lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN(C)[C@H]1CCCC[C@@H]1Nc1cc(F)c(S(=O)(=O)Nc2cccc(F)n2)cc1C1CC1
InChIKey: JCHPFDRJFNLXRK-HKUYNNGSSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following voltage clamp electrophysiology studies were performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAs for Nav1.7 (NM_002977) and Nav1.5 (AC137587) were stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents were measured in the whole-cell configuration using Syncropatch 384PE (NanIon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10l cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 μl. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.For voltage protocol, an holding potential of −50 mV is applied during the whole experiment. A depolarizing step is applied to −10 mV for 10 ms, followed by a hyperpolarization step to −150 mV for 20 ms to allow channel recovery from inactivation. A second depolarizing step is applied from −150 mV to −10 mV for 10 ms, where currents were measured to derive blocking effects of compounds. Inhibition is determined based on 7.5 min of compound incubation.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 450.56, ALogP 4.32, HBA 5, HBD 2, PSA 74.33, QED 0.61
Compound (SMILES): Cn1ccc(C(=O)Nc2ccc(-c3n[nH]c(=O)c4ccccc34)cc2)n1
InChIKey: WCJLHBRQUDVGPS-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 345.36, ALogP 2.58, HBA 5, HBD 2, PSA 92.67, QED 0.6
Compound (SMILES): C#CCNC(=O)C[C@@H]1CCc2cc(C(O)(C(F)(F)F)C(F)(F)F)ccc2N1S(=O)(=O)c1ccc(F)cc1
InChIKey: FPYMBYPOUSZDTI-KRWDZBQOSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Spa Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 50 nM RORγ (A262-S507) is incubated with test compound at varying concentrations for 15 min at room temperature in PBS buffer containing 0.5% fatty acid free BSA. 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 15 min. 4 mg/mL of Ysi Copper HIS-TAG SPA Beads (Perkin Elmer) are added, and the mixture is incubated for 30 min. The reaction is read on a MicroBeta Trilux scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 18.9 nM (pChEMBL 7.71999979019165)
Descriptors: MW 552.47, ALogP 3.79, HBA 4, HBD 2, PSA 86.71, QED 0.42
Compound (SMILES): Cc1ncc(-c2ccc3nc(-c4nc5ccccc5[nH]4)oc3c2)cc1NS(=O)(=O)CC(F)(F)F
InChIKey: YPNACPKQRZIVHY-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay (PI3K-γ): [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from Perkin-Elmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from Sigma-Aldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 487.46, ALogP 5.05, HBA 6, HBD 2, PSA 113.77, QED 0.36
Compound (SMILES): C[C@@H](O)CNS(=O)(=O)c1ccc(-c2c[nH]c3cc(F)ccc23)cc1
InChIKey: HAVXYUYSVFFDAF-LLVKDONJSA-N
Target: Tryptophan 2,3-dioxygenase (SINGLE PROTEIN), Homo sapiens
UniProt: P48775
Assay: Assay for TDO2 enzymatic activity determination: To measure the TDO2 activity, the procedure described in Dolusic et al. J. Med. Chem.; 2011, 54, 5320-533 was adapted: the reaction mixture contained (final concentrations) potassium phosphate buffer (50 mM, pH 7.5), ascorbic acid (0.25 M), methylene blue (0.125 μM), catalase (40 units/mL, from bovine liver, Sigma), and human recombinant TDO2 enzyme (prepared as described in Dolusic et al. J. Med. Chem.; 2011, 54, 5320-5334; 0.9 μg) without or with the compounds of the present invention at the indicated concentrations (total volume 112.5 μL). The reaction was initiated by the addition of 37.5 μL of L-Trp (final concentration 1 mM) at room temperature. The reaction was conducted at room temperature during one hour and stopped by the addition of 30 μL of 30% (w/v) trichloroacetic acid.To convert N-formylkynurenine into kynurenine, the