lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CS(=O)(=O)Nc1nnc2cc(O[C@@H]3CCCN(Cc4ccc(F)cc4Cl)C3)c(C3CC3)cn12
InChIKey: VPMXUTIDQYAEDO-QGZVFWFLSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): The following patch voltage clamp electrophysiology studies were performed on representative compounds of the invention using human embryonic kidney cells (HEK), permanently transfected with an expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit, grown in culture media containing 10% FBS, 1% PSG, and 0.5 mg/mL G418 at 37° C. with 5% CO2. HEK cells used for the electrophysiology (EP) recordings had a passage number of less than 40 for all studies and were used within three days from the time of plating. NaV1.7 and NaV1.5 cDNAs (NM_002977 and AC137587; SCN5A, respectively) were stably expressed in HEK-293 cells. The β1 subunit was coexpressed in both the NaV1.7 and NaV1.5 cell lines.Sodium currents were measured using the patch clamp technique in the whole-cell configuration using either a PatchXpress automated voltage clamp or manually using an Axopatch 200B (Axon Instruments) or Model 2400 (A-M systems) amplifier. The manual voltage clamp protocol was as follows: Borosilicate glass micropipettes were fire-polished to a tip diameter yielding a resistance of 2-4 Mohms in the working solutions. The pipette was filled with a solution comprised of: 5 mM NaCl, 10 mM CsCl, 120 mM CsF, 0.1 mM CaC12, 2 mM MgCl2, 10 mM HEPES, 10 mM EGTA; and adjusted to pH 7.2 with CsOH. The external solution had the following composition: 140 mM NaCl, 5 mM KCl, 2 mM CaC12, 1 mM MgCl2, 10 mM HEPES; and adjusted to pH 7.4 with NaOH. In some studies, the external sodium was reduced by equimolar replacement with choline. Osmolarity in the CsF internal and NaCl external solutions was adjusted to 300 mOsm/kg and 310 mOsm/kg with glucose, respectively. All recordings were performed at ambient temperature in a bath chamber with a volume of 150 μL. Control sodium currents were measured in 0.5% DMSO. Controls and representative compounds of the invention were applied to the recording chamber through a 4-pinch or 8-pinch valve bath perfusion system manufactured by ALA Scientific Instruments.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 493.99, ALogP 3.81, HBA 7, HBD 1, PSA 88.83, QED 0.54
Compound (SMILES): CCc1nc2ccc(C(O)(c3cnnn3C)C3CN(C(C)=O)C3)cc2c(Cl)c1OCC1CC1
InChIKey: MSXKYUAZLPVWIB-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor is a fluorescence based assay that estimates ligand binding affinities by measuring the effect of a ligand on protein thermal stability (Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R. (2001) High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen 6, 429-40, and Matulis, D., Kranz, J. K., Salemme, F. R., and Todd, M. J. (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor. Biochemistry 44, 5258-66). This approach is applicable to a wide variety of systems, and rigorous in theoretical interpretation through quantitation of equilibrium binding constants (KD).In a ThermoFluor experiment where protein stability is monitored as the temperature is steadily increased, an equilibrium binding ligand causes the midpoint of an unfolding transition (Tm) to occur at a higher temperature. The shift in the melting point described as a ΔTm is proportional to the concentration and affinity of the ligand. The compound potency may be compared as a rank order of either ΔTm values at a single compound concentration or in terms of KD values, estimated from concentration response curves.
Measurement: Kd = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 469.97, ALogP 3.08, HBA 7, HBD 1, PSA 93.37, QED 0.57
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2ccc(-c3cc(B4OC(C)(C)C(C)(C)O4)cnc3N)cc2)c1
InChIKey: DFYOFNHNKPQJOJ-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: Biochemical Assay: Biochemical PDGFRβ kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 μL reaction volumes containing 36 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 μl per well of kinase buffer containing 200-300 ng purified cytoplasmic domain PDGFR-b protein (Millipore). Following a 60 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 μl of O-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm. IC50 values for compound inhibition were calculated directly from graphs of optical density (arbitrary units) versus compound concentration following subtraction of blank values.
