lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): NCC1(C(=O)Nc2ccccc2)CCCN(c2ccc(C(N)=O)c3ncncc23)C1
InChIKey: BPJGWADXXYQSHV-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 404.47, ALogP 1.91, HBA 6, HBD 3, PSA 127.23, QED 0.6
Compound (SMILES): Cc1c(N2C(=O)O[C@@H](C)[C@@H]2[C@H](O)C(F)(F)F)ccc(C#N)c1Cl
InChIKey: AJSPBOQQLUICJK-BPTDKIDVSA-N
Target: Androgen receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P15207
Assay: Biological Assay: In order to demonstrate the utility of the compounds of this invention, an androgen receptor binding assay was performed wherein many of the compounds of this invention are shown to demonstrate significant affinity for the androgen receptor. The assay was performed as specified by the manufacturer (Invitrogen, Madison, Wis.). Briefly, 1 ul of 10 mM compound was added to 500 ul of AR screening buffer in a 1.5 ml eppendorf tube to make a 2x10E-5M stock. 10-fold serial dilutions of the test compounds were prepared ranging in concentration from 10E-5M to 10E-12M. Each dilution was added in triplicate to a black 384-microtiter plate. The test compounds will be diluted 2-fold in the final reaction. 2x AR-Fluormone complex was prepared with 2 nM Flourmone AL Green and 30 nM AR. 25 ul of 2x complex was aliquoted to each reaction well, such that the final reaction volume was 50 ul per well.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 348.71, ALogP 3.16, HBA 4, HBD 1, PSA 73.56, QED 0.89
Compound (SMILES): COC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(NS(=O)(=O)O)cc1)c1nc(CC(F)(F)F)cs1
InChIKey: UEDXDGTYRBGQSC-PMACEKPBSA-N
Target: Receptor-type tyrosine-protein phosphatase beta (SINGLE PROTEIN), Homo sapiens
UniProt: P23467
Assay: Inhibition of recombinant HPTPbeta (unknown origin) preincubated for 10 mins followed by substrate addition measured after 15 mins by DiFMUP probe-based fluorescence assay
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 586.61, ALogP 3.83, HBA 7, HBD 4, PSA 146.72, QED 0.25
Compound (SMILES): CC(=O)Nc1ccc(CN2CCC(CC#N)(n3cc(C(N)=O)c(Nc4ccccc4)n3)CC2)cc1
InChIKey: HWGJBFHFRNFJMV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 471.57, ALogP 3.59, HBA 7, HBD 3, PSA 129.07, QED 0.46
Compound (SMILES): Cc1ncc(-c2cc(C(=O)N3CCC(c4ccc(C#N)cc4)CC3)ccc2C)[nH]1
InChIKey: LGQRJEALSUOBFF-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 245 nM (pChEMBL 6.610000133514404)
Descriptors: MW 384.48, ALogP 4.59, HBA 3, HBD 1, PSA 72.78, QED 0.72
Compound (SMILES): COC(=O)c1cnc(N2CCN(c3nnc(-c4ccc(F)c(Cl)c4)c(C)c3C)C[C@H]2C)cn1
InChIKey: QHYINPMHPSJHOL-CYBMUJFWSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 1 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 470.94, ALogP 3.84, HBA 8, HBD 0, PSA 84.34, QED 0.53
Compound (SMILES): COC(=O)Nc1nc2ccc(C3(C)c4ccccc4C(=O)N3c3cccc(Br)c3)cc2[nH]1
InChIKey: CUALBYWSEVCZPI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 491.35, ALogP 5.43, HBA 4, HBD 2, PSA 87.32, QED 0.4
Compound (SMILES): N#Cc1c[nH]c(C(=O)Nc2ccc(C3(CN4CCOCC4)CCOCC3)cc2C2=CCCCC2)n1
InChIKey: PTIISTLFPJGFMO-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Mus musculus
UniProt: P09581
Assay: Fluorescence Polarization Competition Immunoassay: An autophosphorylation, fluorescence polarization competition immunoassay was used to determine the potency for c-fms inhibition exhibited by selected compounds of Formula I. The assay was performed in black 96-well microplates (LJL BioSystems). The assay buffer used was 100 mM 4-(2-hydroxyethyl)piperazine 1-ethanesulfonic acid (HEPES), pH 7.5, 1 mM 1,4-dithio-DL-threitol (DTT), 0.01% (v/v) Tween-20. Compounds were diluted in assay buffer containing 4% dimethylsulfoxide (DMSO) just prior to the assay. To each well, 5 μL of compound were added followed by the addition of 3 μL of a mix containing 33 nM c-fms (Johnson & Johnson PRD) and 16.7 mM MgCl2 (Sigma) in assay buffer. The kinase reaction was initiated by adding 2 μL of 5 mM ATP (Sigma) in assay buffer. The final concentrations in the assay were 10 nM c-fms, 1 mM ATP, 5 mM