lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@H](Nc1ncc(F)c(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1ccc(Cl)c(Cl)c1
InChIKey: WFOPXBRBZHRGKH-ATEUNZGCSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Fluorescence Biochemical Assay: The IDH1 (R132H) mutant catalyzes the reduced form of NADP+ (NADPH) and α-ketoglutarate (α-KG) to form nicotinamide adenine dinucleotide phosphate (NADP+) and R (−)-2-hydroxyglutarate (2HG). The reaction can be monitored kinetically by following the oxidation of NADPH to NADP+ which is measured using fluorescence, excitation at 355 nm and emission at 530 nm. Reactions were monitored using the Perkin-Elmer Envision, Model 2101. More specifically, the biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Cat. No. 781076) using a final reaction volume of 20 μL and the following assay buffer conditions: 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM DTT, 0.02% BSA, 0.02% Tween-20, 10 μM NADPH and 100 μM α-KG. The final reaction mixture contained 2.5% DMSO and test compounds with concentrations ranging 0.0000008-25 μM. The IDH1 (R132H) enzyme was used at a final concentration of 10 nM.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 415.25, ALogP 3.8, HBA 6, HBD 2, PSA 87.58, QED 0.77
Compound (SMILES): O=C1NCCN1c1cc(Cc2n[nH]c(=O)c3c2CCCC3)ccc1F
InChIKey: PIVKEORYLUBFQJ-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 342.37, ALogP 1.91, HBA 3, HBD 2, PSA 78.09, QED 0.89
Compound (SMILES): O=C(NC1CCN(Cc2ccc(OC3CCNCC3)c(Br)c2)CC1)c1cc2ccc(-c3ccc(F)cc3)cc2s1
InChIKey: VHYPEZOCPFHYFF-UHFFFAOYSA-N
Target: Urotensin-2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKP6
Assay: Displacement of europium-labeled urotensin-2 from human recombinant urotensin-2 receptor expressed in HEK293 cell membranes after 90 mins by filtration based time resolved fluorometric assay
Measurement: IC50 = 74 nM (pChEMBL 7.130000114440918)
Descriptors: MW 622.6, ALogP 7, HBA 5, HBD 2, PSA 53.6, QED 0.23
Compound (SMILES): CC1(C)c2cc(OCC(N)=O)ccc2C(=O)c2c1[nH]c1cc(C#N)ccc21
InChIKey: METUHGKGBJAKIH-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Inhibition Assay: ALK-inhibiting activity was measured by following an activity of inhibiting phosphorylation by biotinylated peptide (EGPWLEEEEEAYGWMDF). For the detection of phosphorylation of the biotinylated peptide, time-resolved fluorescence measurement was performed using an anti-phosphorylated tyrosine antibody labeled with europium cryptate and streptavidin conjugated to XL665, i.e., an allophycocyanin derivative.
Measurement: IC50 = 17.15 nM (pChEMBL 7.769999980926514)
Descriptors: MW 359.39, ALogP 2.77, HBA 4, HBD 2, PSA 108.97, QED 0.75
Compound (SMILES): Nc1ccccc1NC(=O)CCCCCNC(=O)c1cscn1
InChIKey: VXKWFYGUKQIMKR-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: HDAC enzyme inhibitionAssay: HDAC enzyme inhibition assays were performed using purified HDACs 1-10 essentially as described in Beckers et al., 2007, Int. J. Cancer., 121:1138-48 and Perez-Balado et al., 2007, J. Med. Chem., 50:2497-2505. Inhibition assays using nuclear extract were performed essentially as described in Herman et al., 2006, Nat. Chem. Biol., 2:551-558. Briefly, the purified HDACs or nuclear extract were incubated with an acetylated substrate in the absence of the compound to be assayed and with increasing concentrations of the compound. The rate of substrate deacetylation was measured under each condition, and half-maximal inhibitory concentration with regard to each HDAC was determined by standard means.
