lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CN1CCN(c2cc(C(N)=O)nc(-c3ccc(Oc4ccc(F)cc4)cc3)n2)C(C(N)=O)C1
InChIKey: BBOCRFBJZWHFGM-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FLIPR Assay: In vitro assays were performed in a recombinant cell line expressing cDNA encoding the alpha subunit (Nav1.7, SCN9a, PN1, NE) of human Nav1.7 (Accession No. NM002977). The cell line was provided by investigators at Yale University (Cummins et al, J. Neurosci. 18(23): 9607-9619 (1998)). For dominant selection of the Nav1.7-expressing clones, the expression plasmid co-expressed the neomycin resistance gene. The cell line was constructed in the human embryonic kidney cell line, HEK293, under the influence of the CMV major late promoter, and stable clones were selected using limiting dilution cloning and antibiotic selection using the neomycin analogue, G418. Recombinant beta and gamma subunits were not introduced into this cell line. Additional cell lines expressing recombinant Nav1.7 cloned from other species can also be used, alone or in combination with various beta subunits, gamma subunits or chaperones.
Measurement: IC50 = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 450.47, ALogP 1.78, HBA 7, HBD 2, PSA 127.67, QED 0.59
Compound (SMILES): Oc1ncn(-c2ccc(-c3c(/C=C/c4ccc5ccccc5n4)nc4c(N5CCOCC5)ccnn34)cc2)n1
InChIKey: INYLSLWNPQZRRW-FMIVXFBMSA-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 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 516.57, ALogP 4.24, HBA 10, HBD 1, PSA 106.49, QED 0.36
Compound (SMILES): COCCN(C)c1nc2c(c(Nc3ccc(C(F)(F)F)cn3)n1)CCN(c1ncccc1C(F)(F)F)CC2
InChIKey: IPXIHJVYANUFAO-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: O35433
Assay: Functional Assay: The assay was performed as described below, using HEK293 cells transfected with rat TRPV1 (rTRPV1/HEK293). These cells had a geneticin selection marker and were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% fetal bovine serum, penicillin/streptomycin (50 units/mL), and 500 μg/mL geneticin in 5% CO2 at 37° C. Cells were passaged frequently, every 3-5 days, to avoid overgrowth, depletion of essential medium components, or acidic medium exposure. Cells were passaged using a brief wash in 0.05% trypsin with 1 mM EDTA, followed by dissociation in divalent-free phosphate-buffered saline (Hyclone #SH30028.02). Dissociated cells were seeded onto poly-D-lysine coated black-walled 96-well plates (Biocoat; Becton Dickinson #354640) at about 40,000 cells per well and grown for approximately 1 day in culture medium to near confluency. The assay buffer was composed of 130 mM NaCl, 2 mM KCl, 2 mM MgCl2, 10 mM HEPES, 5 mM glucose, and either 2 mM or 20 μM CaCl2. On the day of the experiment, the culture medium was replaced with 2 mM calcium assay buffer using an automated plate washer (ELx405; Biotek, VT). The cells were incubated in 100 μL/well Fluo-3/AM (2 μM; TEFLabs #0116) with Pluronic F127 (100 μg/mL; Sigma #P2443) for 1 h at rt in the dark. After loading the cells, the dye solution was replaced with 50 μL/well of 20 μM calcium assay buffer using the ELx405 plate washer. Test compounds (50 μL/well) were added to the plate and incubated for 30 min. Intracellular Ca2+ levels were subsequently assayed using a Fluorometric Imaging Plate Reader (FLIPR™ instrument, Molecular Devices, CA) to simultaneously monitor Fluo-3 fluorescence in all wells (λexcitation=488 nm, λemission=540 nm) during challenge with agonist (capsaicin). The IC50 values were determined. Cells were challenged with 150 nM capsaicin and the fluorescence counts were captured following agonist addition at a sampling rate of 0.33 Hz. The contents of the wells were mixed 3 times (40 μL mix volume) immediately after the additions were made. Concentration dependence of block was determined by exposing each well of cells in duplicate rows of a 96 well plate to a serial dilution of test compound. The concentration series usually started at 10 Î¼M with a three-fold serial decrement in concentration. The magnitude of the capsaicin response was determined by measuring the change in fluo3 fluorescence before and 100 seconds after the addition of the agonist.