MgCl2, 2% DMSO. Control reactions were ran in each plate: in positive and negative control wells, assay buffer (made 4% in DMSO) was substituted for the compound; in addition, positive control wells received 1.2 μL of 50 mM ethylenediaminetetraaceticacid (EDTA). The plates were incubated at room temperature for 45 min. At the end of the incubation, the reaction was quenched with 1.2 μL of 50 mM EDTA (EDTA was not added to the positive control wells at this point; see above). Following a 5-min incubation, each well received 10 μL of a 1:1:3 mixture of anti-phosphotyrosine antibody, 10×, PTK green tracer, 10× (vortexed), FP dilution buffer, respectively (all from PanVera, cat. #P2837). The plate was covered, incubated for 30 min at room temperature and the fluorescence polarization was read on the Analyst. The instrument settings were: 485 nm excitation filter; 530 nm emission filter; Z height: middle of well; G factor: 0.93. Under these conditions, the fluorescence polarization values for positive and negative controls were approximately 300 and 150, respectively, and were used to define the 100% and 0% inhibition of the c-fms reaction.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 475.59, ALogP 3.87, HBA 6, HBD 2, PSA 103.27, QED 0.66
Compound (SMILES): CC(CC(N)=O)(NC(=O)c1cc(OCC(F)(F)F)c(C2CC2)cn1)C1CC1
InChIKey: HWFRYNQHOHFLGR-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×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 385.39, ALogP 2.67, HBA 4, HBD 2, PSA 94.31, QED 0.72
Compound (SMILES): O=C(N/N=C/c1cccc([N+](=O)[O-])c1)c1cccc(NC(=O)c2cccc([N+](=O)[O-])c2)c1
InChIKey: XYPRRGOGPZTHOR-LPYMAVHISA-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 = 18400 nM (pChEMBL 4.739999771118164)
Descriptors: MW 433.38, ALogP 3.52, HBA 7, HBD 2, PSA 156.84, QED 0.33
Compound (SMILES): O=C(O)Cc1ccc(-c2ccc(F)c3c2CN(C(=O)CC2CCOc4ccccc42)CC3)c(OCC(F)(F)F)c1
InChIKey: PFPZJLZTELYSNX-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 3.5 nM (pChEMBL 8.460000038146973)
Descriptors: MW 557.54, ALogP 5.9, HBA 4, HBD 1, PSA 76.07, QED 0.37
Compound (SMILES): O=C(Nc1cc(-c2ccc(F)cc2)on1)c1ccc2cc3n(c2c1)CCCNC3=O
InChIKey: DNWADSCQPSZJGE-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 155 nM (pChEMBL 6.809999942779541)
Descriptors: MW 404.4, ALogP 3.82, HBA 5, HBD 2, PSA 89.16, QED 0.54
Compound (SMILES): O=C(Nc1ccc2[nH]nc(-c3ccc(F)cc3)c2c1)c1cccnc1
InChIKey: SJDJAJCYFNOHRI-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 332.34, ALogP 4.02, HBA 3, HBD 2, PSA 70.67, QED 0.6
Compound (SMILES): CC(C)c1c(N(CCC2CC2)S(=O)(=O)c2ccccc2)sc2ccccc12
InChIKey: MLZGRSSLWOMHCR-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 = 28.9 nM (pChEMBL 7.539999961853027)
Descriptors: MW 399.58, ALogP 6.02, HBA 3, HBD 0, PSA 37.38, QED 0.49
Compound (SMILES): COc1cc(CCN2CCN(C)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: IJHIXUNOBPCLFN-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human GST-rTEV-tagged IGF1R (957 to 1367 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 674.76, ALogP 6.53, HBA 9, HBD 2, PSA 99.92, QED 0.17
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: FLSIHAOQACZRTM-KQUOGIPMSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 2430.82
Compound (SMILES): C[C@@]1(c2cc(Nc3ncnc4cc(C#N)cnc34)cnc2F)C[C@@H](C(F)(F)F)OC(N)=N1
InChIKey: KJBKHJJJGKNGTR-UGSOOPFHSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE 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 = 368 nM (pChEMBL 6.429999828338623)
Descriptors: MW 446.37, ALogP 3.06, HBA 9, HBD 2, PSA 134.99, QED 0.46
Compound (SMILES): C#Cc1cccc(N2C(=O)c3ccccc3C2(O)c2ccc3[nH]c(NC(=O)OC)nc3c2)c1
InChIKey: YUNGGCFGLSXIDG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 438.44, ALogP 3.58, HBA 5, HBD 3, PSA 107.55, QED 0.42
Compound (SMILES): Fc1ccc(Nc2n[nH]c3scnc23)cc1Cl
InChIKey: LTYZNZOXSOBCID-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Receptor Activity Assay: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGluR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured.