Measurement: IC50 = 1039 nM (pChEMBL 5.980000019073486)
Descriptors: MW 332.43, ALogP 2.65, HBA 5, HBD 3, PSA 97.11, QED 0.51
Compound (SMILES): C[C@@H](NC(=O)c1nn(-c2ccc(Cl)cc2Cl)c2c1CCC/C2=C\c1ccc(Cl)cc1)c1ccccn1
InChIKey: GJRORIVCMARHTK-VXNZKSKFSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Inverse agonist activity at human cannabinoid receptor 1 expressed in HEK293 cells assessed as increase in forskolin-induced cAMP production preincubated with cells followed by forskolin stimulation measured after 30 mins in presence of D2-labeled cAMP and Eu3+-TBP-NHS cryptate by HTRF assay
Measurement: EC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 537.88, ALogP 7.6, HBA 4, HBD 1, PSA 59.81, QED 0.29
Compound (SMILES): CC1(C)CN(C(=O)Cn2c(=O)c(C(=O)NCc3ccc(Cl)cc3)cc3c(Oc4cccnc4)ccnc32)C1
InChIKey: OAVXQKUGKJCDCN-UHFFFAOYSA-N
Target: DNA polymerase catalytic subunit (SINGLE PROTEIN), Human cytomegalovirus (strain AD169) (HHV-5) (Human herpesvirus 5)
UniProt: P08546
Assay: HCMV Polymerase LANCE TR-FRET Assay: The assay conditions are the following: 10 mM HEPES pH 7.5, 25 mM KCl, 7.5 mM NaCl, 5 mM MgCl2, 0.2 mg BSA/mL, 1 mM TCEP, 1.5% glycerol, 5% DMSO, 235 nM dATP, 350 nM dCTP, 350 nM dGTP, 235 nM dTTP, 12 nM biotin-16-dUTP, 23.5 nM Dig-primer/template, 2 nM GST-UL54. The assay volume is 10 μL. Each reagent is added as follow: 4 μL a+3 μL b+3 μL c; a: compound diluted in compound dilution buffer to obtain 12.5% DMSO; b: enzyme (GST-UL54) in 10 mM Hepes pH 7.5, 25 mM KCl, 5 mM MgCl2, 25 mM NaCl, 5% Glycerol, 0.67 mg BSA/mL, 1 mM TCEP w/o DMSO (2 nM GST-UL54 is present in the assay); c: substrate in 10 mM HEPES pH 7.5, 25 mM KCl, 5 mM MgCl2, 1 mM TCEP, 783 nM dATP, 1166 nM dCTP, 1166 nM dGTP, 783 nM dTTP, 40 nM biotin-16-dUTP, 78 nM Dig-primer (5'-/Dig/AGC TCG TTT AGT GAA CC-3')/template (5'-GAG GTC AAA ACA GCG TGG ATG GCG TCT CCA GGC GAT CTG ACG GTT CAC TAA ACG AGC T-3') w/o DMSO. The primer and template are annealed in 10 mM Tris-HCl pH 7.5, 50 mM NaCl at a respective concentration of 50 μM. They are incubated at 95 °C. for 5 min in a dry batch block. The block is removed from the dry bath and allowed to cool to RT. Aliquots are made and stored at −20 °C. To perform the assay, 3 μL of the enzyme solution is added to columns 2-12 and 14-24. The enzyme is substituted by the blank solution (b solution without enzyme) for columns 1 and 13 (blanks). The plate is centrifuged at 200×g for 30 sec. 3 μL of substrate solution is added to each well. The plate is centrifuged at 200×g for 30 sec. Plates are incubated at 37 °C. for 30 min. 5 μL of conjugate solution is added (25 mM Hepes pH 7.5, 0.1 M NaCl, 0.25% Tween-20, 1 mg/mL BSA, 12 mM EDTA, 24 nM Sreptavidin-APC, 342 ng/mL Anti-Dig-Europium). The plates are incubated at RT for at least 120 min. The signal is read on the Envision plate reader (Perkin-Elmer) or equivalent.
Measurement: IC50 = 280 nM (pChEMBL 6.550000190734863)
Descriptors: MW 532, ALogP 4.04, HBA 7, HBD 1, PSA 106.42, QED 0.39
Compound (SMILES): O=S(=O)(Nc1ccc(F)c(Nc2ncccc2-c2ncnc3[nH]cnc23)c1F)c1ccc(OC(F)(F)F)cc1
InChIKey: IARQQKDGLMSINT-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Enzyme Assay: To check the B-Raf kinase inhibitory activity of the compounds of the present invention, the following experiments were conducted.(1) Serial Signaling: 20 ul of diluting solvent (20 mM MOPS, pH 7.2, 25 mM Î²-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM dithiothreitol) and 10 ul of Mg/ATP mixed solution (500 uM ATP, 75 mM magnesium chloride) were added into a centrifuge tube, the derivative compound of Formula 1 was added or the compounds of the Examples were not added as a control group, and then 1 ng of activated B-Raf, 0.4 ng of inactivated MEK1, and 1 ug of inactivated MAPK2 were added. The solutions in the tube were collected on the bottom via centrifugation and were reacted at 30° C. for 30 minutes. After taking 4 ul of the mixed solution, the test proceeded to the next step.(2) Phosphorylation of Matrix Protein MBP by MAPK2: 10 ul of diluting solvent, 20 ug of MBP used as a matrix.
