lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)C(F)(F)F.O=c1[nH]nc([C@]2(Cc3cccc(Nc4cc[nH]n4)n3)CC[C@@H]([S+]([O-])c3cccc(Cl)c3Cl)CC2)o1
InChIKey: KRVQZGWRVCEHPX-RDEZRPTCSA-N
Target: Aurora kinase A (SINGLE PROTEIN), Homo sapiens
UniProt: O14965
Assay: Inhibition of human N-terminal His-tagged Aurora A expressed in Escherichia coli BL21-Codon-Plus(DE3)-RIL cells using 5-carboxy-fluorescein-y-aminobutyric acid-Ala-Leu-Arg-Arg-Ala-Ser-Leu-Gly-NH2 as substrate after 120 mins by fluorescence polarization assay
Measurement: IC50 = 0.31 nM (pChEMBL 9.510000228881836)
Descriptors: MW 533.44, ALogP 4.76, HBA 7, HBD 3, PSA 135.55, QED 0.29
Compound (SMILES): COC(=O)C[C@H](C)n1c(=O)[nH]/c(=N\c2ccc(OC(C)C)c(C)c2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: OZWRXXHHPQYXIO-KRWDZBQOSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37 C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 500.98, ALogP 3.49, HBA 8, HBD 1, PSA 107.68, QED 0.48
Compound (SMILES): CN1CCN(c2ccc(-c3ccc4[nH]c5nccc(Cl)c5c4c3)cc2)CC1
InChIKey: ZUQQEJBCJAGIIZ-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: ELISA-Based In Vitro Kinase Assay: GST-tagged recombinant wild-type or L1196M mutated ALK kinase (rALK) was expressed in Sf9 insect cells using the pBacPAK baculovirus vector system (Clontech) and purified using Glutathione Sepharose 4B affinity beads (GE Healthcare). Recombinant 3C protease was used to remove the GST tag. Purified ALK was used to screen inhibitors in the ELISA-based kinase assay, as follows: Nunc-Immuno 96-well plates were incubated overnight at 30° C. with coating solution containing 2 μg of a specific ALK peptide substrate (ARDIYRASFFRKGGCAMLPVK) in PBS. Wells were then washed with 200 μL of wash buffer (PBS-Tween 0.05%) and incubated with 4% BSA in PBS for at least 2 h at 30° C. The kinase reaction was performed in the presence of 50 mM Tris pH 7.5, 5 mM MnCl2, 5 mM MgCl2, 0.3 mM ATP and purified rALK in a total volume of 100 μL/well at 30° C. for 15 min. For inhibitor testing the reaction mix was preincubated with inhibitor or vehicle for 10 min at room temperature before transferring to the ELISA plate. After the reaction, the wells were washed 5 times with 200 μL of wash buffer. Phosphorylated peptide was detected using 100 μL/well of a mouse monoclonal anti-phosphotyrosine antibody (clone 4G10 UpstateBiotech Ltd) diluted 1:2000 in PBS+4% BSA. After 30 min incubation at room temperature the antibody was removed and wells were washed as described above. 100 μL of a secondary antibody (anti-mouse IgG, Horseradish Peroxidase linked whole antibody, Amersham Pharmacia Biotech) diluted 1:1000 in PBS+4% BSA was added to each well and the plate was incubated again for 30 min at room temperature before washing as above. The plate was developed using 100 μL/well TMB Substrate Solution (Pierce) and the reaction was stopped by adding an equal volume of 1M H2SO4. Finally, the absorbance was read at 450 nm using an ELISA plate reader (Bio-Rad).
Measurement: IC50 = 2100 nM (pChEMBL 5.679999828338623)
Descriptors: MW 376.89, ALogP 4.79, HBA 3, HBD 1, PSA 35.16, QED 0.55
Compound (SMILES): COc1ccc2c(c1)CC1(CCN([C@H]3CCN(Cc4nc5c(c(=O)[nH]4)COCC5)C3=O)CC1)C2=O
InChIKey: FMURKSCWRQGCFO-NRFANRHFSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H2K2
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 uL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 uM, 20 nM of purified enzyme, and 250 uM of p-NAD+ in the 1x Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 uL of 5x quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 uL of the 5x quenching solution per well was added prior to the addition of p-NAD+. The % Inhibition was calculated as: (Control-Sample)/(Control-Background)*100. "Control" is the average value of 8 wells without compound; and "Background" is the average of 8 wells mixed with 5x quenching solution measured prior to initiation of the reaction.