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 462.33
Compound (SMILES): C[C@@H](O)[C@@H](CO)NC(=O)CSc1nonc1/C(=N/O)Nc1ccc(F)c(Br)c1
InChIKey: IMQYUMBZMNJMQG-RDDDGLTNSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 uM delta aminolevulinic acid for 48 h at 16° C. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 uM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 uM) or buffer alone (30 uM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at RT for 30 min. Afterwards, 10 uL of 400 uM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at RT for 60 min and reactions were quenched by addition of 10 uL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 h or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 min at 1000 ug. The resulting fluorescence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 53.9 nM (pChEMBL 7.269999980926514)
Descriptors: MW 478.3, ALogP 1.17, HBA 9, HBD 5, PSA 153.1, QED 0.12
Compound (SMILES): CNC(=O)c1cc(Oc2ccc(/N=c3\[nH]c(=O)[nH]c(=O)n3Cc3ccc(C)cc3)cc2)ccn1
InChIKey: WHVYHZBJJFUTKM-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 458.48, ALogP 2, HBA 7, HBD 3, PSA 134.23, QED 0.41
Compound (SMILES): C=CC(=O)N1CCC(Oc2cc3c(Nc4ccc(OC(F)(F)F)c(Cl)c4)ncnc3cc2OC)CC1
InChIKey: SPBBVFQOXOQDHG-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 522.91, ALogP 5.49, HBA 7, HBD 1, PSA 85.81, QED 0.41
Compound (SMILES): COc1ccc2c(c1)[C@]1(C[C@H]1c1ccc3c(-c4ccc(OCCN(C)C)cc4)n[nH]c3c1)C(=O)N2
InChIKey: TVJIKXZQJVMALM-FIPFOOKPSA-N
Target: Serine/threonine-protein kinase PLK4 (SINGLE PROTEIN), Homo sapiens
UniProt: O00444
Assay: Inhibition Assay: PLK4 activity was measured using an indirect ELISA detection system. Dephosphorylated GST-PLK4 (4 nM) was incubated in the presence of 15 μM ATP (Sigma cat#A7699), 50 mM HEPES-Na2+ pH 7.4, 10 mM MgCl2, 0.01% Brij 35 (Sigma cat#03-3170), in a 96 well microtitre plate pre-coated with MBP (Millipore cat#30-011). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (50 mM TRIS-C1 pH 7.4 and 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat#9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat#1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat#T0440). The colourimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N sulphuric acid), and quantified by detection at 450 nm with either a monochromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 468.56, ALogP 4.56, HBA 5, HBD 2, PSA 79.48, QED 0.42
Compound (SMILES): CC(C)(C)CNC(=O)c1cnn2c(C3CCNCC3)cc(=O)[nH]c12
InChIKey: BCIZEXWHUYUFDO-UHFFFAOYSA-N
Target: Tissue-type plasminogen activator (SINGLE PROTEIN), Homo sapiens
UniProt: P00750
Assay: Plasma-based Clot Lysis Assay: The clot-lysis test system configures the kinetics of clot formation and degradation in vitro and allows quantifying modulation of the process by selected test compounds.The test compounds were dissolved in 1% acetic acid and further complemented with an equal volume of DMSO. The resulting stock solutions were serially diluted in 0.5% acetic acid/50% DMSO. 1 μL aliquots of these solutions were placed into 384 well microplates (Greiner, black, transparent bottom), followed by 30 μL of diluted human citrated plasma (platelet-poor, final concentration: 5%; supplemented with fibrinogen, final concentration: 3 μM; dilution buffer: 20 mM HEPES, 150 mM NaCl, 0.01% Brij (pH 7)). The reactions were started by addition of 20 μL of CaCl2 (final concentration: 10 mM), and tPA (tissue plasminogen activator, final concentration: 0.2 nM) in dilution buffer, followed by an additional volume of 20 μL dilution buffer for improved mixing. The reactions were incubated at 37° C. Clot formation and degradation was monitored spectrophotometrically by kinetic optical density measurements at 405 nm. IC50 values were determined by comparing the resulting time courses with the time course of a blank control reaction.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 331.42, ALogP 1.27, HBA 5, HBD 3, PSA 91.29, QED 0.79