Measurement: IC50 = 610 nM (pChEMBL 6.210000038146973)
Descriptors: MW 563.47, ALogP 5.14, HBA 8, HBD 3, PSA 134.78, QED 0.23
Compound (SMILES): C[C@@H]1CC[C@@H]([C@@H](C)C(=O)Nc2ccc(Cl)c(-c3ccccn3)c2)[C@H]2Cc3c(c4ccccc4n3C)C[C@H]21
InChIKey: RXVLUIAXECSAFS-NGQKKBAQSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of Smo-mediated Hh signalling pathway in mouse Shh Light2 cells by Gli-luciferase reporter gene assay
Measurement: IC50 = 476 nM (pChEMBL 6.320000171661377)
Descriptors: MW 512.1, ALogP 7.55, HBA 3, HBD 1, PSA 46.92, QED 0.31
Compound (SMILES): COc1cc(F)ccc1C(=O)N1CCC(N2CC(CC#N)(n3cc(-c4ncnc5[nH]ccc45)cn3)C2)CC1
InChIKey: XXZUBCPXZFVIPQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 514.57, ALogP 3.2, HBA 8, HBD 1, PSA 115.96, QED 0.42
Compound (SMILES): CC1CC(=O)NN=C1c1ccc2nc(-c3ccc(N4CCOCC4)nc3Cl)oc2c1
InChIKey: JPXHMLGSNZNILO-UHFFFAOYSA-N
Target: Glucose-dependent insulinotropic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDV5
Assay: AlphaScreen cAMP Assay: MIN6 cells [Miyazaki J et al. Endocrinology. 1990 July; 127(1):126-32] were stably transfected with an expression vector for human GPR119 cDNA (Acc. No. NP-848566). Min-6/hGPR119 cells are cultured in DMEM, 10% FBS, 50 uM beta-mercaptoethanol, 0.3 mg/mL Geniticin, 2 mM GlutaMAX at 37 C. 5% CO2. For the assay, the cells are placed in Optiplates (white, 384-well, 160W-barcoded, TC, sterile with lid, Cat. No. #6007688 (Perkin Elmer); 10000 cells/well; 50 ul). The plates covered with lids are then incubated for 24 hours at 37 C./5% CO2.After the medium has been sucked out of the wells completely, 10 ul of the test compound are added, the compounds are diluted beforehand with stimulating buffer (140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 1.5 mM CaCl2, 10 mM Hepes, 5 mM NaHCO3; pH 7.4. 0.5 mM IBMX and 0.1% BSA, the final DMSO concentration is 1%). After 45 minutes' incubation at RT, the cAMP concentrations are determined using the AlphaScreen cAMP Assay.