Measurement: IC50 < 8.6 nM (pChEMBL 8.065999984741211)
Descriptors: MW 478.55, ALogP 1.47, HBA 7, HBD 1, PSA 104.83, QED 0.71
Compound (SMILES): COc1cccc(/C=C/c2ccccc2F)c1
InChIKey: PIVNDVZGTKYESE-MDZDMXLPSA-N
Target: Nuclear factor erythroid 2-related factor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16236
Assay: Activation of Nrf2 (unknown origin) expressed in human HepG2 cells after 5 hrs by ARE-driven luciferase reporter gene assay
Measurement: EC50 = 3800 nM (pChEMBL 5.420000076293945)
Descriptors: MW 228.27, ALogP 4, HBA 1, HBD 0, PSA 9.23, QED 0.72
Compound (SMILES): Cc1ccc(-c2ccc(O[C@@H]3CN[C@H](C(=O)N[C@@H](Cc4ccccc4)C(=O)NS(=O)(=O)c4ccc(Cl)c([N+](=O)[O-])c4)C3)cc2)cc1
InChIKey: RZBNTOGKGQMNSL-NSGJQZOKSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Inhibition of 5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG binding to Mcl-1 (unknown origin) preincubated for 30 mins followed by FAM-labeled peptide addition measured after 20 mins by fluorescence polarization assay
Measurement: Ki = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 663.15, ALogP 4.57, HBA 8, HBD 3, PSA 156.74, QED 0.16
Compound (SMILES): CC(C)Nc1cc(-n2ncc3cc(C#N)cnc32)ncc1C(=O)NCCC1CC1
InChIKey: AYXVFXQHYMLPMP-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 4.1 nM (pChEMBL 8.390000343322754)
Descriptors: MW 389.46, ALogP 3.04, HBA 7, HBD 2, PSA 108.52, QED 0.64
Compound (SMILES): CC(c1ccccc1-c1ccc(Cl)cc1)N1CCN(c2ccc(C(=O)NS(=O)(=O)c3ccc(NCC4CCOCC4)c([N+](=O)[O-])c3)c(Oc3ccc4[nH]ccc4c3)c2)CC1
InChIKey: DOIRGIGOPKQZNI-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Mus musculus
UniProt: P10417
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 = 0.077 nM (pChEMBL 10.109999656677246)
Descriptors: MW 849.41, ALogP 9.03, HBA 10, HBD 3, PSA 159.14, QED 0.08
Compound (SMILES): CCCOc1ccc(N2CCC(Oc3ccc([C@H](C)NC(C)=O)cc3)C2)c(C#N)c1
InChIKey: SWRNZLHDCKJWAT-NVHKAFQKSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 200 μM f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 155 nM (pChEMBL 6.809999942779541)
Descriptors: MW 407.51, ALogP 4.2, HBA 5, HBD 1, PSA 74.59, QED 0.71
Compound (SMILES): COc1nc(-c2nc3n(n2)CCC[C@H]3c2ccc(N3CCCCC3)cc2Cl)ccc1-n1cnc(C)c1
InChIKey: GUCMKFKJZFAVDZ-NRFANRHFSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Rattus norvegicus
UniProt: P08592
Assay: Inhibition of Abeta42 production in Wistar rat fetus brain primary neuronal cultures incubated for 3 days by ELISA
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 504.04, ALogP 5.41, HBA 8, HBD 0, PSA 73.89, QED 0.36
Compound (SMILES): NCCOc1c(-c2ccc(Br)cc2)[nH]c(=O)c2cc(F)ccc12
InChIKey: IZPURWNXWPTMHH-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
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 377.21, ALogP 3.43, HBA 3, HBD 2, PSA 68.11, QED 0.73
Compound (SMILES): COc1cc(Nc2ncc3c(n2)CCN(C(=O)c2cccnc2)C3)cc(OC)c1
InChIKey: OBYUARSXZKLKQF-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: PKR KinaseGlo Assay: Commercially available recombinant human GST-PKR (SignalChem, Canada; 1.5 uM-2 uM stock) is diluted to 500 nM in assay buffer (20 mM Tris-HCl, pH 7.2, 10 mM KCl, 10 mM MgCl2, 10% glycerol). Preactivated PKR is dispensed to 384/96-well black plates at 3.125/12.5 uls/well using the liquid handler Janus. Appropriate dilutions of inhibitors are added to 384/96-well plate followed by 6.6 uM ATP (final) and incubated for 10 minutes at room temperature. The remaining ATP/well is determined by adding 6.25/25 uls/well Kinase-Glo assay mix (Promega) and luminescence is measured on EnVision luminescence plate reader (integration time, 0.2 sec; Perkin-Elmer, Massachusetts, USA).