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 541.5, ALogP 4.74, HBA 8, HBD 1, PSA 79.3, QED 0.43
Compound (SMILES): Cn1cc(-c2c[nH]c3ncnc(N4CCC(F)CC4)c23)cn1
InChIKey: HZOIUUMKYGXMKG-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 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 300.34, ALogP 2.3, HBA 5, HBD 1, PSA 62.63, QED 0.79
Compound (SMILES): COc1cc(-c2ccc(F)c(Cl)c2)c(F)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: NRMWKXGYSBBJDG-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 1255 nM (pChEMBL 5.900000095367432)
Descriptors: MW 543.94, ALogP 5.39, HBA 7, HBD 1, PSA 103.43, QED 0.31
Compound (SMILES): COc1cccc(OC)c1C1CCCCC(=O)N1Cc1ccc(-c2ccccc2)cc1
InChIKey: QYACYWJYVVSSHL-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 188 nM (pChEMBL 6.730000019073486)
Descriptors: MW 415.53, ALogP 6.01, HBA 3, HBD 0, PSA 38.77, QED 0.49
Compound (SMILES): CCOc1ccc(-c2nc3ccc([C@@H]4CNCCO4)cc3o2)cn1
InChIKey: KTWURAHJKZUHLR-INIZCTEOSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q923Y9
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000xg for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized.
Measurement: Ki = 87.3 nM (pChEMBL 7.059999942779541)
Descriptors: MW 325.37, ALogP 2.95, HBA 6, HBD 1, PSA 69.41, QED 0.8
Compound (SMILES): O=C(Nc1cccc(Cl)c1)c1ccccc1Sc1ccc2c(/C=C/c3ccccn3)n[nH]c2c1
InChIKey: PGOFCHZWKNKRQH-SDNWHVSQSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Inhibition of human phosphorylated FGFR1 L457V/C488A/C584S mutant expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using poly(E4Y1) as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 483, ALogP 7.19, HBA 4, HBD 2, PSA 70.67, QED 0.27
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N2CCOCC2)nc(S(C)(=O)=O)n1
InChIKey: BDMRXFODCGLAFM-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Raf Activity: Raf and biotinylated Mek, kinase dead, were combined at 2× final concentrations in assay buffer (50 mM Tris, pH 7.5, 15 mM MgCl2, 0.01% BSA and 1 mM DTT) and dispensed 5 ml per well in assay plates (Greiner white 384 well assay plates #781207) containing 0.25 ml of 40× of a Raf kinase inhibitor test compound diluted in 100% DMSO. The plate was incubated for 60 minutes at room temperature. The Raf kinase activity reaction was started by the addition of 5 mL per well of 2×ATP diluted in assay buffer. After 3 hours (b-Raf(V600E)) or 1 hour (c-Raf). The reactions were stopped and the phosphorylated product was measured using a rabbit anti-p-MEK (Cell Signaling, #9121) antibody and the Alpha Screen IgG (ProteinA) detection Kit (PerkinElmer #6760617R), by the addition of 10 mL to the well of a mixture of the antibody (1:2000 dilution) and detection beads (1:2000 dilution of both beads) in Stop/bead buffer (25 mM EDTA, 50 mM Tris, pH 7.5, 0.01% Tween20). The additions were carried out under dark conditions to protect the detection beads from light. A lid was placed on top of the plate and incubated for 1 hour at room temperature, then the luminescence was read on a PerkinElmer Envision instrument. The concentration of each compound for 50% inhibition (IC50) was calculated by non-linear regression using XL Fit data analysis software.
