lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(O)(c1nccs1)c1cccc(-c2ncnc3cc(N4CCOCC4)ccc23)c1F
InChIKey: BYOYXEYQONGYBZ-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: DNA-PK Assay (Biochemical): The kinase assay was carried out in streptavidin-coated 348-well microtitre flashplates. To this end, 1.5 μg of DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK-biotin-NH2 (biotin-DNA-PK peptide), were incubated for 90 min at room temperature in a total volume of 36.5 μl (34.25 mM HEPES/KOH; 7.85 mM Tris HCl; 68.5 mM KCl; 5 μM ATP; 6.85 mM MgCl2; 0.5 mM EDTA; 0.14 mM EGTA; 0.69 mM DTT; pH 7.4) with 500 ng of DNA from calf thymus, 0.1 μCi of 33P-ATP and 1.8% of DMSO per well with and without the test compound. The reaction was stopped using 50 μl/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 μl of 0.9% saline solution. A nonspecific reaction (blank value) was determined using 10 μM of an innate kinase inhibitor. The radioactivity measurement was carried out using a TopCount. IC50 values were calculated in RS1.
Measurement: IC50 > 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 436.51, ALogP 3.98, HBA 7, HBD 1, PSA 71.37, QED 0.52
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CCN(C(=O)CN5CCCC5)C[C@H]4C)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: JSDZUPMCUNPQOJ-LJQANCHMSA-N
Target: Toll-like receptor 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR96
Assay: TLR7/8/9 Inhibition Reporter Assays: 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. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 5560 nM (pChEMBL 5.25)
Descriptors: MW 514.68, ALogP 4.15, HBA 7, HBD 1, PSA 85.66, QED 0.43
Compound (SMILES): Cc1ccc(Cn2c(=O)n(C[C@H](C)C(=O)O)c(=O)[nH]/c2=N\c2ccc(OC(C)C)cc2)cc1
InChIKey: AQMWXWWTVFUXNJ-KRWDZBQOSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 452.51, ALogP 2.44, HBA 7, HBD 2, PSA 118.68, QED 0.54
Compound (SMILES): NCc1ccc(CNc2nccc3c2ccn3Cc2csc(-c3ccccc3)n2)cc1
InChIKey: OCACJQIBMJEKIH-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibitory Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stürzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 8100 nM (pChEMBL 5.090000152587891)
Descriptors: MW 425.56, ALogP 5.28, HBA 6, HBD 2, PSA 68.76, QED 0.37
Compound (SMILES): COC[C@@H](C)Oc1nc(C(=O)N[C@@H]2CCC[C@@H]2O)cc(N2CCC(c3n[nH]c4nccc(OC)c34)CC2)n1
InChIKey: HKYDPYVPFPTXAX-SUMDDJOVSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL enzyme inhibition (% inhibition, Kiapp and Ki values) by small molecule inhibitors was evaluated using a fluorescence-based microfluidic mobility shift assay. AXL catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-30 (5-FAM-KKKKEEIYFFF-CONH2, 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. Human wild-type receptor tyrosine kinase protein Axl comprising residues 505-811 was produced in-house using the baculoviral expression vector system that incorporated a hexahistidine affinity tag into the protein (LJIC-1916B1.1). The enzyme was preactivated by auto-phosphorylation of 34 uM non-activated enzyme in the presence of 2 mM ATP, 4 mM MgCl2, 50 mM NaCl and 1 mM TCEP in 20 mM HEPES, pH 7.3 at 4° C. for 30 minutes. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 120 μM ATP (ATP Km=70.4 μM), 0.01% Tween-20, 3 μM FL-Peptide-30, and 0.5 nM phosphorylated AXL enzyme in 100 mM HEPES buffer at pH 7.3. 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 30 minutes at 25° C. by the addition of 50 μL of 200 mM EDTA, pH 7.5. The Ki values were determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable. (See, Morrison, J. F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et biophysica acta 185, 269-286; and Murphy, D. J. (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design, Analytical biochemistry 327, 61-67.)
