lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): NC[C@H]1CC[C@H](C(=O)N2C[C@@H](N3CCN(S(=O)(=O)C4CC4)CC3)C[C@H]2C(=O)Nc2ccc(C(=O)O)cc2)CC1
InChIKey: KPHTWWMYMFXYAQ-MWYJAUPDSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: In Vitro Assay: Human Factor Xa (American Diagnostica Inc.) and human thrombin (Sigma) activities were measured at the enzyme concentrations of 0.18 U/mL and 0.12 U/mL, respectively in the same buffer containing 150 mM NaCl, 2 mg/mL PEG6000, 50 mM Tris-HCl (pH7.4), except that the reactions were started with 300 μM S-2222 (phenyl-Ile-Glu-Gly-Arg-pNA, Chromogenix) and 300 μM S-2366, respectively.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 561.71, ALogP 1.17, HBA 7, HBD 3, PSA 153.35, QED 0.43
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2ncnc3c(C)nn(C)c23)cc(C#Cc2ccc(-n3cnc(C)c3)cc2)n1
InChIKey: WELBXKPSYNELDG-UHFFFAOYSA-N
Target: Baculoviral IAP repeat-containing protein 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13490
Assay: XIAP BIR3 & cIAP1 BIR3 Binding Assays (DELFIA): 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% TritonX100) for one hour at room temperature in the presence of inhibitory 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-N1-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 fluorescence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 505.59, ALogP 3.17, HBA 9, HBD 2, PSA 115.44, QED 0.35
Compound (SMILES): N#Cc1nc(-c2ccnc(Nc3ccc(NC(=O)CN4CC[C@H](F)C4)nc3)n2)ccc1N1CC[C@H](O)C1
InChIKey: AHEAUBHTIQRMTK-WMZOPIPTSA-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 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 503.54, ALogP 2.1, HBA 10, HBD 3, PSA 143.19, QED 0.44
Compound (SMILES): c1cc2c(ncn3nnc(C4CC5CCC4C5)c23)[nH]1
InChIKey: SNLKNTUIVFHPNV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 253.31, ALogP 2.51, HBA 4, HBD 1, PSA 58.87, QED 0.72
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1cnc(C(F)(F)F)cn1)c1ncccc1Br
InChIKey: HHOJQPPWOPIDQE-QWRGUYRKSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 430.23, ALogP 3.42, HBA 5, HBD 2, PSA 79.8, QED 0.78
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1Cl)c1nc2c(Cl)ccc(Cl)c2c(=O)n1-c1ccc(N)nc1
InChIKey: INFYRHWEYYBOMD-ZETCQYMHSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 492.76, ALogP 3.45, HBA 10, HBD 4, PSA 163.65, QED 0.33
Compound (SMILES): CC(C)(C)Nc1cnc2cccc(-c3cc4c([nH]3)CCNC4=O)c2n1
InChIKey: LXUFTOZVBIOJCM-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 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 335.41, ALogP 3.12, HBA 4, HBD 3, PSA 82.7, QED 0.67
Compound (SMILES): C[C@H](NC(=O)C(=O)NCc1cccc2ccccc12)C(=O)N[C@@H](CC(=O)O)C(=O)COP(=O)(c1ccccc1)c1ccccc1
InChIKey: UYLOQGSFWYOIGW-DWACAAAGSA-N
Target: Caspase-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P42574
Assay: Inhibition of recombinant CPP32 (unknown origin) pre-incubated for 60 mins with Acetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin substrate followed by enzyme addition by fluorescence based assay
Measurement: Ki = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 629.61, ALogP 2.44, HBA 7, HBD 4, PSA 167.97, QED 0.13
Compound (SMILES): COc1ccc(C2CC(c3ccc(OC)c(OC)c3)=NN2c2ccc(C(=O)O)cc2)cc1
InChIKey: GGUPJZMHXUDMOC-UHFFFAOYSA-N
Target: Prostaglandin G/H synthase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23219
Assay: Colorimetric Assay: The activities of COX-1 and COX-2 were measured after the addition of arachidonic acid and incubation at 25° C. for 5 min by absorbance at 590 nm as specified by the manufacturer.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 432.48, ALogP 4.77, HBA 6, HBD 1, PSA 80.59, QED 0.58
Compound (SMILES): COc1cc(C(=O)NCc2c(F)ccc(OC)c2F)ccc1-c1cn[nH]c1
InChIKey: YKCPIOFQNXOLHH-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity.