reaction mixture was incubated at 65° C. for 30 min. Then 150 μL of the reaction mixture was mixed with 120 μL of 2.5% (w/v) 4-(dimethylamino)-benzaldehyde in acetic acid and incubated for 5 min at room temperature. Kynurenine concentrations were determined by measuring the absorbance at 480 nm. A standard curve was made with pure kynurenine. The TDO activity was measured as described above using ten serial concentrations of the compounds of the present invention. Data were fitted using the Prism software (GraphPad Software, Inc.) using standard parameters.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 348.4, ALogP 2.63, HBA 3, HBD 3, PSA 82.19, QED 0.66
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cnn(C5CCN(C(C)=O)C5)c4)cc3C3(CC3)C2=O)c1F
InChIKey: MRDCFVVSPLVYAF-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: FGFR Enzymatic Assay 1: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 1 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 4 hours.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 523.54, ALogP 3.61, HBA 7, HBD 0, PSA 89.79, QED 0.51
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(OCCN3CCN(C(C)=O)CC3)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)C1
InChIKey: FFJZZMMXJGTYKF-LITSAYRRSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 625.72, ALogP 3.24, HBA 8, HBD 2, PSA 104.2, QED 0.34
Compound (SMILES): CCC(C(=O)Nc1ccc(C(=O)OC[C@@H](C(=O)O)N2CCCC2)cc1)n1cc(OC)c(-c2cc(Cl)ccc2C#N)cc1=O
InChIKey: WRBDXQGVLBWOER-AMVUTOCUSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). 1 μl of the diluted substance solutions is placed into each of the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride; 5 mM of calcium chloride; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 mM of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBz1)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 607.06, ALogP 4.34, HBA 9, HBD 2, PSA 150.96, QED 0.3
Compound (SMILES): N#C/C(=C\c1ccc2cn[nH]c2c1)C(N)=O
InChIKey: XLGYCNBBWHAJDG-YCRREMRBSA-N
Target: Serine/threonine-protein kinase Nek2 (SINGLE PROTEIN), Homo sapiens
UniProt: P51955
Assay: Nek2 Inhibition Assay: For RSK2 kinase assays, see Example 64 above. Full-length human T175A NEK2 (referred to as NEK2) was expressed and purified as previously described (Knapp S. et al. J. Biol. Chem., 2007, 282: 6833-6842). The C22V mutant of NEK2 was generated by Quikchange mutagenesis (Stratagene) and was indistinguishable from NEK2 in kinase activity assays. NEK2 kinases (60 nM) in 20 mM Hepes, pH 7.6, 10 mM MgCl2, 1 mM EDTA, 0.2 mg/mL BSA, and 100 μM ATP were pre-incubated with inhibitors (8-10 concentrations, in duplicate) for 30 min at room temperature. Kinase reactions were initiated by the addition of 0.4 μCi/μL of γ-32P-ATP (6000 Ci/mmol, NEN) and 2.37 mg/mL β-casein (Sigma) and incubated for 30 minutes at room temperature.
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 212.21, ALogP 0.96, HBA 3, HBD 2, PSA 95.56, QED 0.57
Compound (SMILES): C=CC(=O)N1CCOc2c1ccc1ncnc(Nc3ccc(Oc4ccccn4)cc3)c21
InChIKey: AQHZJMAJXAKVPW-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of EGFR and HER2 Kinase Activity Assay: The assay for the inhibition of EGFR and HER2 kinase activity by small molecular compounds was carried out using the method as follows:1) Dilution of the compoundsIn a 96-well plate a, the compounds were diluted with DMSO using a 3-fold gradient dilution to form 11 concentrations, the 12th concentration is pure DMSO (as a positive control); and in a new 96-well plate b the above solutions were diluted 25 times with ultrapure water (DMSO concentration is 4%).2) Transferring the compounds to 384-well plateThe compound solutions diluted with ultrapure water in