Measurement: Ki = 0.109 nM (pChEMBL 9.960000038146973)
Descriptors: MW 525.61, ALogP 2.2, HBA 10, HBD 3, PSA 147.61, QED 0.38
Compound (SMILES): NC(=O)c1cccnc1O[C@H]1CC2(C[C@H](Nc3nc(-c4ccccc4Cl)cs3)C2)C1
InChIKey: BMWOUKACOGDJIM-PLWNSFSXSA-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) (SEQ ID No. 1). 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 = 1698 nM (pChEMBL 5.769999980926514)
Descriptors: MW 440.96, ALogP 4.76, HBA 6, HBD 2, PSA 90.13, QED 0.58
Compound (SMILES): C#CC(=O)N1CC(N(C)c2ccc(C(N)=O)c3[nH]c(C)cc23)C1
InChIKey: JIWRZXNQUJLZPL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) : The in-house BTK corresponds to recombinant human catalytic domain (aa 393-659), which was expressed in SF9 cells with an N-terminal his tag and purified by immobilized metal affinity chromatography. BTK was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 0.2 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4 and 0.001 mM ATP. After about 60 mM incubation at room temperature, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 mM at room temperature, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 310.36, ALogP 0.86, HBA 3, HBD 2, PSA 82.43, QED 0.82
Compound (SMILES): CN1CCN(c2ccc(Nc3ncc4ccn(-c5ccc(NS(=O)(=O)C(C)(C)C)cc5)c4n3)cc2)CC1
InChIKey: OUTVETGRDINKAY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: AlphaLisa Assay: 1. Add 20 μl of the diluted compound to each well of the 384-well opti plate2. Tap the plate gently3. Add 10 μl of the enzyme to each well of the opti plate4. Cover with plate sealer5. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.6. Incubate the plate at room temperature for 10 minutes7. Add 10 μl of the substrate8. Cover with plate sealer9. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.10. Cover with plate sealer and incubate at room temperature for 1 hour11. After incubation, transfer 10 μl of reaction mix to fresh wells of a 384-well plate.12. Add 10 μl acceptor bead from working stock.13. Cover with plate sealer14. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.15. Cover with plate sealer and incubate at room temperature for 30 minutes16. Add 10 μl donor bead from working stock (in dark).17. Cover with plate sealer18. Spin the plate briefly (640 rpm for 30 s) in a plate centrifuge.19. Cover with plate sealer and incubate at room temperature for 15-30 minutes20. Read the plate in pherastar (alpha screen protocol)
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 519.68, ALogP 4.46, HBA 8, HBD 2, PSA 95.39, QED 0.39
Compound (SMILES): O=C(N[C@@H]1CC[C@@]2(S(=O)(=O)c3ccc(F)cc3)c3ccc(C(F)(C(F)(F)F)C(F)(F)F)cc3CC[C@@H]12)N1C[C@@H](O)[C@@H](O)C1
InChIKey: ZBULNPNWPXJEIA-BTPPTMBQSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: General RORgamma Gal4 Reporter Assay: Inverse agonist activity of potential ligands to RORγ was measured by inhibition of luminescence in a Gal4-luciferase reporter assay in Jurkat cells.Jurkat cells stably over-expressing the RORγ receptor, Jurkat pEx/Gal/hRORγ CLBD/HYG pG5luc/blast, were plated at a concentration of 10,000 cells/well in a 384-well solid white cell culture plate (Perkin Elmer #6007899) in assay buffer RPMI 1640 (Gibco 11875-085 1L) containing 0.1% BSA, 100×HEPES (Gibco 15360-080), 100 mM sodium pyruvate (Gibco 11360-040), 50 mg/mL Hygromycin B (Invitrogen 10687-010) and 10 mg/mL blasticidin (Invitrogen R210-01). 100 nL of test compound in a 3-fold serial dilution, with final concentrations ranging from 40 μM to 0.67 nM, were added to the cells which were then incubated overnight.The following day, cells were lysed with 10 μL of Steady-Glo Luciferase Assay System (Promega Cat. No. EZ550), and analyzed immediately. IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce luciferase activity by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 642.57, ALogP 4.26, HBA 5, HBD 3, PSA 106.94, QED 0.34
Compound (SMILES): COc1ccc(CC(=O)[C@H]2CC[C@H]3[C@@H]4CC[C@@H]5C[C@](C)(O)CC[C@]5(C)[C@H]4CC[C@]23C)cc1
