lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1cnc2c(-c3ccc(OCCC4CCN(C(=O)CO)CC4)c(C(F)(F)F)c3)nc(C#N)nc21
InChIKey: ZYHKTSBONLNQNK-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Homo sapiens
UniProt: P25774
Assay: Inhibition Assay: 5 μL of a human cathepsin S enzyme (R&D1183-CY-010) was added to a 96-well plate so as to be 20 ng/well. Next, 10-fold dilution of the test compound (a 10 mM DMSO solution) was serially performed with a buffer solution for assay (a solution of 50 mM sodium methoxide, 250 mM sodium chloride and 5 mM dithiothreitol (DTT), adjusted to pH=4.5 with 1 N hydrochloric acid) six times such that the final concentration becomes 0.1 nM to 10 μM then, 20 μL of each of the solutions was added to the wells (the final DMSO concentration was 0.1%), and 25 μL of synthetic substrate VVR-AMC (3211-V manufactured by PEPTIDE INSTITUTE, INC.) was added thereto such that the final concentration becomes 40 μM, thereby starting a enzyme reaction. A fluorescence intensity (an excitation wavelength: 380 nm and a fluorescent wavelength: 460 nm) was measured at 37° C. for 10 minutes immediately after the start of the reaction using a fluorescence spectrophotometer (SPECTRA MAX GEMINI, manu
Measurement: IC50 = 6.1 nM (pChEMBL 8.210000038146973)
Descriptors: MW 488.47, ALogP 2.92, HBA 8, HBD 1, PSA 117.16, QED 0.57
Compound (SMILES): O=c1cc(NC2CCN(S(=O)(=O)C(F)(F)F)CC2)c2cc(C(Cc3ccc(F)cc3)c3ccc(Cl)cc3)ccc2[nH]1
InChIKey: PHKMBHSVEAYADC-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 608.06, ALogP 6.42, HBA 4, HBD 2, PSA 82.27, QED 0.24
Compound (SMILES): COc1nc2ccc(C(O)(c3ccc(C(F)(F)F)nc3)c3cncn3C)cc2c(Cl)c1Cc1ccc(S(C)(=O)=O)cc1
InChIKey: BUNRTAJYWAGDHW-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1 °C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20. Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 617.05, ALogP 5.32, HBA 8, HBD 1, PSA 107.2, QED 0.26
Compound (SMILES): COCCNS1(O)C[C@@](C)(c2cc(NC(=O)c3ccc(Cl)cn3)ccc2F)NC(=N)C1(C)C
InChIKey: BZXZUMDZEXZKSC-QFIPXVFZSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: HTRF FRET Assay: Varying concentrations of inhibitors at 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window.
Measurement: Ki = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 498.02, ALogP 4.13, HBA 6, HBD 5, PSA 119.36, QED 0.37
Compound (SMILES): Cc1ccc(C(=O)Nc2cc(C(C)(C)C)on2)cc1-n1cc(-c2cncc(N3CCC(CN(C)C)CC3)c2)nn1
InChIKey: NDWPFNZZWDYAPN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: B-RAF (V600E) Kinase Assay: In a dilution series 10 uL/well of test substance solution are placed in a multiwell plate. The dilution series is selected so that generally a range of concentrations of 2 uM to 0.119 nM or 0.017 nM is covered. If necessary the initial concentration of 2 uM is changed to 50 uM, 10 uM or 0.4 uM or 0.2857 uM and further dilution is carried out accordingly. The final concentration of DMSO is 5%. 