lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(C)c1cccc(-n2c3nc(Nc4ccc5c(c4)CNCC5)ncc3c(=O)n2CC(F)(F)F)n1
InChIKey: QDCNHGJGNJUEDT-UHFFFAOYSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: LanthaScreen TR-FRET Assay: IC50 values of compounds against WEE1 kinase enzyme were determined by LanthaScreen Terbium Labeled TR-FRET assay. Kinase assays were performed in 1× kinase buffer (#PV6135, Invitrogen, Life Technologies Grand Island, N.Y.) where total reaction volume was 10 μL in low-volume 384-well plates (#4511, Corning). Serially diluted compounds (3-fold) were incubated with WEE1 Enzyme (1 nM) (#PR7373A, Invitrogen, Life Technologies Grand Island, N.Y.) for 10 min, following which a mixture of ATP (10 μM) (#A1852, Sigma, St-Louis, Mo.) and fluorescent-PolyGT substrate (200 nM) (#PV3610, Invitrogen, Life Technologies Grand Island, N.Y.) was added and incubated in dark at room temperature for 1 h. After 1 h, 10 L stop solution containing Terbium labeled antibody (4 nM) (#PV3529, Invitrogen, Life Technologies Grand Island, N.Y.) and EDTA (#E5134, Sigma, St-Louis, Mo.) (20 mM) in TR-FRET dilution buffer (#PV3574, Invitrogen, Life Technologies Grand Island, N.Y.) was added. Readings were taken in a Synergy Neo Plate reader (BioTek, Winooski) at single excitation of 340 nm and Dual emission at 495 nm and 520 nm respectively.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 497.53, ALogP 4.23, HBA 8, HBD 2, PSA 89.66, QED 0.44
Compound (SMILES): Cc1nsc2nc(Nc3cnn(C4CCOCC4)c3)nc(O[C@H]3CC[C@H](N4CCOCC4)CC3)c12
InChIKey: FHHXBLDYYVCLGR-KESTWPANSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: In Vitro Enzymatic Activity Evaluation: The inhibitory activity of the test compounds against human IRAK4 was evaluated by measuring IC50 values in a 33P-labeled kinase activity assay (Reaction Biology Corp).Buffer conditions: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO.Procedures: The test compound was dissolved in DMSO at room temperature to prepare a 10 mM solution for later use. The substrate was dissolved in fresh buffer, to which the kinase was added and mixed well. The DMSO solution containing the test compound was added to the above mixed reaction system by an acoustic technique (Echo 550). The concentrations of the compounds in reaction system were 10 μM, 3.33 μM, 1.11 μM, 0.370 μM, 0.123 μM, 41.2 nM, 13.7 nM, 4.57 nM, 1.52 nM, and 0.508 nM. After 15 minutes of incubation, the reaction was started by adding 33P-ATP (activity: 0.01 μCi/μL; the corresponding concentration is listed in Table 1). Supplier's catalog number, lot number and concentration information in the reaction system for IRAK4 and its substrate are listed in Table 1. After 120 minutes of reaction at room temperature, the resulting solution was loaded on P81 ion exchange chromatography paper sheet (Whatman #3698-915). After repeated washing with 0.75% phosphoric acid solution, the radioactivity of the phosphorylated substrate residue on the paper sheet was measured. The kinase activity data are shown as a comparison of the kinase activity of the test compound and the kinase activity of the blank (DMSO only) and a curve was fitted using Prism4 software (GraphPad) to give IC50 values.
