lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(-c2cccc(C(F)(F)F)c2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: YFIBWLODXJUCGW-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 2554 nM (pChEMBL 5.590000152587891)
Descriptors: MW 559.5, ALogP 5.61, HBA 7, HBD 1, PSA 103.43, QED 0.27
Compound (SMILES): O=C(NO)c1ccc(NCCOc2ccc3cc2COC/C=C/COCc2cccc(c2)-c2ccnc(n2)N3)cc1
InChIKey: ZDNWRPFSJGRPNH-OWOJBTEDSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 (1 to 1215 residues) expressed in sf9 cells preincubated with enzyme followed by fluorogenic Arg-His-Lys-Lys(Ac)-AMC substrate addition measured after 2 hrs by fluorescence assay
Measurement: IC50 = 395 nM (pChEMBL 6.400000095367432)
Descriptors: MW 553.62, ALogP 5.1, HBA 9, HBD 4, PSA 126.86, QED 0.11
Compound (SMILES): O=c1cc(NC2CCN(S(=O)(=O)C(F)(F)F)CC2)c2cc(C(c3ccc(Cl)cc3)c3ccc(Cl)cc3)cc(Br)c2[nH]1
InChIKey: XZBWAKUKMRWBNR-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 689.38, ALogP 7.5, HBA 4, HBD 2, PSA 82.27, QED 0.21
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCC(NCC2CCCCC2)CC1
InChIKey: YRSNVUIDUDNSDI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 153 nM (pChEMBL 6.820000171661377)
Descriptors: MW 453.57, ALogP 3.98, HBA 7, HBD 3, PSA 121.65, QED 0.52
Compound (SMILES): CCOc1cc(N2CCN(CCS(C)(=O)=O)CC2)c(Cl)cc1Nc1nccc(-c2c(-c3ccc(OCC)c(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: LPRBNEUFUZWEEI-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 831.35, ALogP 7.35, HBA 12, HBD 2, PSA 143.29, QED 0.11
Compound (SMILES): c1ccc(-c2cc(CNc3nc(Nc4cc(C5CC5)[nH]n4)cc(N4CCOCC4)n3)on2)nc1
InChIKey: SDWDOHSEWUGWHZ-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 459.51, ALogP 3.32, HBA 10, HBD 3, PSA 129.91, QED 0.36
Compound (SMILES): COc1ccc(-c2cccc(OCC(O)CN3CCc4ccccc4C3)c2)c(Cl)c1
InChIKey: FHYHECDEOUNXFL-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 423.94, ALogP 4.81, HBA 4, HBD 1, PSA 41.93, QED 0.59
Compound (SMILES): COc1ccc(-c2c3ccccc3cc3c(=O)cc(N4CCOCC4)oc23)cc1
InChIKey: IQUGIKIYSFPJHS-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of p110alpha (unknown origin) assessed as decrease in ATP consumption after 60 mins by Kinase-Glo assay
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 387.44, ALogP 4.46, HBA 5, HBD 0, PSA 51.91, QED 0.49
Compound (SMILES): Cc1c(Cc2ccccc2)nnc(N2CCN(Cc3ccccc3)CC2)c1C
InChIKey: REUQIRONFPYTOT-UHFFFAOYSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 372.52, ALogP 4.01, HBA 4, HBD 0, PSA 32.26, QED 0.68
Compound (SMILES): CCNC(=O)c1cc2c(-c3cc(S(C)(=O)=O)ccc3Oc3ccc(F)cc3F)cn(C)c(=O)c2[nH]1
InChIKey: GLPRVXBSUHOVMP-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: TR-FRET Assay: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus#6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe.The final concentration of 1× assay mixture contained 2% DMSO, 12 nM His tagged BRD4 (BDI_K57-E168) and 100 nM probe or 4 nM His tagged BRD4 (BDII_E352-M457) and 30 nM probe, and 1 nM Europium-conjugated anti-His-tag antibody, and compound concentrations in the range of: 49.02 μM-0.61 nM or 0.98 μM-0.15 nM.After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 15.5 nM (pChEMBL 7.809999942779541)
Descriptors: MW 501.51, ALogP 3.76, HBA 6, HBD 2, PSA 110.26, QED 0.42
Compound (SMILES): C#CCCCCC(=O)Nc1nc2nn(C)cc2c2nc(-c3ccco3)nn12
InChIKey: QCDXAQOOYGGZCE-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Radioligand Binding Assays: Radioligand binding assays at hA1, hA2A, and hA3ARs were performed according to the procedures described in Gao, Z. G., et al., Biochem Pharmacol. 2003; 65: 1675-84, using the radiolabeled agonists [3H]R-PIA from Moravek Biochemicals (Brea, Calif.) or [3H]CGS21680 and [125I]I-AB-MECA from PerkinElmer (Waltham, Mass.) for the hA1, hA2AAR, and hA3AR assays, respectively.