InChIKey: FVXMJSUKTPANLP-QGBMZPLUSA-N
Target: Gamma-aminobutyric acid type B receptor subunit 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9Z0U4
Assay: TBD: TBD
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 438.65, ALogP 6.22, HBA 3, HBD 1, PSA 46.53, QED 0.61
Compound (SMILES): C=C1CC(c2nc3c(C)c(C)ccc3[nH]2)N(C(=O)c2nc(C3CC3)sc2-c2cccc(C)c2)C1
InChIKey: MYSKBZWJKAYHAI-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.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 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M 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.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 468.63, ALogP 6.63, HBA 4, HBD 1, PSA 61.88, QED 0.34
Compound (SMILES): CCc1nc2ccc(N3CCC4(CNC(=O)O4)C3)nn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: OTIXTXGLBKOWMN-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: Compound IC50 values are determined in a fluorescent hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal. 10 uL of a dilution series of compound, starting from 20 uM highest concentration, dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 ug/mL or 0.64 ug/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 uL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 uL of 0.75 uM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 493.57, ALogP 4.01, HBA 9, HBD 1, PSA 87.89, QED 0.45
Compound (SMILES): CN1C(=O)C(Cc2ccc(-c3ccncc3)cc2)NC(=O)c2ccccc21
InChIKey: LJOQPYLDIOBLQD-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Inhibition Assay: In a final reaction volume of 25 μL, ROCK-II (h, amino acids 11-552) (5-10 mU) was incubated with 50 mM Tris pH 7.5, 0.1 mM EGTA, 30 μM KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, 10 mM magnesium acetate and [γ-32P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction was initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of 5 μL of a 3% phosphoric acid solution. 10 μL of the reaction was then spotted onto a P30 filter-mat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
Measurement: IC50 = 800 nM (pChEMBL 6.099999904632568)
Descriptors: MW 357.41, ALogP 3.07, HBA 3, HBD 1, PSA 62.3, QED 0.78
Compound (SMILES): CC(=O)NCc1ncccc1C1COc2c(-c3c(C)noc3C)ccc3[nH]c(=O)n1c23
InChIKey: ZZEDEQPBFBBRSD-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 419.44, ALogP 2.61, HBA 7, HBD 2, PSA 115.04, QED 0.53
Compound (SMILES): CN1C(=O)N(C(C)(C)C)Cc2c1cnc1[nH]c(-c3ccc(CN4CCC(S(C)(=O)=O)CC4)cc3)c(-c3ccc(C(=O)NCC4(C#N)CC4)cc3)c21
InChIKey: XADKTRJBBRUWOQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 LanthaScreen JH1 Binding Assay: JAK2 JH1 binding assay utilizes catalytic domain (JH1, amino acids 826-1132) of human JAK2 expressed as N-terminal FLAG-tagged, biotinylated protein in a baculovirus expression system (Carna Biosciences, Product #08-445-20N). The assay was conducted in black 384-well polystyrene plates in a final reaction volume of 20 μL. JAK2 JH1 (1.5 nM) was incubated with compounds (100 nL serially diluted in DMSO) in the presence of 50 nM Fluorescent JAK2-JH1 Tracer and 0.5 nM Streptavidin-Tb cryptate (Cisbio Part #610SATLB) in assay buffer (50 mM Tris, pH=7.5, 10 mM MgCl, 0.01% Brij-35, 0.1% BSA, 1 mM EGTA, 5% Glycerol and 5 mM DTT). Non-specific binding was accessed in the presence of 2 mM ATP. After incubation for 2 hours at 25° C., LanthaScreen signals were read on a PHERAstar FS plate reader (BMG LABTECH). Data was analyzed with IDBS XLfit and GraphPad Prism 5.0 software using a four parameter dose response curve to determine IC50 for each compound.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 707.9, ALogP 6.11, HBA 7, HBD 2, PSA 142.5, QED 0.23
Compound (SMILES): [C-]#[N+]c1ncc(C#C)cc1S[C@H]1OC(CO)[C@H](O)[C@H](n2cc(-c3cc(F)c(Cl)c(F)c3)nn2)C1OC