Measurement: EC50 = 1180 nM (pChEMBL 5.929999828338623)
Descriptors: MW 268.7, ALogP 3.56, HBA 4, HBD 2, PSA 53.6, QED 0.75
Compound (SMILES): COc1cncc(C2CC[C@H]3[C@@H]4CCC5=CC(=O)NCC[C@]5(C)[C@H]4CC[C@]23C)c1
InChIKey: CFUADGMSZIMWFG-DRLDVTDQSA-N
Target: Steroid 17-alpha-hydroxylase/17,20 lyase (SINGLE PROTEIN), Homo sapiens
UniProt: P05093
Assay: Inhibition of CYP17 in human testicular microsomes using 17alpha-hydroxypregnenolone as substrate assessed as decrease in DHEA levels after 90 mins by ELISA
Measurement: IC50 <= 11 nM (pChEMBL 7.959000110626221)
Descriptors: MW 394.56, ALogP 4.86, HBA 3, HBD 1, PSA 51.22, QED 0.78
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C1CCN(C(=O)/C=C/CN2CCOCC2)CC1
InChIKey: NIKYELXINWPOQX-QPJJXVBHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: HotSpot kinase assay: IC50s were determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 μM ATP.). For enzyme inhibition assays, compounds were tested in range of ten concentrations from 10 uM to 0.0005 uM using purified enzymes and the Hotspot kinase assay. Reaction conditions were 1 uM ATP, one hour incubation with inhibitor, and kinase activity detected using 33-ATP phosphorylation of an appropriately selected peptide substrate.
Measurement: IC50 = 4.07 nM (pChEMBL 8.390000343322754)
Descriptors: MW 539.64, ALogP 3.92, HBA 9, HBD 1, PSA 111.63, QED 0.35
Compound (SMILES): Cc1noc(C)c1-c1cc(C(O)C2CCCC2)c2nc(C3CC3)[nH]c2c1
InChIKey: QMXGHJWYWHBEKK-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Homogeneous Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Binding of the two tandem bromodomains, BRD4-1 and BRD4-2, to an acetylated histone H4 peptide was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay. The synthetic peptide containing amino acids 1-18 of histone H4 was acetylated at lysine 5, 8, 12, 16 and conjugated to biotin (SGRGACKGGACKGLGACKGGAACKRH-GSGSK-biotin[SEQ ID NO:1]) was purchased from Millipore. BRD4-1 and BRD4-2 were expressed and purified from Escherichia coli as N-terminal His6-tagged proteins. An XL665 labeled anti-His antibody (Cisbio) was used to specifically bind BRD4 and a cryptate labeled streptavidin protein was used because it specifically recognized the biotinylated H4 peptide. Binding of BRD4 to the peptide resulted in an increase in FRET signal whereas disruption of this protein-peptide interaction with a small molecule inhibitor resulted in a decrease in FRET signal. Assays were performed in 50 mM Hepes (pH 7.5), 150 mM NaCl, 0.1 mg/ml BSA, 0.01% (v/v) Brij, 0.5% (v/v) DMSO and 200 nM H4 peptide at the following concentrations for each BRD4 isoform: 60 nM BRD4-1 and 120 nM BRD4-2. After an assay reaction time of 60 minutes at 25° C., binding was measured with 2 nM crytptate labeled streptavidin and 10 nM anti-His-XL665 antibody. TR-FRET signal was detected on an Envision plate reader (Ex: 320 nm; Em: 615/665 nm; 100 As delay and 200 Î¼s read window). Data were normalized based on a positive (2 Î¼M I-BET) and negative (DMSO) controls and IC50values were calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 95.2 nM (pChEMBL 7.019999980926514)
Descriptors: MW 351.45, ALogP 4.94, HBA 4, HBD 2, PSA 74.94, QED 0.7
Compound (SMILES): CCC(C)(C)NC(=O)C1CCN(Cc2cccc(NC(=O)c3cccc(C(F)(F)F)[n+]3[O-])c2)CC1
InChIKey: DJQJHFKVRJGZGC-UHFFFAOYSA-N
Target: Atypical chemokine receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P25106