Compound (SMILES): O=C1CCc2cc(/C=C/C(=O)N3CC4C=C(c5ccco5)CC4C3)cnc2N1
InChIKey: MSRQRSNPKYEGCJ-ZZXKWVIFSA-N
Target: Enoyl-[acyl-carrier-protein] reductase [NADPH] FabI (SINGLE PROTEIN), Staphylococcus aureus
UniProt: Q6GI75
Assay: Enzyme Inhibition Assay: FabI enzyme inhibition assays were carried out in half-area, 384-well microtitre plates. Compounds were evaluated in 40-μl assay mixtures containing 100 mM NaADA, pH 6.5 (ADA=N-[2-acetamido]-2iminodiacetic acid), 250 μM crotonoyl-CoA, 625 μM NADH and 50 μg/ml S. aureus ATCC 29213 FabI. Inhibitors were typically varied over the range of 50 to 0.39 μM. The reaction mixtures were incubated for 30 minutes at room temperature and the reaction was stopped by adding 200 mM Tris buffer (pH 9.0) to create a pH-shift. The consumption of NADH was monitored by measuring the change in absorbance at 340. By comparing sample readings to those of negative (absence of compound) and positive (absence of enzyme) controls, the percent inhibition of enzymatic activity of the compounds was determined. A best-fit curve is fitted by a minimum of squares method.
Measurement: IC50 = 1650 nM (pChEMBL 5.78000020980835)
Descriptors: MW 375.43, ALogP 3.13, HBA 4, HBD 1, PSA 75.44, QED 0.84
Compound (SMILES): CN1CCN(C(=O)c2ccc(-c3cn(C)c(=O)c4[nH]ccc34)cc2)CC1
InChIKey: KQAKWCUGTBVOMY-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 350.42, ALogP 1.92, HBA 4, HBD 1, PSA 61.34, QED 0.77
Compound (SMILES): CC(=O)c1nn(CC(=O)N2[C@@H]3C[C@@H]3C[C@H]2C(=O)N[C@@H]2CCC[C@H](C(F)(F)F)C2)c2ccc(OCc3ncccn3)cc12
InChIKey: BFTCULYBMYFCAZ-PCVLGWKGSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Complement Factor D Assay: Method 1: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter. IC50 values are calculated from percentage of inhibition of complement factor D-activity as a function of test compound concentration.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 584.6, ALogP 3.83, HBA 8, HBD 1, PSA 119.31, QED 0.4
Compound (SMILES): OCCCONc1nonc1/C(=N/O)Nc1ccc(F)c(Cl)c1
InChIKey: VLNCPRXDKPHTIE-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Inhibitory Activity Assay: 1. Materials, Kits and EquipmentsSodium L-ascorbate (Cat: A4034-100G, SIGMA)4-(dimethylamino)benzaldehyde (Cat: 156477-25g, SIGMA)Trichloroacetic acid (Cat: T0699-100ML, SIGMA)L-Tryptophan (Cat: T8941-25G, SIGMA)Methylene blue (Cat: M9140-25G, SIGMA)Potassium dihydrogen phosphate (Cat: 10017618, Sinopharm Chemical Reagent)Disodium hydrogen phosphate (Cat: 20040618, Sinopharm Chemical Reagent)Constant temperature water tank (Cat: DK-8D, Shanghai Jinghong Experimental Equipment)Multifunctional microplate reader (Cat: M5, Molecular Devices)96-well reaction plate (Cat: 3590, costar)IDO1 protease (commercially available)Desktop Microplate Reader: SpectraMax M5 Microplate Reader (Molecular Devices)Test compounds: self-madePositive control agent: INCB024360 (commercially available)2. Reagent Preparation100 mM PBS:100 mM disodium hydrogen phosphate and 100 mM potassium dihydrogen phosphate mixed in a ratio of 3:5, pH 6.5IDO1 assay buffer:100 mM PBS containing 400 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue and 1000 U/ml catalase, pH 6.530% trichloroacetic acidddH2 O solution of 30% trichloroacetic acidEhrlich reagent1% (w/v) diluted solution of 4-(dimethylamino) benzaldehyde compoundAll compounds were dissolved with DMSO. During the assay, each compound was diluted to a