InChIKey: FGWHROAUTOYQSM-GMNQVVRASA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: Fluorescence Anisotropy Assay: The affinity of Example 1-71 for galectins were determined by a fluorescence anisotropy assay where the compound was used as an inhibitor of the interaction between galectin and a fluorescein tagged saccharide probe
Measurement: Kd = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 533.94, ALogP 3.23, HBA 9, HBD 2, PSA 106.88, QED 0.28
Compound (SMILES): N#Cc1ccnc(N2C(=O)[C@H](O)C[C@H]2C(=O)N(c2cccc(F)c2)[C@H](C(=O)NC2CCC(F)(F)CC2)c2ccccc2Cl)n1
InChIKey: LHIJKVKSIGTZTB-GVAUOCQISA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: In Vitro Assay: A test compound is prepared as 10 mM stock in DMSO and diluted to 50× final concentration in DMSO, for a 50 μl reaction mixture. IDH enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutaric acid is measured using a NADPH depletion assay. In the assay the remaining cofactor is measured at the end of the reaction with the addition of a catalytic excess of diaphorase and resazurin, to generate a fluorescent signal in proportion to the amount of NADPH remaining. IDH1-R132 homodimer enzyme is diluted to 0.125 μg/ml in 40 μl of Assay Buffer (150 mM NaCl, 20 mM Tris-Cl pH 7.5, 10 mM MgCl2, 0.05% BSA, 2 mM b-mercaptoethanol); 1 μl of test compound dilution in DMSO is added and the mixture is incubated for 60 minutes at room temperature. The reaction is started with the addition of 10 μl of Substrate Mix (20 μl NADPH, 5 mM alpha-ketoglutarate, in Assay Buffer) and the mixture is incubated for 90 minutes at room temperature. The reaction is terminated with the addition of 25 μl of Detection Buffer (36 μg/ml diaphorase, 30 mM resazurin, in 1× Assay Buffer), and is incubated for 1 minute before reading on a SpectraMax platereader at Ex544/Em590.Compounds are assayed for their activity against IDH1 R132C following the same assay as above with the following modifications: Assay Buffer is (50 mM potassium phosphate, pH 6.5; 40 mM sodium carbonate, 5 mM MgCl2, 10% glycerol, 2 mM b-mercaptoethanol, and 0.03% BSA). The concentration of NADPH and alpha-ketoglutarate in the Substrate Buffer is 20 μM and 1 mM, respectively.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 627.02, ALogP 4.08, HBA 7, HBD 2, PSA 139.52, QED 0.4
Compound (SMILES): Cc1ccc(-c2sc(C(=O)N3CCC[C@@H](N)C3)nc2-c2ccc(C#N)cc2)cc1
InChIKey: BJZAFOZWSBOIEG-LJQANCHMSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: n Vitro Enzyme Inhibition Assay LSD-1: This assay determines the ability of a test compound to inhibit LSD1 demethylase activity. E. coli expressed full-length human LSD1 (Accession number 060341) was purchased from Active Motif (Cat#31334).The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1-0.5 nM LSD1, 50 nM H3K4me1-biotin labeled peptide (Anaspec cat #64355), 2 μM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively.The assay reaction was performed according to the following procedure: 2 μL of the mixture of 150 nM H3K4me1-biotin labeled peptide with 2 μL of 11-point serial diluted test compound in 3% DMSO were added to each well of plate, followed by the addition of 2 μL of 0.3 nM LSD1 and 6 μM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 μL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Enzymatic reaction is terminated within 15 minutes if 0.5 LSD1 enzyme is used in the plate. Plates were read by EnVision Multilabel 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 < 100 nM (pChEMBL 7)
Descriptors: MW 402.52, ALogP 4.22, HBA 5, HBD 1, PSA 83.01, QED 0.71
Compound (SMILES): C=CC(=O)N1CC(N(C)C(=O)Cn2ccc3cc(Br)cc(C)c32)C1
InChIKey: WPACVYQOKDXVKK-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (20 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for 20 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 3320 nM (pChEMBL 5.480000019073486)
Descriptors: MW 390.28, ALogP 2.57, HBA 3, HBD 0, PSA 45.55, QED 0.75
Compound (SMILES): COc1cc(C(F)(F)F)cc2c1c(N[C@@H]1C[C@@H]1F)nc(=O)n2-c1ccccc1
InChIKey: YVFPRZVCQUZGFQ-QWHCGFSZSA-N