Measurement: EC50 = 379 nM (pChEMBL 6.420000076293945)
Descriptors: MW 425.88, ALogP 3.24, HBA 7, HBD 1, PSA 92.85, QED 0.65
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(C#CC3CC3)cc1C21N=C(N)N(Cc2cccc(-c3nccs3)c2)C1=O
InChIKey: IDPSMDPGMSDNOT-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 10.3 nM (pChEMBL 7.989999771118164)
Descriptors: MW 536.7, ALogP 5.26, HBA 6, HBD 1, PSA 80.81, QED 0.46
Compound (SMILES): CC(C)(C)OC(=O)COc1cc(NC2CCN(S(=O)(=O)C(F)(F)F)CC2)c2cc(C(c3ccc(Cl)cc3)c3ccc(Cl)cc3)ccc2n1
InChIKey: GKDVWBLMHRYWFT-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
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 724.63, ALogP 8.17, HBA 7, HBD 1, PSA 97.83, QED 0.14
Compound (SMILES): CCNC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3[C@H](C)c3ccccc3)ccc2[nH]1
InChIKey: ZTGFAYYLHSPPAQ-DIERRCTGSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 455.52, ALogP 4.11, HBA 4, HBD 4, PSA 110.35, QED 0.36
Compound (SMILES): O=C(Nc1ccc(C2=CCN(C(=O)C3=CCCC3)CC2)cc1)N1Cc2ccncc2C1
InChIKey: KORCMVPKGZHWQX-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 = 3.26 nM (pChEMBL 8.489999771118164)
Descriptors: MW 414.51, ALogP 4.36, HBA 3, HBD 1, PSA 65.54, QED 0.81
Compound (SMILES): CN(C)C(=O)CCNc1ncnc2[nH]cc(-c3cccc(C#N)c3)c12
InChIKey: CLAQSCOHFCXYES-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 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 334.38, ALogP 2.39, HBA 5, HBD 2, PSA 97.7, QED 0.75
Compound (SMILES): O=c1oc2ccccc2c(N2CCN(C(c3ccccc3)c3ccccc3)CC2)c1CO
InChIKey: OAVSELNRYRSYAX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of human recombinant GST-tagged BRAF catalytic domain (416 to 766 residues) V600E mutant expressed in baculovirus expression system preincubated for 1 hr followed by human full length recombinant N-terminal His-tagged MEK1 substrate addition measured after 25 mins by gel electrophoresis method
Measurement: IC50 = 370 nM (pChEMBL 6.429999828338623)
Descriptors: MW 426.52, ALogP 4.2, HBA 5, HBD 1, PSA 56.92, QED 0.49
Compound (SMILES): N#Cc1ncccc1-c1ccc2c3[nH]ncc3c(=O)n(CC(F)(F)F)c2c1
InChIKey: FNJXFCFTEPMACG-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 = 51.9 nM (pChEMBL 7.289999961853027)
Descriptors: MW 369.31, ALogP 3.37, HBA 5, HBD 1, PSA 87.36, QED 0.59
Compound (SMILES): CC(C)Oc1ccc2[nH]nc(-c3cc(N4CCN(S(C)(=O)=O)CC4)ncn3)c2c1
InChIKey: ZJTLVQZZJYWOSZ-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 = 7.99 nM (pChEMBL 8.100000381469727)
Descriptors: MW 416.51, ALogP 1.89, HBA 7, HBD 1, PSA 104.31, QED 0.68
Compound (SMILES): COc1cc(-c2ccc3c(c2)C2(N=C(N)N(C)C2=O)C2(CCC(OC)CC2)C3)ccn1
InChIKey: WRMCIPDJJHVWPU-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 10.5 nM (pChEMBL 7.980000019073486)
Descriptors: MW 420.51, ALogP 2.87, HBA 6, HBD 1, PSA 90.04, QED 0.82
Compound (SMILES): N#C[C@H](NC(=O)c1ccc(C2CC2)c(OCC2CC2)n1)C1CC1
InChIKey: CTGPIYJODYQPII-INIZCTEOSA-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 = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 311.39, ALogP 2.78, HBA 4, HBD 1, PSA 75.01, QED 0.84
Compound (SMILES): O=C(O)CNc1ccc(-c2c(C#Cc3ccc4ccccc4n3)nc3c(N4CCOCC4)ccnn23)cn1
InChIKey: JQLVWKHGBJODNK-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 505.54, ALogP 3.07, HBA 9, HBD 2, PSA 117.77, QED 0.35
Compound (SMILES): C[C@H](c1nc2c(cnn2C2CCOCC2)c(=O)[nH]1)N1CC(F)(Cc2ccccc2)C1
InChIKey: UUXWBMKQSULUBW-OAHLLOKOSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition of human recombinant PDE9 expressed in insect Sf9 cells using [3H]-cGMP as substrate after 30 mins by scintillation proximity assay
Measurement: IC50 = 50.3 nM (pChEMBL 7.300000190734863)
