lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCCNc1cc(Nc2nccc(-c3cnc(N4CC[C@H](F)C4)c(C#N)c3)n2)ccn1
InChIKey: CVAKBVASVPZLKY-KRWDZBQOSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 15 nM (pChEMBL 7.823999881744385)
Descriptors: MW 434.48, ALogP 3.16, HBA 9, HBD 2, PSA 111.88, QED 0.52
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CC4F)cc12
InChIKey: BGPBFZMUWAKPLT-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 5.486 nM (pChEMBL 8.260000228881836)
Descriptors: MW 630.65, ALogP 6.84, HBA 6, HBD 1, PSA 97.44, QED 0.23
Compound (SMILES): O=C(O)c1ccc(-c2cnc(N3CCOCC3)c3nc([C@H]4C[C@@H](c5ccc6ccccc6n5)C4)cn23)cc1
InChIKey: MGULDWUFWCBTGQ-ZRZAMGCNSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay hPDE10a: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6×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 to 20 μL, of incub
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 505.58, ALogP 5.14, HBA 7, HBD 1, PSA 92.85, QED 0.35
Compound (SMILES): Nc1ncc(-c2ncccc2OC(F)(F)F)nc1C(=O)Nc1ncccc1N1CCC(N)(CO)CC1
InChIKey: AAKDVUMRVXYBST-UHFFFAOYSA-N
Target: Protein kinase C theta type (SINGLE PROTEIN), Homo sapiens
UniProt: Q04759
Assay: In Vitro Inhibition Activity Assay: The compounds of formula I were tested for their activity on different PKC isoforms according to a published method (D. Geiges et al. Biochem. Pharmacol. 1997; 53:865-875) The assay is performed in a 96-well polypropylene microtiterplate (Costar 3794) that has been previously siliconized with Sigmacote (Sigma SL-2). The reaction mixture (50 μL) contains 10 μL of the relevant PKC isozyme together with 25 μL of the PKC inhibitor compound and 15 μL of a mix solution that contains 200 μg/mL protamine sulfate, 10 mM Mg(NO3)2, 10 μM ATP (Boehringer 519987) and 3750 Bq of 33P-ATP (Hartmann Analytic SFC301, 110 TBq/mmol) in 20 mM Tris-buffer pH 7.4+0.1% BSA. Incubation was performed for 15 minutes at 32° C. in a microtiterplate shaking incubator (Biolabo Scientific Instruments). Reaction was stopped by adding 10 μl of 0.5 M Na2EDTA, pH 7.4. 50 μl of mixture are pipetted onto a pre-wetted phosphocellulose paper (Whatmann 3698-915) under gentle pressure.
Measurement: IC50 = 2.1 nM (pChEMBL 8.680000305175781)
Descriptors: MW 504.47, ALogP 1.96, HBA 10, HBD 4, PSA 165.4, QED 0.39
Compound (SMILES): CC(C)=CCN1C[C@H](CO)[C@@H](O)[C@H](O)C1
InChIKey: PQAUOIYEPRBRRD-GMTAPVOTSA-N
Target: Lysosomal acid glucosylceramidase (SINGLE PROTEIN), Homo sapiens
UniProt: P04062
Assay: Enzyme Assay: To 2.5 μl of supernatant (in 96-well plates) was added 17.5 μl reaction buffer (citrate phosphate buffer, pH 4.5, no Triton X-100), and 50 μl of 4-methyl umbelliferone (4-MU)-labeled substrate, β-glucopyranoside, or a labeled negative controls α-glucopyranoside or α-galacatopyranoside). Plates were incubated at 370 for 1 hour, followed by the addition of 70 μl stop buffer (0.4 M glycine-NaOH, pH 10.6). Activity of GCase was determined by measuring the emission at 460 nm by exciting at 355 nm using a 1 second read time per well (Victor2 multilabel counter-Wallac).