10 uL/well of the B-Raf (V600E)-kinase solution are pipetted in (containing 0.5 ng B-Raf (V600E)-kinase (e.g. from Upstate) in 20 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 0.286 mM sodium orthovanadate, 10% glycerol, 1 mg/mL bovine serum albumin, 1 mM dithiothreitol) and the mixture is incubated for 1 h at RT under with shaking. The kinase reaction is started by the addition of 20 uL/well ATP solution [final concentration: 250 uM ATP, 30 mM Tris-HCl pH 7.5, 0.02% Brij, 0.2 mM sodium orthovanadate, 10 mM magnesium acetate, 0.1 mM EGTA, phosphatase cocktail (Sigma, #P2850, dilution recommended by the manufacturer)] and 10 uL/well MEK1 solution [containing 50 ng biotinylated MEK1 (prepared from purified MEK1 according to standard procedure, e.g. with EZ-Link Sulpho-NHS-LC-Biotin reagent, Pierce, #21335)] and carried out for 60 min at RT with constant shaking. The reaction is stopped by the addition of 12 uL/well of a 100 mM EDTA solution and incubation is continued for a further 5 min. 55 uL/well of the reaction solution are transferred into a streptavidin-coated plate (e.g. Streptawell HighBond, Roche, #11989685001) and shaken gently for 1 h at RT, in order to bind biotinylated MEK1 to the plate. After elimination of the liquid the plate is washed five times with 200 uL/well of 1Ã¿PBS and 100 uL/well solution of primary antibody plus europium-labelled secondary antibody [Anti Phospho-MEK (Ser217/221), Cell Signaling, #9121 and Eu-N1 labelled goat-anti-rabbit antibody, Perkin Elmer, #AD0105] is added, the primary antibody is diluted 1:2000 and the secondary antibody is diluted to 0.4-0.5 ug/mL in Delfia Assay Buffer (Perkin Elmer, #1244-111). After 1 h shaking at RT the solution is poured away and washed five times with 200 uL/well Delfia Wash Buffer (Perkin Elmer, #4010-0010/#1244-114). After the addition of 200 uL/well Enhancement Solution (Perkin Elmer, #4001-0010/#1244-105) the mixture is shaken for 10 min at RT and then measured in a Wallac Victor using the program "Delfia Time Resolved Fluorescence (Europium)". IC50 values are obtained from these dosage-activity curves using a software program (GraphPadPrizm).
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 542.69, ALogP 4.95, HBA 9, HBD 1, PSA 105.21, QED 0.35
Compound (SMILES): COc1cc2ncccc2cc1NC(=O)c1csc2cnc(NCC(C)C)nc12
InChIKey: GACWRDLJSVTTSQ-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: In vitro IRAK-1 and IRAK-4 Assay: Purified recombinant IRAK-4 protein was incubated with 250 uM synthetic peptide(KKARFSRFAGSSPSQSSMVAR) in 30 ul of kinase buffer including (20 mM MOPS pH7.2, 25 mM beta glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, 1 mM DTT, 50 uM ATP, 20 mM MgCl2, 10 uCi gamma-33P, 0.1% BSA) for the indicated time. For purified recombinant IRAK-1 protein kinase assay, 50 uM ATP was used. A 25 ul aliquot of the reaction mixture was transferred on to p81 phosphocellulose squares (Upstate Biotechnology, Lake Placid, N.Y.). The assay squares were washed three times with 0.75% phosphoric acid and once with acetone. Enzyme activity was measured by determining the bound radioactivity by liquid scintillation counting.