Descriptors: MW 411.48, ALogP 2.8, HBA 6, HBD 1, PSA 76.04, QED 0.7
Compound (SMILES): N#Cc1ccc2c(c1)CN(C(=O)Nc1ccc(C(=O)NC(CO)CO)cc1)C2
InChIKey: AOBVNTJCFIVTGW-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 = 44.6 nM (pChEMBL 7.349999904632568)
Descriptors: MW 380.4, ALogP 1.19, HBA 5, HBD 4, PSA 125.69, QED 0.62
Compound (SMILES): O=c1[nH]c(N2CCOCC2)nc(NCC(O)CO)c1-c1nc2ccccc2s1
InChIKey: RYVLYWRRFFOWJB-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IMAP technology (Molecular Devices): A 20 ul reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 uM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 ul of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 8100 nM (pChEMBL 5.090000152587891)
Descriptors: MW 403.46, ALogP 0.65, HBA 9, HBD 4, PSA 123.6, QED 0.47
Compound (SMILES): CC1(Oc2ccccc2)CN(C(=O)c2ccc3cc(Oc4ccc(C(F)(F)F)cn4)ccc3n2)C1
InChIKey: KAEJAMZBFMAFRO-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 > 20000 nM (pChEMBL 4.698999881744385)
Descriptors: MW 479.46, ALogP 5.73, HBA 5, HBD 0, PSA 64.55, QED 0.37
Compound (SMILES): CN1Cc2ccncc2N(S(C)(=O)=O)C/C=C/c2cc(ccc2N2CCOCC2)Nc2ncc(C(F)(F)F)c1n2
InChIKey: SQSRYKIYYZPXLV-ONEGZZNKSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
Assay: Inhibition of full length recombinant human N-terminal His6-tagged PYK2 expressed in baculovirus infected sf21 cells
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 575.62, ALogP 3.9, HBA 9, HBD 1, PSA 103.79, QED 0.49
Compound (SMILES): CC(=O)N[C@H]1CCN(c2ccc(-c3cc(C)cc(Nc4cc(C(F)(F)F)ccn4)n3)cn2)C1
InChIKey: YDNIGECNGIEISM-SFHVURJKSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 456.47, ALogP 4.32, HBA 6, HBD 2, PSA 83.04, QED 0.6
Compound (SMILES): COCCOc1cnc2c(Nc3ccc(F)c([C@]4(C(F)F)COCC(N)=N4)c3)nccc2c1
InChIKey: VPJLMCOWNDPWKS-QFIPXVFZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE-1 Inhibition Assay: Recombinant BACE-1 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 uM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals.
Measurement: IC50 = 600 nM (pChEMBL 6.21999979019165)
Descriptors: MW 461.44, ALogP 3.39, HBA 8, HBD 2, PSA 103.88, QED 0.5
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)OCC3CCC3)CC2)C1
InChIKey: DTKQISJPXILNTF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 <= 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 474.57, ALogP 3.15, HBA 8, HBD 1, PSA 115.96, QED 0.58
Compound (SMILES): Cc1nn(Cc2ccc(Oc3ccccc3)cc2)c2nc(CCc3ccccc3)cc(C(=O)O)c12
InChIKey: VIQRLZAPHRFXQB-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 homolog (SINGLE PROTEIN), Mus musculus
UniProt: P97287
Assay: Fluorescence Polarization-Based Binding Assay: Sensitive and quantitative FP-based binding assays were developed and optimized to determine the binding affinities of small-molecule inhibitors to the recombinant Mcl-1, A1/Bfl-1, Bcl-w, Bcl-2, and Bcl-xL proteins. The concentrations of the proteins used in the competitive binding experiments were 10 nM for Mcl-1, 80 nM for Bcl-xL, and 60 nM for Bcl-2. The fluorescent probes, Flu-BID or FAM-BID were fixed at 2 nM for all assays, which binds with Kd values of 2.3 nM, 14.2 nM and 24.1 nM against Mcl-1, Bcl-2 and Bcl-xL respectively. 5 μL of the tested compound in DMSO and 120 μL of protein/probe complex in the assay buffer (20 mM phosphate pH 7.4, 50 mM NaCl, 1 mM EDTA. 0.05% Pluronic F68)s) were added to 96 well black assay plates, incubated at room temperature for 3 h and the polarization values (mP) were measured at an excitation wavelength at 485 nm and an emission wavelength at 530 nm using the plate reader Synergy H1 Hybrid, BioTek.