Measurement: IC50 = 4000 nM (pChEMBL 5.400000095367432)
Descriptors: MW 215.29, ALogP -0.4, HBA 4, HBD 3, PSA 63.93, QED 0.56
Compound (SMILES): C#CCN(Cc1ccc2cccc(O)c2n1)C(C#N)C1CCN(Cc2ccccc2)CC1
InChIKey: PPPLUJWSLKUDBO-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibition of human recombinant MAOB using p-tyramine as substrate preincubated for 30 mins followed by substrate addition measured for 1 hr by horse-radish peroxidase/amplex red-based fluorometric method
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 424.55, ALogP 4.18, HBA 5, HBD 1, PSA 63.39, QED 0.58
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1NC1CCNCC1
InChIKey: RDRLFKLJQDCWPH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 376.46, ALogP 2.38, HBA 5, HBD 4, PSA 94.73, QED 0.56
Compound (SMILES): CC(C)C(O)c1ccc([C@@H]2[C@@H](C/C=C\CCCC(=O)O)[C@H](Cl)C[C@H]2O)cc1
InChIKey: CFCKBYKCOVVZRP-HRGNYIASSA-N
Target: Prostaglandin E2 receptor EP2 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P43116
Assay: Agonist activity at human recombinant EP2 receptor expressed in HEK293 cells assessed as effect on calcium accumulation by Fluo-4 AM dye based FLIPR assay
Measurement: EC50 = 497 nM (pChEMBL 6.300000190734863)
Descriptors: MW 394.94, ALogP 4.65, HBA 3, HBD 3, PSA 77.76, QED 0.32
Compound (SMILES): Cc1c(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)cc(C#N)n1Cc1ccccc1
InChIKey: OHYXBKHOZFSEQF-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: FL5.12 Cellular Assay: The efficacy of the compounds of Formula (I) can also be determined in cell-based killing assays using a variety of cell lines and mouse tumor models. For example, their activity on cell viability can be assessed on a panel of cultured tumorigenic and non-tumorigenic cell lines, as well as primary mouse or human cell populations. In one exemplary set of conditions, mouse FL5.12 cells transfected with Bcl-XL were cultured under standard conditions in RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3). For assaying the compound activity, the cells were exchanged into an IL-3-depleted deprivation media, which was identical to the growth media except for the absence of FBS and WEHI-3B conditional media, for 2 days. Then the cells were exchanged to 3% FBS assay media (RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3).
Measurement: EC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 624.73, ALogP 6.9, HBA 8, HBD 2, PSA 124.14, QED 0.2
Compound (SMILES): O=C(Nc1cn(CC(=O)N2C[C@H]3CN(CCc4ccccc4)C[C@H]3C2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: WQPAFMBWOAFVAR-ZRZAMGCNSA-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 = 0.66 nM (pChEMBL 9.180000305175781)
Descriptors: MW 661.11, ALogP 4.73, HBA 9, HBD 1, PSA 109.89, QED 0.23
Compound (SMILES): CC(C)C[C@H](NC(=O)c1ccc(C2CC2)c(OCC2CC2)n1)C(=O)N(C)C
InChIKey: SWSQBWNJWICAPP-SFHVURJKSA-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 = 678.1 nM (pChEMBL 6.170000076293945)
Descriptors: MW 373.5, ALogP 2.98, HBA 4, HBD 1, PSA 71.53, QED 0.72
Compound (SMILES): CN(C)c1nc(NCc2ccc(NC(=O)c3ccc(F)cc3)cc2)c2cc(CO)ccc2n1
InChIKey: KYJDQGADIRPBDH-UHFFFAOYSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf22C11 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 2986 nM (pChEMBL 5.53000020980835)
Descriptors: MW 445.5, ALogP 4.19, HBA 6, HBD 3, PSA 90.38, QED 0.4
Compound (SMILES): Cc1cccc(C(=O)N2C[C@@H](C)OC[C@H]2Cc2cc(-c3ncccn3)ccc2F)c1-n1nccn1
InChIKey: STUGMTDSOKPRBQ-WIYYLYMNSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: OX Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 ug/mL G418, 100 U/mL penicillin, 100 ug/mL streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37 C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/L and 20 mM HEPES.