Target: S-adenosylmethionine synthase isoform type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31153
Assay: Phosphate Sensor Fluorescence Assay : MAT2A enzyme is incubated with a test compound in DMSO or DMSO and its substrates (L-methionine and ATP) in a microtiter plate. The enzymatic reaction is stopped by the addition of Working Phosphate Sensor Mixture. The plate is analyzed for fluorescence at 450 nm. The high control (DMSO with enzyme and its substrates) gives high fluorescence which represents no inhibition of enzymatic activity while the low control (DMSO with MAT2A substrates and no enzyme) gives low fluorescence which represents full inhibition of enzymatic activity.Materials:Human MAT2A: Cepter, amino acids 1-395Tris, pH 7.5: Invitrogen cat #15567-027KCl: Ambion cat #AM9640GMgCl2: Ambion cat #AM9530GBrij-35: Sigma cat B4184-10MLDTT: Goldbio cat #DTT100BGG: Sigma cat #G5009-25GPNP: Novus Biologicals cat #NBP1-508727-MEG: Cayman Chemical cat #15988L-Methionine: Alfa Aesar cat #J61904ATP: Alfa Aesar cat #J60336Phosphate Sensor: Thermo Fisher cat #PV4407EDTA: Life Tech cat #15575-038Assay plate: 384-well black polypropylene plate: Thomas Scientific cat #1149Q35Final Assay Conditions:Assay Buffer: 50 mM Tris, pH 7.5/50 mM KCl/10 mM MgCl2/0.01% Brij-35/1 mM DTT/0.1% BGG/40 nM PNP/6 uM 7-MEGMAT2A: 10 nM for Cepter clone ID 329, lot 00023-123 before the addition of Working Phosphate Sensor Mixture    5 nM for Cepter clone ID 334, lot 00023-148 before the addition of Working Phosphate Sensor MixtureL-methionine: 500 uM before the addition of Working Phosphate Sensor MixtureATP: 500 uM before the addition of Working Phosphate Sensor MixtureProcedure:For the assay, a mixture of 1 mM L-methionine/1 mM ATP (2× final pre-stopped concentration) in assay buffer; MAT2A (2× final pre-stopped concentration) in Assay Buffer and Working Phosphate Sensor Mixture (1.5 uM Phosphate Sensor/30 mM EDTA in Assay Buffer, which is 3× final concentrations) were prepared. Test compounds or DMSO were added to the appropriate well suing D300e digital dispenser. 5 μl/well of Assay Buffer was added to the wells corresponding to the negative control and 5 μl/well of MAT2A was added to all the wells except for those corresponding to the negative control. After incubating the plate at room temperature for 15 minutes, 5 μl/well of the 1 mM L-methionine/1 mM ATP mixture was added to all wells. The plate was centrifuged at 1000 rpm for 1 minute and then incubated at room temperature for 1 hour. 5 μl of the Working Phosphate Sensor Mixture was added to all wells and the plate was centrifuged at 1000 rpm for 1 minute. The plate was read for fluorescence at 450 nm after exciting at 430 nm.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 393.34, ALogP 3.94, HBA 5, HBD 1, PSA 56.15, QED 0.68
Compound (SMILES): Oc1ccc(Nc2cc(-c3ccc(-n4ccnc4)cc3)[nH]n2)c(C(F)(F)F)c1
InChIKey: NNVRSELEZIFCSI-UHFFFAOYSA-N
Target: TRAF2 and NCK-interacting protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKE5
Assay: ADP-Glo Kinase Assay: The inhibitory properties of compounds were evaluated with TNIK kinase enzyme system and luminescent ADP-GloFigure US11485711-20221101-P00001 Kinase Assay from Promega Corporation according to the manufacturer's protocol. The tested compounds were incubated with TNIK kinases. Reactions were performed in 10 μl kinase buffer supplemented with reaction mixture containing 2 μl ATP, 2 μl MBP protein and 2 μl TNIK at 37° C. for 30 min. The reactions' conditions are provided in Table 5. After kinase reaction, 5 Figure US11485711-20221101-P00002 reaction mixtures were transferred to 384 well assay plate (Greiner, solid white low-binding plates). Next, 5 μl of ADP-Glo Reagent was added to each well and incubated at 37° C. for 30 min. After 45 min, 10 μl Kinase Detection Reagent was added to each well and luminescence signal was detected with the SpectraMax M5e Microplate Reader (Molecular Devices, Menlo Park, Calif.) after 30 min at 37° C. incubation. The IC50 values were calculated using GraphPad Prism software (GraphPad Software Inc., La Jolla, Calif., USA) to determine kit performance. To the cut-off assay, the inhibitory activity of the compounds against TNIK kinase was expressed as the percentage of the kinase inhibitory activity for an indicated concentration of inhibitor.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 385.35, ALogP 4.73, HBA 5, HBD 3, PSA 78.76, QED 0.44