Measurement: Ki = 151 nM (pChEMBL 6.820000171661377)
Descriptors: MW 363.38, ALogP 2.4, HBA 8, HBD 1, PSA 103.14, QED 0.42
Compound (SMILES): Cc1ccc2c(Nc3ccc(F)c(Cl)c3)nccc2c1NC(=O)c1csc2c(N)ncnc12
InChIKey: GNDBDDNLWIOKDN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Kinase Activity: The compounds prepared in Examples were tested for inhibitory activity against three subtypes of RAF, i.e., RAF1 Y340D Y341D (C-RAF), B-RAF normal type and B-RAF.sup.V600E using Kinase Profiling Service (Invitrogen, U.S.) according to the manufacturer's instructions. The levels of enzymatic inhibition of the compounds were calculated as percent inhibition at various concentrations.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 478.94, ALogP 5.92, HBA 7, HBD 3, PSA 105.82, QED 0.3
Compound (SMILES): COc1cc(CCCN2CCN(C)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: UOUIRSAMKHDQKN-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 688.78, ALogP 6.92, HBA 9, HBD 2, PSA 99.92, QED 0.15
Compound (SMILES): CN(C)CC(O)Cn1cc(NC(=O)c2cnn3cccnc23)c(-c2cc(Cl)ccc2OC(F)F)n1
InChIKey: BNIFWZKGIUYSMM-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK 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 = 0.61 nM (pChEMBL 9.210000038146973)
Descriptors: MW 505.91, ALogP 3.02, HBA 9, HBD 2, PSA 109.81, QED 0.36
Compound (SMILES): COC(=O)N[C@H](C(=O)N1CCC[C@H]1c1nc2cc([C@H]3CC[C@H](c4ccc5[nH]c([C@@H]6CCCN6C(=O)[C@@H](NC(=O)OC)C(C)C)nc5c4)N3c3cc(F)c(N4CCCCC4)c(F)c3)ccc2[nH]1)C(C)C
InChIKey: HJCNWJOJPGJAGF-CNGYLHDYSA-N
Target: Nonstructural protein 5A (SINGLE PROTEIN), Hepatitis C virus
UniProt: Q5L478
Assay: Inhibition of Hepatitis C virus genotype 1a H77 replicon NS5A Y93H mutant transfected in human HuH7 cells assessed as decrease in replication of viral replicon after 4 days by luciferase reporter gene assay
Measurement: EC50 = 0.491 nM (pChEMBL 9.3100004196167)
Descriptors: MW 951.13, ALogP 8.88, HBA 10, HBD 4, PSA 181.12, QED 0.09
Compound (SMILES): O=C(c1cccc2ccccc12)n1c(C2CCCC2)nc2ccccc21
InChIKey: YEAPPULZCJSLHC-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Displacement of [3H]CP-55940 from human recombinant CB1 receptor expressed in HEK293 cell membranes incubated for 1.5 hrs
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 340.43, ALogP 5.54, HBA 3, HBD 0, PSA 34.89, QED 0.48
Compound (SMILES): CNC(=O)c1ccnc(C#Cc2ccc(F)c([C@]3(CF)C[C@@H](C(F)(F)F)OC(N)=N3)c2)c1
InChIKey: GKXQYJGONFQFOV-FXAWDEMLSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 > 15600 nM (pChEMBL 4.807000160217285)
Descriptors: MW 452.38, ALogP 2.81, HBA 5, HBD 2, PSA 89.6, QED 0.55
Compound (SMILES): C=CC(=O)Nc1cc(OCCOC)ccc1Nc1cc(N(C)C(=O)Nc2c(Cl)c(OC)cc(OC)c2Cl)ncn1
