lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1ccc(C(=O)Nc2ccc(-c3n[nH]c(=O)c4ccccc34)cc2)n1
InChIKey: WCJLHBRQUDVGPS-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
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 < 100 nM (pChEMBL 7)
Descriptors: MW 345.36, ALogP 2.58, HBA 5, HBD 2, PSA 92.67, QED 0.6
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 delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay (PI3Kδ): [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). PI3Kδ (p110δ/p85α) Recombinant Human Protein was purchased from Eurofins (St Charles, Mo.). 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 PI3Kδ 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, 3.4 nM PI3Kδ. 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): 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 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
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): 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 > 10000 nM (pChEMBL 5)
Descriptors: MW 212.21, ALogP 0.96, HBA 3, HBD 2, PSA 95.56, QED 0.57
Compound (SMILES): C=CC(=O)N(C)Cc1ccc(Nc2nc(Nc3cccc(OCCN4CCCC4=O)c3)ncc2F)cc1
InChIKey: BRQXWVJKMWGRCE-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
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 > 1000 nM (pChEMBL 6)
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), Rattus norvegicus
UniProt: Q63207
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 = 30 nM (pChEMBL 7.519999980926514)
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 = 181 nM (pChEMBL 6.739999771118164)
Descriptors: MW 473.51, ALogP 3.82, HBA 8, HBD 1, PSA 95.91, QED 0.44
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 active 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 active His-tag p38α MAPK used in the ligand binding assays were prepared as described by Bukhtiyarova et al.
Measurement: Ki = 8700 nM (pChEMBL 5.059999942779541)
Descriptors: MW 457.53, ALogP 5.8, HBA 5, HBD 1, PSA 59.42, QED 0.3
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 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UNA0
Assay: In Vitro Assay: TABLE IX: 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 ADAMTS-5.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 hADAMTS-5 (0.63 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 90 min at 37° C. (Excitation 485 nm, emission 530).
Measurement: IC50 = 12.5 nM (pChEMBL 7.900000095367432)
Descriptors: MW 440.45, ALogP 0.87, HBA 6, HBD 2, PSA 100.21, QED 0.61
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 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
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 = 27494 nM (pChEMBL 4.559999942779541)
Descriptors: MW 457.5, ALogP 4.68, HBA 6, HBD 2, PSA 80.32, QED 0.51
Compound (SMILES): COc1c(F)cccc1-c1noc([C@@H]2CCCN2C(=O)c2cccc(F)c2-c2ccn[nH]2)n1
InChIKey: XTBHBVCTQWTHNF-SFHVURJKSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES. On the day of the assay, 50 uL of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/L, 5 mM probenecid (Sigma) and 3 uM of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at rt for 30 min before measurement.Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 uL/well, incubated for 120 min and finally 10 uL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist. The IC50 value (the concentration of compound needed to inhibit 50% of the agonistic response) is determined and may be normalized using the obtained IC50 value of an on-plate reference compound. Optimized conditions were achieved by adjustment of pipetting speed and cell splitting regime. The calculated IC50 values may fluctuate depending on the daily cellular assay performance. Fluctuations of this kind are known to those skilled in the art. In the case where IC50 values have been determined several times for the same compound, the geometric mean has been given.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 451.43, ALogP 4.39, HBA 6, HBD 1, PSA 97.14, QED 0.48
Compound (SMILES): Cc1ncc(-c2ccc3cnc(NC(=O)c4ccc(OCc5ccccc5)cc4)cc3c2)n1C
InChIKey: KLXKWCHLQKYBHK-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK3 beta kinase assay: Briefly, recombinant GSK3β kinase, ATP and Ser/Thr peptide 09 are prepared in 1× Kinase buffer to final concentrations of 0.04 μg/mL, 46 μM, and 4 μM respectively. The mixture is allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 serve as control reactions. After incubation, diluted Development Buffer is added to the reaction and allowed to further incubate for one hour at room temperature. The plate is read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer). The Emission ratio (Em) is calculated as a ratio of the coumarin (C) emission signal (at 445 nm)/Fluorescein (F) emission signal (at 520 nm). The percent phosphorylation is then calculated using the following formula: [1−((Em ratio×F100%)−C100%)/((C0%−C100%)+(Em ratio×(F100%−F0%)))].