Measurement: IC50 = 0.214 nM (pChEMBL 9.670000076293945)
Descriptors: MW 520.53, ALogP 3.96, HBA 7, HBD 1, PSA 101.49, QED 0.51
Compound (SMILES): Cc1cnc(NCc2cc(-c3ccccc3)no2)nc1Nc1cc(C2CC2)n[nH]1
InChIKey: GUXTVYGMICYDEA-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 387.45, ALogP 4.4, HBA 7, HBD 3, PSA 104.55, QED 0.43
Compound (SMILES): Cc1n[nH]c2cc(F)c(C3C(C#N)=C(C(F)F)NC(C(F)F)=C3C#N)cc12
InChIKey: WOWAGKXGSRDYJG-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Kinase Homogeneous Time-Resolved Fluorescence Assay: The N-terminally His6-tagged recombinant kinase domain of the human c-Met (amino acids 960-1390), expressed in insect cells (SF21) and purified by Ni-NTA affinity chromatography and consecutive size exclusion chromatography (Superdex 200), is used. Alternatively, commercially available c-Met (Millipore) can be used. As substrate for the kinase reaction, the biotinylated poly-Glu, Tyr (4:1) copolymer (#61GTOBLC, Cis Biointernational, Marcoule, France) is used. For the assay, 50 mL of a 100-fold concentrated solution of the test compound in DMSO is pipetted into a black low-volume 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). 2 μL of a solution of c-Met in assay buffer [25 mM Hepes/NaOH, pH 7.5; 5 mM MgCl2; 5 mM MnCl2; 2 mM dithiothreitol; 0.1% (v/v) Tween 20 (Sigma); 0.1% (w/v) bovine serum albumin] are added, and the mixture is incubated for 15 min at 22° C. to allow pre-binding of the test compound to the enzyme before the start of the kinase reaction. Then, the kinase reaction is started by the addition of 3 μL of a solution of adenosine triphosphate (ATP, 16.7 μM; final concentration in the 5 μL assay volume is 10 μM) and substrate (2.27 μg/mL, final concentration in the 5 μL assay volume is 1.36 μg/mL Ë¿30 nM) in assay buffer, and the resulting mixture is incubated for a reaction time of 30 min at 22° C. The concentration of c-Met in the assay is adjusted depending on the activity of the enzyme lot and is appropriately chosen to have the assay in the linear range; typical enzyme concentrations are in the range of about 0.03 nM (final concentration in the 5 μL assay volume). The reaction is stopped by the addition of 5 μL of a solution of HTRF detection reagents [40 nM streptavidine-X Lent and 2.4 nM PT66-Eu-chelate, an europium-chelate labelled anti-phosphotyrosine antibody (Perkin-Elmer)] in an aqueous EDTA solution [100 mM EDTA, 0.2% (w/v) bovine serum albumin in 50 mM HEPES/NaOH, pH 7.5]. The resulting mixture is incubated for 1 h at 22° C. to allow the binding of the biotinylated phosphorylated peptide to the streptavidine-XLent and the PT66-Eu-chelate. Subsequently, the amount of phosphorylated substrate is evaluated by measurement of the resonance energy transfer from the PT66-Eu-chelate to the streptavidine-XLent. Therefore, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm are measured in an HTRF reader, e.g. Rubystar (BMG Lab-technologies, Offenburg, Germany) or Viewlux (Perkin-Elmer). The ratio of the emissions at 665 nm and at 622 nm is taken as the measure for the amount of phosphorylated substrate.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 379.29, ALogP 3.78, HBA 4, HBD 2, PSA 88.29, QED 0.79
Compound (SMILES): Cc1cc(OC(F)F)cnc1C(=O)Nc1cc(F)c(F)c([C@]2(C)C[C@@H](C(F)(F)F)OC(N)=N2)c1
InChIKey: XMPNDCVYVQQNTC-DJJJIMSYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 494.37, ALogP 4.4, HBA 6, HBD 2, PSA 98.83, QED 0.6
Compound (SMILES): CC1(C)CC2=NNC(=O)c3cccc4[nH]c(c2c34)C1