Measurement: IC50 = 89.5 nM (pChEMBL 7.050000190734863)
Descriptors: MW 499.64, ALogP 3.71, HBA 11, HBD 1, PSA 99.45, QED 0.54
Compound (SMILES): COC(=O)c1ccnc(O[C@@H]2CC[C@@H](C)N(C(=O)c3ccc(OC)nc3-c3ncccn3)C2)c1
InChIKey: DWXHACJTGPWMRJ-NVXWUHKLSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: TBDIn a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 2109 nM (pChEMBL 5.679999828338623)
Descriptors: MW 463.49, ALogP 2.8, HBA 9, HBD 0, PSA 116.63, QED 0.51
Compound (SMILES): CC(C)CC#Cc1ncc(-c2cc(=O)[nH]c(-c3cc(CNC(=O)C(C)C)ccc3C(F)(F)F)n2)cn1
InChIKey: MLYRAUBKMOCQRA-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 497.52, ALogP 4.58, HBA 5, HBD 2, PSA 100.63, QED 0.49
Compound (SMILES): COc1cc(-c2c(-c3ccc(F)cc3)nc(N)n3nc(Cc4c(F)cccc4F)nc23)ccn1
InChIKey: JUJONFDGLHEEMD-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Binding Affinities to Different Adenosine Receptors: The compounds at different concentrations were incubate with hA1 membrane (from PerkinElmer) and [3H]-8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) for 50 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 L ULTIMA GOLD was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA2a membrane (from PerkinElmer) and [3H]-CGS21680 for 90 min at 25° C., meanwhile 100 μL 0.5% PEI solution was added into UNFILTER-96 GF/B filter plate for 60 min at 4° C., then UNIFILTER-96 GF/B filter plate was washed twice with 50 ml wash buffer, the membrane mix was transferred into UNIFILTER-96 GF/B filter plate, and the filter plate was washed 4 times before incubated at 55° C. for 10 min. At last, 40 L ULTIMA GOLD was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA2b membrane (from PerkinElmer) and [3H]-DPCPX for 60 min at 27° C., and the binding reactions were stopped by rapid filtration through 0.5% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 L of scintillation cocktail was added into each well, and CPM was read by TopCount.The compounds at different concentrations were incubate with hA3 membrane (from PerkinElmer) and [1211]-AB-MECA for 60 min at 27° C., the binding reactions were stopped by rapid filtration through 0.50% BSA coated UNIFILTER-96 GF/C plates using cell harvester. The filter plates were then washed three times with ice cold wash buffer, and dried at 37° C. for 120 min. At last, 50 L of scintillation cocktail was added into each well, and CPM was read by TopCount.
Measurement: IC50 = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 462.44, ALogP 4.45, HBA 7, HBD 1, PSA 91.22, QED 0.42
Compound (SMILES): Cc1cc(Nc2ncnc3cccc(O[C@H]4C[C@H]5CC[C@@H](C4)N5CC(F)F)c23)ccc1Oc1ccn2ncnc2c1
InChIKey: IJKYTLFDLUSRSJ-SGRGSNTQSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition Assay: HER2: The compounds' inhibition of target modulation were determined as follows: BT474 cells were sorted in 96 well plates (20000 cells/well) with the DMEM medium containing 10% FBS overnight and then treated with tested compounds at a series of concentrations (3 μM, 0.3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.001 μM, 0.0001 μM. The plates were incubated for 4 h at 37° C. with 5% CO2 and then the HER2 (Y1248) phosphorylation level of cells in each well were measured with MSD Kit (Phospho-ErbB2 (Tyr1248) Assay Whole Cell Lysate Kit: Cat # K151CLD-3). The assay is a electrochemiluminescent method (MESO SCALE DISCOVERY) for determining both phosphorylated and total HER2 of cells with an MSD SECTOR® Imager and then the ratio of p-HER2/total HER2 can be generated by the machine. The percentage of inhibition was got from formula: % inhibition=100×[1−(ratio of sample well−ratio of Min ctrl well)/(ratio of Max−Ratio of Min ctrl well)]. The IC50 values were further calculated as the compounds concentration required for 50% inhibition in best-fit curves using Prism GraphPad 7.0 or Microsoft Xlfit softwar
Measurement: IC50 = 6.17 nM (pChEMBL 8.210000038146973)
Descriptors: MW 557.61, ALogP 6.16, HBA 9, HBD 1, PSA 89.7, QED 0.24
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C5COC5)CC4)cc3c2C(C)C)c(F)cn1
InChIKey: UALLNMJRLCQLSE-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
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 = 13.8 nM (pChEMBL 7.860000133514404)
Descriptors: MW 407.53, ALogP 5.38, HBA 3, HBD 1, PSA 41.15, QED 0.63
Compound (SMILES): Nc1ncc(-c2ccc(-c3cccnc3Oc3ccccc3)cc2F)cn1
InChIKey: HGLUUVDQCALZMO-UHFFFAOYSA-N
Target: Flap endonuclease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P39748
Assay: Binding Assay: The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 μg/well final for human) are incubated in the presence of the HTRF ligand. [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid] (25 nM final), a terbium labeled anti-His tag antibody (0.5 ng/well final, from Cisbio) and compounds. The reaction is allowed to proceed for two hours after which the plate is read on an Envision plate reader in HTRF mode. Data are expressed as a HTRF ratio. For human FLAP binding assays, data are analyzed with 3DX Explorer software. A ratio is calculated with the relative light units at 520 nm over the relative light units at 495 nm. For analysis, data from multiple runs are averaged and each compound may be tested in 2 to 20 runs. Each run comprises two plates and each plate includes duplicates. Data from each plate is averaged and data are imported into 3DX. The data from multiple runs are aggregated as the average of duplicates of the calculated ratios in order to calculate Ki and IC50 values.