Assay: In Vitro Assay: CHO-K1 CXCR7 b-arrestin cells are detached from culture dishes with a cell dissociation buffer (Invitrogen, #13151-014) and collected in growing medium (F12 HAMS 90% (v/v)/FCS 10% (v/v), Penicilin/streptomycin 1% (v/v)). 5000 cells per well (in 20 μl) are seeded in a 384 well plate (white-walled, clear bottom; BD Falcon #353274). The plate is incubated at 37° C./5% CO2 for 24 hours. Medium is then replaced by 20 μl OPTIMEM (Invitrogen #31985) for 3 to 4 hours. Test compounds are dissolved at 10 mM in DMSO and serially diluted in DMSO to 200× of the final concentration for dose response curves. Compounds are then diluted 1:33.3 in HBSS1×. 5 μl/well of HBSS1×/20 mM HEPES/0.2% BSA are added to the assay plate followed by addition of 5 μl/well of diluted compounds. CXCL12 (Peprotech #300-28A) may be used as a reference agonist. The plate is incubated for 90 minutes at 37° C. 12 μl of detection reagent (Path Hunter Detection Kit, DiscoveRx, #93-0001) is transferred to the assay plate and to the plate is incubated for 1 hour at room temperature. Luminescent signal is read in a microplate reader (FLUOstar Optima, bmg).
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 492.54, ALogP 4.11, HBA 4, HBD 2, PSA 88.38, QED 0.45
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCN(C(=O)OCC2CC2(F)F)CC1
InChIKey: MUPOTNLGQQJGAQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 476.46, ALogP 3.36, HBA 7, HBD 2, PSA 126.27, QED 0.63
Compound (SMILES): CC(C)NC(=O)c1c(O)[nH]/c(=N\c2ccc(OC(C)C)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: BPPURWLTFULGLD-UHFFFAOYSA-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 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 in M MgCl2, 5.0 mM CaCl2, 5.6 D-glucose, 2.5 mM probenecid, 1.0% BSA, and 0.08% Pluronic F-127, pH 7.5) 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 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence inte
Measurement: IC50 = 504 nM (pChEMBL 6.300000190734863)
Descriptors: MW 470.96, ALogP 3.74, HBA 6, HBD 3, PSA 108.71, QED 0.49
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(CC2CCCCC2)C(=O)[C@@H](O)Cc2ccccc2)c1
InChIKey: TXZMNBIPFBSSPK-XJYHXZFBSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Cavia porcellus
UniProt: P41144
Assay: Displacement of [3H]-diprenorphine from guinea pig kappa opioid receptor expressed in CHOK1 cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 517.71, ALogP 4.51, HBA 4, HBD 2, PSA 86.87, QED 0.49
Compound (SMILES): CCOc1ccc(C[C@H]2C[C@H](C(=O)NO)C2)cc1-c1nc2c(CC)nn(C)c2c(=O)[nH]1
InChIKey: WFSWUBOROARLDR-HDJSIYSDSA-N
Target: cGMP-specific 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O76074
Assay: Inhibition of full length recombinant human N-terminal GST-tagged PDE5A1 expressed in baculovirus infected Sf9 cells using cGMP as substrate after 30 mins by HTRF assay
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 425.49, ALogP 2.36, HBA 7, HBD 3, PSA 122.13, QED 0.39
Compound (SMILES): Cc1cc(Nc2cc(C(F)F)ccn2)nc(-c2ccc(C(C)(O)C3CCC(C(=O)OC4CCCCC4)CC3)nc2)c1
InChIKey: ZBTZNZZCVYHUBE-UHFFFAOYSA-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 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 564.68, ALogP 7.42, HBA 7, HBD 2, PSA 97.23, QED 0.27
Compound (SMILES): COc1ccc2c(c1)C(=O)NCCS2
InChIKey: GYYTWZZEOQZZDK-UHFFFAOYSA-N
Target: Ryanodine receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92736
Assay: Induction of FKBP12.6 re-binding to PKA-phosphorylated-RyR2 (unknown origin) after 30 mins in presence of [35S]-FKBP12.6 by liquid scintillation counting