Measurement: Ki = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 463.54, ALogP 6.06, HBA 5, HBD 1, PSA 77.24, QED 0.3
Compound (SMILES): CCCC[C@H](NC(C)=O)C(=O)N[C@H]1CCC(=O)NC[C@@H](C(=O)N[C@@H](Cc2c[nH]c3ccccc23)C(N)=O)NC(=O)[C@H](CCCNC(=N)N)NC(=O)[C@@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC1=O
InChIKey: KPDWCDURFGPKLY-PDBPWWFNSA-N
Target: Melanocortin receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P32245
Assay: Competitive Inhibition Binding Assay: A competitive inhibition binding assay was performed using membrane homogenates prepared from HEK-293 cells that express recombinant hMCR-1a or hMCR-4 (in each instance where the h prefix refers to human), or alternatively membrane homogenates from B16-F10 mouse melanoma cells containing endogenous murine MCR-1. In the examples that follow, all MCR-1 and MCR-4 values are for human recombinant receptors, unless otherwise noted. Assays were performed in 96 well polypropylene round-bottom plates (VWR catalog number 12777-030). Membrane homogenates were incubated with 0.1 nM [I125]-NDP-α-MSH (Perkin Elmer) and increasing concentrations of test peptides of the present invention in buffer containing 25 mM HEPES buffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.3 mM 1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for 90 minutes at 37° C., the assay mixture was filtered onto GF/B Unifilter plates (Perkin-Elmer catalog number 6005177) and washed with 3 mL of ice-cold buffer per well. Filters were air dried and 35 uL of scintillation cocktail added to each well. Plates were counted in a Microbeta counter for bound radioactivity. Non-specific binding was measured by inhibition of binding of [I125]-NDP-α-MSH in the presence of 1 uM NDP-α-MSH. Maximal specific binding (100%) was defined as the difference in radioactivity (cpm) bound to cell membranes in the absence and presence of 1 uM NDP-α-MSH. Each assay was conducted in duplicate and the actual mean values are described, with results less than 0% reported as 0%. Ki values for peptides of the present invention were determined using Graph-Pad Prism curve-fitting software.
Measurement: Ki = 358 nM (pChEMBL 6.449999809265137)
Descriptors: MW 996.14, ALogP -1.82, HBA 11, HBD 14, PSA 382.26, QED 0.03
Compound (SMILES): Cc1ccc(C(=O)Nc2cc(C(C)(C)C)on2)cc1-n1cc(-c2cncc(N3CCN(C4CCCC4)CC3)c2)nn1
InChIKey: URSMWTJTEYWXSV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: B-RAF (V600E) Kinase Assay: In a dilution series 10 uL/well of test substance solution are placed in a multiwell plate. The dilution series is selected so that generally a range of concentrations of 2 uM to 0.119 nM or 0.017 nM is covered. If necessary the initial concentration of 2 uM is changed to 50 uM, 10 uM or 0.4 uM or 0.2857 uM and further dilution is carried out accordingly. The final concentration of DMSO is 5%. 10 uL/well of the B-Raf (V600E)-kinase solution are pipetted in (containing 0.5 ng B-Raf (V600E)-kinase (e.g. from Upstate) in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.286 mM sodium orthovanadate, 10% glycerol, 1 mg/mL bovine serum albumin, 1 mM dithiothreitol) and the mixture is incubated for 1 h at RT under with shaking. The kinase reaction is started by the addition of 20 uL/well ATP solution [final concentration: 250 uM ATP, 30 mM Tris-HCl pH 7.5, 0.02% Brij, 0.2 mM sodium orthovanadate, 10 mM magnesium acetate, 0.1 mM EGTA, phosphatase cocktail (Sigma, #P2850, dilution recommended by the manufacturer)] and 10 uL/well MEK1 solution [containing 50 ng biotinylated MEK1 (prepared from purified MEK1 according to standard procedure, e.g. with EZ-Link Sulpho-NHS-LC-Biotin reagent, Pierce, #21335)] and carried out for 60 min at RT with constant shaking. The reaction is stopped by the addition of 12 uL/well of a 100 mM EDTA solution and incubation is continued for a further 5 min. 55 uL/well of the reaction solution are transferred into a streptavidin-coated plate (e.g. Streptawell HighBond, Roche, #11989685001) and shaken gently for 1 h at RT, in order to bind biotinylated MEK1 to the plate. After elimination of the liquid the plate is washed five times with 200 uL/well of 1Ã¿PBS and 100 uL/well