the 96-well plate b above was transferred to the corresponding wells of a 384-well plate in duplicate.3) Addition of 4×kinase solution: 2.5 μl of the above 4× kinase solution was taken using multichannel pipette and added to the corresponding reaction wells of the 384-well plate, mixed well and pre-reacted at room temperature for 5 minutes.4) Addition of 2×substrate/ATP mixed solution: 5 μl of the above 2×substrate/ATP mixed solution was taken using multichannel pipette and added to the corresponding reaction wells of the 384-well plate.5) Negative control: negative control wells were set in the 384-well plate, and 2.5 μl 4×substrate, 2.5 μl 4×enzyme solution, 2.5 μl 1×Kinase Assay Buffer and 2.5 μl ultrapure water containing 4% DMSO were added to each well.6) Mixed by centrifugation and kept at room temperature for 2 hours in the dark.7) Termination of the enzymatic reaction:5 μl of the above 4× stop solution was pipetted to the corresponding wells of the 384-well plate, centrifuged and mixed, and reacted at room temperature for 5 minutes.8) Development reaction:5 μl of the above 4× detection solution was pipetted into the corresponding wells of the 384-well plate, centrifuged and mixed, and reacted at room temperature for 1 hour.9) The 384-well plate was placed into a microplate reader and the signal was detected using the corresponding program.10) IC50 analysis:Well reading value=10000*EU665 value/EU615 valueInhibition rate=(reading value of positive control well−reading value of experimental well)/(reading value of positive control well−reading value of negative control well)*100%Corresponding IC50s can be calculated by entering the drug concentrations and the corresponding inhibition rates into GraphPad Prism 5.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 425.45, ALogP 4.47, HBA 7, HBD 1, PSA 89.47, QED 0.47
Compound (SMILES): COCc1cn(Cc2cccc(F)c2)c2cc(-c3cc(NC(=O)[C@@H]4CCCNC4)ncc3Cl)cnc12
InChIKey: YRHIHMXZWMOITK-GOSISDBHSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: 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 615665 nm).
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 508, ALogP 5.02, HBA 6, HBD 2, PSA 81.07, QED 0.37
Compound (SMILES): C=CC(=O)N(C)Cc1ccc(Nc2nc(Nc3cccc(OCCN4CCCC4=O)c3)ncc2F)cc1
InChIKey: BRQXWVJKMWGRCE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Omnia Assay Protocol : Briefly, 10× stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13×ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT (DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 27° C. with a 0.5 μL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 μL of the ATP/Tyr-Sox peptide substrate mix and monitored every 30-90 seconds for 60 minutes at λex360/λcm485 in a Synergy4 plate reader from BioTek (Winooski, Vt.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 504.57, ALogP 4.25, HBA 7, HBD 2, PSA 99.69, QED 0.38
Compound (SMILES): NC(=O)c1nn(-c2ccc(OC(F)F)cc2)c2c1CCN(C1CCN(N3CCOCC3=O)CC1)C2=O
InChIKey: JKPYXYOCVLDBJL-UHFFFAOYSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: In Vitro Assay: Factor Xa (human or rat): In the assay for measuring the inhibition of human or rat factor Xa activity, Tris-HCl buffer (50 mM, pH 8.3, 150 mM NaCl) is used. The IC50 is measured by adding dropwise the assay buffer of 50 μL human Factor Xa (Enzyme Research Laboratories, Inc; the final concentration of 8.36 nM) or 50 μL rat Factor Xa (Enzyme Research Laboratories, Inc; the final concentration of 57.5 nM) to the appropriate wells of a Greiner 384 microtiter plate. 2 μL the assay buffer containing 2% (V/V) DMSO (uninhibited control) or various concentrations of the compounds to be tested is diluted in the assay buffer containing 2% (V/V) DMSO, and add the assay buffer of 48 μL substrate S-2222 (Chromogenix; chemical formula: Bz-Ile-Glu(γ-OR)-Gly-Arg-pNA.HCl R═H (50%) and R═CH3 (50%)), the final concentration is 0.172 mM. The assay is performed by pre-incubating the tested compounds with enzyme for 10 min, and then the assay is initiated by adding substrate S-2222 to obtain a final volume of 100 μL.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 504.49, ALogP 0.81, HBA 8, HBD 1, PSA 123.23, QED 0.62