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 391.43, ALogP 2.83, HBA 7, HBD 1, PSA 89.47, QED 0.72
Compound (SMILES): CC(=O)N[C@@H](C)c1ccc(OC2CCN(c3ccc(OCC4CCOC4)cc3)C2)cc1
InChIKey: ZYLYHFYXEHVBQZ-ISKPRUBYSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 200 μM f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 894 nM (pChEMBL 6.050000190734863)
Descriptors: MW 424.54, ALogP 3.96, HBA 5, HBD 1, PSA 60.03, QED 0.7
Compound (SMILES): Cc1nn(C)c(C)c1S(=O)(=O)N1CCC(Oc2ccc(Cl)cc2C#N)CC1
InChIKey: ZQNZGINXIFNSSP-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Biological Assay: Prokineticin receptor 1 (PKR1) antagonists may be functionally assessed by measurement of change in intracellular calcium levels induced by Gq mediated increase in inositol triphosphate (IP3) levels. The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro. Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1xHanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1x Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 uM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following excitation at 485 nM. Readings from wells that do not contain antagonist enable percentage inhibition curves to be plotted using 4-parameter fit algorithm and IC50 values are calculated for each test compound.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 408.91, ALogP 2.79, HBA 6, HBD 0, PSA 88.22, QED 0.78
Compound (SMILES): COc1cc(OC)cc(N(CC#Cc2cc(C#N)ccn2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: WJQLXQXBPJJDTC-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 absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 9.12 nM (pChEMBL 8.039999961853027)
Descriptors: MW 501.55, ALogP 4.5, HBA 9, HBD 0, PSA 101.98, QED 0.31
Compound (SMILES): Cc1c(Cl)cccc1NC(=O)c1cc(NC(=O)c2ccccc2C(F)(F)F)cc2[nH]c(NC3CCCC3)nc12
InChIKey: PIQXJZVJAPXAGM-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition Assay: mPGES-1 microsome fractions were prepared from CHO-K1 cells transiently transfected with plasmid encoding the human mPGES-1cDNA. Microsomes were diluted with potassium phosphate buffer containing reduced glutathione (pH7.4), and DMSO containing test compound or DMSO alone was added (such that DMSO final concentration would be 1% in each) and incubated at 4° C. for 20 minutes. Then, the enzymatic reactions were initiated by the addition of PGH2 substrate (final concentration 1 μM) and incubated at 4° C. for 60 seconds. The reaction was terminated by the addition of a citrate solution (final citrate concentration 50 mM) containing ferric chloride (final concentration 1 mg/mL). PGE2 production in the enzyme reaction aliquot was measured using HTRF kit (Cisbio International, catalogue #62P2APEC).
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 555.99, ALogP 7.4, HBA 4, HBD 4, PSA 98.91, QED 0.2
Compound (SMILES): Cc1c(-c2c(F)cc(C(N)=O)c3[nH]c4c(c23)CC[C@@H](C(C)(C)O)C4)cccc1-n1c(=O)c2cccc(F)c2n(C)c1=O
InChIKey: ZRYMMWAJAFUANM-MRXNPFEDSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition of full length recombinant human His-tagged BTK expressed in baculovirus using fluoresceinated peptide incubated for 60 mins by fluorescent assay
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 572.61, ALogP 4.4, HBA 6, HBD 3, PSA 123.11, QED 0.3
Compound (SMILES): NCc1ccc(Cl)cc1CNC(=O)[C@@H]1CCCN1C(=O)C1NCc2ccccc21
InChIKey: UAMDKBMLZPEFEW-XJDOXCRVSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: In Vitro Assay: Relevant in vitro assays are referenced in Morrissette, et al., Bioorg. Med. Chem. Lett. 2004, 14, 4161-4164 and described in Lewis, et al. Thromb. Res. 1993, 70, 173 (assays of human α-thrombin and human trypsin), and Lewis, et al. Thromb. Haemostasis 1995, 74, 1107-1112. The assays were carried out at 25° C. in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1% PEG. Trypsin assays also contained 1 mM CaC12. In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate were determined, a Thermomax 96-well plate reader was used was used to measure (at 405 nm) the time dependent appearance of p-nitroaniline. sar-PR-pna was used to assay human α-thrombin (Km=125 μM) and bovine trypsin (Km=125 μM). p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm−1M−1.