Measurement: Ki = 13.2 nM (pChEMBL 7.880000114440918)
Descriptors: MW 358.38, ALogP 4.72, HBA 5, HBD 1, PSA 73.92, QED 0.57
Compound (SMILES): COc1ccc([C@H]2C[C@@H]2COc2nc(C)ncc2-c2c[nH]nn2)nc1
InChIKey: IHLVTNKSGOAFQQ-YPMHNXCESA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60;
Measurement: Ki = 1.36 nM (pChEMBL 8.869999885559082)
Descriptors: MW 338.37, ALogP 2.16, HBA 7, HBD 1, PSA 98.7, QED 0.73
Compound (SMILES): Cc1cc2nc(C(CNC(=O)c3ccc(-n4cnnc4)cc3Cl)c3ccc(F)cc3)[nH]c2cc1C
InChIKey: VJIXRSBZGUCQAV-UHFFFAOYSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Determination of Inhibitory Activity Against Factor IXa: Formation of a clot to stem bleeding at a site of blood vessel injury involves the coordinated activity of a group of plasma proteins that initiate and propagate fibrin formation and subsequently protect fibrin from premature degradation. Factor IX is a key component of the plasma system that forms a fibrin clot at a site of vascular injury. The activity of Factor IXa is measured by monitoring the cleavage of the fluorescent peptide, CH3SO2-D-CHG-Gly-Arg-AFC.AcOH (CHG is cyclohexyl-glycine and AFC is trifluoro aminomethyl coumarin). Factor IXa cleaves the amide bond between Arg and AFC, thereby releasing the AFC fluorophore. The free AFC can be detected with a fluorescence detector at an excitation wavelength of 405 nM and emission wavelength of 510 nM.
Measurement: IC50 = 8.42 nM (pChEMBL 8.069999694824219)
Descriptors: MW 488.95, ALogP 5.11, HBA 5, HBD 2, PSA 88.49, QED 0.35
Compound (SMILES): C=CC(=O)Nc1ccc(C(=O)N[C@@H]2CCC[C@@H](Nc3n[nH]c4c3CN(C(=O)N[C@H](CN(C)C)c3ccccc3)C4(C)C)C2)cc1
InChIKey: BMDYRVWBRSFBFF-STROYTFGSA-N
Target: CDK7/Cyclin H/MNAT1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P51948
Assay: Inhibition of Kinase Activity: Compounds of the invention were assayed for activity against a variety of different kinases.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 612.78, ALogP 4.75, HBA 6, HBD 5, PSA 134.49, QED 0.21
Compound (SMILES): NC(=O)Cc1cc2ccc(OCc3cccc(Cl)c3)cc2[nH]c1=O
InChIKey: WROVPIZEGDNQNH-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Enzymatic Assay: The MAO enzymatic assay was performed according to the fluorometric method described by Matsumoto and colleagues (Matsumoto et al., Clin. Biochem. 1985, 18, 126-129) with the following modifications. Human recombinant MAO-A and MAO-B expressed in insect cells were used. For both assays, test compound and/or vehicle were preincubated with purified enzyme in phosphate buffer pH 7.4 for 15 minutes at 37° C. The reaction was initiated by addition of 50 μm kynuramine. Following a 60 minute incubation period, the reaction was terminated by the addition of 6 N NaOH. The amount of 4-hydroxyquinoline that formed was determined by spectrofluorimetrically at 325 nm/465 nm.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 342.78, ALogP 2.79, HBA 3, HBD 2, PSA 85.18, QED 0.75
Compound (SMILES): Cc1cc(F)cc(Cl)c1NC(=O)c1cc(F)c(-n2nc3n(c2=O)CCCOC3)cc1O[C@@H](C)C(F)(F)F
InChIKey: WEDFPOYPQONFHE-LBPRGKRZSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Enzymatic Assay: Assay1: The enzymatic assay couples DHODH activity with bleaching of the dye 2,6-dichlorophenolindophenol (DCIP) (Knecht and Loffler, 1998; Miller et al., 1968). The assay was conducted in buffer containing 50 mM Tris, 0.1% Triton X-100, 150 mM potassium chloride, 2 nM DHODH, 1 mM dihydroorotate, 0.1 mM decylubiquinone, 0.06 mM DCIP, and 2% DMSO at pH 8.0 at 32 degree Celsius. The reaction was initiated by addition of substrates. Enzyme activity was monitored kinetically by the reduction in DCIP absorbance at 600 nm. Purified recombinant human DHODH enzyme was purchased from Novus (cat. no. NBP1-98916). Other chemicals were purchased from Sigma-Aldrich. Absorbance measurements were obtained using a BMG clarion star plate-reading spectrophotometer.