InChIKey: HHHFTURDICHXNW-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: PARP-2 and PARP-3 Enzymatic Assay: PARP-2 and PARP-3 enzymatic assays were conducted using commercial PARP-2/PARP-3 Chemiluminescent Assay Kit (BPS Biosciences) and the protocols with the kits. Briefly, histones were coated in a high binding plate first, and incubated with PARP-2 or PARP-3, and increasing concentrations of compounds for 0.5 h. Then, biotinylated NAD and activated DNA were added to the wells. The biotinylated PARylation product was measured by adding streptavidin-HRP and HRP substrates which produce chemiluminescence.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 253.31, ALogP 2.59, HBA 2, HBD 2, PSA 57.25, QED 0.74
Compound (SMILES): O=C(CCCCCCC(=O)Nc1ccc(NCCOc2ccc3cc2COC/C=C/COCc2cccc(c2)-c2ccnc(n2)N3)cc1)NO
InChIKey: DSAJSILPNCXMSX-AATRIKPKSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Inhibition of full length human recombinant N-terminal GST-tagged HDAC6 (1 to 1215 residues) expressed in sf9 cells preincubated with enzyme followed by fluorogenic Arg-His-Lys-Lys(Ac)-AMC substrate addition measured after 2 hrs by fluorescence assay
Measurement: IC50 = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 680.81, ALogP 6.77, HBA 10, HBD 5, PSA 155.96, QED 0.04
Compound (SMILES): Cc1noc2ccc(-c3ccc(C[C@@H](C#N)NC(=O)[C@@H]4CNCCCO4)cc3)cc12
InChIKey: QCTYSVBOKIUUFU-UGKGYDQZSA-N
Target: Dipeptidyl peptidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P53634
Assay: Inhibition of DPP1 in human U937 cells using Gly-Phe-AFC as substrate preincubated for 60 mins followed by substrate addition by fluorescence assay
Measurement: IC50 = 15.85 nM (pChEMBL 7.800000190734863)
Descriptors: MW 404.47, ALogP 2.73, HBA 6, HBD 2, PSA 100.18, QED 0.68
Compound (SMILES): CCCC[C@H](NC(C)=O)C(=O)N[C@H]1CCC(=O)NCCCC[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: VOSIMUPSRJWOJN-QLBSZNDGSA-N
Target: Melanocyte-stimulating hormone receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q01726
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 = 0.01 nM (pChEMBL 11)
Descriptors: MW 1038.22
Compound (SMILES): NC(=O)c1c(N)ncnc1N1CCC(c2nc(-c3cccc(C(F)(F)F)c3)cn2CCN2CCCC2)CC1
InChIKey: WARQMCTXKWMOOS-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.degree. 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 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 528.58, ALogP 3.52, HBA 8, HBD 2, PSA 119.19, QED 0.48
Compound (SMILES): Cc1cc(-c2cn(CCN3CCC3)c(C3CCN(c4ncnc(N)c4C(N)=O)CC3)n2)ccc1F
InChIKey: PANRGGPPMYRRKF-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.degree. 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 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 478.58, ALogP 2.56, HBA 8, HBD 2, PSA 119.19, QED 0.54
Compound (SMILES): C=CC(=O)N1CC[C@@H](Oc2cc3c(Nc4ccc(F)c(Cl)c4F)ncnc3cc2OC)C[C@@H]1C(=O)OC
InChIKey: QIYKORVSWNMNRD-FZKQIMNGSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-tagged recombinant human EGFR T790M mutant expressed in baculovirus infected Sf21 cells using poly (Glu,Tyr)4:1 as substrate preincubated for 10 mins followed by substrate addition measured after 1 hr by fluorescence polarization assay
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 532.93, ALogP 4.41, HBA 8, HBD 1, PSA 102.88, QED 0.27
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(-c3cc(C#N)cc(C#N)c3)cc1C21N=C(C)C(N)=N1
InChIKey: BSTQSBRVJGRBPC-HDVKRFLQSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: sAPPbeta Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at −140° C. at a concentration of 7.5-9.5×106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 100 μL cell susp/well. The cell plates were then incubated for 7-24 h at 37° C., 5% CO2. The cell medium was removed, followed by addition of 30 μL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37° C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPβ release. MSD sAPPβ plates were blocked in 1% BSA in Tris wash buffer (40 μL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 μL/well). 20 μL of medium was transferred to the pre-blocked and washed MSD sAPPβ microplates, and the cell plates were further used in an ATP assay to measure cytotoxicity. The MSD plates were incubated with shaking at r.t. for 2 h and the media discarded. 10 μL detection antibody was added (1 nM) per well followed by incubation with shaking at r.t. for 2 h and then discarded. 40 μL Read Buffer was added per well and the plates were read in a SECTOR Imager.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 423.52, ALogP 4.21, HBA 6, HBD 1, PSA 107.55, QED 0.78