solution of primary antibody plus europium-labelled secondary antibody [Anti Phospho-MEK (Ser217/221), Cell Signaling, #9121 and Eu-N1 labelled goat-anti-rabbit antibody, Perkin Elmer, #AD0105] is added, the primary antibody is diluted 1:2000 and the secondary antibody is diluted to 0.4-0.5 ug/mL in Delfia Assay Buffer (Perkin Elmer, #1244-111). After 1 h shaking at RT the solution is poured away and washed five times with 200 uL/well Delfia Wash Buffer (Perkin Elmer, #4010-0010/#1244-114). After the addition of 200 uL/well Enhancement Solution (Perkin Elmer, #4001-0010/#1244-105) the mixture is shaken for 10 min at RT and then measured in a Wallac Victor using the program "Delfia Time Resolved Fluorescence (Europium)". IC50 values are obtained from these dosage-activity curves using a software program (GraphPadPrizm).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 554.7, ALogP 5.24, HBA 9, HBD 1, PSA 105.21, QED 0.35
Compound (SMILES): Cc1nc(C(NC(=O)c2ccc(C(F)(F)F)c(OCC3CC3)n2)C2CC2)no1
InChIKey: QJXIDJTYVUOGKY-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 = 52.4 nM (pChEMBL 7.28000020980835)
Descriptors: MW 396.37, ALogP 3.46, HBA 6, HBD 1, PSA 90.14, QED 0.77
Compound (SMILES): CN(C)C1(c2ccccc2)CCC(c2ccccc2)(N(C)C(=O)NCc2ccccc2)CC1
InChIKey: SFLSYHDNPJVERS-UHFFFAOYSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Binding Assay: The compounds were examined with membranes of recombinant CHO-ORL 1 cells in a receptor binding assay with 3H-nociceptin/orphanin FQ. This test system was conducted in accordance with the method outlined by Ardati et al. (Mol. Pharmacol., 51, 1997, pp. 816-824). The concentration of 3H-nociceptin/orphanin FQ amounted to 0.5 nM in these tests. The binding assays were conducted in each case on 20 ug of membrane protein per 200ul of preparation in 50 mM of HEPES, pH 7.4, 10 nM of MgCl2 and 1 mM of EDTA. The binding to the ORL 1-receptor was determined using 1 mg of WGA-SPA beads (Amersham-Pharmacia, Freiburg) in each case by incubating the preparation for one hour at RT and then conducting measurements in the Trilux scintillation counter (Wallac, Finland).
Measurement: Ki = 90 nM (pChEMBL 7.050000190734863)
Descriptors: MW 441.62, ALogP 5.75, HBA 2, HBD 1, PSA 35.58, QED 0.53
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5nc(OC)ccc5n3CC4)cc12
InChIKey: NXCSUJBJLUENHQ-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 206.3 nM (pChEMBL 6.690000057220459)
Descriptors: MW 625.68, ALogP 5.64, HBA 8, HBD 1, PSA 119.56, QED 0.25
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(Br)c3c(N)ncnc32)cc2cccc(C)c2c1=O
InChIKey: VBXHOCIRRABFDD-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 475.35, ALogP 4.14, HBA 7, HBD 1, PSA 91.62, QED 0.42
Compound (SMILES): C[C@H]1[C@H]2Cc3ccc(C(=O)NCCc4ccc(-c5ccc(O)nc5)cc4)cc3[C@@]1(C)CCN2CC1CC1
InChIKey: MJARPNLDLZUFSF-NCPGCRMVSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: null: The Ki (binding affinity) for u opioid receptors was determined using a competitive displacement assay as previously described in Neumeyer (Journal of Med. Chem. 2012, p 3878), which is incorporated herein in its entirety. Briefly, membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed the cloned human u opioid receptor were incubated with 12 different concentrations of the compound set forth herein in the presence of 0.25 nM [3H]DAMGO (see Tiberi et al., Can. J. Physiol. Pharmacol. 1988, Vol. 66, p 1368, which is incorporated by reference herein in its entirety) in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO (see Gulati et al., Life Sci. 1990, Vol. 47, p 159, which is incorporated by reference herein in its entirety). Nonspecific binding was measured by inclusion of 10 uM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber.
Measurement: Ki = 0.68 nM (pChEMBL 9.170000076293945)
Descriptors: MW 495.67, ALogP 5.36, HBA 4, HBD 2, PSA 65.46, QED 0.46
Compound (SMILES): O=C(Nc1nc2ccccc2s1)c1cccc2c1CN(c1ccc(-c3ccn(C(=O)c4ccccc4)c3)c(C(=O)O)n1)CC2
InChIKey: JWUCDNZVXPLSJC-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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 599.67, ALogP 6.36, HBA 8, HBD 2, PSA 117.42, QED 0.23