Measurement: IC50 = 5.35 nM (pChEMBL 8.270000457763672)
Descriptors: MW 373.36, ALogP 3.3, HBA 4, HBD 2, PSA 76.24, QED 0.7
Compound (SMILES): CC1CCC([C@H](NC(=O)c2ccccc2)C(=O)N2CC[C@H]3OCC(=O)[C@H]32)CC1
InChIKey: MZMUFCSCXROGTF-BURCVLKOSA-N
Target: Procathepsin L (SINGLE PROTEIN), Homo sapiens
UniProt: P07711
Assay: In Vitro Inhibition Assay: In vitro inhibition assay using cathepsin.
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 384.48, ALogP 2.18, HBA 4, HBD 1, PSA 75.71, QED 0.86
Compound (SMILES): CN1CC2(CCN(c3cncc4ccccc34)CC2)CC1=O
InChIKey: QYQQAZRYPIKPEC-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition of human CYP11B1-8C7 expressed in chinese hamster V79 cells using 11-deoxycortisol as substrate preincubated for 1 hr followed by substrate addition measured for 3 hrs by HTRF assay
Measurement: IC50 = 1996 nM (pChEMBL 5.699999809265137)
Descriptors: MW 295.39, ALogP 2.68, HBA 3, HBD 0, PSA 36.44, QED 0.81
Compound (SMILES): Cn1cc2c(nc(NC(=O)CCCc3cn(CCCCCCNC(=O)c4ccc(-c5c6ccc(=N)c(S(=O)(=O)O)c-6oc6c(S(=O)(=O)O)c(N)ccc56)c(C(=O)O)c4)nn3)n3nc(-c4ccco4)nc23)n1
InChIKey: XEABBZPOOUCNPW-UHFFFAOYSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
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 = 31.8 nM (pChEMBL 7.5)
Descriptors: MW 1008.02
Compound (SMILES): Cc1nn(C)cc1-c1c[nH]c2ncnc(N3CCOCC3)c12
InChIKey: RGWYNOKIZOFIMS-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 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 298.35, ALogP 1.5, HBA 6, HBD 1, PSA 71.86, QED 0.78
Compound (SMILES): Clc1ccc(CCNC[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)cc1
InChIKey: HJSZIUDMNAXXLP-QAQDUYKDSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 408.94, ALogP 4.37, HBA 5, HBD 2, PSA 70.9, QED 0.46
Compound (SMILES): CCOc1ncc(C(=O)Nc2ccc(OC(F)(F)F)cc2)cc1-c1cncnc1
InChIKey: QDFOKEWPQMANBZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Caliper Assay: All assays were performed in 384-well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, as well as four 8-point serial dilutions of staurosporine as a reference compound, plus 16 high and 16 low controls. Liquid handling and incubation steps were done on a Thermo CatX workstation equipped with Innovadyne Nanodrop Express. Between pipetting steps, tips were cleaned in wash cycles using wash buffer. 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 μL 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 μM sodium orthovanadate, 20 mM MgCl2, 2 mM MnCl2, 4 μM ATP, 4 μM peptide (FITC-Ahx-EAIYAAPFAKKK-NH2)) and 4.5 μL 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 μM 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 μL 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. Briefly, samples from terminated kinase reactions were applied to the chip. Analytes are transported through the chip by constant buffer flow and the migration of the substrate peptide is monitored by the fluorescence signal of its label. Phosphorylated peptide (product) and unphosphorylated peptide (substrate) are separated in an electric field by their charge/mass ratio.