Measurement: IC50 = 506 nM (pChEMBL 6.300000190734863)
Descriptors: MW 472.52, ALogP 3.64, HBA 7, HBD 0, PSA 86.03, QED 0.44
Compound (SMILES): COc1cc(C(F)(F)F)ccc1-c1cccc2cc(S(=O)(=O)Nc3ncccn3)ccc12
InChIKey: XFRLPIMGOMZXNS-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Inhibition of human Nav1.7 expressed in HEK293 cells assessed as inhibition of sodium current at holding potential of -125 mV measured for 3 to 5 mins by whole cell voltage clamp method
Measurement: IC50 = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 459.45, ALogP 5.13, HBA 5, HBD 1, PSA 81.18, QED 0.45
Compound (SMILES): COc1cc(OC)cc(N(CC2COCC(C)(C)N2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: ZAWBYXATEBIOBQ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 488.59, ALogP 3.95, HBA 9, HBD 1, PSA 86.56, QED 0.42
Compound (SMILES): CNc1ccc(CNc2nnc(-c3ccc4[nH]cc(-c5ccc(F)cc5F)c4c3)s2)cc1
InChIKey: KONCPQOVMLAHLO-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 447.51, ALogP 6.29, HBA 5, HBD 3, PSA 65.63, QED 0.29
Compound (SMILES): CC(C)(C)OC(=O)N1CC[C@](C)(N2CCc3c(-c4cnc(N)nc4)nc(N4CCOCC4)nc32)C1
InChIKey: MEWKDLGRKBRJAF-DEOSSOPVSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition of recombinant human N-terminal Myr signal and C-terminal Flag-tagged PI3K p110beta expressed in rat Rat1 cells
Measurement: IC50 = 640 nM (pChEMBL 6.190000057220459)
Descriptors: MW 482.59, ALogP 2.11, HBA 10, HBD 1, PSA 122.83, QED 0.7
Compound (SMILES): Nc1nn2cccnc2c1C(=O)Nc1cn([C@H]2C[C@@H](N)C2)nc1-c1cc(Cl)ccc1OC(F)F
InChIKey: GLQQQPJQXUIPOL-TXEJJXNPSA-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 = 0.07 nM (pChEMBL 10.149999618530273)
Descriptors: MW 488.89, ALogP 3.34, HBA 9, HBD 3, PSA 138.38, QED 0.38
Compound (SMILES): Cc1c(-c2c(F)cc(C(N)=O)c3[nH]c4c(c23)CC[C@H](C(C)(C)O)C4)cccc1-n1c(=O)cc2c(F)cccn2c1=O
InChIKey: OILCFDXBRQHLMC-INIZCTEOSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Assay: To V-bottom 384-well plates were added test compounds, human recombinant Btk (1 nM, Invitrogen Corporation), fluoresceinated peptide (1.5 μM), ATP (20 μM), and assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij 35 surfactant and 4 mM DTT in 1.6% DMSO), with a final volume of 30 μL. After incubating at room temperature for 60 min, the reaction was 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 fluorescent substrate and phosphorylated product.