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 448.53, ALogP 5.78, HBA 5, HBD 1, PSA 69.04, QED 0.36
Compound (SMILES): CCc1c(-c2cc(Cl)ncc2OC)[nH]c2ccc(C3CCN(C4CCN(CC(C)C)CC4)CC3)cc12
InChIKey: BTGWTCCFXPDVSF-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 514 nM (pChEMBL 6.289999961853027)
Descriptors: MW 509.14, ALogP 6.75, HBA 4, HBD 1, PSA 44.39, QED 0.36
Compound (SMILES): Cc1cnc2c(N)c(C(=O)NCCc3ccc(N4C5CCC4CNC5)c(C#N)c3)sc2n1
InChIKey: HDSHOHUYXFVGRQ-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 25 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHP3
Assay: Biochemical Assay: USP25: The assay was performed in a final volume of 9 μL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 3 mM BME (2-Mercaptoethanol; Sigma 63689-25ML-F), 0.03% BGG (0.22 μM filtered. Sigma, G7516-25G), and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO was pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 25 μM to 1.3 nM, top to lowest dose, respectively. Enzyme USP25, construct USP25-His6, (Boston Biochem E-546). Concentration and incubation times were optimized for the maximal signal-to-background while maintaining initial velocity conditions at a fixed substrate concentration. The final concentration of the enzyme in the assay was 75 pM. Final substrate (Ub-Rh110 Ubiquitin-Rhodamine 110, R&D Systems # U-555) concentration was 25 nM with [Ub-Rh110]<<Km. 3 μL of 2× enzyme was added to assay plates (pre-stamped with compound) preincubated with USP25 for 30 minutes and then 3 μL of 2×Ub-Rh110 was added to assay plates. Plates were incubated for 45 minutes at room temperature before addition of 3 μL of stop solution (final concentration of 10 mM citric acid (Sigma, 251275-500G)). Fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 447.57, ALogP 2.37, HBA 8, HBD 3, PSA 119.96, QED 0.55
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(C(=O)N5CCS(=O)(=O)CC5)n4c3)ccc2o1
InChIKey: QUAPETJJFHRDRT-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
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 > 10000 nM (pChEMBL 5)
Descriptors: MW 411.44, ALogP 1.6, HBA 8, HBD 1, PSA 123.8, QED 0.53
Compound (SMILES): O[C@@H]1[C@H](O)CS[C@H]1c1nnc2c(N3CCCCC3)nccn12
InChIKey: DDPWNYOCXAWCHR-GMTAPVOTSA-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 321.41, ALogP 0.62, HBA 8, HBD 2, PSA 86.78, QED 0.84
Compound (SMILES): O=C(Nc1cccc(C(=O)c2ccc3ncc(-c4cccnc4)nc3c2)c1)Nc1ccc(Cl)c(C(F)(F)F)c1
InChIKey: VNHCYXPUZROHCQ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibitory Activity Assay: KDR (VEGFR2): Inhibitory activities of compounds against KDR (VEGFR2) were also measured by Invitrogen using Z′-LYTE® Method as described above with the following modification. The 2× KDR (VEGFR2)/Tyr 01 mixture is prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. The final 10 μL Kinase Reaction consists of 0.5-11.7 ng KDR (VEGFR2) and 2 μM Tyr 01 in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA. After the 1 hour Kinase Reaction incubation, 5 μL of a 1:256 dilution of Development Reagent B is added.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 547.92, ALogP 7.24, HBA 5, HBD 2, PSA 96.87, QED 0.23
Compound (SMILES): Cc1cnc(C(=O)N[C@@H](C)c2cc3cccc(C#Cc4cnn(C)c4)c3c(=O)n2-c2ccccc2)c(N)n1
InChIKey: KRQKZGMXITZFSA-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition Assays: Class I PI3-Ks can be either purchased (p110α/p85α, p110β/p85α, p110δ/p85α from Upstate, and p110γ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay. Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgCl2), and freshly sonicated phosphatidylinositol (100 μg/mL). Reactions are initiated by the addition of ATP containing 10 μCi of γ-32P-ATP to a final concentration of 10 or 100 μM and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 μL 1N HCl followed by 160 μL CHCl3:MeOH (1:1). The biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCl3. This extract is spotted on TLC plates and developed for 3-4 hours in a 65:35 solution of n-propanol:1M acetic acid.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 503.57, ALogP 3.3, HBA 8, HBD 2, PSA 120.72, QED 0.36
Compound (SMILES): Cc1cnc(Cc2nc3cnc4ccc(Cl)cc4c3n2[C@H]2CCOCC2(F)F)cn1
InChIKey: VDSACXZRWKTZKA-SFHVURJKSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat #PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: The compound dose response was prepared by diluting compound to a top concentration of 0.3 mM in 100% DMSO and serial diluted by half-log in DMSO to give an 11 point curve, 100× final assay concentration. Using Echo acoustic dispensing, 60 nL of compound was transferred to a low volume Corning 384-well assay plate. 3 μL of a working solution of substrate (200 nM LRRKtide, 2 mM ATP) prepared in assay buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to the 60 nL compound assay plate. The kinase reaction was started with 3 μL of a working solution of LRRK2 enzyme at a concentration of 4 μg/m L. The final reaction concentrations were 100 nM LRRKtide, 1 mM ATP, 2 μg/mL LRRK2 enzyme and a compound dose response with a top dose of 3 μM. The reaction was allowed to progress at room temperature for 30 minutes and then stopped with the addition of 6 μL of detection buffer (20 mM Tris pH 7.6, 0.01% NP-40, 6 mM EDTA with 2 nM terbium labeled antiphospho-ERM). After an incubation of 1 hour at room temperature, the plate was read on an Envision with an excitation wavelength of 340 nm and a reading emission at both 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data. Inhibition of mutant G2019S LRRK2 (Invitrogen cat #PV4881) was measured in the exact same method. All final concentrations of substrate ATP and enzyme were the same.