Measurement: IC50 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 546.88, ALogP 4.78, HBA 7, HBD 1, PSA 87.38, QED 0.47
Compound (SMILES): CCCCC(Cc1c[nH]c2ccccc12)NC(=O)c1cnc(N2CCN(C3CC3)CC2)s1
InChIKey: CCJUUDMCGOGCOK-UHFFFAOYSA-N
Target: Alpha-synuclein (SINGLE PROTEIN), Homo sapiens
UniProt: P37840
Assay: In-Vitro Fluorescence Polarization Assay: The fluorescence polarization assay tests the ability of compounds to inhibit the self-aggregation of α-synuclein peptide fragments. Peptides were incubated for 120 min at room temperature in the presence or absence of test compounds (compound concentrations were 33.3 to 0.015 μM). Samples were read on a Beckman Coulter DTX 880 plate reader in fluorescence polarization mode using excitation at 485 nm and emission at 520 nm. Data was analyzed using a four-parameter logistic fit (XLFit, IDBS Software). Peptide 4F (CTGFVKKDQLGK (SEQ ID NO: 1)) was prepared by American Peptide. Fresh peptide samples were reconstituted in purified water at 5 mM and diluted into 50 mM HEPES pH 7.4 with 50 mM NaCl to 100 nM final concentration. Solid compounds were dissolved in DMSO (10 mM), and then diluted serially in DMSO (300×) followed by dilution in buffer (1×) to provide solutions with a consistent final DMSO concentration of 0.33%.
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 451.64, ALogP 4.44, HBA 5, HBD 2, PSA 64.26, QED 0.5
Compound (SMILES): COc1cn(Cc2cc(F)c(F)cc2F)c(Nc2cccc(Cl)c2C)nc1=O
InChIKey: DUYWPZKCXCKSPM-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 409.8, ALogP 4.42, HBA 5, HBD 1, PSA 56.15, QED 0.63
Compound (SMILES): O=C1NC2(CCc3ncc(F)cc3[C@H]3[C@@H]4C[C@@H]4CN3c3ccn4ncc1c4n3)CC2
InChIKey: DHIAGBPAFZLDHX-BBEGDGIKSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HotSpot Assay: The HotSpot assay platform was used to measure kinase/inhibitor interactions as described in Anastassiadis et al., Nat Biotechnol. 29:1039-45, 2011. In brief, for each reaction, kinase and substrate were mixed in a buffer containing 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO. Compounds were then added to each reaction mixture. After a 20-min incubation, ATP (Sigma-Aldrich) and [γ-33P] ATP (PerkinElmer) were added at a final total concentration of 100 μM. Reactions were carried out at room temperature for 2 h and spotted onto P81 ion exchange cellulose chromatography paper (Whatman). Filter paper was washed in 0.75% phosphoric acid to remove unincorporated ATP. The percent remaining kinase activity relative to a vehicle-containing (DMSO) kinase reaction was calculated for each kinase/inhibitor pair.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 404.45, ALogP 2.67, HBA 6, HBD 1, PSA 75.42, QED 0.62
Compound (SMILES): Cc1c(COc2ccc(CNC3(CO)CCCC3)cn2)cccc1-c1ccccc1
InChIKey: WAEJPUPLURAWGG-UHFFFAOYSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Homogenous Time-Resolved Fluorescence (HTRF) Binding Assay: All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (withv) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 μl of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and further incubation for 15 m. PD-L1 from either human, cyno, or mouse were used. HTRF detection was achieved using europium crypate-labeled anti-Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 μl was dispensed on top of binding reaction. The reaction mixture was allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between PD-1-Ig/PD-L2-His (20 & 5 nM, respectively), CD80-His/PD-L1-Ig (100 & 10 nM, respectively) and CD80-His/CTLA4-Ig (10 & 5 nM, respectively). Competition studies between biotinylated SEQ ID NO:71 and human PD-L1-His were performed as follows. Inhibitors were pre-incubated with PD-L1-His (10 nM final) for 60 m in 4 μl of assay buffer followed by addition of biotinylated SEQ ID NO:71 (0.5 nM final) in 1 μl of assay buffer. Binding was allowed to equilibrate for 30 m followed by addition of europium crypated labeled Strepatavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reaction was allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer.