Measurement: IC50 = 0.84 nM (pChEMBL 9.079999923706055)
Descriptors: MW 558.59, ALogP 4.16, HBA 6, HBD 3, PSA 122.59, QED 0.31
Compound (SMILES): CC(C)(C)c1nc2ccccc2n1C(=O)c1cccc2ccccc12
InChIKey: YJJXLJXTINUIHK-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 328.42, ALogP 5.18, HBA 3, HBD 0, PSA 34.89, QED 0.48
Compound (SMILES): CCN1CCC(CCOc2ncc(-c3nc(C#N)nc4c3ccn4C)cc2C(F)(F)F)CC1
InChIKey: IMEWSXDKPDPQKM-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Mus musculus
UniProt: O70370
Assay: Inhibition Assay: First, spleen cells collected from C57BL/6 mice were seeded in a 96-well plate so as to be a 1× 105 cells/well, then, 5-fold dilution of 10 mM DMSO solution of the test compound was serially performed with RPMI 1640 medium (including 10% fetal bovine serum (FCS), 5×10^−5 M 2-mercaptoethanol, 50 IU/mL penicillin and 50 μg/mL streptomycin) nine times such that the final concentration becomes 0.026 nM to 10 μM, and the resultant products were added thereto (the final DMSO concentration was 0.1%). At the same time, LPS (Sigma L4005) was added to the wells such that the final concentration becomes 2 μg/mL, and culture was performed at 37° C. for 48 hours in 5% CO2. After culturing, the cells were stained at 4° C. for 20 minutes with PE-labeled streptavidin (BD BIOSCIENCE 554061) and biotin-labeled YAe antibody (EBIOSCIENCE 13-5741-85) together with FITC-labeled anti-mouse B220 antibody (BD BIOSCIENCE 553088), and an expression level (fluorescence intensity of YAe-bioti
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 458.49, ALogP 4.42, HBA 7, HBD 0, PSA 79.86, QED 0.54
Compound (SMILES): C#Cc1cccc(Nc2ncnc3cc(OC)c(NC(=O)CCCCCCC(=O)NO)cc23)c1
InChIKey: MHUVTLXVRJYDAB-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of N-terminal GST-tagged human HER2 (Lys676 to Val1255 residues) expressed in baculovirus expression system using biotinylated FLT3 at Tyr589 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 461.52, ALogP 4.15, HBA 7, HBD 4, PSA 125.47, QED 0.15
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cc(C)cc(C)c3)ccc2[nH]1
InChIKey: MXPXZVZGDXXHMY-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 442.48, ALogP 4.21, HBA 5, HBD 3, PSA 107.55, QED 0.44
Compound (SMILES): C=CC(=O)Nc1cccc(-c2n[nH]c3ccccc23)c1
InChIKey: SYTUFLIGWRGQDS-UHFFFAOYSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: O14733
Assay: Inhibition of MEK7 (unknown origin)
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 263.3, ALogP 3.35, HBA 2, HBD 2, PSA 57.78, QED 0.71
Compound (SMILES): CNCCOc1cccc(C(=O)NCC(O)CN2CCc3ccccc3C2)c1
InChIKey: MNVWMBWUTDPHDJ-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 383.49, ALogP 1.43, HBA 5, HBD 3, PSA 73.83, QED 0.57
Compound (SMILES): N#Cc1cccc(OCC2CN(c3ncnc4[nH]cc(-c5cccc(C#N)c5)c34)CCO2)c1
InChIKey: YXNMHCJGLJBGCK-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 436.48, ALogP 3.65, HBA 7, HBD 1, PSA 110.85, QED 0.51
Compound (SMILES): CCN(CC)CCNC(=O)c1ccc2cc(Nc3nc(C)cc(Nc4cc(C5CC5)n[nH]4)n3)ccc2c1
InChIKey: CKCPXUDGCVMDEI-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 498.64, ALogP 5.1, HBA 7, HBD 4, PSA 110.86, QED 0.23
Compound (SMILES): c1ccc2nc(CCc3nc4c(N5CCOCC5)ccnn4c3-c3ccc(-c4nnn[nH]4)nc3)ccc2c1
InChIKey: WVQKXUXFABBSMW-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 = 1 nM (pChEMBL 9)
Descriptors: MW 504.56, ALogP 3.14, HBA 10, HBD 1, PSA 122.9, QED 0.36
Compound (SMILES): NC[C@@H]1CCN(c2ncc(C(=O)Nc3ccc(OC(F)(F)F)cc3)cc2-c2cncnc2)C1
InChIKey: ZHSJPWWWEJYCPF-AWEZNQCLSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper ABL : The assay plates were prepared by addition of 50 nL per well of compound solution in 90% DMSO. The kinase reactions were started by stepwise addition of 4.5 uL per well of peptide/ATP-solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 uM ATP, 4 uM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 uL per well of enzyme solution (50 mM HEPES, pH 7.5, 1 mM DTT, 0.02% BSA, 0.6% DMSO, 10 mM beta-glycerophosphate, and 10 uM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 3.5 nM ABL (ABL(64-515), produced in-house from E. coli)). Kinase reactions were incubated at 30° C. for 60 minutes and subsequently terminated by addition of 16 uL per well of stop solution (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35). Plates with terminated kinase reactions were transferred to the Caliper LC3000 workstations for reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift technology.