concentration as needed. The compound of each concentration was added to multi-wells, and the final concentration of DMSO was controlled at 1%.3. Test Methoda.) the reaction mixture was prepared by adding 50 nM IDO1 and the desired concentration of the test compound to 100 μL of IDO1 assay buffer. IDO1 and assay buffer need to be preheated to 37° C.b.) The mixture was reacted in a constant temperature water tank at 37° C. for 30 minutes.c.) 50 μL of 30% trichloroacetic acid was added.d.) The above mixture was reacted in a constant temperature water tank at 52° C. for 30 minutes.e.) The reaction mixture was centrifuged at 12000 g for 10 minutes at room temperature.f.) 100 μL of the obtained supernatant and 100 μL of Ehrlich reagent were mixed.g.) the absorbance at 480 nm was measured using an M5 microplate reader.4. Data AnalysisInhibition rate=(ODpostive−ODsample)/(ODpositive−ODnegative)*100%5. Results and Discussion
Measurement: IC50 = 93 nM (pChEMBL 7.03000020980835)
Descriptors: MW 345.72, ALogP 1.84, HBA 8, HBD 4, PSA 125.03, QED 0.2
Compound (SMILES): CC1(C)c2cccc(C(=O)NO)c2CN1c1ccc(C(F)(F)F)c(C#N)n1
InChIKey: LIGHYZKMPYJESI-UHFFFAOYSA-N
Target: Histone deacetylase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96DB2
Assay: In Vitro Histone Deacetylase Assay I: The probe binding HDAC11 assay was performed using a time resolved fluorescence (TRF) assay format. Recombinant N-terminal GST tag full-length human HD AC 11 was expressed and purified from baculovirus in Sf9 insect cells (SignalChem, #H93-30G-1000). Each assay was performed in 1536 black well microplates (Corning, #3936) in a final volume of 8 μL in assay buffer containing 50 mM HEPES (pH 7.5), 50 mM KCl, 50 mM NaCl, 0.5 mM GSH (L-Glutathione reduced, Sigma #G4251), 0.03% BGG (0.22 μM filtered, Sigma, #G7516-25G), and 0.01% Triton X-100 (Sigma, #T9284-10L). 100 nL of 10-point, 3-fold serial dilution in DMSO was pre-dispensed into respective wells of 1536 assay plates for a final test concentration range of 25 μM to 1.3 nM respectively. The final concentration in the assay of HD AC 11 and probe (a fluorescein labeled HD AC 11 inhibitor) was 2.5 nM and 20 nM respectively. 4 μL of 2× probe and 2× anti-GST Terbium (Cisbio, #61GSTXLB) was added to assay plates followed by 4 μL of 2×HDAC11. Plates were incubated for 16 hours at room temperature before time resolved fluorescence was read on the Envision (Excitation at 340 nm, and Emission at 485 nm and 535 nm, Perkin Elmer).Data from HD AC 11 Assays were reported as percent inhibition (inh) compared with control wells based on the following equation: % inh=1−((FLU−AveLow)/(AveHigh−AveLow)) where FLU=measured time resolved fluorescence. AveLow=average time resolved fluorescence of no enzyme control (n=32). AveHigh=average time resolved fluorescence of DMSO control (n=32). IC50 values were determined by curve fitting of the standard 4 parameter logistic fitting algorithm included in the Activity Base software package: IDBS XE Designer Model205. Data is fitted using the Levenburg Marquardt algorithm.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 376.34, ALogP 3.35, HBA 5, HBD 2, PSA 89.25, QED 0.62
Compound (SMILES): O[C@@H]1[C@H](O)CS[C@H]1c1nnc2c(NCCc3c(F)cccc3F)nccn12
InChIKey: ZUUCATNXUITGAB-MGPQQGTHSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Assay for Binding Affinity for Adenosine Receptor A3: HEK-293 cell membrane homogenates (32 μg protein), in which A3 adenosine receptors were expressed, are incubated for 120 min at 22° C. with 0.15 nM [1251]AB-MECA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 2 UI/ml ADA. Nonspecific binding is determined in the presence of 1 μM IB-MECA. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is IB-MECA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 393.42, ALogP 1.57, HBA 8, HBD 3, PSA 95.57, QED 0.61