Measurement: IC50 = 555 nM (pChEMBL 6.260000228881836)
Descriptors: MW 402.54, ALogP 5.03, HBA 4, HBD 2, PSA 54.38, QED 0.55
Compound (SMILES): O=C(NCCC1CCCN1)c1ccc(Nc2nc3c(C4=CCN(C(=O)CCC(F)(F)F)CC4)cccn3n2)cc1
InChIKey: HSCWQVWNPONJHN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.69 nM (pChEMBL 9.15999984741211)
Descriptors: MW 555.61, ALogP 4.3, HBA 7, HBD 3, PSA 103.66, QED 0.36
Compound (SMILES): N#Cc1ccc(CN2CCC(NC(=O)c3ccc(C(=O)N4CCC(Oc5ccc(S(=O)(=O)C(F)(F)F)cc5)CC4)nc3)[C@H](F)C2)cc1
InChIKey: BFKLKEDGPVUXEU-QXPUDEPPSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P54646
Assay: Enzyme-Linked Immunosorbent Assay: Compounds were assayed for their ability to activate AMPK using an enzyme-linked immunosorbent assay. Reagents and procedures for measuring AMPK activation are well known and kits for AMPK activation assays are commercially available.
Measurement: EC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 673.69, ALogP 4.27, HBA 8, HBD 1, PSA 132.7, QED 0.35
Compound (SMILES): Cc1ccccc1[C@]1(O)C[C@H]2CN(CC(O)c3cccc(C(F)(F)F)c3)C[C@H]2C1
InChIKey: DKYSKBITAXEZBH-SFJHNWJUSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: FDSS Assay: To each well of the plate, 10 μL test compound, control (MK801) or HHnoCa buffer was added to a final concentration of 10 μM with a final concentration of DMSO of 0.1%. Following 10 minutes pre-incubation in the dark, plates are loaded onto the Hamamatsu FDSS 6000. After collecting baseline fluorescence images, 3 μM glutamate, 3 glycine, and 1 mM Ca2+ in HHnoCa buffer is added to each well, and Ca2+ is recorded for 3 minutes. Data were processed by computing ratio of fluorescence at the end of data collection to baseline fluorescence to assess degree of Ca′ influx inhibition relative to that observed in MK801.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 405.46, ALogP 4.28, HBA 3, HBD 2, PSA 43.7, QED 0.8
Compound (SMILES): C=CC(=O)N1CCC(Oc2cc3c(Nc4ccc(Cl)c(Cl)c4)c(C#N)cnc3cc2OC)CC1
InChIKey: ARTKYIYACREMAY-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Cell Viability Assay and IC50 Estimation: Cell viability was determined using the Cell Titer Glo assay (Promega) as previously described. Briefly, 2000-3000 cells per well were plated in 384-well plates (Greiner Bio-One) in technical triplicate. Cells were treated with seven different concentrations of tyrosine kinase inhibitors or vehicle alone at a final volume of 40 μL per well. After 3 days hours, 11 μL of Cell Titer Glo was added to each well. Plates were shaken for 15 minutes, and bioluminescence was determined using a FLUOstar OPTIMA multi-mode micro-plate reader (BMG LABTECH). Bioluminescence values were normalized to DMSO treated cells, and normalized values were plotted in GraphPad Prism using non-linear regression fit to normalized data with a variable slope. IC50 values were calculated by GraphPad Prism at 50% inhibition.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 497.38, ALogP 5.72, HBA 6, HBD 1, PSA 87.48, QED 0.44
Compound (SMILES): CC(C)(C)C(=O)Nc1cn2nc(-c3c(-c4ccc(F)cc4)ncn3Cc3cccnc3)ccc2n1
InChIKey: MKSBWWWWHCKWMR-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 7.79 nM (pChEMBL 8.109999656677246)
Descriptors: MW 469.52, ALogP 4.83, HBA 7, HBD 1, PSA 90, QED 0.4
Compound (SMILES): COc1cc(Nc2ccc3ncc(C4=CCOCC4)nc3c2C#N)ccc1OCc1ccc(C)nc1
InChIKey: KWYWNCVVBWZTBC-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: Kinase Assay: CSF-1R: Inhibition of CSF-1R kinase activity by a test compound disclosed herein was estimated by AlphaScreen (PerkinElmer). Standard assay conditions were 1.25 ng of recombinant CSF-1R kinase (SignalChem) with 10 ng Biotin-conjugated Poly-(Glu 4:Tyr 1) (Cisbio) in the assay buffer (10 μM ATP, 10 mM MOPs, pH7.0, 0.21 mM EDTA, 0.5% glycerol, 1 mg/ml BSA, 0.01% 2-mercaptoethanol, 0.001% Brij35) in a final volume of 25 μL. Reactions were incubated at 30° C. for 45 min and stopped by adding 5 μL of 50 mM EDTA. Analysis of resulting product using an AlphaScreen kit, and counted with Enspire Alpha (PerkinElmer). The readout from control reaction (complete reaction mixture) was designated as 0% inhibition and the readout for the reaction without enzyme as 100% inhibition. The IC50 values of quinoxaline compounds of this invention against CSF-1R kinase were determined using the GraphPad Prism 5 software.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 479.54, ALogP 5.34, HBA 8, HBD 1, PSA 102.18, QED 0.38