Measurement: EC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 209.27, ALogP 1.53, HBA 3, HBD 1, PSA 38.33, QED 0.76
Compound (SMILES): Cc1c(N(Cc2ccc(OC(F)(F)F)cc2)S(=O)(=O)c2ccsc2)sc2ccccc12
InChIKey: VNQZCYFQRFPHCU-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 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 483.56, ALogP 6.57, HBA 5, HBD 0, PSA 46.61, QED 0.31
Compound (SMILES): NS(=O)(=O)NCc1nnc(C(=O)c2nc3ccc(-c4ccc(F)cc4)cc3s2)o1
InChIKey: RNPVUJKRQIQJFN-UHFFFAOYSA-N
Target: Hepatic triacylglycerol lipase (SINGLE PROTEIN), Homo sapiens
UniProt: P11150
Assay: Inhibition of human HL expressed in human COS7 cells using A10070 as substrate preincubated for 20 min followed by DMPG vesicle doped substrate addition and measured at 20 secs interval for 10 mins by fluorescence assay
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 433.45, ALogP 2.01, HBA 8, HBD 2, PSA 141.07, QED 0.44
Compound (SMILES): CC[C@@]1(C(=O)Nc2cc(C)cc(C(F)(F)F)c2)CN(c2c(C)cccc2C)C(=O)[C@H]1C
InChIKey: JILYNYYLSQLSQK-UZUQRXQVSA-N
Target: Chemerin-like receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99788
Assay: Displacement of [125I]-chemerin from ChemR23 (unknown origin) transfected in mouse BaF3 cells after 3 hrs by scintillation counting method
Measurement: IC50 <= 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 432.49, ALogP 5.65, HBA 2, HBD 1, PSA 49.41, QED 0.68
Compound (SMILES): CC[C@@H](NC(=O)c1cc(C(=O)N2CCC[C@@H]2C)n2c1COCC2)c1ccc(OC)c(Cl)c1
InChIKey: YAAUULCOJSKINF-HNAYVOBHSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 459.97, ALogP 4.19, HBA 5, HBD 1, PSA 72.8, QED 0.7
Compound (SMILES): Cc1nc(C)n(CC(=O)N2CCN(c3sc(C(F)(F)F)nc3-c3nc4ccc(OC5CNC5)cc4[nH]3)C[C@H]2C)n1
InChIKey: MKVNPZBCHHFZPL-CYBMUJFWSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 575.62, ALogP 3, HBA 10, HBD 2, PSA 117.09, QED 0.36
Compound (SMILES): O=C(Nc1ccncc1)C(=O)c1ccc2[nH]c3ccccc3c2c1
InChIKey: SPFJTPFLHPMWOX-UHFFFAOYSA-N
Target: Pancreatic triacylglycerol lipase (SINGLE PROTEIN), Sus scrofa
UniProt: P00591
Assay: Inhibition of porcine pancreatic lipase assessed as reduction in 4-nitrophenol formation from 4-nitrophenyl butyrate substrate preincubated for 30 mins followed by substrate addition measured after 5 mins by spectrophotometer
Measurement: IC50 = 29350 nM (pChEMBL 4.53000020980835)
Descriptors: MW 315.33, ALogP 3.54, HBA 3, HBD 2, PSA 74.85, QED 0.45
Compound (SMILES): Cc1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)c2ccc(C(=O)O)cc2F)sc2ccccc12
InChIKey: IEGKHNDKIZNQDU-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 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 541.52, ALogP 6.6, HBA 4, HBD 1, PSA 74.68, QED 0.28
Compound (SMILES): COc1nc(-c2nc3n(n2)CCC[C@H]3c2ccc(N3CCCCCC3)cc2)ccc1-n1cnc(C)c1
InChIKey: XSAZFWPYYZZHKH-QHCPKHFHSA-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 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 483.62, ALogP 5.15, HBA 8, HBD 0, PSA 73.89, QED 0.39
Compound (SMILES): C[C@H](Nc1ncnc(N)c1-c1nnn[nH]1)c1nn2cccc2c(=O)n1-c1ccccc1
InChIKey: XZUMCDZMSAJYES-NSHDSACASA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition of PI3Kdelta (unknown origin) preincubated for 30 mins followed by insulin stimulation for 5 mins by Western blot analysis
Measurement: IC50 = 0.2 nM (pChEMBL 9.699999809265137)
Descriptors: MW 415.42, ALogP 1.21, HBA 11, HBD 3, PSA 157.59, QED 0.38