Compound (SMILES): CCOc1cnc2c(-c3nc4cc(F)c(O[C@@H]5CC[C@@H]5OC(=O)Nc5cnc(OCCO)nc5)cc4s3)cc(Cl)cc2c1
InChIKey: LSHFZHKMWJWKQU-PKTZIBPZSA-N
Target: Proteinase-activated receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI0
Assay: FLIPR Assay in PAR4-Expressing HEK293 Cells: FLIPR-based calcium mobilization assay in HEK293 cells was used to measure PAR4 antagonism agonism, and selectivity against PAR1. The activity of the PAR4 antagonists of the present invention were tested in PAR4 expressing cells by monitoring H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-Val-Lys-Asn-Gly-NH2-induced intracellular calcium mobilization. Counter screens for agonist activity and PAR1 antagonist activity were also performed. Briefly, PAR/PAR4-expressing HEK293 cells were grown in DMEM (Life Technology, Grand Island, N.Y.) containing 10% heat-inactivated FBS, 1% Penicillin-Streptomycin, 10 μg/mL blasticidin, and 100 μg/mL Zeocin at 37° C. with 5% CO2. Cells were plated overnight prior to the experiment in a black 384-well Purecoat Amine clear bottom plate (Becton Dickinson Biosciences, San Jose, Calif.) at 10,000 cells/well in 30 μL growth medium and incubated in a humidified chamber at 37° C. with 5% CO2 overnight. Prior to compound addition, the cell medium was replaced with 40 μL of 1× calcium and magnesium-containing Hank's Balanced Saline Solution (HBSS) (with 20 mM HEPES) and 1:1000 diluted fluorescent calcium indicator (Codex Biosolutions, Gaithersburg, Md.). After a 30 minute incubation period at 37° C. and a further 30 minute incubation and equilibration period at room temperature, 20 μL test compound (diluted in 1×HBSS buffer) was added at various concentrations at 0.17% dimethyl sulfoxide (DMSO) final concentration. Changes in fluorescence intensity were measured using a Functional Drug Screening System (FDSS, Hamamatsu, Japan) to determine agonist activities. The cells were then incubated for 30 minutes at room temperature followed by addition of 20 μL of agonist peptide for antagonist activity measurement. The PAR4 agonist peptide (H-Ala-Phe(4-F)-Pro-Gly-Trp-Leu-V al-Lys-Asn-Gly-NH2) and the PAR1 agonist peptide (SFFLRR) were routinely tested to ensure a proper response at the EC50 value in the assay (5 μM for PAR4 agonist peptide and 2 μM for PAR1 agonist peptide). Compound potency was derived from 11-point concentration-response curves.
Measurement: IC50 = 9.2 nM (pChEMBL 8.039999961853027)
Descriptors: MW 626.07, ALogP 6.02, HBA 11, HBD 2, PSA 137.81, QED 0.19
Compound (SMILES): Cc1cc(-n2c(C(=O)NC3CC3)nc3cccnc32)c(Cl)cc1F
InChIKey: WVSKTYGNRIZTKH-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Scintillation Proximity Assay (SPA): The PDE4B and 4D assays use scintillation proximity assay (SPA) technology to measure the inhibition of human recombinant PDE4B and PDE4D enzyme activity by compounds in vitro. The PDE4B and 4D assays are run in parallel using identical parameters, except for the concentration of enzyme (32 pM PDE4B and 16 pM PDE4D). The assays are performed in a 384-well format with 50 μL assay buffer (50 mM Tris pH 7.5; 1.3 mM MgCl2; 0.01% Brij) containing enough PDE4B and PDE4D to convert 20% of substrate (1 μM cAMP consisting of 20 nM 3H-cAMP+980 μM cold cAMP) and a range of inhibitors. Reactions are incubated for 30 min at 25° C. The addition of 20 μL of 8 mg/mL yitrium silicate SPA beads (PerkinElmer) stops the reaction. The plates are sealed (TopSeal, PerkinElmer) and the beads are allowed to settle for 8 hrs, after which they are read on the Trilux Microbeta overnight.