Compound (SMILES): COc1cc(OC)cc(N(CC2CC2)c2ccc3ncc(-c4cnn(CCNS(=O)(=O)N(C)C)c4)nc3c2)c1
InChIKey: JCBRKJGZGWDDLQ-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 551.67, ALogP 3.45, HBA 9, HBD 1, PSA 114.71, QED 0.28
Compound (SMILES): C[C@H](Oc1cc(C(=O)NC2(CC(N)=O)CS(=O)(=O)C2)ncc1C1CC1)C(F)(F)F
InChIKey: IDVGLNUYQRWZHG-VIFPVBQESA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×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 > 10000 nM (pChEMBL 5)
Descriptors: MW 435.42, ALogP 1.06, HBA 6, HBD 2, PSA 128.45, QED 0.66
Compound (SMILES): CCc1cc(OCCc2nc(-c3ccc(F)cc3)oc2C)ccc1CCC(=O)O
InChIKey: YFRXOWDDODRUGC-UHFFFAOYSA-N
Target: Peroxisome proliferator-activated receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P37231
Assay: Kinase Activity Assay: After 24 h treatment with the medicament, the cells were lysed with a lysis solution (Cell Culture Lysis buffer, Promega) and centrifuged, and the supernatant was collected. The supernatant was reacted with a fluorescence assay kit (Promega) and counted by a fluorometer (Ascent Fluoroskan FL reader, Thermo Labsystems, Finland), and the relative intensity of luciferase was determined. For the assay of the β-galactosidase activity used in the experiment as the internal reference (the internal reference for calibrating the transfection efficiency), 50 μl of the supernatant was transferred into a fresh microplate, treated with a Promega kit, and the value was read with a microplate reader at a wavelength of 405 nm (Bio-tech Instruments Inc., Winooski, Vt., USA) (Sauerberg, P.; Olsen, G. S.; Jeppesen, L.; Mogensen, J. P. et al., J. Med. Chem., 2007, 50, 1495-1503).
Measurement: EC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 397.45, ALogP 4.99, HBA 4, HBD 1, PSA 72.56, QED 0.55
Compound (SMILES): NC(=O)[C@@H](NC(=O)c1cnc(C2CC2)c(OCC2CC2)n1)c1ccccc1
InChIKey: ONOFWQOMNXXKBI-INIZCTEOSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×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 10s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 366.42, ALogP 2.1, HBA 5, HBD 2, PSA 107.2, QED 0.74
Compound (SMILES): CC[C@@H](CN1CCN(S(=O)(=O)C2CC2)CC1)N1C(=O)[C@@](C)(CC(=O)O)C[C@H](c2cccc(Cl)c2)[C@H]1c1ccc(Cl)cc1
InChIKey: UIPNLSUAIFATGC-QQMOYWOFSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Homogenous Time-Resolved Fluorescence Assay (HTRF2 Assay): The standard assay conditions for the in vitro HTRF assay consisted of a 50 ul total reaction volume in black 384-well Costar polypropylene plates in 1×PBS buffer pH 7.4, 1 mM DTT, 0.1% BSA, 2.5 nM GST-hMDM2 (aa 1-188), 5 nM biotinylated-p53 (aa 1-83), 1.8 nM SA-XLent (Cisbio; Bedford, Mass.), 0.6 nM anti-GST cryptate monoclonal antibody (Cisbio; Bedford, Mass.) and 200 mM KF. Amino acid residues 1-188 of human MDM2 were expressed as an amino-terminal glutathione-S-transferase (GST) fusion protein (GST-hMDM2) in Escherichia coli. Residues 1-83 of human p53 were expressed as an amino-terminal AviTag™-TrxA-6×His fusion protein (biotinylated p53) in E. coli. Each protein was purified from cell paste by affinity chromatography.Specifically, 10 uL of GST-hMDM2 was incubated with 10 ul of diluted compound (various concentrations, serially diluted) in 10% DMSO for 20 minutes at room temperature. 