InChIKey: KSHKPQNNKTXRLX-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Kinase HotSpotâ¿¢ Assay: Compounds were profiled for FGFR inhibition activity at Reaction Biology Corporation (Malvern, Pa.) with their Kinase HotSpot assay. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.Recombinant FGFR1 (2.5 nM), FGFR2 (1 nM), FGFR3 (5 nM), or FGFR4 (12 nM) (Invitrogen) was prepared as a mixture with substrate KKKSPGEYVNIEFG (SEQ ID NO:1) (20 uM, FGFR1 substrate); and Poly [E,Y]4:1 (0.2 mg/ml, FGFR2,3,4 substrate)] in kinase reaction buffer (20 mM HEPES-HCl, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij35, 0.1 mM Na3VO4, 0.02 mg/ml BSA, 2 mM DTT, and 1% DMSO). Compound was added to the enzyme/substrate mixture using acoustic technology (Labcyte Echo 550, Sunnyvale, Calif.) (see, Olechno et al., 2006, Improving IC50 results with acoustic droplet ejection. JALA 11, 240-246) and pre-incubated for 0, 15, or 60 minutes at room temperature. After compound pre-incubation, a mixture of ATP (Sigma-Aldrich) and 33P-γ-ATP (PerkinElmer) was added to a final concentration of 10 uM to initiate kinase reactions. Reactions were incubated for 120 minutes at room temperature and then spotted onto Whatman P81 ion exchange filter paper. Unbound phosphate was removed by extensively washing filters in 0.75% phosphoric acid. See, Anastassiadis et al., 2011, Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity. Nat Biotechnol 29, 1039-1045.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 591.45, ALogP 5.36, HBA 9, HBD 3, PSA 136.17, QED 0.19
Compound (SMILES): O=C(O)CCN1CCC(Nc2cc(=O)[nH]c3ccc(C(c4ccc(Cl)cc4)c4ccc(Cl)cc4)cc23)CC1
InChIKey: MJEBXCKCUQSZIP-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 550.49, ALogP 6.37, HBA 4, HBD 3, PSA 85.43, QED 0.22
Compound (SMILES): Cn1cc(-c2cc(NS(=O)(=O)C3CC3)ccc2Oc2ccc(F)cc2F)c2cc[nH]c2c1=O
InChIKey: WZDGTJOAZLDFEB-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Bromodomain Domain Binding Assay: A time-resolved fluorescence resonance energy transfer (TR-FRET) assay was used to determine the affinities of compounds of the Examples listed in Table 1 for each bromodomain of human BRD4. His-tagged first (BD1: amino acids K57-E168) and second (BD2: amino acids E352-E168) bromodomains of human BRD4 were expressed and purified. An Alexa647-labeled BET-inhibitor was used as the fluorescent probe in the assay. Compound dilution series were prepared in DMSO via a 3-fold serial dilution from 2.5 mM to 42 nM. Compounds were then diluted 6:100 in assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) to yield 3× working solutions. Six microliters (L) of the working solution was then transferred to white, low-volume assay plates (Costar #3673). A 1.5× assay mixture containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and the Alexa-647-conjugated probe molecule was also prepared. Twelve L of this solution were added to the assay plate to reach a final volume of 18 L. The final concentration of 1× assay buffer contains 2% DMSO, 50 M-0.85 nM compound, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively). After a one-hour incubation at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 471.49, ALogP 4.51, HBA 5, HBD 2, PSA 93.19, QED 0.43