Measurement: IC50 = 16600 nM (pChEMBL 4.78000020980835)
Descriptors: MW 344.78, ALogP 3.41, HBA 4, HBD 1, PSA 59.81, QED 0.79
Compound (SMILES): CCc1nc2ccc(N3CCN(CC(=O)N4CC(O)C4)CC3)nn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: QCNMBIBDGOKMBW-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: Compound IC50 values are determined in a fluorescent hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal. 10 uL of a dilution series of compound, starting from 20 uM highest concentration, dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 ug/mL or 0.64 ug/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 uL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 uL of 0.75 uM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 550.66, ALogP 2.65, HBA 10, HBD 1, PSA 93.34, QED 0.38
Compound (SMILES): CC[C@@H]1CCC[C@@H](NC(=O)[C@@H]2C[C@@]3(CO)C[C@H]3N2C(=O)Cn2nc(C(N)=O)c3ccccc32)C1
InChIKey: AZGKNMCASBBINL-FFDDBACDSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Human Complement Factor D Assay: Method 1: Recombinant human factor D (expressed in E. coli and purified using standard methods) at 10 nM concentration is incubated with test compound at various concentrations for 1 hour at room temperature in 0.1 M Hepes buffer, pH 7.5, containing 1 mM MgCl2, 1 M NaCl and 0.05% CHAPS. A synthetic substrate Z-Lys-thiobenzyl and 2,4-dinitrobenzenesulfonyl-fluoresceine are added to final concentrations of 200 μM and 25 μM, respectively. The increase in fluorescence is recorded at excitation of 485 nm and emission at 535 nm in a microplate spectrofluorimeter. IC50 values are calculated from percentage of inhibition of complement factor D-activity as a function of test compound concentration.
Measurement: IC50 = 403 nM (pChEMBL 6.389999866485596)
Descriptors: MW 467.57, ALogP 1.57, HBA 6, HBD 3, PSA 130.55, QED 0.57
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(N5CCN(C(=O)C6CCCN6)CC5)nc4c3)ccc2o1
InChIKey: OZKWIUJHEVVDIK-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 443.51, ALogP 2.42, HBA 8, HBD 2, PSA 113.41, QED 0.5
Compound (SMILES): C=CC(=O)N[C@@H]1CCCC[C@@H]1Nc1ncc2c(n1)N(Cc1ccccc1)C(=O)N(c1c(Cl)c(OC)cc(OC)c1Cl)C2
InChIKey: WFHLORBBLUARRC-RTWAWAEBSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Omnia Assay: Omnia Assay Protocol for Potency Assessment Against FGFR 4 Enzyme. a) [FGFR4-WT]=10 nM, [ATP]=300 uM, [Y10-Sox]=10 uM (ATP KMapp 300 uM): A 10x stock solution of FGFR4-WT (PR4380C), (from Invitrogen, Carlsbad, Calif.) corresponding to method a in Table 7, was prepared as described below. Alternatively, a 10x stock solution of FGFR4-WT (F01-11G), (SignalChem, Richmond, BC) corresponding to method b in Table 7, was prepared as described below. A solution of 1.4xATP (AS001A) and 5x Tyr-Sox conjugated peptide substrate (KNZ3101) were prepared in 1x kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10x stock, KB002A) and 0.2 mM DTT (DS001A). 5 uL of FGFR4 was pipetted into a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.), containing a 0.5 uL volume of 100% DMSO. The serially diluted compounds were prepared on a Tecan EVO100. A second addition of 10 ul of Tyr-Sox FGFR4 substrate was added to each well and the kinase reactions were started with the addition of 35 uL of 1.4xATP. The reactions were monitored every 71 seconds for 240 minutes at λex360/λem485 in a Synergy plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics (R2, 95% confidence interval, absolute sum of squares). Initial velocity (0 minutes to ~60 minutes) from each reaction was determined from the slope of a plot of relative fluorescence units vs time (seconds) and then plotted against inhibitor concentration to estimate IC50 from log [Inhibitor] vs Response (Variable Slope model in GraphPad Prism from GraphPad Software (San Diego, Calif.)).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 611.53, ALogP 5.97, HBA 7, HBD 2, PSA 108.92, QED 0.29