Measurement: IC50 = 39.7 nM (pChEMBL 7.400000095367432)
Descriptors: MW 429.86, ALogP 4.52, HBA 6, HBD 0, PSA 65.72, QED 0.48
Compound (SMILES): COc1cc(OC)c(Cl)c(-c2cc3cnc(Nc4ccc(N5CCC(N6CCOCC6)CC5)cc4)nc3n3cnnc23)c1Cl
InChIKey: ATZFXJNVHKPZHC-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Kinase Assay: The inhibitory activity of some compounds of the present disclosure against FGFR1, FGFR2, FGFR3, FGFR4, KDR was tested by mobility shift assay (concentration of ATP is Km value).Test method:Reagent: basic kinase buffer: 50 mM HEPES (pH 7.5); 0.0015% Brij-35Stop buffer: 100 mM HEPES (pH 7.5); 0.015% Brij-35; 0.2% Coating Reagent #3; 50 mM EDTAPreparing of compounds: dilute test compounds to specific concentration using 100% DMSOReaction process:    1) preparing of 2.5× enzyme solution    adding kinase to 1× basic kinase buffer    2) preparing of 2.5× peptide solution    adding FAM-labeled peptide and ATP to 1× basic kinase buffer  3) preparing of analysis board    transferring 10 μL of test compound to 384-well plate, adding 904, of 1× basic kinase buffer    4) adding 104, of 2.5× enzyme solution to each well of the assay plate and incubating for 10 min at room temperature    5) adding 104, of 2.5× enzyme solution to each well of the assay plate and incubating for specific time at 28° C. 6) stop the reaction by adding 254, of stop solution to each well    7) reading data with Caliper and calculating IC50 value.
Measurement: IC50 = 6.7 nM (pChEMBL 8.170000076293945)
Descriptors: MW 635.56, ALogP 5.71, HBA 11, HBD 1, PSA 102.17, QED 0.24
Compound (SMILES): CNC(=O)N1CCn2c(C3CCS(=O)(=O)CC3)nc(-c3ccc4c(c3)c(-c3cnn(C)c3)nn4C)c2C1
InChIKey: NUSNGACZDVZJGL-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaScreen Assay: The binding of 2.5 nM of BRD4(49-170) to 10 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) was assessed following the same procedure described for the CBP assay. The standard dose response curves were fitted by Genedata Screener software using the variable-slope model:Signal=Signalnegative control+(SignalDMSO control−Signalnegative control)/(1+(IC50/Dose){circumflex over ( )}Hill slope).