Compound (SMILES): CO[C@H]1CN(c2ncc(C(=O)Nc3ccc(OC(F)(F)F)cc3)cc2-c2cncnc2)C[C@@H]1O
InChIKey: HSZKZOAFPZIIMI-OALUTQOASA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Radio ABL1 (64-515) Assay: For determination of ABL kinase activity, the radiometric filter-binding assay was used. The assay was performed by mixing 10 uL of the compound pre-diluted with 10 uL of ATP (20 uM ATP with 0.1 uCi [γ-33P]-ATP) with the phospho-acceptor peptide poly[Ala6Glu2LysHBr5Tyr1]=polyAEKY) in 20 mM Tris/HCl pH 7.5, 1 mM DTT, 10 mM MgCl2, 0.01 mM Na3VO4, 50 mM NaCl. 10 uL of enzyme (ranging between 5 nM to 20 nM) was added to initiate the reaction. Pre-incubation of enzyme with compounds (when stated) was performed by exposing the enzyme to compounds prior to addition of the substrate mixture (ATP and/or peptide substrate). After 15 mM at room temperature, the reaction was stopped by the addition of 50 uL 125 mM EDTA, and the peptide-bound 33P separated on filter-plates (PVDF or MAIP; Millipore, Volketswil, Switzerland) prepared according to the manufacturer's instructions. Filter-plates were washed 3x with 0.5% H3PO4, followed by addition of 30 uL scintillation cocktail (Microscint, Perkin Elmer) per well and then analysed in a TopCount NXT scintillation counter (Perkin Elmer).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 475.43, ALogP 2.89, HBA 8, HBD 2, PSA 109.7, QED 0.56
Compound (SMILES): CNC(=O)CC1COc2cc(Cl)ccc2N1C(=O)c1ccc2c(c1)NC(=O)CO2
InChIKey: PLOQCPGEMKIJBX-UHFFFAOYSA-N
Target: Mineralocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08235
Assay: Binding Assay: Test A1: Briefly, in Test A1 the assay was run in 384 well format in 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA 20 mM NaMoO4, 0.1 mM DTT and 10% glycerol at rt. Compounds were tested in a 7 (Test A1) or 10 (Test A2) concentration response curve ranging from 10 nM to 10 μM (Test A1) or 1 nM to 37 μM (Test A2). Compounds were spotted at the bottom of a well of a 384-well PE Opti-Plate to yield final DMSO concentrations in the assay of 2%. Pre-made MBP-MR/P23 lysate: 3H-aldosterone mix (final assay concentration 7 μg/mL MBP-MR LBD/P23 lysate; 5 nM aldosterone) was added onto the top of the spotted compound and preincubated for 1 h at RT. After 1 h an equal volume anti-rabbit SPA beads coupled with rabbit anti-MBP were added to the assay mixture and incubated for 3 hrs at rt. The inhibition of the scintillation signal by displacement of the bound 3H-aldosterone by test compounds is measured by scintillation counting using a Microbeta Trilux (Wallac).