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 458.44, ALogP 3.47, HBA 7, HBD 2, PSA 106.26, QED 0.58
Compound (SMILES): O=C(O)c1cc2ccccc2nc1/C=C/c1cn2nc(Cl)cc(N3CCOCC3)c2n1
InChIKey: YEBKTRXUFIPKJV-AATRIKPKSA-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 = 1 nM (pChEMBL 9)
Descriptors: MW 435.87, ALogP 3.64, HBA 7, HBD 1, PSA 92.85, QED 0.52
Compound (SMILES): O=c1cc(C(F)(F)F)c2ccc(OCCCCn3cc(CCCCN4C[C@H](O)[C@@H](O)[C@H](O)[C@H]4CO)nn3)cc2o1
InChIKey: BDLBRPFDEIFVRK-RRJCBXKSSA-N
Target: Sucrase-isomaltase, intestinal (SINGLE PROTEIN), Rattus norvegicus
UniProt: P23739
Assay: Inhibition of rat small intestinal brush border membrane sucrase assessed as reduction in production of D-glucose using disaccharides as substrate incubated for 10 to 30 mins
Measurement: IC50 = 950 nM (pChEMBL 6.019999980926514)
Descriptors: MW 570.57, ALogP 1.34, HBA 11, HBD 4, PSA 154.31, QED 0.19
Compound (SMILES): COCCn1cc(-c2cnc(N)c(C(=O)Nc3ccnc4ccoc34)c2)cn1
InChIKey: FIRCCCGMXJDUAZ-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: IKKepsilon Flashplate Assay: The kinase assay is performed either as 384-well Flashplate assay (for e.g. Topcount measurement). 1 nM IKKepsilon, 800 nM biotinylated IkappaBalpha(19-42) peptide (Biotin-C6-C6-GLKKERLLDDRHDSGLDSMKDEE) SEQ ID NO: 1 and 10 uM ATP (spiked with 0.3 uCi 33P-ATP/well) are incubated in a total volume of 50 ul (10 mM MOPS, 10 mM Mg-acetat, 0.1 mM EGTA, 1 mM Dithiothreitol,0.02% Brij35, 0.1% BSA, 0.1% BioStab, pH 7.5) with or without test compound for 2 hours at 30° C. The reaction is stopped with 25 ul 200 mM EDTA. After 30 Min at room temperature the liquid is removed and each well washed thrice with 100 ul 0.9% sodium chloride solution. Non-specific reaction is determined in presence of 3 uM MSC2119074 (BX-795). Radioactivity is measured with Topcount (PerkinElmer).