Measurement: IC50 = 9.4 nM (pChEMBL 8.029999732971191)
Descriptors: MW 404.35, ALogP 4.09, HBA 6, HBD 1, PSA 86.23, QED 0.67
Compound (SMILES): C[C@@H]1CN(c2sc(C(F)(F)F)nc2-c2nc3ccccc3[nH]2)CCN1C(=O)Cn1cc(C2CCN(C)CC2)cn1
InChIKey: MDOLKXWWPFVDTC-QGZVFWFLSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Antagonist activity at human recombinant CXCR3 expressed in CHO-K1 cells measured after 10 mins in presence of CXCL10 by FLIPR analysis
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 572.66, ALogP 4.45, HBA 8, HBD 1, PSA 86.18, QED 0.38
Compound (SMILES): CC(C)CCNC(=O)c1ccc(NC(=O)N2Cc3ccc(F)cc3C2)cc1
InChIKey: GBBURVMXNCSFRE-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Inhibition of C-terminal His-tagged human recombinant NAMPT using FK866 or isoindoline urea-based Oregon green (488) probe incubated for 3 hrs by TR-FRET assay
Measurement: Ki = 7.9 nM (pChEMBL 8.100000381469727)
Descriptors: MW 369.44, ALogP 4.15, HBA 2, HBD 2, PSA 61.44, QED 0.83
Compound (SMILES): Cn1nc(-c2ccc(F)cc2)c(-c2ccc3nc(NC(=O)c4ccnc(F)c4)cn3n2)c1N1CCN(Cc2ccon2)CC1
InChIKey: YLZRLYXUAQSEHW-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 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 596.6, ALogP 4.03, HBA 11, HBD 1, PSA 122.51, QED 0.27
Compound (SMILES): O=CNC(CNC(=O)Cn1nc(-c2ccc(Cl)cc2)n(C[C@H](O)C(F)(F)F)c1=O)c1cccc(C(F)(F)F)c1
InChIKey: YBICINNGDZZTIZ-ZVAWYAOSSA-N
Target: Vasopressin V2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P30518
Assay: Cellular in Vitro Assay: On the day before the assay, the cells are plated out in culture medium (DMEM, 10% FCS, 2 mM glutamine, 10 mM HEPES) in 384-well microtitre plates and kept in a cell incubator (96% humidity, 5% v/v CO2, 37° C.). On the day of the assay, the culture medium is replaced by a Tyrode solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM glucose, 20 mM HEPES), which additionally contains the cofactor coelenterazine (50 μM), and the microtitre plate is then incubated for a further 3-4 hours. The test substances in various concentrations are placed for 10 to 20 minutes in the wells of the microtitre plate before the agonist [Arg8]-vasopressin is added, and the resulting light signal is measured immediately in the luminometer.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 579.89, ALogP 2.91, HBA 7, HBD 3, PSA 118.25, QED 0.25
Compound (SMILES): COc1cc2ncnc(N3CCN(C(=S)Nc4ccc(S(=O)(=O)Nc5ncccn5)cc4)CC3)c2cc1OC
InChIKey: MUQXLPQDKAJNMQ-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P16234
Assay: Inhibition of PDGFRA in human PANC1 cells measured after 4 days by sulforhodamine B assay
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 566.67, ALogP 2.76, HBA 10, HBD 2, PSA 134.7, QED 0.32
Compound (SMILES): CN1CCN(CCNc2ccc(S(=O)(=O)NC(=O)c3ccc(N4CCN(CC5=C(c6ccc(Cl)cc6)CC(C)(C)CC5)CC4)cc3Oc3ccc4[nH]ccc4c3)cc2[N+](=O)[O-])CC1
InChIKey: OZZNCUWTNDYZJX-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki = 15.7 nM (pChEMBL 7.800000190734863)
Descriptors: MW 895.53, ALogP 8.09, HBA 11, HBD 3, PSA 156.39, QED 0.07
Compound (SMILES): Cc1c(Cc2ccccc2)nnc(N2CCN(c3cnc(C(=O)NCC(C)O)cn3)[C@H](C)C2)c1C
InChIKey: RGTBFZDODFBZGR-QNSVNVJESA-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 = 100 nM (pChEMBL 7)
Descriptors: MW 475.6, ALogP 2.3, HBA 8, HBD 2, PSA 107.37, QED 0.54