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 415.83, ALogP 2.21, HBA 5, HBD 2, PSA 96.97, QED 0.8
Compound (SMILES): N#Cc1nc(C(=O)NCC(C(=O)O)c2ccccc2F)c(O)c2ccc(Oc3ccccc3)cc12
InChIKey: RRUOOJXGLKPFSE-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 11930 nM (pChEMBL 4.920000076293945)
Descriptors: MW 471.44, ALogP 4.34, HBA 6, HBD 3, PSA 132.54, QED 0.37
Compound (SMILES): Cc1nc(C(C)(C)NC(=O)c2ccc(N3CCCC3)c(OCC3CC3)n2)no1
InChIKey: GMIAFWFGCHEJDG-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 385.47, ALogP 2.83, HBA 7, HBD 1, PSA 93.38, QED 0.78
Compound (SMILES): O=C(O)CCCOc1ccc(-c2cc(OC(F)(F)F)ccc2F)cc1
InChIKey: UXXNWFQAORSGLZ-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Mus musculus
UniProt: Q7TMA4
Assay: Agonist activity at mouse GPR120 expressed in CHOK1 cells measured after 60 mins by IP1-HTRF assay
Measurement: EC50 = 432 nM (pChEMBL 6.369999885559082)
Descriptors: MW 358.29, ALogP 4.63, HBA 3, HBD 1, PSA 55.76, QED 0.58
Compound (SMILES): O=C(O)C1CN(C(c2ccccc2)c2ccccc2)CCN1C(=O)CC(c1ccccc1)c1ccccc1
InChIKey: ADXUUSOTCRBZGI-UHFFFAOYSA-N
Target: Voltage-dependent N-type calcium channel subunit alpha-1B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q02294
Assay: Antagonist activity at rat alpha1B N-type Ca2+ channel expressed in HEK293 cells assessed as 0.2 Hz current frequency dependent channel blocking for 2 mins in presence of Ba2+ by whole-cell patch clamp assay
Measurement: IC50 = 140 nM (pChEMBL 6.849999904632568)
Descriptors: MW 504.63, ALogP 5.6, HBA 3, HBD 1, PSA 60.85, QED 0.34
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2ncnc3nc(C)n(C)c23)cc(NC(=O)c2ccc(Cl)cn2)n1
InChIKey: IJFBEHQRNFJTIY-UHFFFAOYSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: DELFIA Assay: R3 domains of human XIAP (covering amino acids 241 to 356; XIAP BIR3) and cIAPl (covering amino acids 256 to 363; cIAPl BIR3) were expressed and purified from Ecoli as GST-fusion proteins. Peptide AVPIAQKSE-Lys(Biotin), representing the N-terminus of mature human SMAC (SMAC peptide), was used as interaction partner in the protein- peptide interaction assay.BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonXlOO) for one hour at room temperature in the presence of inhibitiory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-Nl- antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluoresecence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 479.93, ALogP 2.58, HBA 9, HBD 3, PSA 139.61, QED 0.38
Compound (SMILES): CC(C)(C)NC(=O)c1ccc(OC/C(=C\F)CN)cc1
InChIKey: OWNSXNXYELKDSO-FLIBITNWSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Recombinant SSAO/VAP-1 Inhibtion Assay: Briefly, HMEC cell expressing human SSAO/VAP-1 were grown in several 10 cm petri dishes, once the cells reached 100% confluency, cells were harvested and homogenates were prepared. Cells were washed twice with 5 mL of chilled HES buffer (20 mM HEPES, 1 mM EDTA, 250 mM sucrose, pH 7.4). HES buffer containing 1× protease inhibitor (Sigma Aldrich) and added and cells were incubated on ice for 3 min. Buffer was removed and cells were scraped and transferred to a centrifuge tube. Cell lysates were prepared by passing through 23 G needle for, 10 times and followed by 27 G needle for 10 times. Alternatively the cell lysates were prepared by using IKA Ultra-Turrax T 10 homogenizer for 3 min for every 10 mL of cell suspensions. Cells were then spun for 5 min at 300×g. The clear supernatant was transferred to new centrifuge tube and stored at −80 °C. until colorimetric assay was performed. Prior to the assay, 0.5 mM pargyline was added in order to inhibit any residue MAO activities. Briefly, 50 μL of cell lysate was incubated with test compounds for 30 min at 37 °C. Reaction mixtures were added and kinetic was read as described in detail in Example 5: Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M NaPO4 buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-9 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37 °C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M NaPO4 buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37 °C. excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).
Measurement: IC50 < 100000 nM (pChEMBL 4)
Descriptors: MW 280.34, ALogP 2.41, HBA 3, HBD 2, PSA 64.35, QED 0.87
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1cccs1
InChIKey: WUZLLRMBAPHKQG-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 664 nM (pChEMBL 6.179999828338623)
Descriptors: MW 331.44, ALogP 3.26, HBA 4, HBD 3, PSA 84.22, QED 0.51
Compound (SMILES): COCc1cc(OC)nc(N2CC3C(c4ccccc4)(C2)NC(=N)N(C)S3(=O)=O)c1
InChIKey: LELOSQBFNBDVQT-UHFFFAOYSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 were determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3x the desired final concentration in 1x BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO were pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1x BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30 C. The assay was initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 uM for 4 uM for autoBACE-2) prepared in 1x BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C.