Compound (SMILES): CC(C)=CCN1CC[C@]2(C)c3cc(C(N)=O)ccc3C[C@@H]1C2C
InChIKey: ASEDTRKYKLTHTA-UCPOHXDKSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Opioid Receptor Binding Assay: The Ki (binding affinity) for opioid receptors was determined using a competitive displacement assay as previously described in Neumeyer (Journal of Med. Chem. 2012, p 3878), which is incorporated herein in its entirety. Briefly, membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed the cloned human opioid receptor were incubated with 12 different concentrations of the compound set forth herein in the presence of 0.25 nM [3H]DAMGO (see Tiberi et al., Can. J. Physiol. Pharmacol. 1988, Vol. 66, p 1368, which is incorporated by reference herein in its entirety) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO (see Gulati et al., Life Sci. 1990, Vol. 47, p 159, which is incorporated by reference herein in its entirety). Nonspecific binding was measured by inclusion of 10 M naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. IC50 values were calculated by least squares fit to a logarithm-probit analysis. Ki values of unlabelled compounds were calculated from the equation Ki=(IC50)/1+S where S=(concentration of radioligand)/(Kd of radioligand) (Cheng and Prusoff, 1973).
Measurement: Ki = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 312.46, ALogP 3.28, HBA 2, HBD 1, PSA 46.33, QED 0.87
Compound (SMILES): CC[C@@H](NC(=O)c1cc(OC2CCOCC2)cc(-c2ncc(C)s2)c1)c1cnc(C(F)(F)F)nc1
InChIKey: ULSBLOPPTNALIT-HXUWFJFHSA-N
Target: P2X purinoceptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBL9
Assay: Intracellular Calcium Measurement to Assess Antagonist Activity at Human P2X3 and Human P2X2/3 Receptors: A fluorescent imaging plate reader (FLEX/FLIPR station; Molecular Devices) was used to monitor intracellular calcium levels using the calcium-chelating dye Fluo-4 (Molecular Probes). The excitation and emission wavelengths used to monitor fluorescence were 470-495 nm and 515-575 nm, respectively. Cells expressing purinergic receptors P2X3 (human) or P2X2/3 (human) were plated at a density of 15,000 cells/well in collagen-coated 384-well plates approximately 20 hours before beginning the assay. On the day of the assay, 20 μl of loading buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, 2 μM Fluo-4, and 5 units/mL, hexokinase, pH=7.4) was added and cells dye-loaded for 90 min at 37° C. The dye supernatant was removed and replaced with 45 μl probenecid buffer (Hank's balanced salt solution, 20 mM HEPES, 0.5 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA, 5 mM probenecid, 10 mM D-glucose monohydrate, pH=7.4). The test compound was added in a volume of 5 μl and allowed to incubate for 30 min at 37° C. The final assay DMSO concentration is 1%. The agonist, α,β-Me-ATP, was added in a volume of 20 μl at a concentration representing the EC80 value. The fluorescence was measured for an interval of 90 sec at 2 sec intervals and analyzed based on the increase in peak relative fluorescence units (RFU) compared to the basal fluorescence. Peak fluorescence was used to determine the response to agonist obtained at each concentration of test compound by the following equation: % Response=100*(RFU(test compound)−RFU(control))/(RFU(DMSO)−RFU(control))The Examples were tested in triplicates per plate and mean values were plotted in Excel XLFit to determine IC50 values at the human P2X3 and human P2X2/3 receptors, percentage of maximal inhibition and the Hill coefficients.