20 uL of biotinylated-p53 was added to the GST-hMDM2+compound mixture, and then incubated at room temperature for 60 min. 10 uL of detection buffer consisting of SA-XLent, anti-GST cryptate antibody and KF was added to GST-hMDM2, biotinylated-p53 and compound reaction and left at room temperature to reach equilibrium for >4 hrs. The final concentration of DMSO in the reaction was 2%. Time-resolved fluorescence readings were measured on a microplate multilabel reader. Percentage of inhibition was calculated relative to nutlin-3. All assay conditions remained the same as described above, with the exception of the following changes in reagent concentrations: 0.2 nM GST-hMDM2 (1-188), 0.5 nM biotinylated-p53 (1-83), 0.18 nM SA-XLent, and 100 mM KF.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 636.64, ALogP 5.42, HBA 5, HBD 1, PSA 98.23, QED 0.37
Compound (SMILES): COc1cc(-c2cccc(Cl)c2)c(Cl)cc1-n1c(=O)ccc2cc(S(=O)(=O)Nc3ccon3)ccc21
InChIKey: QBEMEVOJUUAPKA-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 In Vitro PX Assay: HEK 293 cells stably transfected with human Nav1.7 were recorded in whole cell voltage clamp mode with the PatchXpress automated electrophysiology system (Molecular Devices, LLC, Sunnyvale, Calif.). Compound effects were measured on a partially inactivated state of the sodium channel. Cells were clamped to a holding potential yielding 20 to 50% inactivation. To elicit sodium current, channels were activated by pulsing to -10 mV for 20 msec. This voltage protocol was repeated at a rate of 0.1 Hz throughout the experiment. A single concentration of test compound was applied to cells for a duration of 3 minutes.
Measurement: IC50 = 1757 nM (pChEMBL 5.75)
Descriptors: MW 542.4, ALogP 5.76, HBA 7, HBD 1, PSA 103.43, QED 0.29
Compound (SMILES): CC(=O)N1C2CCC1CN(C(=O)CC1([C@H]3CO[C@H]4CCC[C@H]4N3S(=O)(=O)c3ccc(Cl)cc3)CC1)C2
InChIKey: YBYCWWWTOAXRLV-SZDHHTISSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of recombinant human CYP3A4 expressed in baculovirus infected insect cells preincubated for 30 mins using fluorescent substrate
Measurement: IC50 = 15000 nM (pChEMBL 4.820000171661377)
Descriptors: MW 536.09, ALogP 3.04, HBA 5, HBD 0, PSA 87.23, QED 0.58
Compound (SMILES): OC(c1ccc(-c2cnc(N3CCOCC3)c3nc(/C=C/c4ccc5ccccc5n4)cn23)cc1)(C(F)(F)F)C(F)(F)F
InChIKey: PXMRWROOVAUHPL-VAWYXSNFSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay hPDE10a: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6×His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added to 20 μL, of incub
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 599.54, ALogP 6.26, HBA 7, HBD 1, PSA 75.78, QED 0.24
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2CCOCC2)c(S(=O)(=O)C(F)(F)F)c1)c1ccc(N2CCN(Cc3ccccc3-c3ccc(Cl)cc3)CC2)cc1Oc1ccccc1
InChIKey: OLQVQUIHNOVTJY-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The inhibition constant (Ki) is the dissociation constant of an enzyme-inhibitor complex or a protein/small molecule complex, wherein the small molecule is inhibiting binding of one protein to another protein or peptide. Where the Ki for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-XL) is greater than the limits of detection of the assay used. Where the binding selectivity ratio for a compound is represented as > (greater than) a certain numerical value, it is intended to mean that the selectivity of a particular compound for Bcl-2 over Bcl-XL is at least as great as the number indicated. Where the Ki for a compound is represented as < (less than) a certain numerical value, it is intended to mean that the binding affinity value (e.g., for Bcl-2) is lower than the limit of detection of the assay used. Inhibition constants were determined using Wang's equation (Wang Z-X).