Measurement: IC50 = 271 nM (pChEMBL 6.570000171661377)
Descriptors: MW 407.5, ALogP 4.57, HBA 7, HBD 2, PSA 89.03, QED 0.49
Compound (SMILES): N#CCC(=O)N1CCC(C(=O)N2CC[C@](c3ccc(C(OCc4c(F)cccc4F)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: XHXKWETVSJEZEG-YTTGMZPUSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Scintillation Proximity Assay (SPA): The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromatography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 uM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat#G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat #RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer). The competition data of the test compound over a range of concentrations was plotted as percentage inhibition of radioligand specifically bound in the absence of test compound (percent of total signal). After correcting for non-specific binding, IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce [3H] 25-hydroxycholesterol specific binding by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 221 nM (pChEMBL 6.659999847412109)
Descriptors: MW 775.69, ALogP 6.69, HBA 6, HBD 0, PSA 107.78, QED 0.18
Compound (SMILES): O=C(/C=C/c1cc2ccccc2[nH]1)c1cc2ccc(Br)cc2[nH]1
InChIKey: IAEKSWFKQKJYDU-BQYQJAHWSA-N
Target: Pyruvate kinase (SINGLE PROTEIN), Staphylococcus aureus (strain MRSA252)
UniProt: Q6GG09
Assay: Inhibition of recombinant N-terminal His-tagged methicillin-resistant Staphylococcus aureus MRSA252 pyruvate kinase expressed in Escherichia coli BL21(DE3) using PEP as substrate measured after 5 mins by LDH-coupled assay
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 365.23, ALogP 5.31, HBA 1, HBD 2, PSA 48.65, QED 0.38
Compound (SMILES): COc1n[nH]c2cc(NC(=O)NCc3cccnc3N(C)C)nc(CO)c12
InChIKey: ZOPYRBCIGXJUNH-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 544.3 nM (pChEMBL 6.260000228881836)
Descriptors: MW 371.4, ALogP 1.24, HBA 7, HBD 4, PSA 128.29, QED 0.51
Compound (SMILES): O=C(COc1cccc2c1CCCC2)NCC(O)CN1CCc2ccccc2C1
InChIKey: DYDNJWNWZWCBDY-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 394.52, ALogP 2.48, HBA 4, HBD 2, PSA 61.8, QED 0.76
Compound (SMILES): O=C(N[C@H]1CCNC1)c1cc(NC(=O)c2cc(-c3ncccc3F)c(F)cc2Cl)n(-c2ccccc2)n1
InChIKey: LARFYHZGUHPKJV-HNNXBMFYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 522.94, ALogP 4.21, HBA 6, HBD 3, PSA 100.94, QED 0.35
Compound (SMILES): CS(=O)(=O)c1cc(C#N)ccc1C1C2=C(CCC2=O)N(c2cccc(C(F)(F)F)c2)C(=O)N1CC(=O)N1CC2CC2C1
InChIKey: KSNJWWHCQPSYQI-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Nuetrophil Elastase Acitivty Assay: Various concentrations of the neutrophil elastase inhibitor are incubated with plasma. Subsequently, the enzyme activity is measured using the fluorogenic substrate MeOSuc-Ala-Ala-Pro-Val-AMC (Bachem Cat. No. 1-1270, substrate concentration: 250 uM, pH 7.5, 25 mM TRIS buffer, 250 mM NaCl) in analogous fashion as described for the human neutrophil assay.
Measurement: EC50 < 1 nM (pChEMBL 9)
Descriptors: MW 598.6, ALogP 4.06, HBA 6, HBD 0, PSA 118.86, QED 0.51
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=NC2(c2nc(NC(=O)c3coc(CF)n3)cs2)CO1
InChIKey: ABEZBNOKPIPPHW-IJLCECLYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37 °C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 411.48, ALogP 2.53, HBA 9, HBD 2, PSA 115.63, QED 0.79
Compound (SMILES): N=C(N)Nc1ccc(C(=O)Oc2ccc(CC(=O)N[C@@H](Cc3ccccc3)C(=O)O)c(C(F)(F)F)c2)cc1
InChIKey: KZRPAQOWLGHARN-NRFANRHFSA-N
Target: Serine protease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P07477
Assay: Inhibition Assay: The compound was dissolved in dimethyl sulfoxide (DMSO), and diluted to an arbitrary concentration (A). A was 100-fold diluted with a buffer (0.1 M Tris (pH 8.0), 0.15 M NaCl, 10 mM CaCl2, 0.05% Brij38) (B). The r-h trypsin was diluted with a buffer to 0.088 ug/mL, and the m-trypsin was diluted with a buffer to 1/50 (C). The dilution ratio of the m-trypsin (1/50) was set to exhibit the same activity as the 0.088 ug/mL r-h trypsin as determined by kinetic analysis. The substrate solution of a substrate for the enzyme reaction, BZiPAR, (Rhodamine Reference Substrate) was diluted with a buffer to 5 umol/L (D). B; 5 uL, C; 5 uL, and D; 10 uL were added to a 384-plate, and incubated at room temperature for 30 minutes. The fluorescent signals were detected with Ex/Em = 497/520 using Tecan Safire Fluorometer. The compound was reviewed from 2500 nM to its 3-fold value, 0.0075 nM, at 12 concentrations, and the inhibitory rate of each compound was calculated.