Compound (SMILES): CCc1c(Cl)[nH]c2ncc(-c3ccc(N)c(C(=O)N(C)C)c3F)cc12
InChIKey: QEZUQGHVFWJIKD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Inhibition Activity Assay: The presently disclosed compounds find use in inhibiting the activity of the enzyme HPK1. HPK1, also referred to as mitogen activated protein kinase kinase kinase kinase 1 or MAP4K1, is a member of the germinal center kinase subfamily of Ste20-related serine/threnonine kinases. HPK1 functions as a MAP4K by phosphorylating and activating MAP3K proteins, including MEKK1, MLK3 and TAK1, leading to the activation of the MAPK Jnk.
Measurement: Ki = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 360.82, ALogP 3.87, HBA 3, HBD 2, PSA 75.01, QED 0.7
Compound (SMILES): CS(=O)(=O)NC(=O)c1cc(C(F)(F)F)c2cc(OCC34CCC(OCc5c(-c6c(Cl)cncc6Cl)noc5C5CC5)(CC3)CC4)ccc2n1
InChIKey: JQAWTOGHWFIYHD-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/ml Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 739.6, ALogP 7.87, HBA 9, HBD 1, PSA 133.51, QED 0.17
Compound (SMILES): Cc1nc(C(=O)N2CC(F)(F)C[C@@H](C)C2CNc2ncc(C(F)(F)F)cn2)c(-c2ccc(F)cn2)s1
InChIKey: HJCNUQFYEDFLNC-ZVDHGWRTSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Orexin Receptor Cell-Based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHOOX1 or CHOOX2 cells were seeded into black-walled clear-base 384-well plates (Corning, catalog #3712) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and then incubated in a 5% CO2, 37° C. incubator overnight to reach 90% confluency. The cells were incubated with equal volume of calcium 6 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 2 h, followed by test compounds (dose-range 0.1 nM-10 μM) for another 30 min. The plates were then placed into a FLIPR (Molecular Devices, Inc.) to monitor fluorescence (λ excitation 488 nm, λ emission 540 nm) before and after the addition of EC90 of [OX].
Measurement: IC50 > 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 530.5, ALogP 5.06, HBA 7, HBD 1, PSA 83.9, QED 0.46
Compound (SMILES): CC(C)N1CCN(c2cccc([C@H]3[C@H](C(=O)NO)[C@@H]3c3ccccc3)c2)CC1
InChIKey: RUMUGZXUAHCWCB-YPAWHYETSA-N
Target: Histone deacetylase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P56524
Assay: Inhibition Assay: 5 μl of each solution of 1:20 diluted compound from above is transferred to a clear bottomed, black, 384-well assay plate using the Bravo or the Janus (384-well MDT head from Perkin Elmer). Using a 16-channel Matrix multi-channel pipette, 35 pII of the working solution of HDAC4 catalytic domain enzyme (0.86 μg/ml in assay buffer) is transferred to the assay plate. The assay is then started by adding 10 μl of 5× (50 μM) substrate to the assay plates using either the Bravo, Janus or 16-channel Matrix multi-channel pipette. The assay plate is then shaken for two minutes on an orbital shaker at 900 rpm (rotations per minute). Next the plate is incubated for 15 minutes at 37° C. The reaction is stopped by adding 25 μl of 3× (30 μM) developer/stop solution to the assay plates using either the Bravo, Janus or a 16-channel Matrix multi-channel pipette. Assay plates are then shaken for 5 minutes on an orbital shaker at 1200 rpm. Next, the assay plates are incubated at 37° C. for 1 hour in a tissue culture incubator. Finally, the fluorescence is measured (Excitation: 355 nm, Emission: 460 nm) using PerkinElmer EnVision in top read mode.