Measurement: Ki > 660 nM (pChEMBL 6.179999828338623)
Descriptors: MW 883.41, ALogP 8.37, HBA 10, HBD 2, PSA 134.35, QED 0.12
Compound (SMILES): Cc1ccccc1-n1c(Cn2nc(-c3cccc(O)c3)c3c(N)ncnc32)cc2cccc(F)c2c1=O
InChIKey: JDRCCHYIRIYYTG-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect 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 492.51, ALogP 4.58, HBA 8, HBD 2, PSA 111.85, QED 0.37
Compound (SMILES): Cc1cccc2cc(Cn3nc(-c4ccc5oc(N)nc5c4)c4c(N)ncnc43)n(-c3ccccc3)c(=O)c12
InChIKey: OWLYXAGENLRSKD-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 514.55, ALogP 4.46, HBA 10, HBD 2, PSA 143.67, QED 0.35
Compound (SMILES): COc1cc(C(=O)N2CCCC2)cc(N(CCCO)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: XIGQDVFIOYNOFH-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 486.58, ALogP 3.8, HBA 8, HBD 1, PSA 96.61, QED 0.4
Compound (SMILES): C#CCN(Cc1ccc2cccc(O)c2n1)C(C#N)CC1CCN(Cc2ccccc2)CC1
InChIKey: MFWCFENQSTYMSU-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibition of human recombinant MAOB using p-tyramine as substrate preincubated for 30 mins followed by substrate addition measured for 1 hr by horse-radish peroxidase/amplex red-based fluorometric method
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 438.58, ALogP 4.57, HBA 5, HBD 1, PSA 63.39, QED 0.52
Compound (SMILES): C=Cc1c(N)ncnc1N1CCC(c2nc(-c3ccc(F)c(C(F)(F)F)c3)cn2CCN2CCCCC2)CC1
InChIKey: QYOOPWNXGCHVME-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 543.61, ALogP 5.59, HBA 7, HBD 1, PSA 76.1, QED 0.39
Compound (SMILES): COc1nc2ccc(C(O)(c3cncn3C)C3CCN(C(C)=O)CC3)cc2c(Cl)c1Cc1ccc(-n2cccn2)cc1
InChIKey: MAJOOHMQVBMIGI-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Inverse agonist activity at GAL4 DBD-fused wild type human RORgammat LBD (850 to 1635 residues) expressed in HEK293T cells assessed as inhibition of transcriptional activity after 24 hrs by dual-glo luciferase reporter gene assay
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 585.11, ALogP 4.9, HBA 8, HBD 1, PSA 98.3, QED 0.29
Compound (SMILES): N#CCC1(n2cc(-c3ccnc4[nH]ccc34)cn2)CN(C2CCN(C(=O)C3CCC(F)(F)CC3)CC2)C1
InChIKey: GQMRHFSVNVKOJI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
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 > 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 507.59, ALogP 4.17, HBA 6, HBD 1, PSA 93.84, QED 0.56
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCN(Cc2ccco2)CC1
InChIKey: ANCLYIWHTJQVEU-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 423.45, ALogP 2.76, HBA 8, HBD 2, PSA 126, QED 0.56
Compound (SMILES): NC(=O)c1c(N)ncnc1N1CCC(c2nc(-c3ccc(OC(F)F)cc3)cn2CCN2CCC2)CC1
InChIKey: RBLDRSHGAAWOKD-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 15000 nM (pChEMBL 4.820000171661377)
Descriptors: MW 512.57, ALogP 2.71, HBA 9, HBD 2, PSA 128.42, QED 0.45
Compound (SMILES): CCOc1ccc(-c2nc3ccccn3c2-c2ccnc(Nc3ccc(N4CCN(S(C)(=O)=O)CC4)cc3OC)n2)cc1C(=O)Nc1c(F)cccc1F
InChIKey: OFRBTEGWYCEAEP-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 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 754.82, ALogP 6.22, HBA 11, HBD 2, PSA 143.29, QED 0.16
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(CN4CCN(CCCCC(=O)NO)CC4)cc3)cc12)c1ccccc1
InChIKey: KJTCUAWNUJWJQJ-JOCHJYFZSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of N-terminal GST-tagged human HER2 (Lys676 to Val1255 residues) expressed in baculovirus expression system using biotinylated FLT3 at Tyr589 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA method
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 527.67, ALogP 4.59, HBA 7, HBD 4, PSA 109.41, QED 0.13