Measurement: IC50 = 0.29 nM (pChEMBL 9.539999961853027)
Descriptors: MW 528.49, ALogP 3.58, HBA 5, HBD 5, PSA 154.6, QED 0.12
Compound (SMILES): Cc1nc2cccc(-c3cc4c([nH]3)CCNC4=O)c2nc1NC1CNC1
InChIKey: MLHKMTKJGFNNJQ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 348.41, ALogP 1.6, HBA 5, HBD 4, PSA 94.73, QED 0.58
Compound (SMILES): CN(C)CC(c1ccc(C(F)(F)F)cc1)N1CCN(c2ncnc(N)c2-c2ccc(N)nc2)CC1
InChIKey: OSKPKUBBIPVDEA-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: Enzyme Assay: P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction.
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 486.55, ALogP 3.15, HBA 8, HBD 2, PSA 100.43, QED 0.55
Compound (SMILES): CCN(C)C1CN(C[C@H]2CC[C@H](c3nnn4cnc5[nH]ccc5c34)CC2)C1
InChIKey: HXKUQMNJEYIBQI-SHTZXODSSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 44 nM (pChEMBL 7.360000133514404)
Descriptors: MW 367.5, ALogP 2.52, HBA 6, HBD 1, PSA 65.35, QED 0.75
Compound (SMILES): CCCn1cc(C(=O)NCc2ccc(S(C)(=O)=O)cn2)c(=O)c(-c2cccc(C(F)(F)F)c2)c1C
InChIKey: QBWJZCOMAAXXNJ-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA. Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 Î¼L substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 507.53, ALogP 3.98, HBA 6, HBD 1, PSA 98.13, QED 0.52
Compound (SMILES): CNC(=O)c1cccc(Nc2nc3c(c(Nc4ccccc4S(=O)(=O)NC(C)C)n2)CCN3)c1
InChIKey: OFQMYGWFTGXFND-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 481.58, ALogP 2.98, HBA 8, HBD 5, PSA 137.14, QED 0.33
Compound (SMILES): Cc1cc(N=S(C)(C)=O)cc2ncnc(Nc3ccc(F)cc3OCC3(C#N)CCC3)c12
InChIKey: LIKUKUMHCCDNCZ-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In vitro Kinase Assay: The inhibition of kinase activity of MNK2a was assessed using pre-activated GST-MNK2a. The white, 384-well OptiPlate F plates were purchased from PerkinElmer. The ADP-Glo Kinase Assay (including ultra pure ATP) was purchased from Promega (V9103). Activated MNK2a was obtained as described in WO2011/104340. The unlabeled eIF4E peptide (NH2-TATKSGSTTKNR-CONH2), differing from Seq. ID No. 5 of WO 2011/104340 by the C-terminal CONH2 group, was purchased from Thermo Fisher Scientific. All other materials were of highest grade commercially available. Compounds are tested in either serial dilutions or single dose concentrations. The compound stock solutions are 10 mM in 100% DMSO The serial compound dilutions are prepared in 100% DMSO followed by 1:27.3 intermediate dilution in assay buffer. The final DMSO concentration in assay will be <3%.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 453.54, ALogP 5.25, HBA 7, HBD 1, PSA 100.26, QED 0.55
Compound (SMILES): O=C(Nc1ccc(C(=O)N2CCN(c3cccc(F)c3)CC2)cc1)N1Cc2ccccc2C1
InChIKey: FVAIDOUTUBTNGE-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1251667.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 27.8 nM (pChEMBL 7.559999942779541)
Descriptors: MW 444.51, ALogP 4.34, HBA 3, HBD 1, PSA 55.89, QED 0.65
Compound (SMILES): NCC(c1ccc(C(F)(F)F)cc1)C1CCN(c2ncnc(N)c2-c2cn[nH]c2)CC1
InChIKey: CBGPEGDCORSVLT-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: Enzyme Assay: P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction.