Measurement: Ki = 6.4 nM (pChEMBL 8.1899995803833)
Descriptors: MW 431.52, ALogP 1.12, HBA 7, HBD 2, PSA 107.85, QED 0.73
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(N(Cc2nccs2)S(=O)(=O)Cc2ccccc2)ccc1Oc1ccc(F)cc1F
InChIKey: FFPPVADXQWDVDF-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method C: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.047 mM to 0.78 nM or 5-fold serial dilution from Assay method A. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 44.4 nM (pChEMBL 7.349999904632568)
Descriptors: MW 623.7, ALogP 6.51, HBA 8, HBD 0, PSA 90.73, QED 0.17
Compound (SMILES): Oc1nc(O)c(-c2ccc(-c3c(CCc4ccc5ccccc5n4)nc4c(N5CCOCC5)ccnn34)cc2)o1
InChIKey: UXPNCYCPIFADNH-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 534.58, ALogP 4.63, HBA 10, HBD 2, PSA 122.04, QED 0.31
Compound (SMILES): COc1cn(C(CC2(C)CCOCC2)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C#N
InChIKey: PAESNLCGOZMSMT-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 550.01, ALogP 5.13, HBA 7, HBD 2, PSA 130.65, QED 0.4
Compound (SMILES): C[C@@H]1CN(Cc2ccccc2)c2nc(Cl)ccc2[C@H](C)N1C(=O)NCc1ccccc1
InChIKey: JEEPAZPOYVQOTG-MOPGFXCFSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Channel blocking activity at recombinant human CaV 3.2 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 434.97, ALogP 5.42, HBA 3, HBD 1, PSA 48.47, QED 0.56
Compound (SMILES): Cc1nc(N2CC(c3nc4ccccc4n3C)C2)cc(-n2nc(C)nc2C(O)C(F)(F)F)n1
InChIKey: KTOHNYWHPPXADM-UHFFFAOYSA-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. A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 nL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/1600 dilution from aliquots; sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP PDE from Molecular Devices (product # R7506), at a final concentration of 50 nM are made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). The enzyme and the substrate are then added to the assay plates in two consecutive additions of 10 μL, and then shaken to mix. The reaction is allowed to proceed at room temperature for 30 minutes. A binding solution is then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction is stopped by addition of 60 μL of the binding solution to each well of the assay plates and the plates are sealed and shaken for 10 seconds. The plate was incubated at room temperature for at least one hour prior to determining the fluorescence polarization (FP). The parallel and perpendicular fluorescence of each well of the plate was measured using a Perkin Elmer EnVision™ plate reader (Waltham, Mass.).