Compound (SMILES): CC(C)(O)COc1cc(-c2ccc(N3CC(Oc4ccccc4F)C3)nc2)c2c(C#N)cnn2c1
InChIKey: XFEWYYSZSVEGQW-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 = 25.3 nM (pChEMBL 7.599999904632568)
Descriptors: MW 473.51, ALogP 3.82, HBA 8, HBD 1, PSA 95.91, QED 0.44
Compound (SMILES): CN(c1ccc2c(c1)CCC[C@H]2CNc1cnccc1C(=O)O)c1ccc2ccoc2c1
InChIKey: VBXRXRXLJLWQAZ-IBGZPJMESA-N
Target: Lysine-specific demethylase 4C (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3R0
Assay: JMJD2C Assay: The ability of test compounds to inhibit the activity of JMJD2C was determined in 384-well plate format under the following reaction conditions: 0.3 nM JMJD2C, 300 nM H3K9me3-biotin labeled peptide (Anaspec cat #64360), 2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, and 2 μM ammonium iron(II) sulfate. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-di-methylated histone H3 lysine 9 (H3K9me2) antibody (PerkinElmer) in the presence of 5 mM EDTA in LANCE detection buffer (PerkinElmer) at a final concentration of 50 nM and 1 nM, respectively.The assay reaction was initiated by the following: 2 μl of the mixture of 900 nM H3K9me3-biotin labeled peptide and 6 μM alpha-ketoglutaric acid with 2 μl of 11-point serial diluted inhibitor in 3% DMSO were added to each well of the plate, followed by the addition of 2 μl of 0.9 nM JMJD2C to initiate the reaction. The reaction mixture was incubated at room temperature for 30 minutes, and terminated by the addition of 6 μl of 5 mM EDTA in LANCE detection buffer containing 100 nM Phycolink Streptavidin-allophycocyanin and 2 nM Europium-anti-H3K9me2 antibody. Plates were read by EnVisionMultilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature. A ratio was calculated (665/615) for each well and fitted to determine inhibition constant (IC50).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 427.5, ALogP 5.83, HBA 5, HBD 2, PSA 78.6, QED 0.4
Compound (SMILES): COc1cc2c(cc1OC)[C@H]1C[C@@H](COC(=O)CC(C)(C)n3cccc3)[C@H](CC(C)C)CN1CC2
InChIKey: RQIBOUPCLWPFPT-GIFXNVAJSA-N
Target: Synaptic vesicular amine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q05940
Assay: Determining human Vmat2 Inhibitory Activity of a Compound: The human Ki's for the compounds listed in Table 11 were determined using a slightly modified procedure shown below (see data in column under the heading Ki nM). In a total volume of 0.15 mL in low-binding 96-well plates (Corning #3605), twelve concentrations of Compound 1-1 and R,R,R-DHTBZ were competed against 10 nM 3H-dihydrotetrabenezine (American Radiolabeled Chemicals, Kd 2.6 nM) on rat forebrain homogenate (100 μg membrane protein per well) or human platelet homogenate (15 μg membrane protein per well) in VMAT2 binding buffer (Dulbecco's phosphate buffered saline, 1 mM EDTA, pH 7.4). Following incubation at 25° C. for 90 minutes, bound radioligand was collected by rapid filtration onto GF/B glass fiber filters using a Unifilter-96 Harvester (PerkinElmer). Filter plates were pre-treated with 0.1% polyethylenimine and allowed to dry overnight, and following harvesting the filter plates were washed with 800 d VMAT2 binding buffer. Bound radioligand was quantified by scintillation counting using a Topcount NXT (PerkinElmer).