Measurement: IC50 = 21939 nM (pChEMBL 4.659999847412109)
Descriptors: MW 522.64, ALogP 2.28, HBA 9, HBD 1, PSA 119.94, QED 0.44
Compound (SMILES): c1cc2c(cn1)CN(c1nccc(Nc3ccc4[nH]ncc4c3)n1)C2
InChIKey: IUGSGIWTJFDBSX-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: Rho-kinase inhibition assay for ROC2 and ROCK1: Dose response curves for Rho-kinase inhibition were derived from a Invitrogen Z′-LYTE Kinase Assay Kit (Invitrogen catalog number PV3793). Purified active ROCK1 and ROCK2 were obtained from Invitrogen (catalog numbers ROCK1, PV3691 and ROCK2, PV3759). The kit components include a coumarin and fluorescein labeled peptide based on myosin light chain 2 (KKRPQRRYSNVF), a proprietary protease containing development reagent and a proprietary Stop buffer used to terminate the development reaction. The inhibitory activities of compounds are measured according to the manufactures protocol. Briefly, decreasing concentrations of test compounds or the known ROCK inhibitor Y-27963, are added, from 10 uM to 2.56×10−5 uM to reaction buffer containing 50 mM HEPES pH 7.5, 10 mM MgCl2, 5 mM EGTA, and 0.05% Brij-35 and of ROCK1 at 0.18 ug/mL or ROCK2 at 0.8 ug/mL in assay dilution buffer. This mixture is overlayed into a white 96-well half area plate and the reaction is initiated with the addition of 5 uM ATP for ROCK1 or 12 uM ATP for ROCK2. The assay proceeds at room temperature for 1 hour followed by the addition of development reagent, and further incubation for 1 hour at room temperature. STOP reagent is then added and the reaction and immediately the coumarin and fluorescein emission signals are read on a Tecan Infinite M1000 fluorescence plate reader (excitation: 400 nm; emission 445 and 520 nm, respectively). By comparing the emission ratios of the test samples against control samples, percent phosphorylation values are calculated and the concentration of inhibitor that produces ½ inhibition of kinase activity (IC50) is determined using Prism. Table 1 provides IC50 concentrations for compounds of the above examples. Several of the compounds also demonstrated activity in a preliminary assay that measured inhibition of myosin light chain phosphorylation (pMLC). For compounds marked ND, activity was not determinable under the test conditions employed.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 329.37, ALogP 3.01, HBA 6, HBD 2, PSA 82.62, QED 0.6
Compound (SMILES): CCOc1cc(-c2ccc(N3CC4CC(C3)N4C(=O)c3ccc(OCCOC)nc3)nc2)c2c(C#N)cnn2c1
InChIKey: CGHLSTDPQZMZEQ-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 = 2978 nM (pChEMBL 5.53000020980835)
Descriptors: MW 539.6, ALogP 3.19, HBA 10, HBD 0, PSA 118.11, QED 0.3
Compound (SMILES): O=C(OC(C(F)(F)F)C(F)(F)F)N1CCC2(CCCN2Cc2ccc(Cl)cc2N2CC3CCC(C2)O3)CC1
InChIKey: WHWOFAUFSRWEHX-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction or cell lysates) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP¿Rh (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (50 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL, ABHD6 and FAAH using ImageJ 1.43u software. Data from this assay is shown in Table 2 (% inhibition at 1 μM).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 569.97, ALogP 5.77, HBA 5, HBD 0, PSA 45.25, QED 0.43
Compound (SMILES): COCCC(C(=O)Nc1ccc(C(=O)O)cc1)n1cc(OC)c(-c2cc(Cl)ccc2C2=NOCC2)cc1=O
InChIKey: LOUUMSPZPZILMW-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Activity Assay: To determine the plasma kallikrein inhibition of the substances according to the invention, a biochemical test system is used which utilizes the reaction of a peptidic plasma kallikrein substrate to determine the enzymatic activity of human plasma kallikrein. Here, plasma kallikrein cleaves from the peptic plasma kallikrein substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions is placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM Tris/HCl pH 7.4; 100 mM sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of plasma kallikrein from Kordia (0.6 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the substrate H-Pro-Phe-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).
