lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)n1cc(C(=O)c2ccnc(NC(=O)Cc3nc(C4CC4)oc3C3CC3)c2)c2cnccc21
InChIKey: JJWBRBYBCJVJIP-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 26.1 nM (pChEMBL 7.579999923706055)
Descriptors: MW 469.55, ALogP 5.17, HBA 7, HBD 1, PSA 102.91, QED 0.36
Compound (SMILES): CC(C)C(=O)Nc1ccccc1-c1ccc2c3[nH]ncc3c(=O)n(CC(F)(F)F)c2c1
InChIKey: YLKYRMWSHZKFGM-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Enzymatic Screening Assay: The enzymatic test is a test in two steps.In a first step, it consists in placing in contact the compound according to the invention, the dialysed MetAP2 protein and the substrate (Met-Pro-Arg-pNa peptide synthesized by Neosystem), the N-terminal methionine of which can be cleaved with MetAP2, and which bears at the C-terminal end a para-nitroaniline (pNa) chromophore, which can itself be released by another peptidase only when the N-terminal methionine has been cleaved beforehand.Consequently, the second step consists in reacting the peptides cleaved in the preceding step with a second peptidase in order to release the chromophore. The peptidase used in this second step is cathepsin, which comes from the TagZyme DAPase kit (Quiagen, 34366).The MetAP2 activity is proportional to the amount of para-nitroaniline released, which is measured by absorbance at 405 nm.
Measurement: IC50 = 25.4 nM (pChEMBL 7.590000152587891)
Descriptors: MW 428.41, ALogP 4.7, HBA 4, HBD 2, PSA 79.78, QED 0.49
Compound (SMILES): CN1CCN(c2cc(-c3n[nH]c4ccc(-c5cnn(C)c5)cc34)ncn2)CC1
InChIKey: QXJXXGMUXGGKKC-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 2.85 nM (pChEMBL 8.539999961853027)
Descriptors: MW 374.45, ALogP 2.17, HBA 7, HBD 1, PSA 78.76, QED 0.59
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=N[C@@]2(c2nc(NC(=O)c3cnc(OCC4CC4)cn3)cs2)CO1
InChIKey: NTZLXCBNRLXSKA-LOOLNKMNSA-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 = 161 nM (pChEMBL 6.789999961853027)
Descriptors: MW 460.59, ALogP 2.66, HBA 10, HBD 2, PSA 124.61, QED 0.67
Compound (SMILES): Cn1c(NC(=O)N2CCC(N3CC(CC#N)(n4cc(-c5ncnc6[nH]ccc56)cn4)C3)CC2)nc2ccccc21
InChIKey: LTSCRPSSSDSHSQ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 535.62, ALogP 3.33, HBA 9, HBD 2, PSA 136.58, QED 0.35
Compound (SMILES): CN1CCN(c2cc(Nc3cc(C4CC4)[nH]n3)nc(NCc3cc(-c4ccoc4)no3)n2)CC1
InChIKey: SRAPMPXWCRVOPP-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 461.53, ALogP 3.43, HBA 10, HBD 3, PSA 124.17, QED 0.36
Compound (SMILES): O=c1c2ccccc2[se]n1-c1ccccc1O
InChIKey: RETMDRSAJYWULY-UHFFFAOYSA-N
Target: Methionine aminopeptidase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P50579
Assay: Non-covalent/mixed type inhibition of recombinant human MetAP2 using Met-AMC as substrate preincubated for 30 mins followed by substrate addition by spectrofluorimetric method
Measurement: IC50 = 10200 nM (pChEMBL 4.989999771118164)
Descriptors: MW 290.18
Compound (SMILES): COc1cc(/C=C/C(=O)CC(=O)/C=C/c2ccc3ccccc3c2)ccc1O
InChIKey: QVRAESASNNQBSC-MKICQXMISA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Inhibition of amyloid beta (1 to 42 residues) (unknown origin) aggregation after 24 hrs by thioflavin T fluorescence assay