Measurement: IC50 = 710 nM (pChEMBL 6.150000095367432)
Descriptors: MW 506.55, ALogP 5.37, HBA 7, HBD 1, PSA 86.23, QED 0.46
Compound (SMILES): CCn1c(-c2cnc(C)nc2)nc2c(O[C@H]3CCN(C(=O)N4CC(F)(F)C4)C3)ncnc21
InChIKey: WHALQHANBBETNA-AWEZNQCLSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Biochemical Assay: The PI3-Kinase biochemical assay was optimized using the HTRF kit provided by Upstate (Millipore). The assay kit contains six reagents: 1) 4× Reaction Buffer; 2) native PIP2 (substrate); 3) Stop (EDTA); 4) Detection Mix A (Streptavidin-APC); 5) Detection Mix B (Eu-labeled Anti-GST plus GST-tagged PH-domain); 6) Detection Mix C. In addition, the following items were obtained or purchased; PI3Kinase (alpha 14-602, beta 14-603, gamma 14-558 and delta 14-604 from Upstate; Millipore), dithiothreitol (Sigma, D-5545), Adenosine-5′ triphosphate (InVitrogen, Cat#AS001A), native PIP3 (PI(3,4,5)P3, diC8, H+, CELLSIGNALS, INC. Cat #907) DMSO (Sigma, 472301).PI3Kinase Reaction Buffer is prepared by dilution the stock 1:4 with de-ionized water. DTT, PIP2 and Biotin-PIP3 were added to 1536 assay plate at a final concentration of 5 mM, 5 mM and 25 nM on the day of use. Enzyme addition and compound pre-incubation are initiated by the addition of 1.25 ul of PI3K (at twice its final concentration) in the 1× reaction buffer to all wells using a BioRaptor. Plates are incubated at room temperature for 15 minutes. Reactions are initiated by addition of 1.25 ul of 2× substrate solution (PIP2 and ATP in 1× reaction buffer) using BioRaptor. Plates are incubated in humidified chamber at room temperature for one hour. Reactions are quenched by addition of 0.625 uL of stop solution to all wells using the BioRaptor. The quenched reactions are then processed to detect product formation by adding 0.625 uL of Detection Solution to all wells using the BioRaptor (Detection mix C, Detection Mix A, and Detection Mix B combined together in an 18:1:1 ratio prepared 2 hours prior to use). Following a one hour incubation in the dark, the HTRF signal is measured on the Envision plate reader set for 330 nm excitation and dual emission detection at 620 nM (Eu) and 665 nM (APC).
Measurement: IC50 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 444.45, ALogP 2.14, HBA 8, HBD 0, PSA 102.16, QED 0.61
Compound (SMILES): CC(=O)N1CCN(c2cccc(-c3cc(-c4cnn(C[C@@H](O)C(F)(F)F)c4)c4c(N)ncnn34)c2)C(=O)C1(C)C
InChIKey: DDUNKXHHKIKBGE-OAQYLSRUSA-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 556.55, ALogP 2.74, HBA 9, HBD 2, PSA 134.88, QED 0.39
Compound (SMILES): CN(C)c1cccc2cc(S(=O)(=O)[C@H]3OC(CO)[C@H](O)C(n4cc(-c5cc(F)c(F)c(F)c5)nn4)[C@@H]3O)ccc12
InChIKey: SEWXANCFUWWJSI-CERWPVKCSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: Fluorescence Anisotropy Assay: The assay was adapted to be able to measure the high affinity of compounds for galectin-3 by using the below probe constructed to have high affinity for galectin-3 which made it possible to use a low concentration of galectin-3 (50 nM). 100 nM albumin was included as a carrier to prevent protein loss at such low concentration of galectin.