Measurement: IC50 = 1430 nM (pChEMBL 5.840000152587891)
Descriptors: MW 379.5, ALogP 3.22, HBA 4, HBD 2, PSA 55.81, QED 0.62
Compound (SMILES): COc1cc(-c2cnc(N)c(-c3ccc(NC(=O)c4cn(CC5(C)CCOCC5)cc(-c5ccc(F)cc5)c4=O)cc3F)c2)ccc1OC[C@H]1COCCO1
InChIKey: PXNSUHQVXVDKFQ-WJOKGBTCSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Cell-Free Axl Kinase Inhibitory Activity: A kinase dilution solution containing 170 ng/ml AXL (the 464th to 885th amino acids of the intracellular domain of human AXL expressed as a fusion protein with glutathione transferase in a baculovirus expression system and purified by glutathione Sepharose chromatography; Carna Biosciences, Inc., catalog No. 08-107) was prepared using a kinase reaction buffer solution (100 mM HEPES (pH 7.4), 0.003% Brij-35, 0.004% Tween-20, 1 mM DTT, and 10 mM MgCl2) and added at 19 μl/well to a 384-well plate.Next, each test compound was diluted with DMSO, and this dilution solution was added at 1 μl/well to the plate.After preincubation at room temperature for 30 minutes, a solution containing a substrate peptide (FL-Peptide 30 (5FAM-KKKKEEIYFFF-CONH2), Caliper Life Sciences, catalog No. 760430) and ATP at 1.5 μM and 10 μM, respectively, was prepared and added at 5 μl/well to the plate to start kinase reaction. The plate was incubated at 28° C. for 1.5 hours, and the reaction was terminated by the addition of a termination buffer solution (100 mM HEPES (pH 7.4), 0.015% Brij-35, 40 mM EDTA, and 0.1% Coating Reagent 3) at 40 μl/well.The substrate peptide and the phosphorylated peptide in the reaction mixture were separated and quantified using EZ Reader II (Caliper Life Sciences).The kinase reaction was evaluated on the basis of a product ratio (P/(P+S)) calculated from the peak height (S) of the substrate peptide and the peak height (P) of the phosphorylated peptide.The rate of inhibition (Inhibition) was determined according to the following expression (automatically calculated using the software of the EZ Reader II system).
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 752.82, ALogP 6.98, HBA 10, HBD 2, PSA 136.16, QED 0.15
Compound (SMILES): CN1CC[C@@H](COc2nc(N3CC4CCC(C3)N4)c3cnc(-c4cccc5cccc(Cl)c45)c(F)c3n2)C1
InChIKey: BQBYMDKGQCSDKJ-QHGAOEGBSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Binding Assay: Table 2: The ability of a compound to bind to KRAS G12D was measured using a TR-FRET displacement assay. Biotinylated GDP-loaded recombinant human KRAS G12D (corresponding to amino acids 1:169, produced at Array BioPharma) was incubated with a custom-made Cy5 labelled tracer, europium labelled streptavidin and compound (2% DMSO final) in buffer (50 mM HEPES [pH 7.5], 5mM MgCl2, 0.005% Tween-20 & 1 mM DTT). After a 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using a concentration of control compound that completely inhibits binding of the tracer to KRAS. The POC values were fit to a 4-parameter logistic curve and the IC50 value was determined as the concentration where the curve crosses 50 POC.