Measurement: Ki = 77 nM (pChEMBL 7.110000133514404)
Descriptors: MW 412.92, ALogP 2.25, HBA 4, HBD 3, PSA 87.46, QED 0.7
Compound (SMILES): O=C(Nc1ccc(-c2cn(Cc3ccc(N4CCOCC4)cc3)nn2)cc1)N1Cc2ccccc2C1
InChIKey: HNDQRZVIZVWAIA-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 507 nM (pChEMBL 6.289999961853027)
Descriptors: MW 480.57, ALogP 4.38, HBA 6, HBD 1, PSA 75.52, QED 0.46
Compound (SMILES): COCCN(C)C(=O)c1ccc(NC(=O)N2Cc3ccc(F)cc3C2)cc1
InChIKey: GUXJNXZRIXVTOB-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 60.7 nM (pChEMBL 7.21999979019165)
Descriptors: MW 371.41, ALogP 3.09, HBA 3, HBD 1, PSA 61.88, QED 0.88
Compound (SMILES): O=C(CN1CCC(c2n[nH]c3cc(F)ccc23)CC1)NCc1nc2c(c(=O)[nH]1)COCC2
InChIKey: RZPNNTCBQSMUAY-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The autoparsylation activity of the TNKS 1/2 or PARP1/2 enzymes was measured by the liquid chromatography-mass spectrometry (LC/MS) detection of nicotinamide as readout. Compound activity in inhibiting the TNKS and PARP autoparsylation was evaluated by IC50 measurements. In the compound screening assays, the reaction is composed of 5 μL of compound in 8-point serial dilutions with concentrations ranging from 0.0086 to 18.75 μM, 20 nM of purified enzyme, and 250 μM of β-NAD+ in the 1× Assay Buffer. After 60 min incubation at room temperature, the reactions were quenched by the addition of 10 μL of 5× quenching solution (20% formic acid and 500 nM [d]-nicotinamide in water). For the background control wells, 10 μL of the 5× quenching solution per well was added prior to the addition of β-NAD+.
Measurement: IC50 = 1210 nM (pChEMBL 5.920000076293945)
Descriptors: MW 440.48, ALogP 1.35, HBA 6, HBD 3, PSA 116, QED 0.55
Compound (SMILES): COC(=O)C(C)(C)NC(=O)c1ccc(C)c(OCC(F)(F)F)n1
InChIKey: ZZMFXRUCFZHIFN-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
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 = 77.2 nM (pChEMBL 7.110000133514404)
Descriptors: MW 334.29, ALogP 2.01, HBA 5, HBD 1, PSA 77.52, QED 0.83
Compound (SMILES): O=C(O)/C=C/c1ccc2cccc(N3CCN(CCc4ccc(OCCCN5CCCCCC5)cc4)CC3)c2n1
InChIKey: NFCYQBPNSWBYEF-DTQAZKPQSA-N
Target: Histamine H1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35367
Assay: Antagonist activity at human Histamine H1 receptor expressed in CHO cells assessed as inhibition of histamine-induced calcium flux preincubated for 30 mins followed by histamine addition by Fluo-4-AM dye based FLIPR assay
Measurement: Ki = 199.53 nM (pChEMBL 6.699999809265137)
Descriptors: MW 542.72, ALogP 5.34, HBA 6, HBD 1, PSA 69.14, QED 0.26
Compound (SMILES): CN1c2nc(C3CCCNC3)ccc2N(CC2CC2(F)F)S1(=O)=O
InChIKey: HOHKQQOZBQXJFV-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Fluorescence Laser Imaging Plate Reader (FLIPR) Based Assay: The compounds of the present invention may be tested in a fluorescence laser imaging plate reader (FLIPR) based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr-cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with dose responses of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity. The potentiation response is monitored after a subsequent addition of an EC20 concentration of glutamate (900 nM).