Measurement: IC50 = 590 nM (pChEMBL 6.230000019073486)
Descriptors: MW 372.42, ALogP 4.81, HBA 4, HBD 1, PSA 63.6, QED 0.48
Compound (SMILES): CS(=O)(=O)NCCNc1nonc1/C(=N/O)Nc1ccc(F)c(C(F)(F)F)c1
InChIKey: BOMZTIHNXJGGQT-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: Human indoleamine 2,3-dioxygenasae (IDO) with an N-terminal His tag was expressed in E. coli and purified to homogeneity. IDO catalyzes the oxidative cleavage of the pyrrole ring of the indole nucleus of tryptophan to yield N′-formylkynurenine. The assays were performed at room temperature as described in the literature using 95 nM IDO and 2 mM D-Trp in the presence of 20 mM ascorbate, 5 μM methylene blue and 0.2 mg/mL catalase in 50 mM potassium phosphate buffer (pH 6.5). The initial reaction rates were recorded by continuously following the absorbance increase at 321 nm due to the formation of N′-formlylkynurenine (See: Sono, M., et al., 1980, J. Biol. Chem. 255, 1339-1345).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 426.35, ALogP 1.44, HBA 8, HBD 4, PSA 141.74, QED 0.13
Compound (SMILES): CC(=O)N1CCC(C(N)=O)(C(=O)N2CC[C@](c3ccc(C(O)(C(F)(F)F)C(F)(F)F)cc3)(S(=O)(=O)c3ccc(F)cc3)C2)CC1
InChIKey: HFLZPPATCDUYOD-VWLOTQADSA-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 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 667.6, ALogP 3.15, HBA 6, HBD 2, PSA 138.08, QED 0.28
Compound (SMILES): CC(CC(=O)N1CCc2c(F)ccc(-c3ccc(CC(=O)O)cc3OCC(F)(F)F)c2C1)c1ccccn1
InChIKey: TYKWHZPXSIBASE-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 530.52, ALogP 5.53, HBA 4, HBD 1, PSA 79.73, QED 0.39
Compound (SMILES): COCCOc1n[nH]c2cc(NC(=O)N[C@@H](c3ccccc3)C(C)(C)O)ncc12
InChIKey: DGAIMGBFXYHJQY-KRWDZBQOSA-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 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 399.45, ALogP 2.62, HBA 6, HBD 4, PSA 121.39, QED 0.43
Compound (SMILES): O=C(NCc1ccc(S(=O)(=O)C2CCNCC2)cn1)c1cc2ccncc2o1
InChIKey: ZFEFLVGMIJINGA-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Biochemical Inhibition Assay: The NAMPT Enzymatic Reaction. The NAMPT enzymatic reactions were carried out in Buffer A (50 mM Hepes pH 7.5, 50 mM NaCl, 5 mM MgCl2, and 1 mM THP) in 96-well V-bottom plates. The compound titrations were performed in a separate dilution plate by serially diluting the compounds in DMSO to make a 100x stock. Buffer A (89 uL) containing 33 nM of NAMPT protein was added to 1 uL of 100x compound plate containing controls (e.g. DMSO or blank). The compound and enzyme mixture was incubated for 15 min at rt, then 10 uL of 10x substrate and co-factors in Buffer A were added to the test well to make a final concentration of 1 uM NAM, 100 pM 5-Phospho-D-ribose 1-diphosphate (PRPP), and 2.5 mM Adenosine 5'-triphosphate (ATP). The reaction was allowed to proceed for 30 min at rt, then was quenched with the addition of 11 uL of a solution of formic acid and L-Cystathionine to make a final concentration of 1% formic acid and 10 uM L-Cystathionine. Background and signal strength was determined by addition (or non-addition) of a serial dilution of NMN to a pre-quenched enzyme and cofactor mix.