Measurement: Ki = 260 nM (pChEMBL 6.579999923706055)
Descriptors: MW 482.67, ALogP 5.46, HBA 6, HBD 0, PSA 52.93, QED 0.44
Compound (SMILES): COc1cc(OCc2ccccc2)cc2c1nc(O[C@H]1CC[C@H](OC[C@H](C)NC(C)=O)CC1)n2C
InChIKey: LQEOZDJPRQUBTN-NYVOZVTQSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 298 nM (pChEMBL 6.53000020980835)
Descriptors: MW 481.59, ALogP 4.39, HBA 7, HBD 1, PSA 83.84, QED 0.46
Compound (SMILES): Cc1nnc2c(NCCCCNC(=O)OC(C)(C)C)nc3ccc(F)cc3n12
InChIKey: GRJQZDWBVDUVPE-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Evaluation of BRD4 binding inhibition ability: The following experiment was performed to evaluate the ability of [1,2,4]triazolo[4,3-a]quinoxaline derivative of the present invention to inhibit the interaction between BRD4 (BD1+BD2) bromodomain, one of BET protein family, and tetraacetylated histone H4 peptide.The compound was serially diluted at the ratio of 1:5 in assay buffer from 10 mM stock in DMSO (initial concentration: 100 μM) on white OptiPlate-384 (PerkinElmer). A mixture comprising 100 nM GST-BRD4 (BD1+BD2) and 100 nM biotinylated acetyl-histone H4 (Lys5,8,12,16) peptide was prepared in assay buffer (50 mM HEPES pH 7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). After adding 6 μl of the mixture to the diluent, 6 μl of the pre-mixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads (PerkinElmer, 10 μg/ml in assay buffer, respectively) was added thereto. The samples were incubated in the dark at room temperature for 30 minutes (shaking at 300 rpm). Then, the signals were measured with PerkinElmer Envision HTS Multilabel Reader using an alpha screen protocol of PerkinElmer. Each plate contained the negative control in which biotinylated acetyl-histone H4 peptide and GST-BRD4 (BD1+BD2) were replaced by assay buffer. In the case of using the software GraphPad Prism for calculation, the negative control point was input as a low standard value. The positive control (probe molecule l-BET762 containing protein/peptide mixture) proceeded to pipetting. IC50 value was determined by using GraphPad Prism 3.03 software (or an updated version thereof).
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 388.45, ALogP 3.44, HBA 7, HBD 2, PSA 93.44, QED 0.63
Compound (SMILES): COC(=O)c1ccccc1-c1c(C#CCCO)sc(-c2ccc(F)cc2)c1-c1ccncc1
InChIKey: HOSLIQKTPAYUBV-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: Fluorescence polarisation (FP) assay for inactive p38alpha MARK: FP signals were measured with a PHERAstar microplate reader (BMG Labtech) using black, low-binding half-area 96 well plates (Corning). Each binding data point was carried out in duplicate or triplicate. Data were recorded in millipolarisation (mP) units and measured at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Tris(hydroxymethyl)aminomethane (Tris) assay buffer was used in all binding experiments and consisted of 10 mM Tris, 50 nM potassium chloride, and 3.5 mM 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate (Chaps) at pH 8.0. The recombinant inactive His-tag p38α MAPK used in the ligand binding assays were prepared as described by Bukhtiyarova et al.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 457.53, ALogP 5.8, HBA 5, HBD 1, PSA 59.42, QED 0.3
Compound (SMILES): CC(C)n1cnc2cc(-c3ccc4c(c3)N([C@H]3C[C@@H](N5CCCCC5)C3)C(=O)C43CCN(C(=O)C(C)(C)O)CC3)nc(Nc3ccncc3F)c21