Compound (SMILES): CCC(C(=O)Nc1ccc(C2CCNC2)cc1)c1ccccc1
InChIKey: OYEZXPCVBHKJLG-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 60.6 nM (pChEMBL 7.21999979019165)
Descriptors: MW 308.42, ALogP 3.9, HBA 2, HBD 2, PSA 41.13, QED 0.88
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCN2CC[C@@]3(C[C@H]3c3ccc(F)cc3F)C2)n1C
InChIKey: XIHOJUYAGODVFH-HTAPYJJXSA-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 = 257.04 nM (pChEMBL 6.590000152587891)
Descriptors: MW 445.54, ALogP 4.42, HBA 7, HBD 0, PSA 59.98, QED 0.4
Compound (SMILES): Cc1ccc2ncccc2c1-c1cc(-c2c(C)noc2C)cc2nc(C3CC3)[nH]c12
InChIKey: KHQQNXFBTALTQF-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 78.6 nM (pChEMBL 7.110000133514404)
Descriptors: MW 394.48, ALogP 6.24, HBA 4, HBD 1, PSA 67.6, QED 0.39
Compound (SMILES): Cn1nc(C2CCN(C(=O)CC(=O)O)CC2)nc1-c1cnc(N)c(-c2nnc(C(C)(C)C)o2)n1
InChIKey: LLGDRAUZZHUOQT-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: In Vitro Enzyme Inhibition Assay: The inhibition of PI3K-beta, PI3K-alpha, PI3K-gamma and PI3K-delta was evaluated in a Kinase Glo based enzyme activity assay using human recombinant enzymes. Compounds in 100% DMSO were added to assay plates by acoustic dispensing. PI3K enzyme was added in a Tris buffer (50 mM Tris pH7.4, 0.05% CHAPS, 2.1 mM DTT, and 10 mM magnesium chloride) and allowed to preincubate with compound for 20 minutes prior to addition of substrate solution containing PIP2 and ATP. The enzyme reaction was stopped after 80 minutes by the addition of Kinase Glo detection solution containing Lucferin and Luciferase (from Kinase Glo® Plus Luminecent Kinase Assay kit (Promega #V3772). Plates were left for 30 minutes at room temperature then read on a Pherastar Instrument with a standard Luminescence filter block. The final concentration of DMSO, ATP and PIP2 in the assay were, 1%, 8 uM, and 80 uM respectively.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 469.51, ALogP 1.38, HBA 11, HBD 2, PSA 179.04, QED 0.52
Compound (SMILES): COc1cc(/C=N/NC(=O)CCn2cc(C)c3c(C)cccc32)cc2cccnc12
InChIKey: CDIDCUYYRZSZGW-VULFUBBASA-N
Target: Transient receptor potential cation channel subfamily M member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ8
Assay: Inhibition of human TRPM5 transfected in HEK293 cells assessed as inhibition of KCl-stimulated membrane potential by FLIPR assay
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 400.48, ALogP 4.36, HBA 5, HBD 1, PSA 68.51, QED 0.39
Compound (SMILES): COCc1ccc(C(C)=O)c2sc(C(=O)Nc3cc(CN4CC[C@H](O)C4)c4ccccc4n3)c(C)c12
InChIKey: UXOLHRWLSKHKNL-FQEVSTJZSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Activity Assay: A human acute lymphoblastic lymphoma T cell line MOLT-4 (which can be purchased under ATCC No. CRL-1582 from ATCC) was cultured in an RPMI1640 medium containing 10% fetal bovine serum to obtain 5108 MOLT-4 cells. The cells were recovered by centrifugation and suspended in 10 ml of buffer solution A (25 mM Tris-HCl, 5 mM 2-mercaptoethanol, 2 mM benzamidine, 2 mM EDTA, and 0.1 mM 4-(2-aminoethyl)benzenesulfonyl hydrochloride, pH 7.5). The cells were homogenized using a Polytron homogenizer and centrifuged (4° C., 25,000 g, 10 minutes). Then, the supernatant was further ultracentrifuged (4° C., 100,000 g, 60 minutes). The obtained supernatant was filtered through a 0.2um filter to obtain a