Compound (SMILES): N#Cc1cc(-c2cc3c(-c4ccc(OC5CCOCC5)c(C#N)c4)ccnc3[nH]2)ccc1OC1CCOCC1
InChIKey: JASHHRMOVFCTDG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Biological Assay for TBK1 and IKKs: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phopshorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKs were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2× Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 24.1 nM (pChEMBL 7.619999885559082)
Descriptors: MW 520.59, ALogP 5.76, HBA 7, HBD 1, PSA 113.18, QED 0.34
Compound (SMILES): C[C@H](O)C(=O)N1CC[C@H](Oc2ccc(-c3ncnc(Nc4ccc(O[C@H]5CCN(C)C[C@H]5F)cc4)n3)cc2C#N)[C@H](F)C1
InChIKey: IHWPMUHWLMAGRM-PJWMMJPUSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 593.64, ALogP 3.27, HBA 10, HBD 2, PSA 136.73, QED 0.4
Compound (SMILES): Cc1cc2c(cc1C(=O)O)nc(Cc1ccc(OC(F)(F)F)cc1)n2[C@H]1C[C@@H](C)CC(C)(C)C1
InChIKey: CCTMIENZLLMTEM-QAPCUYQASA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Mutant IDH1 R132H Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μL and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μL of detection mix (0.75 μg/mL Luciferase, 0.02 U/mL Oxidoreductase, 4 μg/mL FMN, 2 μL/mL decanal/ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 474.52, ALogP 6.92, HBA 4, HBD 1, PSA 64.35, QED 0.44
Compound (SMILES): Cc1nc(-c2cccc(C(F)(F)F)c2)[se]c1C(=O)O
InChIKey: MFFJRDFTNYKHRZ-UHFFFAOYSA-N
Target: Xanthine dehydrogenase/oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P47989
Assay: Inhibition Assay: The inhibition of the activity of xanthine oxidase (XO) is tested through a coupled enzymatic reaction of xanthine oxidase, horseradish peroxidase (HRP) and a substrate thereof. First, xanthine oxidase oxidizes hypoxanthine to produce xanthine and hydrogen peroxide, and further oxidizes xanthine to produce uric acid and hydrogen peroxide. Then, hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Ampliflu Red) under catalytic action of horseradish peroxidase so as to produce resorufin, a compound with strong fluorescence. The fluorescence intensity of resorufin is determined by using a fluorescence microplate, which is in direct proportion to the activity of xanthine oxidase.
Measurement: IC50 = 25.4 nM (pChEMBL 7.590000152587891)
Descriptors: MW 334.15
Compound (SMILES): CCNc1nc(Nc2cc(C34C[C@@H]3COC4=O)nn2C)ncc1C(F)(F)F
InChIKey: UBODIAHOSAKGSZ-ARAKYBCZSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay: Materials:LRRK2 G2019S enzymeSubstrate (LRRKtide)ATPTR-FRET dilution bufferpLRRKtide antibody384-well assay plateDMSOEnzyme Reaction Conditions50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35.2 mM DTT5 nM LRRK2134 μM ATP60 minute reaction time23° C. reaction temperature10 μL total reaction volumeDetection Reaction Conditions1× TR-FRET dilution buffer10 mM EDTA2 nM antibody23° C. reaction temperature10 μL total reaction volumeCompounds were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.To perform the enzyme reaction, 5 μL of LRRKtide substrate and kinase mixture in assay buffer was added to all wells of the assay plate. The plate was centrifuged to concentrate the mixture at the bottom of the wells. The assay plate was incubated at 23° C. for 20 minutes. Following incubation, 5 μL of 2×ATP in assay buffer was added to each well, and plates were centrifuged to concentrate the mixture at the bottom of the wells. The plate was incubated at 23° C. for 60 minutes.To perform the detection of the reaction, EDTA completely mixed in TR-FRET dilution buffer was added to antibody reagent. 10 μL of detection reagent was added to all wells of each well of the assay plate and the plate was centrifuged to concentrate the mixture at the bottom of the wells. The plate was then incubated at 23° C. for 60 minutes. Plates were read on Perkin Elmer Envision 2104 instrument in TR-FRET mode using 340 nm excitation filter, 520 nm fluorescence emission filter, and 490 or 495 nm terbium emission filter.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 382.35, ALogP 2.22, HBA 8, HBD 2, PSA 93.96, QED 0.77