Measurement: EC50 = 359.2 nM (pChEMBL 6.449999809265137)
Descriptors: MW 358.41, ALogP 1.7, HBA 4, HBD 1, PSA 65.54, QED 0.89
Compound (SMILES): CC(C)OC(=O)c1ccc(C#Cc2c(N)ncnc2N[C@@H](C)c2nc3cccc(Cl)c3c(=O)n2-c2ccccc2)nc1
InChIKey: DBWKOVCXXRYQHM-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM 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 isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (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; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 580.05, ALogP 4.94, HBA 10, HBD 2, PSA 137.91, QED 0.21
Compound (SMILES): CCN1CCCCC1c1nc(COc2cc3ncnc(Nc4ccc(Cl)c(Cl)c4)c3cc2OC)cs1
InChIKey: WVXLPNDNCDLCQW-UHFFFAOYSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 10500 nM (pChEMBL 4.980000019073486)
Descriptors: MW 544.51, ALogP 7.27, HBA 8, HBD 1, PSA 72.4, QED 0.25
Compound (SMILES): CCOc1ccc2[nH]nc(-c3cc(N4CCN(S(C)(=O)=O)CC4)ncn3)c2c1
InChIKey: BZKKQQPSWQGCMH-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 15.38 nM (pChEMBL 7.809999942779541)
Descriptors: MW 402.48, ALogP 1.5, HBA 7, HBD 1, PSA 104.31, QED 0.69
Compound (SMILES): O=S(=O)(c1ccc(-c2nnn[nH]2)cc1)N(CCCCC(F)(F)F)c1sc2ccccc2c1Cl
InChIKey: HNKODGQAXPAOME-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 22.1 nM (pChEMBL 7.659999847412109)
Descriptors: MW 515.97, ALogP 5.66, HBA 6, HBD 1, PSA 91.84, QED 0.31
Compound (SMILES): NC1=N[C@@]2(CCO1)c1cc(NC(=O)c3ccc(Cl)cn3)ccc1Oc1c2cc(C2=CCOCC2)nc1F
InChIKey: RHRDPZBXIDGLJQ-SANMLTNESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The beta secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 521.94, ALogP 4.41, HBA 8, HBD 2, PSA 120.95, QED 0.49
Compound (SMILES): CC(=O)N1CCc2c(C(=O)N3CCC(c4cccc(F)c4Cl)CC3)n[nH]c2C1
InChIKey: UZSKQBUZGPQZTH-UHFFFAOYSA-N
Target: Retinol-binding protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P02753
Assay: Scintillation Proximity Binding Assay: Untagged human RBP4 purified from urine of tubular proteinuria patients was purchased from Fitzgerald Industries International. It was biotinylated using the EZ-Link Sulfo-NHS-LC-Biotinylation kit from Pierce following the manufacturer's recommendations. Binding experiments were performed in 96-well plates (OptiPlate, PerkinElmer) in a final assay volume of 100 ul per well in SPA buffer (IX PBS, pH 7.4, 1 mM EDTA, 0.1% BSA, 0.5% CHAPS). The reaction mix contained 10 nM 3H-Retinol (48.7 Ci/mmol; PerkinElmer), 0.3 mg/well Streptavidin-PVT beads, 50 nM biotinylated RBP4 and a test compound. Nonspecific binding was determined in the presence of 20 uM of unlabeled retinol. The reaction mix was assembled in the dark under dim red light. The plates were sealed with clear tape (TopSeal-A: 96-well microplate, PerkinElmer), wrapped in the aluminum foil, and allowed to equilibrate 6 hours at room temperature followed by overnight incubation at +4° C. Radiocounts were measured using a TopCount NXT counter (Packard Instrument Company).
