC1=O
InChIKey: VISUOJIFTHZXFP-NRFANRHFSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: A Thallium Flux Assay was performed on the compounds of the Examples. This assay has been described previously; see, e.g., PCT Published Application WO 2013/062900.Data collected for compounds in the Examples of the present invention using the Thallium Flux Assay are shown in Table 3 below. All of the tested final product compounds in the Examples (diastereomeric mixtures and individual diastereomers) had IC50 potencies less than 1 μM in the Thallium Flux Assay.
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 478.55, ALogP 1.25, HBA 9, HBD 1, PSA 113.68, QED 0.65
Compound (SMILES): N#Cc1cncc(N2CCC(C(=O)NCCC3CCN(Cc4ccccc4)CC3)CC2)c1
InChIKey: ASLJSAVYLCQAHC-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 431.58, ALogP 3.59, HBA 5, HBD 1, PSA 72.26, QED 0.73
Compound (SMILES): Cc1nnc(C(C)NC(=O)[C@@H]2CN(C3CCCCC3)CC[C@H]2NC(=O)c2cc(-c3ccc(F)cc3F)on2)o1
InChIKey: MPZYRIUZFDHEOD-GNCLGYCDSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: Tango CXCR7-bla U2OS cells are detached from culture dishes with 0.05% trypsin-EDTA and collected in growing medium (McCoy's 5A 90% (v/v), dialyzed FCS 10% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), 1 mM sodium pyruvate, P/S 1% (v/v) 50 μg/ml Hygromycin, 100 μg/ml Geneticin, 200 μg/ml Zeocin), spinned down and resuspended in assay medium (McCoy's 5A 90% (v/v), dialyzed FCS 1% (v/v), 0.1 mM NEAA, 25 mM HEPES (pH7.3), P/S 1% (v/v)). 10′000 cells per well (in 30 μl) are seeded in a 384 well plate (black-walled, clear bottom). The plate is incubated at 37° C./5% CO2 for 24 hours. Test compounds are dissolved to 10 mM in DMSO and serially diluted in DMSO to 500× of the final concentration for dose response curves. Compounds are then diluted 1:100 in assay medium to 5× of the final concentration. 10 μl/well of diluted compounds are added to the assay plate and incubated for 15 minutes at 37° C. Thereafter CXCL12/SDF1-α is diluted in assay medium to 5× of the final concentration (its EC80 value for receptor activation) and 10 μl/well are added to the assay plate. The agonist leads to activation of the receptor and therefore to b-arrestin recruitment. Compounds acting as antagonists reduce this activation. The plate is incubated for 22 hrs at 37° C. 10 μl/well of detection reagent (LiveBLAzer-FRET BIG (CCF4-AM) substrate) is transferred to the assay plate and the plate is incubated for 2 hours at room temperature protected from light. Fluorescent counts are determined (Scan1: Ex 409/20 nm, Em 460/30 nm, Scan 2: Ex 409/20 nm, Em 530/30 nm).
Measurement: IC50 = 268 nM (pChEMBL 6.570000171661377)
Descriptors: MW 542.59, ALogP 3.94, HBA 8, HBD 2, PSA 126.39, QED 0.46
Compound (SMILES): CCCCNc1ncc2c(-c3ccc(NS(=O)(=O)c4ccc(C)cc4)cc3)nn(C3CCC(O)CC3)c2n1
InChIKey: QOGMZXVIBDZEMA-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Inhibition Assay: The ectopic expression of Mer receptor tyrosine kinase (Mer) has been identified as a tumor cell survival gene product in Acute Lymphoblastic Leukemia (ALL) cells and a potential cause of ALL chemoresistance. Hence, we investigated whether the development of small molecule Mer inhibitors was possible.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 534.69, ALogP 5.29, HBA 8, HBD 3, PSA 122.03, QED 0.25
Compound (SMILES): O=C(O)c1ccc(NC(=O)C2(O)C[C@H](F)c3cc(-c4cc(Cl)ccc4-n4cnnn4)cnc32)cc1
InChIKey: VDZBJKMWKAGNPT-XNUZUHMRSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIa 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 room temperature 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. Factor XIa 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; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 40 pM (Sekisui Diagnostics) and the synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 100 μM.