Measurement: IC50 = 5.2 nM (pChEMBL 8.279999732971191)
Descriptors: MW 431.47, ALogP 3.43, HBA 6, HBD 3, PSA 109.74, QED 0.57
Compound (SMILES): [C-]#[N+]c1c(F)ccc([C@H]2CN3CCN(C(=O)Cc4ncc(-n5cnnn5)cc4Cl)C[C@H]3CO2)c1C
InChIKey: UEQUHHLKXMDMBK-HRAATJIYSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Thallium Flux Assay: The ROMK channel functional thallium flux assay is performed in 384 wells, using the FLIPR-Tetra instrument. HEK-hKir1.1 cells are seeded in Poly-D-Lysine microplates and kept in a 37° C.-10% CO2 incubator overnight. On the day of the experiment, the growth media is replaced with the FluxOR™ reagent loading buffer and incubated, protected from light, at ambient temperature (23-25° C.) for 90 min. The loading buffer is replaced with assay buffer±test compound followed by 30 min incubation at ambient temperature, where the Thallium/Potassium stimulant is added to the microplate. The fluorescence intensity of wells containing 3 μM of a standard control ROMK inhibitor of the present invention is used to define the ROMK-sensitive component of thallium flux. Fluorescence in the presence of test compounds is normalized to control values to provide % fluorescence change. IC50 values represent the concentration of compound that inhibits 50% of the ROMK thallium flux signal.
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 496.93, ALogP 2.54, HBA 8, HBD 0, PSA 93.63, QED 0.51
Compound (SMILES): COc1ccc(CNC(=O)[C@@]2(C)[C@@H](OC(C)=O)CC[C@]3(C)[C@H]4[C@@H](O)c5c(cc(-c6cccnc6)oc5=O)O[C@]4(C)[C@@H](OC(=O)c4ccc(C#N)cc4)C[C@H]32)c(OC)c1
InChIKey: YBPLVXSCROVJTQ-GEGUFZPXSA-N
Target: Sterol O-acyltransferase 2 (SINGLE PROTEIN), Mus musculus
UniProt: O88908
Assay: Inhibition of ACAT2 in mouse hepatic microsomal membranes assessed as decrease in formation of [14C]cholesteryl oleate from [1,14C]oleoyl co-enzyme A after 5 mins by bio-image analyzer
Measurement: IC50 < 0.5 nM (pChEMBL 9.300999641418457)
Descriptors: MW 791.85, ALogP 5.69, HBA 13, HBD 2, PSA 196.51, QED 0.2
Compound (SMILES): COc1ccc2c(c1)CN(C(=O)c1nc(N)nc3ccc(C)cc13)C2
InChIKey: FIUXPTAYPDCBOR-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Recombinant Hsp90 [His-Tev-huHsp90-alpha (9-236)] was immobilized on a Biacore CM5 chip at 25 degrees C and a flow rate of 30 uL/min using amine coupling at pH 4.50 according to Biacore standard protocol. Hsp90 was applied at a concentration of 20 ug/mL with 50uM 17-DMAG.Data sets were processed and analyzed using the software Biacore 4000 Evaluation. Solvent corrected and double-referenced association and dissociation phase data were fitted to a simple 1:1 interaction model with mass transport limitations
Measurement: Kd = 2827 nM (pChEMBL 5.550000190734863)
Descriptors: MW 334.38, ALogP 2.69, HBA 5, HBD 1, PSA 81.34, QED 0.78
Compound (SMILES): COc1cc(NC(=O)N2CCCc3ccc(C=O)nc32)ncc1C#N
InChIKey: ALHYZSHKUANXLF-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Inhibition of TEL-fused FGFR4 cytoplasmic/juxta membrane domain (unknown origin) expressed in mouse Ba/F3 cells assessed as reduction in FGFR4 autophosphorylation after 1 hr by ELISA