Compound (SMILES): CC(C)CCc1ccc2c(c1)N(C)C(=O)C(NC(=O)c1cc(Oc3ccccc3)ccn1)CO2
InChIKey: QFFBMIQCNYGFCC-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: biochemical assay using the ADP-Glo technology: ADP-Glo (Promega, Madison, Wis., USA) reagents were thawed at ambient temperature. Kinase Detection Reagent was prepared by mixing kinase detection buffer with the lyophilized kinase detection substrate.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 μM 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 μl 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 column 1) add 2× rxn 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 5.0ul.The plate was quickly centrifuged and the reaction was incubated at 30° C. for 30 minutes. Adding 5 μl of ADP-Glo terminated the reaction. The plate was quickly centrifuged and the reaction was incubated at room temperature for 40 minutes. Kinase Detection Reagent was then added and incubated at room temperature for 30 minutes. The relative light unit (RLU) of kinase reaction was determined by luminescent (Luminescence 0.1 s) using a Wallac Victor2 Luminometer (PerkinElmer, Waltham, Mass., USA).
Measurement: IC50 = 314 nM (pChEMBL 6.5)
Descriptors: MW 459.55, ALogP 4.62, HBA 5, HBD 1, PSA 80.76, QED 0.56
Compound (SMILES): O=C(c1cccc(Oc2ccccc2)c1)N1CCN2C[C@@H](c3ccc(Cl)cc3)CC[C@@H]2C1
InChIKey: LSUSDOCKIJTLBH-LADGPHEKSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: Inhibition Assay: In one embodiment, the present invention provides compounds of formula (Ie) and their pharmaceutically acceptable salts as described herein, wherein said compounds of formula (Ie) and their pharmaceutically acceptable salts have an IC50 for MAGL below 25 μM, preferably below 10 μM, more preferably below 5 μM as measured in an assay comprising the steps of: list-style a) providing a solution of a compound formula (e), or a pharmaceutically acceptable salt thereof, in DMSO; b) providing a solution of MAGL (recombinant wild-type) in assay buffer (50 mM tris(hydroxymethyl)aminomethane; 1 mM ethylenediaminetetraacetic acid); c) adding 1 μL of compound solution from step a) to 19 μL of MAGL solution from step b); d) shaking the mixture for 1 min at 2000 rpm; e) incubating for 15 min at RT; f) adding 20 μL of a solution of 4-nitrophenlyacetate in assay buffer (50 mM tris(hydroxymethyl)aminomethane; 1 mM ethylenediaminetetraacetic acid, 6% EtOH); g) shaking the mixture for 1 min at 2000 rpm;h) incubating for 5 min at RT; i) measuring the absorbance of the mixture at 405 nm a first time; j) incubating a further 80 min at RT;k) measuring the absorbance of the mixture at 405 nm a second time; l) substracting the absorbance measured under i) from the absorbance measured under k) and calculating the slope of absorbance; wherein: i) the concentration of the compound of formula (Ie), or the pharmaceutically acceptable salt thereof in the assay after step f) is in the range of 25 μM to 1.7 nM; ii) the concentration of MAGL in the assay after step f) is 1 nM; >iii) the concentration of 4-nitrophenylacetate in the assay after step f) is 300 μM; and iv) steps a) to 1) are repeated for at least 3 times, each time with a different concentration of the compound of formula (Ie), or the pharmaceutically acceptable salt thereof.
Measurement: IC50 = 1160 nM (pChEMBL 5.940000057220459)
Descriptors: MW 446.98, ALogP 5.84, HBA 3, HBD 0, PSA 32.78, QED 0.5
Compound (SMILES): Cc1cccc(OCC(C)C)c1-n1nc2c(c1-c1ccc(NC(N)=O)cc1)CN(C(=O)OC(C)(C)C)CC2
InChIKey: IFWVYORULMQTLI-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 519.65, ALogP 5.67, HBA 6, HBD 2, PSA 111.71, QED 0.44