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 378.39, ALogP 2.57, HBA 8, HBD 2, PSA 121.09, QED 0.53
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCN(Cc2cnn3ccccc23)CC1
InChIKey: FIGWIWKCKWMFMI-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 = 503 nM (pChEMBL 6.300000190734863)
Descriptors: MW 473.52, ALogP 2.81, HBA 9, HBD 2, PSA 130.16, QED 0.39
Compound (SMILES): COCCOCCOCCOCCOCCO[C@H]1CC[C@@]2(O)[C@H]3Cc4ccc(O)c5c4[C@@]2(CCN3CC2CCC2)C1O5
InChIKey: PHZKNFKDLUYKPR-VSJOCJANSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Binding of PEG-Nalbuphine assays: Specific binding is determined by subtraction of the cpm bound in the presence of 50-100× excess of cold ligand. Binding data assays were analyzed using GraphPad Prism 4.0 and IC50 is generated by non-linear regression from dose-response curves. Ki values were calculated using the Cheng Prusoff equation using the Kd values from saturation isotherms as follows: Ki=IC50/(1+[Ligand]/Kd).
Measurement: Ki = 35.56 nM (pChEMBL 7.449999809265137)
Descriptors: MW 591.74, ALogP 2.44, HBA 10, HBD 2, PSA 108.31, QED 0.25
Compound (SMILES): NC(=O)[C@H](CC1CC1)NC(=O)c1ccc(C2CC2)c(-c2cccc(Cl)c2)n1
InChIKey: SJPKKZTWMYDWGV-SFHVURJKSA-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 383.88, ALogP 3.66, HBA 3, HBD 2, PSA 85.08, QED 0.76
Compound (SMILES): CN(C)CCCc1ccc(Nc2cc(-n3cnc4ccccc43)ncn2)nc1
InChIKey: NYPBFUANCHGSJU-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Inhibition of recombinant full-length human C-terminal His6-tagged CDK2/recombinant full-length human N-terminal GST-tagged cyclin E using fluorescein-QSPKKG-CONH2 as substrate after 60 mins by off-chip mobility shift assay
Measurement: IC50 = 690 nM (pChEMBL 6.159999847412109)
Descriptors: MW 373.46, ALogP 3.45, HBA 7, HBD 1, PSA 71.76, QED 0.53
Compound (SMILES): C[C@H](NC(=O)[C@@H](N)CCCNC(=N)N)C(=O)N[C@H]1CSSC[C@@H](C(=O)O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](CCCNC(=N)N)NC1=O
InChIKey: WOVYKVDSWWPJMK-ODKJCKIQSA-N
Target: C-X-C chemokine receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P61073
Assay: Inhibition of PE-conjugated-12G5 anti-CXCR4 antibody binding to CXCR4 in human CEM-CCRF cells preincubated for 30 mins followed by antibody addition by FACS Canto II cytofluorometric analysis
Measurement: IC50 = 6200 nM (pChEMBL 5.210000038146973)
Descriptors: MW 900.1, ALogP -2.27, HBA 12, HBD 14, PSA 361.72, QED 0.03
Compound (SMILES): CCCc1c(-c2csc(-c3cc(C(=N)N)sc3SC)n2)cnn1-c1ccccn1
InChIKey: BCSYIGQLBKEYFG-UHFFFAOYSA-N
Target: Complement C1s subcomponent (SINGLE PROTEIN), Homo sapiens
UniProt: P09871
Assay: Binding affinity to human C1S assessed as inhibition constant
Measurement: Ki = 70 nM (pChEMBL 7.159999847412109)
Descriptors: MW 440.62, ALogP 5.08, HBA 8, HBD 2, PSA 93.47, QED 0.24
Compound (SMILES): Cn1cc(-c2cc(S(N)(=O)=O)cnc2OC2CCOC2)c2cc[nH]c2c1=O
InChIKey: NFHNPMIGVNUEGG-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 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 390.42, ALogP 0.74, HBA 7, HBD 2, PSA 129.3, QED 0.68
Compound (SMILES): CN1CCN(c2nc3c(c(NCc4ccc(Cl)c(Cl)c4)n2)CCN(c2ncccc2C(F)(F)F)CC3)CC1
InChIKey: LNSUVNXLUJKSFW-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 > 6670 nM (pChEMBL 5.176000118255615)