Measurement: IC50 = 87.2 nM (pChEMBL 7.059999942779541)
Descriptors: MW 533.05, ALogP 4.91, HBA 7, HBD 1, PSA 66.41, QED 0.39
Compound (SMILES): C[C@@H](COCCC(=O)N1CCC(c2ncc(C(F)(F)F)cn2)CC1)Nc1cn[nH]c(=O)c1C(F)(F)F
InChIKey: UPMLIEPSAGPWDZ-LBPRGKRZSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay for Inhibition of PARP7: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 μL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6xHis and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM Example 190 were used to calculate the % inhibition as described below:% ⁢ ⁢ inhibition = 100 × TRF cmpd - TRF min TRF max - TRF minwhere TRFcmpd is the TR-FRET ratio from the compound treated well, TRFmin is the TR-FRET ratio from the Example 190-treated positive control well and TRFmax is the TR-FRET ratio from the DMSO-treated negative control well.The % inhibition values were plotted as a function of compound concentration and the following 4-parameter fit was applied to derive the IC50 the values:Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ Coefficientwhere top and bottom are normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient is normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and x is the compound concentration.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 522.45, ALogP 3.21, HBA 7, HBD 2, PSA 113.1, QED 0.4
Compound (SMILES): C=CC(=O)N1CCN(C(=O)c2cc(Sc3cnc(NC(=O)c4cnn(C)c4)s3)c(C)cc2C)CC1
InChIKey: XFLVXPLUYCMHIV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 510.65, ALogP 3.37, HBA 8, HBD 1, PSA 100.43, QED 0.51
Compound (SMILES): CCOc1cc2ncc(C#N)c(Nc3ccc(OCc4ncccc4C)cc3C)c2cc1NC(=O)CCCN(C)C
InChIKey: RIBQOSJJDUEEMV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13043
Assay: MST1/MST2 Biochemical LanthaScreen Eu Kinase Binding Assay: MST1 and MST2 biochemical LanthaScreen Eu Kinase Binding Assay was based on the binding and displacement of kinase tracer to the kinase of interest. Compounds in 1000×DMSO stock solution was dispensed using automated dispensing system (Labcyte) to 384 well Corning Microplate at 15 nL, 5 uL of Kinase buffer A was added to each well. Plates were shaken and incubated for 1 min to ensure well dissolution of compounds. Kinase/Antibody mixture was added at a final concentration of 5 nM and 2 nM and kinase tracer 222 solution at a final concentration of 100 nM in a total volume of 20 uL. Plates were incubated for 1.5 hrs in dark at room temperature and assay plates were scanned on Envision plate reader with excitation: 340 nM and kinase Tracer Emission at 665 nM.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 552.68, ALogP 6.12, HBA 8, HBD 2, PSA 112.4, QED 0.22
Compound (SMILES): Cc1cc(F)cc(-c2cnc3ncc(-c4cc(F)cc(C#N)c4O)cc3c2N2CCC(N)CC2)c1
InChIKey: TZCICCIEPWIPJS-UHFFFAOYSA-N
Target: Somatostatin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P30874
Assay: cAMP Assay : Four days prior to the assay, 5,000 Chinese hamster ovary cells (CHO-K1, ATCC #CCL-61) stably expressing the human SSTR2 are plated in each well of a 96-well tissue culture-treated plate in Ham's F12 growth media (ThermoFisher #10-080-CM) supplemented with 10% donor bovine serum (Gemini Bio-Products #100-506), 100 U/mL penicillin; 100 ug/mL streptomycin; 2 mM L-glutamine (Gemini Bio-Products #400-110) and 0.2 mg/mL hygromycin B (GoldBio #31282-04-9). The cells are cultured at 37° C., 5% CO2 and 95% humidity. On the day of the assay, the media is aspirated and the cells are treated with 50 μL of 1.6 μM NKH477 (Sigma #N3290) plus various dilutions of compounds of the invention in assay buffer [1× Hank's Balanced Salt Solution (ThermoFisher #SH3058802), 0.5 mM HEPES pH 7.4, 0.1% bovine serum albumin, 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX, VWR #200002-790)]. The cells are incubated for 20 minutes at 37° C. (the final concentration of the compounds of the invention are typically 0-10,000 nM). The cells are treated with 50 μL of lysis buffer (HRTF cAMP kit, Cisbio). The lysate is transferred to 384-well plates and cAMP detection and visualization antibodies are added and incubated for 1-24 hours at room temperature. The time-resolved fluorescent signal is read with a Tecan M1000Pro multiplate reader. The intracellular cAMP concentrations are calculated by regression to a standard curve and are plotted vs. the concentration of the compounds of the invention and the EC50 of the compounds are calculated using standard methods. All data manipulations are in GraphPad Prism v6.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 471.51, ALogP 5.06, HBA 6, HBD 2, PSA 99.06, QED 0.43