Measurement: IC50 = 72 nM (pChEMBL 7.139999866485596)
Descriptors: MW 337.34, ALogP 2.15, HBA 6, HBD 1, PSA 108.21, QED 0.86
Compound (SMILES): Cc1n[nH]c(C)c1S(=O)(=O)N1CCC(=C(F)c2ccc(Cl)c(F)c2)CC1
InChIKey: YAXYBAJRIAAJBG-UHFFFAOYSA-N
Target: Prokineticin receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TCW9
Assay: Antagonist activity at human PKR1 expressed in RBL2H3 cells assessed as inhibition of prokineticin1-mediated intracellular calcium release preincubated for 10 mins followed by PK1 addition by FLIPR assay
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 401.87, ALogP 3.98, HBA 3, HBD 1, PSA 66.06, QED 0.85
Compound (SMILES): C[C@@H]1/C=C\C(=O)[C@@H](O)[C@@H](O)C/C=C/c2cc(NCCN3CCN(C)CC3)cc(O)c2C(=O)O[C@H]1C
InChIKey: YRQWFJSQGRIOEF-LPNBEJBMSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: Inhibition of MEK1 (unknown origin) using ERK2 as substrate after 40 mins by ELISA
Measurement: IC50 = 471 nM (pChEMBL 6.329999923706055)
Descriptors: MW 487.6, ALogP 1.5, HBA 9, HBD 4, PSA 122.57, QED 0.47
Compound (SMILES): O=C(O)c1ccc(-n2nc(Sc3c(Cl)cccc3Cl)c3cc(C(=O)N4CCCC4)ccc32)cc1
InChIKey: FQSIMRQZAGJJBW-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Inverse agonist activity at GAL4 DBD-fused RORgammat LBD (unknown origin) expressed in HG5LN cells after 18 hrs by luciferase reporter gene assay
Measurement: IC50 = 2600 nM (pChEMBL 5.579999923706055)
Descriptors: MW 512.42, ALogP 6.42, HBA 5, HBD 1, PSA 75.43, QED 0.33
Compound (SMILES): NCCN1CCN(C(=O)c2cc(NC(=O)c3cc(-c4ncccc4F)c(Cl)cc3Cl)n(-c3ccccc3)n2)CC1
InChIKey: DFIDTMBBTQFSBE-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 211 nM (pChEMBL 6.679999828338623)
Descriptors: MW 582.47, ALogP 4.35, HBA 7, HBD 2, PSA 109.38, QED 0.34
Compound (SMILES): C[C@H]1CN(C(=O)C2COC2)C[C@H](C)N1c1nc2cc(-c3nc(=O)o[nH]3)nc(-c3cncc(Cl)c3)c2n1C[C@H]1CC[C@H](C)CC1
InChIKey: GZHNAJYYLQVOBI-MUGJNUQGSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: Methods: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (SEQ ID NO:1: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards the p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1x Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1x assay buffer recombinant HDM2 protein, peptide and Lumi-4-Tb Cryptate-conjugate mouse.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 621.14, ALogP 4.39, HBA 10, HBD 1, PSA 135.27, QED 0.33
Compound (SMILES): Cc1ccc(C2=C(c3nn[nH]n3)C(=O)NC(c3ccc(OCCCCCC(F)(F)F)c(C)c3)(C(F)(F)F)C2)cc1
InChIKey: SEORFEJEWYTTFW-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in CORNING Falcon 96-well Polypropylene plates, in a total volume of 60 uL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 uM 2-oleoylglycerol, 15 uM oleoyl-Coenzyme A and 0.0013 ug/uL Human or Mouse MGAT-2 or 0.0026 ug/uL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 uL of ice cold methanol containing 1 ug/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50x2.1 mm column, was used for the chromatography of enzyme products.