Measurement: IC50 = 4.842 nM (pChEMBL 8.3100004196167)
Descriptors: MW 404.87, ALogP 3.13, HBA 3, HBD 1, PSA 69.3, QED 0.84
Compound (SMILES): CC1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccc(C#N)cc2NC1=O
InChIKey: BKSCSZWESYMLPD-KKFPZRRJSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: In Vitro Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 93.25 nM (pChEMBL 7.03000020980835)
Descriptors: MW 547.01, ALogP 6.67, HBA 4, HBD 1, PSA 86.09, QED 0.36
Compound (SMILES): CC(=O)N1CCN(c2cc(-c3n[nH]c4ccc(-c5cnn(C)c5)cc34)ncn2)CC1
InChIKey: NJSISWNBGQTGKN-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 3.15 nM (pChEMBL 8.5)
Descriptors: MW 402.46, ALogP 2.09, HBA 7, HBD 1, PSA 95.83, QED 0.56
Compound (SMILES): CN[C@H](C(=O)N[C@@H]1C(=O)N(Cc2c(OC)ccc3cc(Br)ccc23)c2ccc(C)cc2O[C@H]1C)[C@H](C)O
InChIKey: LKIFTWKEGXUQGG-FRSFCCSCSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 82 nM (pChEMBL 7.090000152587891)
Descriptors: MW 570.48, ALogP 3.69, HBA 6, HBD 3, PSA 100.13, QED 0.4
Compound (SMILES): O=C(CCc1ccccc1)Nc1cccc(CNC(=O)c2cccc(-c3ccccc3)c2)c1
InChIKey: LUSAYUIFORKKLE-UHFFFAOYSA-N
Target: Sentrin-specific protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P0U3
Assay: Inhibition of SENP1 catalytic domain (419 to 644 residues) (unknown origin) expressed in Escherichia coli BL21 (DE3) assessed as reduction in deSUMOylation preincubated for 10 mins followed by addition of RanGAP-SUMO as substrate measured after 25 mins by coomassie staining-based SDS-PAGE analysis
Measurement: IC50 = 8500 nM (pChEMBL 5.070000171661377)
Descriptors: MW 434.54, ALogP 5.85, HBA 2, HBD 2, PSA 58.2, QED 0.37
Compound (SMILES): Cn1ncc(NC(=O)c2csc(-c3cccc(F)c3-c3cccc(F)c3Cl)n2)c1[C@@H]1CC[C@@H](N)[C@@H](F)CO1
InChIKey: FUTZWPQTVQNZFB-UEXGIBASSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Assay: LC3K assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO. PIM reactions were carried out in a final volume of 10 μL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 μL 2×ATP and test compound to 5 μL of 2× enzyme and FAM-peptide, contained 20 pM PIM1, 50 pM PIM2, or 55 pM PIM3, 1 μM FAM-peptide, and 10 μM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 μL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000® (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of −500V, an upstream voltage of −2150V, and a screening pressure of −1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm.
Measurement: Ki = 630 nM (pChEMBL 6.199999809265137)
Descriptors: MW 562.02, ALogP 5.91, HBA 7, HBD 2, PSA 95.06, QED 0.32
Compound (SMILES): CC(C)(O)C(=O)N1CCC(CNc2ccc3ncc(-c4cccc(OC(F)(F)F)c4)n3n2)CC1
InChIKey: FPOOBLUDOKRHTK-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase-Glo Assay: These assays are set up in duplicate 50 ul volumes in white, flat bottom 96 well plates. Inhibitors are added to the solution of 1× kinase buffer, 10 uM ATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, and water in serial dilutions ranging from micromolar to nanomolar concentrations. This solution is incubated at 30 degrees Celsius at 360 rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagent is added to each well, including all positive and negative control wells, and incubated at room temperature for 15 minutes. The plate is then read by the Luminoskan Ascent instrument and the results displayed with the Ascent Software version 2.6.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 477.49, ALogP 3.72, HBA 7, HBD 2, PSA 91.99, QED 0.56
Compound (SMILES): COc1cccc(CN(C(=O)c2ccccc2)[C@H](C)C(N)=O)c1
InChIKey: OVXYDEFBJIUMAF-CYBMUJFWSA-N
Target: Transient receptor potential cation channel subfamily M member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z2W7
Assay: Antagonist activity at human recombinant TRPM8 expressed in HEK293 cells assessed as inhibition of menthol-induced calcium increase by Fluo-4 AM based assay
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 312.37, ALogP 2.21, HBA 3, HBD 1, PSA 72.63, QED 0.89
Compound (SMILES): O=c1c2cn[nH]c2c2ccc(-c3ncccc3Cl)cc2n1CC(F)(F)F