Descriptors: MW 566.46, ALogP 5.17, HBA 7, HBD 1, PSA 60.42, QED 0.46
Compound (SMILES): COc1cc2c(F)cc1[C@@H](C)COC(=O)Nc1ccc(S(=O)(=O)C3CC3)c(c1)CN(C)C(=O)[C@@H]2Nc1cc2ccnc(N)c2cc1F
InChIKey: BZFPMRWPYYLDPZ-NOVUIFNWSA-N
Target: Coagulation factor VII (SINGLE PROTEIN), Homo sapiens
UniProt: P08709
Assay: In Vitro Assay: Tissue kallikrein-1 (37° C) activity was determined in reactions containing 0.05 nM enzyme and 90 μM substrate (H-D-Val-Leu-Arg-AFC) in buffer (0.1M sodium phosphate pH 7.4, 0.2 M NaCl, 0.5% PEG 8000, and 1% DMSO). Assays were performed using 96-well microtiter plates (COSTAR 3600, CORNING, NY, USA) and a thermostatic temperature controlled plate reader (SPECTRAMAX® Gemini, Molecular Devices, Sunnyvale, Calif., USA). Fluorescence was monitored using 400 nm excitation and 505 nm emission wavelengths.
Measurement: Ki = 270 nM (pChEMBL 6.570000171661377)
Descriptors: MW 665.72, ALogP 5.52, HBA 9, HBD 3, PSA 152.95, QED 0.25
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3C4CO)cc12
InChIKey: ZVTSTMMOWMDFAK-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 1.806 nM (pChEMBL 8.739999771118164)
Descriptors: MW 628.66, ALogP 5.71, HBA 7, HBD 2, PSA 117.67, QED 0.24
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3ccn[nH]3)n2)cnc1N1CC[C@H](F)C1
InChIKey: CAELDERPSRNSPW-ZDUSSCGKSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 350.36, ALogP 2.43, HBA 7, HBD 2, PSA 106.41, QED 0.74
Compound (SMILES): NCCOc1c(-c2ccc(Cl)c(Cl)c2)[nH]c(=O)c2cc(F)ccc12
InChIKey: BFJMBXAFUUTYIR-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase PARP3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y6F1
Assay: Biochemical Assay: The identification of compounds capable of binding several PARP proteins is carried out through a screening method including the steps of  a) providing a reaction mixture containing:  b) comparing the polarization signal generated in the absence of the test compound with the one generated in the presence of different concentrations of the test compound, and  c) evaluating the ability of the test compound to displace the compound of formula (IP) as defined above indicated from a decreased fluorescence polarization level.The polarization signal can be measured, e.g., by a plate reader such as the Saphire2 (Tecan).
Measurement: Kd = 10 nM (pChEMBL 8)
Descriptors: MW 367.21, ALogP 3.98, HBA 3, HBD 2, PSA 68.11, QED 0.73
Compound (SMILES): COCCOc1cc2c(Nc3ccc(F)c(Cl)c3)ncnc2cc1OCCCCCCC(=O)NO
InChIKey: WVFGVCPVDJVODM-UHFFFAOYSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of N-terminal GST-tagged human HER2 (Lys676 to Val1255 residues) expressed in baculovirus expression system using biotinylated FLT3 at Tyr589 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 506.96, ALogP 5.03, HBA 8, HBD 3, PSA 114.83, QED 0.16
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCN2CC[C@@]3(C[C@@H]3c3ccccc3)C2)n1C
InChIKey: AAPXNHMQKBDDJN-XMSQKQJNSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Displacement of [3H]-spiperone from human dopamine D2 receptor expressed in CHO-K1 cell membranes coexpressing Galpha16 after 120 mins by liquid scintillation counting
Measurement: Ki = 707.95 nM (pChEMBL 6.150000095367432)
Descriptors: MW 409.56, ALogP 4.14, HBA 7, HBD 0, PSA 59.98, QED 0.43