Measurement: IC50 = 204 nM (pChEMBL 6.690000057220459)
Descriptors: MW 400.46, ALogP 1.68, HBA 7, HBD 2, PSA 114.19, QED 0.67
Compound (SMILES): N#Cc1ccc(CNC(=O)CCOCCN2CCN(c3ncccc3-c3ccc(F)cc3)CC2)cc1
InChIKey: DZHFAWUKQABZMD-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P34969
Assay: Displacement of [3H]-5-CT from human 5-HT7b receptor expressed in HEK293 cell membranes after 1 hr by microbeta counting analysis
Measurement: Ki = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 487.58, ALogP 3.6, HBA 6, HBD 1, PSA 81.49, QED 0.44
Compound (SMILES): COc1ccc(CNC(=O)C2CCN(S(=O)(=O)c3cn(C(C)C)cn3)CC2)cc1
InChIKey: KEFJLNGGOCYAQT-UHFFFAOYSA-N
Target: Dihydrolipoyl dehydrogenase (SINGLE PROTEIN), Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)
UniProt: P9WHH9
Assay: Inhibition Assay: The assay was performed in a manner similar to that described in Bryk et al., Biochemistry (2010) 49:1616-1627 and modified for an online robotics screening system.A solution containing Lpd (100 nM) and 100 mM sodium phosphate buffer (pH 7.0) was prepared. Another solution containing the substrate lipoamide (2 mM), 100 mM sodium phosphate buffer (pH 7.0), EDTA (4 mM), and NADH (200 uM) was also prepared. The detection reagent, DTNB was dissolved in DMSO and diluted to a concentration of 375 uM with a 100 mM sodium phosphate buffer (pH 7.0).4 uL/well of Lpd solution was dispensed into 1536-well clear bottom plates. Next, 50 nL of each test compounds (1 mM dissolved in DMSO) was added into each well. After one hour of incubation, 4 uL/well of lipoamide (LPA) solution was added, so that in each well, the concentrations of Lpd, LPA, and the test compound were 50 nM, 1 mM, and 6.25 uM, respectively.
Measurement: IC50 = 3330 nM (pChEMBL 5.480000019073486)
Descriptors: MW 420.54, ALogP 2.19, HBA 6, HBD 1, PSA 93.53, QED 0.74
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(NC(=O)c3ncc(Cl)cc3C)cc1C21N=C(C)C(N)=N1
InChIKey: LUDSHWAKEHVLGG-SSEMJGATSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Diluted TR-FRET Assay: TR-FRET AssayThe beta-secretase enzyme used in the TR-FRET is prepared as follows: The cDNA for the soluble part of the human beta-Secretase (AA 1-AA 460) was cloned using the ASP2-Fc10-1-IRES-GFP-neoK mammalian expression vector. The gene was fused to the Fc domain of IgG1 (affinity tag) and stably cloned into HEK 293 cells. Purified sBACE-Fc was stored in -80 C. in Tris buffer, pH 9.2 and had a purity of 40%.The enzyme (truncated form) was diluted to 6 ug/mL (stock 1.3 mg/mL) and the substrate (Europium)CEVNLDAEFK(Qsy7) to 200 nM (stock 120 uM) in reaction buffer (NaAcetate, chaps, triton x-100, EDTA pH4.5). The robotic systems Biomek FX and Velocity 11 were used for all liquid handling and the enzyme and substrate solutions were kept on ice until they were placed in the robotic system. Enzyme (9 ul) was added to the plate then 1 ul of compound in dimethylsulphoxide was added, mixed and pre-incubated for 10 minutes.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 465.99, ALogP 4.41, HBA 6, HBD 2, PSA 101.96, QED 0.7
Compound (SMILES): O=C(CN1CCCCC1)Nc1cc(-c2ccc(F)cc2F)cc(-n2cnc3cc(-n4cccn4)ccc32)c1
InChIKey: WLSBHQSTSBFHLK-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: TR-FRET Assay: Compounds were screened in the TR-FRET assay with FGFR1 kinase. 5 ng of FGFR1 [Upstate, USA] kinase was used for assay. The compound was incubated with the kinase for 30 minutes at RT. After the incubation, substrate mix [40 nM Ultra light poly GT (Perkin Elmer, USA) and 13 μM ATP (Sigma)] was added. The above reaction was stopped by the addition of 40 mM EDTA after the 30 mM kinase reaction. The Eu-labelled antiphospho-tyrosine antibody [Perkin Elmer, USA] was added at 0.5 nM and the fluorescence emission at 615 nm/665 nm [excitation at 340 nm] was measured. The compounds were initially screened at 100 nM and 1 μM concentrations. The compounds with >50% inhibition at 100 nM of FGFR1 were taken for the full dose response studies. The final DMSO concentration in the assay was 1%.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 512.56, ALogP 5.58, HBA 6, HBD 1, PSA 67.98, QED 0.32