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 539.97, ALogP 4.22, HBA 8, HBD 2, PSA 128.45, QED 0.39
Compound (SMILES): Cn1cc(-c2cc3ncccc3c(-c3ccn(C4(CC#N)CN(S(C)(=O)=O)C4)n3)n2)cn1
InChIKey: OXLPBAOSYPLHSY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% BSA, 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 1377 nM (pChEMBL 5.860000133514404)
Descriptors: MW 448.51, ALogP 1.78, HBA 9, HBD 0, PSA 122.59, QED 0.46
Compound (SMILES): Cc1cc(C(=O)N[C@@H](c2ccc(Cl)cc2)c2ccc(Cl)cc2Br)ccc1C#N
InChIKey: UDGCFJMLADZOFX-NRFANRHFSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: TBD: TBD
Measurement: IC50 = 113 nM (pChEMBL 6.949999809265137)
Descriptors: MW 474.19, ALogP 6.46, HBA 2, HBD 1, PSA 52.89, QED 0.47
Compound (SMILES): COc1cnc(CN2C3CC2CN(c2ccc(-c4cc(OC[C@@H](C)O)cn5ncc(C#N)c45)cn2)C3)cn1
InChIKey: KCIBKUCXJUBCNQ-PPNDLDFTSA-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 = 88.3 nM (pChEMBL 7.050000190734863)
Descriptors: MW 512.57, ALogP 2.29, HBA 11, HBD 1, PSA 124.93, QED 0.38
Compound (SMILES): COc1cc(OC)c(Cl)c(-c2ccc3c(NC(=O)c4ccncc4)n[nH]c3n2)c1Cl
InChIKey: JGFIMFGPDMLSCC-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Enzyme Kinase Activity Assay: The enzyme reaction substrate Poly(Glu,Tyr) 4:1 was diluted with PBS without potassium ion (10 mM sodium phosphate buffer, 150 mM NaCl, pH7.2-7.4) to 20 μg/mL, 125 μL/well to coat the enzyme plate, and reacted at 37° C. for 12-16 hours. After the liquid was removed from the wells, the plate was washed three times with 200 μL/well of T-PBS (PBS containing 0.1% Tween-20) for 5 minutes each. The enzyme plate was dried in a 37° C. oven for 1-2 hours.50 μL of the ATP solution diluted with the reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added into each well at a 5 μM final concentration. The compounds were diluted to the appropriate concentration in DMSO, 1 μL/well or containing the corresponding concentrations of DMSO (negative control wells). The reaction was initiated by addition of each kinase domain recombinant protein diluted with 49 μL of reaction buffer. Two control wells without ATP were set in each experiment. The reaction mixtures were placed on a shaker (100 rpm) to react at 37° C. for 1 hour. The plates were washed with T-PBS for three times. 100 μL/well of the primary antibody PY99 dilution was added, and reacted on a shaker (100 rpm) at 37° C. for 0.5 hour. The plates were washed with T-PBS for three times. 100 μL/well of the secondary anti-horseradish peroxidase-labeled goat anti-mouse IgG dilution was added, and reacted on a shaker at 37° C. for 0.5 hour. The plates were washed with T-PBS for three times. 100 μL/well of 2 mg/mL OPD developing solution (diluted with 0.1M citric acid-sodium citrate buffer containing 0.03% H2O2 (pH=5.4)), and reacted in dark for 1-10 minutes at 25° C. (Ultrasound is needed in OPD dissolution, and the developing solution should be prepared on the site). The reaction was quenched with 50 μL/well of 2M H2SO4, and read out at 490 nm using a tunable microplate microplate reader SPECTRA MAX 190.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 444.28, ALogP 4.6, HBA 6, HBD 2, PSA 102.02, QED 0.47
Compound (SMILES): C[C@]1(O)CCCC1n1c(=O)ccc2cnc(NC3CCN(S(=O)(=O)C(F)(F)F)CC3)nc21
InChIKey: NPMHHWNUNSEYKD-IBYPIGCZSA-N
Target: CDK6/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: CDK6/Cyclin D1 Mobility Shift Assay: The purpose of the CDK6/Cyclin D1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) in the presence of small molecule inhibitors by using a fluorescence based microfluidic mobility shift assay. CDK6/Cyclin D1 catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide 5-FAM-Dyrktide (5-FAM-RRRFRPASPLRGPPK) (SEQ ID NO:2). 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. Typical reaction solutions contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 2 mM ATP, 0.005% Tween 20 (TW-20), 3 μM 5-FAM-Dyrktide, 3 nM (active sites) CDK6/Cyclin D1 in 40 mM HEPES buffer at pH 7.5.Inhibitor Ki determinations for non-phosphorylated CDK6/CyclinD1 (LJIC-2003A2/1865) were initiated with the addition of ATP (50 μL final reaction volume), following a twelve minute pre-incubation of enzyme and inhibitor at 22° C. in the reaction mix. The reaction was stopped after 35 minutes by the addition of 50 μL of 25 mM EDTA. Ki determinations were made from a plot of the fractional velocity as a function of inhibitor concentration fit to the Morrison equation with the enzyme concentration as a variable.
Measurement: Ki = 11.3 nM (pChEMBL 7.949999809265137)
Descriptors: MW 475.49, ALogP 1.99, HBA 8, HBD 2, PSA 117.42, QED 0.69
Compound (SMILES): CC(C)(C)c1nc(N2CCC(F)(F)C2)c2ncn(CC(=O)c3ccccc3Cl)c2n1
InChIKey: WKPDQJKEYKBGBN-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1x HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 433.89, ALogP 4.51, HBA 6, HBD 0, PSA 63.91, QED 0.57