InChIKey: QYKLEMGBYGJDSU-MKPDMIMOSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Enzymatic Activity Assay: Recombinant HPK1 kinase domain produced via baculovirus infection of insect cells was obtained from Proteros (Proteros Biostructures #PR-0322) and was pre-activated in the presence of 2 mM ATP (Sigma-Aldrich, cat #GE27-2056-01) and 2 mM magnesium chloride for 16 hours at 4° C. The protein reaction mixture was then loaded to a desalting column (Thermo Fisher Scientific, Cat #89889) to remove excess ATP. HPK1 was eluted with buffer containing 20 mM Tris (2-Amino-2-(hydroxymethyl)propane-1,3-diol) pH 8.0, 150 mM NaCl, 2 mM dithiothreitol and 5% glycerol, and was frozen at −80° C. for later use. HPK1 dual phosphorylation was confirmed by mass spectrometry.Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 694.86, ALogP 5.95, HBA 9, HBD 2, PSA 119.72, QED 0.24
Compound (SMILES): O=S(=O)(c1ccc(-c2ccccn2)cc1)N1CCN=C1N1CCN(C2CCC2)CC1
InChIKey: LNZWOODYYLNFHP-UHFFFAOYSA-N
Target: Lysine-specific demethylase 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NHM5
Assay: KDM2B TR-FRET Assay: Full length KDM2B was cloned, expressed, and purified to homogeneity. Compound inhibition of KDM2B demethylase activity was assessed by monitoring the methylation status of a biotin-H3K36me2 peptide substrate (H2N-RKSAPATGGV(KMe2)KPHRYRPGTV-NTPEGBiot; New England Peptide) in the presence of α-keotglutarate (2-OG) and iron (Fe2+) using the TR-FRET assay technology (Cisbio). Specifically, in a 384 well ProxiPlate KDM2B (5 mM final), ascorbate (500 μM final) and DTT (2 mM final) were combined with the biotin-H3K36me2 peptide substrate (200 nM final), 2-OG (0.3 μM or 6 μM final; Sigma K2010) and Fe2+ (100 μM final; Sigma F1543) in 50 mM HEPES (pH 6.5) and 0.01% Triton-X 100 either in the presence of DMSO (final 0.25% DMSO) or compound dilution series in DMSO and mixed. After a two hour incubation at room temperature, a mixture of EU-anti-H3K36mel antibody (2 nM final; Cisbio #64CUSKAZ), and Streptavidin-d2 (50 nM final; Cisbio #64CUS000) in 200 mM KF, 200 mM EDTA, 0.1% BSA and 50 mM HEPES (pH 6.5) was added. After 1 hour incubation, the plates were read on an Envision instrument, the readouts were transformed into % inhibition, and IC50 values were generated using a four parameter logistic model (XLFIT5). The KDM2B TR-FRET Assay described above represents an additional embodiment of the invention.
Measurement: IC50 = 0.102 nM (pChEMBL 9.989999771118164)
Descriptors: MW 425.56, ALogP 2.28, HBA 6, HBD 0, PSA 69.11, QED 0.75
Compound (SMILES): CC(=O)N1CCN(c2cccc(-c3cc(-c4ccnn4C4CCOCC4)c4c(N)ncnn34)c2)C(=O)C12CC2
InChIKey: UCDKGAACDFBWCZ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 526.6, ALogP 2.92, HBA 9, HBD 1, PSA 123.88, QED 0.43
Compound (SMILES): COC[C@]1(C[C@H](C)C(=O)N2CCN(c3cc(F)c(F)c(OC)c3)CC2)NC(=O)NC1=O
InChIKey: RDWFDWWQRNYJGA-YUNKPMOVSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O75173
Assay: In Vitro Assay: TABLE VI: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID NO: 2) by human ADAMTS4.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, ⅕ dilution in DMSO further diluted 1 in 10 in water corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH 7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS) containing hADAMTS4 (0.38 ng/μL) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 180 min at 37° C. (Excitation 485 nm, emission 535).