Measurement: IC50 = 219 nM (pChEMBL 6.659999847412109)
Descriptors: MW 494.87, ALogP 3.32, HBA 8, HBD 3, PSA 143.12, QED 0.38
Compound (SMILES): C[C@H]1CC[C@H](Cn2c(C(=O)c3ccccc3)nc3nc(-c4noc(=O)[nH]4)nc(-c4cccc(Cl)c4)c32)CC1
InChIKey: UBYPAHMRZDLSGE-QAQDUYKDSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 72.68 nM (pChEMBL 7.139999866485596)
Descriptors: MW 529, ALogP 5.55, HBA 8, HBD 1, PSA 119.56, QED 0.29
Compound (SMILES): CCCCCCc1cccc([C@H](NCCc2ccc(C#N)cc2)C(=O)Nc2ccc(-c3cnn(C)c3)cn2)c1
InChIKey: UOIREGCXKYILFI-HKBQPEDESA-N
Target: Histone acetyltransferase p300 (SINGLE PROTEIN), Homo sapiens
UniProt: Q09472
Assay: Biochemical Assay: The full length p300 SPA assay was run following the same protocol as p300 HAT SPA assay, but used 6 nM purified full length p300 (purchased from Active Motif) in place of the purified p300-HAT domain.Select compounds were also evaluated in a H3K18Ac MSD cellular assay that measures the ability of compounds to inhibit the the acetylation of chromatin at H3K18, a process catalyzed by p300 and CBP. In a typical experiment the p300 HAT inhibitory activity inside cells of the compounds described herein was determined in accordance with the following experimental method. 20 k HCT-116 cells per well are plated in 75 μL RPMI+10% FBS media the night before treatment. Compounds plated in DMSO at 4× final concentration are resuspended in 30 μL RPMI+10% FBS, then 25 μL is combined with corresponding wells containing cells. Treated cells are incubated for 2 hr at 37° C., then lysed in 500 μL final volume and frozen at −80° C. MSD plates (Meso Scale Discovery) are coated overnight at 4° C. with 60 μL 1:500 α-total histone antibody (Millipore MAB3422) in PBS. Plates are then blocked with 5% BSA in TBST shaking at RT for 1 hr, washed, and 30 μL lysate added to each well for 2 hr shaking at RT. Plates are washed and 25 μL 1:216 α-H3K18ac antibody (CST 9675) in PBS added, then incubated for 1 hr shaking at RT. Plates are washed again, then 25 μL 1:1000 Sulfo-Tag goat α-rabbit antibody (Meso Scale Discovery R32Ab-1) in PBS is added for 1 hr shaking at RT. Plates are washed once more, then 150 μL 1× Read Buffer (MSD #R92TD-3) is added to all wells and read on MSD SECTOR Imager 2400 using the conventional read setup.
Measurement: IC50 > 2000 nM (pChEMBL 5.698999881744385)
Descriptors: MW 520.68, ALogP 5.99, HBA 6, HBD 2, PSA 95.63, QED 0.21
Compound (SMILES): CCCCc1nc(O)c(-c2nnc(Cc3noc4ccccc34)o2)c(O)c1-c1ccccc1
InChIKey: VXVRFFGAZKXOCT-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyr1)apelin-13) peptide, which was set to 100%.The examples disclosed below were tested in the APJ in vitro assays described above and were found having human APJ cyclic AMP (hcAMP) activity. The EC50 value of each compound is presented at the end of the example description.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 442.48, ALogP 5.28, HBA 8, HBD 2, PSA 118.3, QED 0.35
Compound (SMILES): C[C@@H]1C[C@H](N)C[C@H](c2ccncc2NC(=O)c2ccc(F)c3cc(N4CCC(C)(O)CC4)cnc23)C1
InChIKey: JZMTWKROJYCQEE-OTWHNJEPSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 491.61, ALogP 4.6, HBA 6, HBD 3, PSA 104.37, QED 0.49
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CN(C(=O)CCOC)c2ccccc2N(Cc2c(OC)ccc3cc(Br)ccc23)C1=O
InChIKey: OSTWQFDDJBZQBY-MBSDFSHPSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 597.51, ALogP 3.62, HBA 6, HBD 2, PSA 100.21, QED 0.39
Compound (SMILES): C[C@H](Nc1nn(C)c(=O)c2ccccc12)[C@H](c1ccc(O)cc1)N(C)C
InChIKey: YBLAMBJXTDAGPO-SCLBCKFNSA-N
Target: Histone acetyltransferase KAT2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q92831
Assay: PCAF AlphaLisa Binding Assay: His/Flag epitope tagged PCAF719-832 bromodomain was cloned, expressed and purified to homogeneity in-house. PCAF bromodomain binding and inhibition of the compounds disclosed herein was assessed by monitoring the engagement of biotinylated small molecule ligand (known to bind to the PCAF bromodomain) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate PCAF bromodomain (225 nM final) was combined with the biotinylated small molecule ligand (6 nM final) in 50 mM HEPES (pH 7.5), 75 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 15 minute incubation at room temperature AlphaLisa streptavidin acceptor beads and AlphaLisa anti-histidine donor beads were added to a final concentration of 12.5 μg/mL each. After 90 minutes of equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 352.44, ALogP 2.74, HBA 6, HBD 2, PSA 70.39, QED 0.74