Measurement: IC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 567.53, ALogP 6.21, HBA 5, HBD 2, PSA 92.79, QED 0.24
Compound (SMILES): CCCOc1cccc(-c2c[nH]c3ccc(-c4nnc(N)s4)cc23)n1
InChIKey: RZVRZDUJJWCVMN-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 15.23 nM (pChEMBL 7.820000171661377)
Descriptors: MW 351.44, ALogP 4.12, HBA 6, HBD 2, PSA 89.71, QED 0.56
Compound (SMILES): CN1CCC2(CCN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)CC2)C1
InChIKey: SILAGMOAQIHSOD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.56 nM (pChEMBL 9.25)
Descriptors: MW 599.04, ALogP 4.04, HBA 9, HBD 1, PSA 109.89, QED 0.34
Compound (SMILES): O=c1nc(Nc2ccc(-c3ccon3)cc2)n(Cc2ccc(Cl)cc2)c(=O)n1CCO
InChIKey: IBYTZLXTXPKYSB-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 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 931 nM (pChEMBL 6.03000020980835)
Descriptors: MW 439.86, ALogP 2.5, HBA 9, HBD 2, PSA 115.18, QED 0.45
Compound (SMILES): Cc1cnc2c(NS(=O)(=O)c3ccccc3)cccc2c1
InChIKey: WXQOVUFFURRFPI-UHFFFAOYSA-N
Target: Ferroportin (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NP59
Assay: Antagonist activity at C-terminal halo-tag fused human ferroportin expressed in MDCK cells assessed as inhibition of hepcidin-induced ferroportin internalization after overnight incubation by Draq5 staining-based fluorescence assay
Measurement: IC50 = 610 nM (pChEMBL 6.210000038146973)
Descriptors: MW 298.37, ALogP 3.34, HBA 3, HBD 1, PSA 59.06, QED 0.81
Compound (SMILES): CC#C[C@]1(O)CC[C@H]2[C@@H]3CCc4cc(C(=O)Nc5cccnc5C)ccc4[C@H]3[C@@H](OCc3ccc(Cl)cc3)C[C@@]21C
InChIKey: QEEBMYAFXXDBRD-LUOXVKAHSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Antagonist activity at GR in human HepG2 cells assessed as inhibition of protein mediated-transcriptional activity by MMTV-promoter driven luciferase reporter gene assay
Measurement: IC50 = 0.07 nM (pChEMBL 10.149999618530273)
Descriptors: MW 569.15, ALogP 7.1, HBA 4, HBD 2, PSA 71.45, QED 0.32
Compound (SMILES): CC(C)(C)COC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccccc3)n2)CC1
InChIKey: GZOZHMUFNKBKTK-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 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 438.53, ALogP 3.61, HBA 7, HBD 2, PSA 126.27, QED 0.71
Compound (SMILES): O=C(O)COC[C@H]1CC[C@H](COC(=O)N(c2ccccc2)c2cccc(F)c2)CC1
InChIKey: UMQLASXGJFGXFL-IYARVYRRSA-N
Target: Prostacyclin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43119
Assay: Agonist activity at human recombinant prostacyclin receptor expressed in CHO-K1 cells assessed as induction of cAMP accumulation incubated for 1 hr by HTRF assay
Measurement: EC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 415.46, ALogP 5.01, HBA 4, HBD 1, PSA 76.07, QED 0.66
Compound (SMILES): COC(=O)Nc1nc2cc(C3(O)c4ccccc4C(=O)N3c3cccc(N)c3)ccc2[nH]1
InChIKey: PADJXXVNKOEZEG-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 429.44, ALogP 3.18, HBA 6, HBD 4, PSA 133.57, QED 0.37
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CCC(S(C)(=O)=O)C2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: IHIQKRYMXUFAGS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 12.59 nM (pChEMBL 7.900000095367432)
Descriptors: MW 550.69, ALogP 3.69, HBA 10, HBD 0, PSA 102.68, QED 0.29