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 458.45, ALogP 2.76, HBA 9, HBD 1, PSA 97.78, QED 0.5
Compound (SMILES): CC(=O)N1CCC(N(C(=O)c2ccc(N3CC(F)(F)C3)c(OCC3CC3)n2)C2CC2)CC1
InChIKey: POUQMLMBJYPWGE-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37 °C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30 °C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30 °C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 448.51, ALogP 2.94, HBA 5, HBD 0, PSA 65.98, QED 0.64
Compound (SMILES): COc1cc(O)ccc1-c1ccc2c(-c3nc4cc(N(C)C)ccc4[nH]3)n[nH]c2c1
InChIKey: HHCXBAIPQMHOLU-UHFFFAOYSA-N
Target: CDK4/Cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: P30281
Assay: Inhibition of human CDK4/cyclin D3 expressed in Escherichia coli using retinoblastoma (386 to 928 residues) as substrate after 20 mins by gamma32P-ATP based scintillation counting analysis
Measurement: Ki = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 399.45, ALogP 4.55, HBA 5, HBD 3, PSA 90.06, QED 0.41
Compound (SMILES): Cn1nc(-c2ccc(F)cc2)c(-c2ccc3nc(NC(=O)c4ccnc(F)c4)cn3n2)c1C1CCN(C(=O)c2ccon2)CC1
InChIKey: JJCAJGXJXSDRQX-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul 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 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®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 uM ATP, 1.5 uM 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. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 609.6, ALogP 4.73, HBA 10, HBD 1, PSA 136.34, QED 0.27
Compound (SMILES): COC(=O)CCCCCC[C@H]1C(=O)C[C@@H](O)[C@@H]1c1ccc(C(C)(C)C)cc1
InChIKey: IRVWZTVUXFVHPI-CZTZKLFOSA-N
Target: Prostaglandin E2 receptor EP3 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43115
Assay: Agonist activity at human recombinant EP3A receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 374.52, ALogP 4.53, HBA 4, HBD 1, PSA 63.6, QED 0.54
Compound (SMILES): C#CC(=O)Nc1cccc(-c2cnc3[nH]cc(Cl)c3c2)c1
InChIKey: URAHJBBXVHNRTQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: In Vitro Inhibition Assay: ITK and JAK3 Kinase assay procedures:Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μL of 3% phosphoric acid. Volume was increased to 100 μL by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μL 0.8% phosphoric acid and dried for an hour at 60° C. 100 μL scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 295.73, ALogP 3.46, HBA 2, HBD 2, PSA 57.78, QED 0.71
Compound (SMILES): Cc1nc(C(F)(F)F)ccc1C(=O)Nc1cn2nc(-c3cn(C)nc3-c3ccc(F)cc3)ccc2n1
InChIKey: CCBIRAQXPXEBTH-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul 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 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®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 uM ATP, 1.5 uM 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. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 6.5 nM (pChEMBL 8.1899995803833)
Descriptors: MW 495.44, ALogP 4.91, HBA 7, HBD 1, PSA 90, QED 0.36
Compound (SMILES): Cc1cc(C)cc(C2=N[C@H](NC(=O)[C@H](CCC(F)(F)F)[C@H](CCCC(F)(F)F)C(N)=O)C(=O)Nc3c(Cl)cccc32)c1
InChIKey: IALGOODQMCARJD-HOOSLVGPSA-N
Target: Neurogenic locus notch homolog protein 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM47
Assay: Biological Assay: The pharmacological properties of the compounds of this invention may be confirmed by a number of biological assays.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 619.01, ALogP 5.98, HBA 4, HBD 3, PSA 113.65, QED 0.28
Compound (SMILES): Fc1ccc(-c2noc(CCC3(c4ccccc4)CCCCC3)n2)cc1F
InChIKey: RCELRZXMKQDORQ-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: GTPgammaS Binding Assay: Compounds were loaded in a 384 Falcon v-bottom plate (0.5 ul/well in a 11 point, 3-fold dilution). Membranes prepared from S1P1/CHO cells or EDG3-Ga15-bla HEK293T cells were added to the compound plate (40 ul/well, final protein 3 ug/well) with MULTIDROP. [35S]GTP (1250 Ci/mmol, Perkin Elmer) was diluted in assay buffer: 20 mM HEPES, pH7.5, 10 mM MgCl2, 150 mM NaCl, 1 mM EGTA, 1 mM DTT, 10 uM GDP, 0.1% fatty acid free BSA, and 10 ug/ml Saponin to 0.4 nM. 40 ul of the [35S] GTP solution was added to the compound plate with a final concentration of 0.2 nM. The reaction was kept at room temperature for 45 min. At the end of incubation, all the mixtures in the compound plate were transferred to Millipore 384-well FB filter plates via the VELOCITY11 Vprep liquid handler. The filter plate was washed with water 4 times by using the manifold Embla plate washer and dried at 60° C. for 45 min. MicroScint 20 scintillation fluid (30 ul) was added to each well.
Measurement: EC50 = 5900 nM (pChEMBL 5.230000019073486)
Descriptors: MW 368.43, ALogP 5.85, HBA 3, HBD 0, PSA 38.92, QED 0.57