Measurement: IC50 = 37.5 nM (pChEMBL 7.429999828338623)
Descriptors: MW 440.45, ALogP 0.87, HBA 6, HBD 2, PSA 100.21, QED 0.61
Compound (SMILES): CN(CC(F)F)C(=O)[C@H]1CCc2c(sc3ncnc(Nc4cc5ccnn5cc4Cl)c23)C1
InChIKey: KGQPFBGJPIPAGJ-NSHDSACASA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BUB5
Assay: MKNK1 Kinase High ATP Assay: A recombinant fusion protein of Glutathione-S-Transferase (GST, N-terminally) and human full-length MKNK1 (amino acids 1-424 and T344D of accession number BAA 19885.1), expressed in insect cells using baculovirus expression system and purified via glutathione sepharose affinity chromatography, was purchased from Carna Biosciences (product no 02-145) and used as enzyme. As substrate for the kinase reaction the biotinylated peptide biotin-Ahx-IKKRKLTRRKSLKG (SEQ ID NO: 1, C-terminus in amide form) was used, which can be purchased e.g. from the company Biosyntan (Berlin-Buch, Germany).For the assay 50 nL of a 100 fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384 well microtiter plate (Greiner Bio-One, Frickenhausen, Germany), 2 μL of a solution of MKNK1 in aqueous assay buffer [50 mM HEPES pH 7.5, 5 mM MgCl2, 1.0 mM dithiothreitol, 0.005% (v/v) Nonidet-P40 (Sigma)] was added and the mixture was incubated for 15 min at 22° C. to allow pre-binding of the test compounds to the enzyme before the start of the kinase reaction. Then the kinase reaction was started by the addition of 3 μL of a solution of adenosine-tri-phosphate (ATP, 3.3 mM=>final conc. in the 5 μL assay volume is 2 mM) and substrate (0.1 μM=>final conc. in the 5 μL assay volume is 0.06 μM) in assay buffer and the resulting mixture was incubated for a reaction time of 30 min at 22° C. The concentration of MKNK1 was adjusted depending of the activity of the enzyme lot and was chosen appropriate to have the assay in the linear range, typical concentrations were in the range of 0.003 μg/mL. The reaction was stopped by the addition of 5 μL of a solution of TR-FRET detection reagents (5 nM streptavidine-XL665 [Cisbio Bioassays, Codolet, France] and 1 nM anti-ribosomal protein S6 (pSer236)-antibody from Invitrogen [#44921G] and 1 nM LANCE EU-W1024 labeled ProteinG [Perkin-Elmer, product no. AD0071]) in an aqueous EDTA-solution (100 mM EDTA, 0.1% (w/v) bovine serum albumin in 50 mM HEPES pH 7.5).The resulting mixture was incubated for 1 h at 22° C. to allow the formation of complex between the phosphorylated biotinylated peptide and the detection reagents. Subsequently the amount of phosphorylated substrate was evaluated by measurement of the resonance energy transfer from the Eu-chelate to the streptavidine-XL. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET reader, e.g. a Rubystar (BMG Labtechnologies, Offenburg, Germany) or a Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm was taken as the measure for the amount of phosphorylated substrate. The data were normalised (enzyme reaction without inhibitor=0% inhibition, all other assay components but no enzyme=100% inhibition).
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 476.94, ALogP 4.56, HBA 7, HBD 1, PSA 75.42, QED 0.46
Compound (SMILES): COCc1ccc(F)c([C@H](C)Nc2cc(F)c(S(=O)(=O)Nc3cscn3)c(F)c2)c1
InChIKey: TXMXPBHXNBMQRI-NSHDSACASA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 1944 nM (pChEMBL 5.710000038146973)
Descriptors: MW 457.5, ALogP 4.68, HBA 6, HBD 2, PSA 80.32, QED 0.51
Compound (SMILES): CS(=O)(=O)N1CCC(Nc2ncc3ccc(=O)n([C@@H]4CC[C@@H](O)C4)c3n2)CC1
InChIKey: QPUBZAAAHLUCJC-HUUCEWRRSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: CDK2/Cyclin E1 Mobility Shift Assay: The purpose of the CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2) (SEQ ID NO:1). (CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type full length CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Mat1 enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (± inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA, pH 7.5. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 1.44 nM (pChEMBL 8.84000015258789)
Descriptors: MW 407.5, ALogP 0.71, HBA 8, HBD 2, PSA 117.42, QED 0.77
Compound (SMILES): Cc1cccc(OCC(C)C)c1-n1nc2c(c1-c1ccc(NC(N)=O)cc1)CN(CC1(C(F)(F)F)CC1)CC2
InChIKey: JVWGPQLDTPUDDI-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 541.62, ALogP 6.07, HBA 5, HBD 2, PSA 85.41, QED 0.36
Compound (SMILES): CCc1nn(CC2(COC(=O)c3ccccc3)COC2)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: JIXFTTFKJDGDFI-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: Enzyme Activity Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [5380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 453.54, ALogP 2.4, HBA 7, HBD 1, PSA 91.68, QED 0.68