Compound (SMILES): Cc1cc(-c2c[nH]c3ncnc(N4CCOCC4)c23)sn1
InChIKey: KISKHUBNHHURGR-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 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 301.38, ALogP 2.23, HBA 6, HBD 1, PSA 66.93, QED 0.79
Compound (SMILES): Cc1cc(Nc2cc(C3CC3)[nH]n2)nc(NCc2nc(-c3ccccc3)cs2)n1
InChIKey: VACLMZQVSKMNCV-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 403.51, ALogP 4.86, HBA 7, HBD 3, PSA 91.41, QED 0.41
Compound (SMILES): NC(=O)c1ccc2[nH]nc(-c3cccc(NC(=O)c4cccs4)c3)c2c1
InChIKey: BHRZULMEEUGQGS-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 362.41, ALogP 3.64, HBA 4, HBD 3, PSA 100.87, QED 0.52
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(OCC3CCC3)cc1C21N=C(N)N(C(C)C)C1=O
InChIKey: QDCFAECEEQJIMP-SSEMJGATSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: 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).
Measurement: IC50 = 34.3 nM (pChEMBL 7.460000038146973)
Descriptors: MW 425.57, ALogP 3.76, HBA 5, HBD 1, PSA 77.15, QED 0.78
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(/C=C/c2ncon2)s1)c1ccc(N2CCN(Cc3ccccc3-c3ccc(Cl)cc3)CC2)cc1
InChIKey: SPZMXLLBHGMIIA-FYWRMAATSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Representative compounds were serially diluted in dimethyl sulfoxide (DMSO) starting at 50 μM (2× starting concentration; 10% DMSO) and 10 μL were transferred into a 384-well plate. Then 10 μL of a protein/probe/antibody mix was added to each well at final concentrations listed in TABLE 1. The samples are then mixed on a shaker for 1 minute and incubated for an additional 3 hours at room temperature. For each assay, the probe/antibody and protein/probe/antibody were included on each assay plate as negative and positive controls, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak peptide) and 495/510 nm (Tb-labeled anti-Histidine antibody) emission filters.
Measurement: Ki = 393.36 nM (pChEMBL 6.409999847412109)
Descriptors: MW 646.19, ALogP 6.06, HBA 9, HBD 1, PSA 108.64, QED 0.21
Compound (SMILES): COc1cn(C(CC2CC2(F)F)C(=O)Nc2ccc(C(=O)O)cc2)c(=O)cc1-c1cc(Cl)ccc1C#N
InChIKey: PPOMRFJZWPHFPA-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). In each case 1 μl of the diluted substance solutions are placed into the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride solution; 5 mM of calcium chloride solution; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm).
Measurement: IC50 = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 527.91, ALogP 4.97, HBA 6, HBD 2, PSA 121.42, QED 0.43
Compound (SMILES): CN1CCN([C@@H]2CC[C@H](NS(=O)(=O)c3ccccc3-c3ccc(-c4cnc(N)cn4)c(F)c3)[C@H]2O)CC1
InChIKey: AFGVAPLQECOQOG-POGFHVCPSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: Homogeneous Time Resolved Fluorescence Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBacl (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator [5-[({[2-(2-{3-[3-(tert-Butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid]. The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 ug/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid].
Measurement: Ki = 201 nM (pChEMBL 6.699999809265137)
Descriptors: MW 526.64, ALogP 1.95, HBA 8, HBD 3, PSA 124.68, QED 0.45
Compound (SMILES): CC(C)(CNC(=O)c1nc(C#N)c2cc(-c3ccccc3)ccc2c1O)C(=O)O
InChIKey: JYCKALTXSDGLRG-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 1130 nM (pChEMBL 5.949999809265137)
Descriptors: MW 389.41, ALogP 3.32, HBA 5, HBD 3, PSA 123.31, QED 0.61