soluble fraction.HiTrap Q HP column (5 ml2, GE Healthcare Japan Corp.) equilibrated with buffer solution A was charged with the obtained soluble fraction. Phosphodiesterase was eluted with 300 ml of buffer solution A containing a linear gradient solution of 0 to 0.8 M NaCl to recover sixty 5-ml fractions. Each fraction was tested for cAMP-metabolizing phosphodiesterase activity. In each fraction, a fraction eluted as an active peak centered around 250 mM NaCl was collected from fractions that had cAMP metabolic activity and did not lose the metabolic activity by 10uM rolipram (PDE4 selective inhibitor) and 10uM milrinone (PDE3 selective inhibitor). In order to confirm whether or not PDE10A mRNA was expressed in the MOLT-4 cells, total RNA was prepared from the MOLT-4 cells according to a standard method and analyzed by RT-PCR using PDE10A gene-specific primers (PDE10A sense primer: 5'-TGCTCCATGGTGGAAGTGGA-3' (SEQ ID NO: 1) and PDE10A antisense primer: 5'-CAACTGGAAGCATGCGGTCA-3' (SEQ ID NO: 2)). As a result, PDE10A mRNA was detected from the total RNA of the MOLT-4 cells. On the other hand, total RNA was prepared from Jurkat cells, one type of human T cell, and similarly subjected to RT-PCR. As a result, PDE10A mRNA was rarely detected. An active peak centered around 250 mM NaCl was not observed in fractions obtained by the treatment of Jurkat cells in the same way as above. Eur J Biochem. 1999, 266 (3), 1118-2 has reported that PDE10A from the rat striatum and testis is eluted as an active peak centered around 250 mM NaCl.
Measurement: IC50 = 0.23 nM (pChEMBL 9.640000343322754)
Descriptors: MW 503.62, ALogP 4.93, HBA 7, HBD 2, PSA 91.76, QED 0.35
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1N[C@@H]1CCCNC1
InChIKey: FAUDAQJUMHDILO-CYBMUJFWSA-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 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 376.46, ALogP 2.38, HBA 5, HBD 4, PSA 94.73, QED 0.56
Compound (SMILES): CN(c1ccc(NS(=O)(=O)Cc2ccccc2F)cn1)[C@@H]1CCN(c2ncnc3[nH]ccc23)C1
InChIKey: KJEPSVAGAQPMTH-GOSISDBHSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzymatic Assay: JAK1/2/3 kinase assay are performed in vitro using Kit-Tyr 6 Peptide (Invitrogen, Cat. No. PV4122). TYK2 kinase assay are performed in vitro using Z′-LYTE Kinase Assay Kit-Tyr 3 Peptide (Invitrogen, Cat. No. PV3192). Recombinant human JAK1/2/3 or TYK2 catalytic domains are from Invitrogen (Cat No. PV4774/PV4210/PV3855/PV4790); All reactions (20 μL) are started by adding 2.5 μL of the testing compound in 4% DMSO solution, 5 μL of Kinase/Peptide substrate Mixture (3.2, 0.04, 0.2 or 8 μg/mL for Recombinant human JAK1/2/3 catalytic domains, 4 μM for Z-LYTE Tyr 6 peptide or Z-LYTE Tyr 3 peptide) or Phospho-Peptide solution (Invitrogen, Cat. No. PV3192, diluted with 1.33× Kinase Buffer), 2.5 μL ATP Solution (300/100/40/100 μM, JAK1/JAK2/JAK3/TYK2) or 1.33× Kinase Buffer (Invitrogen, Cat. No. PV3189, 5× diluted with distilled water). The 384-well assay plate (Corning, Cat. No. 3575) is mixed and incubated at room temperature for 1 hour. 5 μL of the Development Solution (Dilute Development Reagent A (Cat. No. PV3297) is diluted to 1/64 with Development Buffer (Cat. No. PV3127) for JAK1, JAK2 and JAK3 assay; Development Reagent A (Cat. No. PV3297) is diluted to 1/2048 with Development Buffer (Cat. No. PV3127) for TYK2 assay. The diluted Development Solution is then added to each well, mixed and incubated at room temperature for another 1 hour. The kinase reaction is then stopped by adding 5 μL of the Stop Reagent (Invitrogen, Cat. No. PV3094), and the plate is read with Wallac 1420 VICTOR3 Multilabel Counter (PerkinElmer) at 445 nm and 520 nm fluorescence. All compounds are initially tested at 8 different concentrations (1 μM down to 0.0003 μM) using a 1:3 serial dilution scheme.