InChIKey: KIJAUPGROTXDCS-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzymatic Screening Assay: The enzymatic test is a test in two steps.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 8.83 nM (pChEMBL 8.050000190734863)
Descriptors: MW 378.74, ALogP 4.16, HBA 4, HBD 1, PSA 63.57, QED 0.57
Compound (SMILES): CC(C)[C@H]1CC[C@]23C[C@@H]4[C@H](C)C(=O)[C@H]5O[C@]54[C@@]2(C)CC(=O)[C@]13C
InChIKey: XTHSBZKJZOMAJE-PGDSUDNMSA-N
Target: Signal transducer and activator of transcription 1-alpha/beta (SINGLE PROTEIN), Homo sapiens
UniProt: P42224
Assay: Inhibition of Stat1 (unknown origin) expressed in LPS/INF-gamma-stimulated human MONO-MAC-6 cells assessed as reduction in GAS dependent transcription activity measured after 4 hrs by luciferase reporter gene assay
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 316.44, ALogP 3.4, HBA 3, HBD 0, PSA 46.67, QED 0.7
Compound (SMILES): Cn1c(-c2cc3ccccc3n2CC(F)(F)F)nc2cc(C(=O)N3CCC[C@@H](N)C3)ccc21
InChIKey: XCHVAFMDSDWGKU-QGZVFWFLSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Inhibition of GST-tagged recombinant human PAD4 (1 to 663 residues) expressed in Escherichia coli using N-a-benzoyl-L-arginine ethyl ester as substrate preincubated for 30 mins followed by substrate addition after 100 mins by DTT-based fluorescence assay
Measurement: IC50 = 158.49 nM (pChEMBL 6.800000190734863)
Descriptors: MW 455.48, ALogP 4.32, HBA 5, HBD 1, PSA 69.08, QED 0.5
Compound (SMILES): O=C(Nc1nc2ccccc2s1)c1cccc2c1CN(c1ccc(-c3ccnc(OC4CCCCC4)c3)c(C(=O)O)n1)CC2
InChIKey: POSGOGLWVNQEAX-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 605.72, ALogP 6.98, HBA 8, HBD 2, PSA 117.54, QED 0.2
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(-c3cncc(C(F)F)c3)cc1C21N=C(C)C(N)=N1
InChIKey: WUJRLQGOZRVKDS-AHLJTMJASA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: TR-FRET Assay: The β-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human β-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc 10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in −80° C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 μg/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 μM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 μl) was added to the plate then 1 μl of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes. Substrate (10 μl) was then added, mixed and the reaction proceeded for 15 minutes at r.t. The reaction was stopped with the addition of Stop solution (7 μl, NaAcetate, pH 9). The fluorescence of the product was measured on a Victor II plate reader with an excitation wavelength of 340 nm and an emission wavelength of 615 nm. The assay was performed in a Costar 384 well round bottom, low volume, non-binding surface plate (Corning #3676). The final concentration of the enzyme was 2.7 g/ml; the final concentration of substrate was 100 nM (Km of 250 nM). The dimethylsulphoxide control, instead of test compound, defined the 100% activity level and 0% activity was defined by wells lacking enzyme (replaced with reaction buffer). A control inhibitor was also used in dose response assays and had an IC50 of 150 nM.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 424.5, ALogP 4.8, HBA 5, HBD 1, PSA 72.86, QED 0.77
Compound (SMILES): CCc1cc2nc(NCc3cccnc3)nc(C(=O)c3cccs3)c2s1
InChIKey: FARDIRBBHGVIGJ-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Fluorometric Imaging Plate Reader (FLIPR) Asssay: The use of a Fluorometric Imaging Plate Reader (FLIPR) to measure calcium flux in Adenosine-receptor expressing cells is a well-established technique. In this assay calcium flux is triggered by receptor activation and measured through the fluorescence of an incorporated calcium-sensitive dye. The potencies shown were determined using expressed human adenosine A2B receptors in mammalian cell lines. Selectivity values were obtained by using mammalian cell lines expressing the human adenosine A1, A2A and A3 receptors.
Measurement: IC50 = 216 nM (pChEMBL 6.670000076293945)
Descriptors: MW 380.5, ALogP 4.55, HBA 7, HBD 1, PSA 67.77, QED 0.5