Measurement: Kd = 960 nM (pChEMBL 6.019999980926514)
Descriptors: MW 578.57, ALogP 2.04, HBA 10, HBD 3, PSA 138.01, QED 0.29
Compound (SMILES): Cn1ccc2c(c(-c3cc(Cl)c(O)c(C(N)=O)c3)cn2CC(=O)Nc2cc(N3CCOCC3)ncc2Cl)c1=O
InChIKey: DTMBFSKDBOVSGU-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Surface Plasmon Resonance (SPR) Assay: SPR studies were performed using a Biacore™ T200 instrument (GE Health Sciences Inc.). The BCL6 BTB protein used in the FP assay was biotinylated using the site specific biotinylating enzyme BirA, and then cleaved with TEV protease to produce the BCL6 BTB domain (biotin-thrombin-BCL6 amino acids 1-129; 17 kd) that were use in SPR. This protein was stably captured (1000RU) to streptavidin coupled SA chips (BR-1005-31, GE Health Sciences Inc.) according to the manufacture's protocol. Compounds were dissolved in 100% DMSO at 10 mM and 2-fold serial dilutions were done in 100% DMSO. For SPR analysis the serially titrated compounds were diluted 1/20 into buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.05% Tween-20, 3 mM EDTA) giving a final concentration of 5% DMSO. The Biacore flow rate was set at 100 ul/min. For KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 seconds. Curve fitting and KD calculations were done with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 265 nM (pChEMBL 6.579999923706055)
Descriptors: MW 571.42, ALogP 2.99, HBA 9, HBD 3, PSA 144.71, QED 0.32
Compound (SMILES): COc1cccc2c1nc(N)n1nc([C@@H]3CCCN(CC(C)C)C3)nc21
InChIKey: ZLPYPKKMUPOAAT-CYBMUJFWSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Competition Binding Assay: Binding affinities of compounds of the invention for the human A2A receptor were determined in a competition binding assay using Scintillation Proximity technology. Thus, 1^μg (^ denotes 1 μg of membrane/well), or preferably 0.25 μg of membranes from HEK293 cells expressing the human A2a receptor were incubated with a compound of the invention at concentrations ranging from 3000 nM to 0.15 nM in a reaction mixture containing also 0.5 nM of a tritiated form of 5-amino-7-[2-phenethyl]-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (the tritiated compound) and 25 μg of wheat germ agglutin-coated yttrium silicate SPA beads for one hour at room temperature with agitation. The beads were then allowed to settle to the bottom of the wells for 1 hr, after which the membrane-associated radioactivity was determined by scintillation counting in a TopCount microplate reader. Ki values were determined using the Cheng-Prusoff equation.
Measurement: Ki = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 354.46, ALogP 2.7, HBA 7, HBD 1, PSA 81.57, QED 0.78
Compound (SMILES): Cc1c2c(O[C@H](C)[C@H]3CNC(=O)C3)cc(-c3cnn(C4CC4)c3)cc2nn1C
InChIKey: WPDYELPRTNSJHU-UKRRQHHQSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: SYK Kinase Inhibition Test: Recombinant human SYK (amino acids 342-635) was expressed as a fusion protein with an N-terminal GST tag. The test compounds were dissolved in 100% DMSO at a concentration of 10 mM and diluted in DMSO to a concentration of 1 mM. Serial Dilution is done in 100% DMSO. All further dilutions of the substances were carried out with test buffer (25 mM HEPES pH7.5; 25 mM MgCl2; 5 mM MnCl2; 50 mM KCl; 0.2% HSA; 0.01% CHAPS; 100 μM Na3VO4; 0.5 mM DTT). Dilution steps and concentration range were adapted according to need. 7 μl aliquots of these dilutions were transferred into a 384-well Optiplate (Perkin Elmer, #6007290). GST-SYK was diluted to 12 nM in the test buffer and 5 μl of this dilution were used in the kinase test (final concentration of SYK=4 nM in a total volume of 15 μl). After 15 minutes incubation at room temperature 3 μl of a mixture of 750 nM ATP and 100 μg/ml poly (L-Glutamic acid L-Tyrosine 4:1), Fluka #81357) in test buffer were added to each well and the incubation was continued for a further 60 minutes at room temperature.
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 379.46, ALogP 2.98, HBA 6, HBD 1, PSA 73.97, QED 0.74
Compound (SMILES): Cn1ccc2c(-c3ncc(Nc4ccc(C5(O)CCOCC5)cn4)c4c3CNC4=O)ccnc21
InChIKey: RHYFBRHJMLMBGO-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 456.51, ALogP 3.02, HBA 8, HBD 3, PSA 114.19, QED 0.43