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 481.56, ALogP 3.15, HBA 7, HBD 2, PSA 107.11, QED 0.42
Compound (SMILES): O=C(Nc1ccc(C2CCCNC2)cc1)c1ccc(Cl)cn1
InChIKey: FUSOYBOVDDVOIU-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing rat TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 46.6 nM (pChEMBL 7.329999923706055)
Descriptors: MW 315.8, ALogP 3.45, HBA 3, HBD 2, PSA 54.02, QED 0.91
Compound (SMILES): O=c1cc(NC2CCN(S(=O)(=O)C3CC3)CC2)c2cc(C(c3ccc(Cl)cc3)c3ccc(Cl)cc3)ccc2[nH]1
InChIKey: VYQRETDTLVQXEG-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 582.55, ALogP 6.38, HBA 4, HBD 2, PSA 82.27, QED 0.25
Compound (SMILES): N#Cc1nc(Nc2ccc([N+](=O)[O-])cc2)nc(NC2CCCCC2)n1
InChIKey: KJSQAFPATOUENW-UHFFFAOYSA-N
Target: Procathepsin L (SINGLE PROTEIN), Homo sapiens
UniProt: P07711
Assay: Inhibition of human cathepsin L using Z-Phe-Arg-AMC as substrate after 30 mins by spectromicrofluorimetric method
Measurement: IC50 = 136 nM (pChEMBL 6.869999885559082)
Descriptors: MW 339.36, ALogP 3.14, HBA 8, HBD 2, PSA 129.66, QED 0.63
Compound (SMILES): OC[C@H]1CN(CC2CCNCC2)C[C@@H](O)[C@@H]1O
InChIKey: RYBXNLJWPSALOR-IJLUTSLNSA-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 = 1070 nM (pChEMBL 5.96999979019165)
Descriptors: MW 244.34, ALogP -1.37, HBA 5, HBD 4, PSA 75.96, QED 0.49
Compound (SMILES): CC1(N)CCN(c2cccnc2NC(=O)c2nc(-c3nccc(CO)c3C(F)(F)F)cnc2N)CC1
InChIKey: NSFBPGFZYOFNAV-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 = 2.9 nM (pChEMBL 8.539999961853027)
Descriptors: MW 502.5, ALogP 2.6, HBA 9, HBD 4, PSA 156.17, QED 0.41
Compound (SMILES): Cc1ccc(C(=O)N2CC(c3ccc(C#N)cc3)C2)cc1-c1[nH]nc(Cl)c1C
InChIKey: TYPIWPUQWNSMIH-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 390.87, ALogP 4.46, HBA 3, HBD 1, PSA 72.78, QED 0.72
Compound (SMILES): Cc1sc2c(-c3cnc(N)nc3)nc(N3CCOCC3)nc2c1CN1CCN(CCO)CC1
InChIKey: ATTUWTCNNWPSRL-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzymatic Inhibitory Activity Assay:   1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.  2. Compound was formulated in 100% DMSO in a concentration gradient, deposited to a 384-well plate to make final DMSO concentration of 1%.  3. PI3Kα and PI3Kδ enzymes were diluted to be the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT. Transferred to a 384-well plate and incubated with the compound for a certain time.  4. Substrate was diluted to an optimum concentration with following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, Km ATP. The reaction was performed in a 384-well plate for 1 h at room temperature for PI3Kα and 2 hrs at room temperature for PI3Kδ.  5. Read the conversion rate using Caliper Reader, and calculate the inhibition rate as the average of two tests.
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 470.6, ALogP 0.99, HBA 11, HBD 2, PSA 116.76, QED 0.54