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3cccc(C(=O)NC4CCNCC4)c3)n2)cnc1N1CC[C@H](F)C1
InChIKey: MROFQFMOZXUUGU-FQEVSTJZSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 30 nM (pChEMBL 7.5229997634887695)
Descriptors: MW 486.56, ALogP 3.18, HBA 8, HBD 3, PSA 118.86, QED 0.49
Compound (SMILES): Cc1ccc(C(=O)N2CCO[C@@H](C)[C@H]2Cc2cccc(-n3nccn3)c2)c(-n2nccn2)c1
InChIKey: WOQOCTXBEIMJQF-PGRDOPGGSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: OX Inhibition Assay: 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 ug/mL G418, 100 U/mL penicillin, 100 ug/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.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 443.51, ALogP 2.63, HBA 8, HBD 0, PSA 90.96, QED 0.47
Compound (SMILES): O=C(Nc1ccc(NS(=O)(=O)c2ccc(F)cc2)cc1)N1Cc2ccncc2C1
InChIKey: JLTGFXWUCKACAO-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 76.4 nM (pChEMBL 7.119999885559082)
Descriptors: MW 412.45, ALogP 3.57, HBA 4, HBD 2, PSA 91.4, QED 0.69
Compound (SMILES): CC1(C)CN(c2ccc3cccc(Cl)c3n2)CC[C@@H]1C(=O)N[C@H]1CC[C@@H](CO)CC1
InChIKey: FSHAZAGZDCHQMH-NZSAHSFTSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition of human mPGES-1 expressed in 293E cells assessed as reduction in conversion of PGH2 to PGE2 after 1.5 min by LC/MS analysis
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 429.99, ALogP 4.41, HBA 4, HBD 2, PSA 65.46, QED 0.76
Compound (SMILES): CCOc1ccc(N(C(=O)OC[C@H]2CC[C@H](COCC(=O)O)CC2)c2cccc(F)c2)cc1
InChIKey: ZCBHEQWCBIRSQH-WGSAOQKQSA-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 459.51, ALogP 5.41, HBA 5, HBD 1, PSA 85.3, QED 0.51
Compound (SMILES): CC(C)Oc1ccc2[nH]nc(-c3cc(N4CCn5nc(C6CC6)nc5C4)ncn3)c2c1
InChIKey: NNPOQGMYTSYUNR-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 18.2 nM (pChEMBL 7.739999771118164)
Descriptors: MW 416.49, ALogP 3.3, HBA 8, HBD 1, PSA 97.64, QED 0.53
Compound (SMILES): O=C(Nc1ccc(-c2cnn(Cc3ccccc3)c2)cc1)N1Cc2ccccc2C1
InChIKey: UYOCQGZXQURYMC-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 = 98.8 nM (pChEMBL 7)
Descriptors: MW 394.48, ALogP 5.15, HBA 3, HBD 1, PSA 50.16, QED 0.52
Compound (SMILES): COc1ccc(Nc2ccc(S(=O)(=O)NCC3CC3)c(C)c2)c(OC)c1
InChIKey: GGGQKRXVKATGKO-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Binding Assay: The binding assays were performed by testing the ability of the new compounds to displace the radiolabeled synthetic non-selective cannabinoid agonist [3H]CP55940 (168 Ci/mmol; PerkinElmer) from the human CB1 (hCB1) or human CB2 (hCB2) receptors on membranes derived from stably transfected HEK-293 cells (PerkinElmer). Membranes were diluted in assay buffer (50 mM Tris-HCl, 1 mM EDTA, 1 mM MgCl2, 1 mg/ml BSA, pH=7.4). The amount of membrane was determined for each batch of membranes according to protein binding assay. The minimum amount of membrane that gave 50% specific binding was used for the binding assay. In most assays, binding was tested using 8 μg and 4 μg protein of hCB1 and hCB2 membranes, respectively. The tested compounds were dissolved in DMSO and diluted in assay buffer to a final concentration of 0.1% solvent. Total binding of [3H]CP55940 was evaluated with 1.5 nM to hCB1 and with 0.5 nM to hCB2, according to Kd affinity of [3H]CP55940 for the respective membranes. The ability of the tested compounds to displace [3H]CP55940 was evaluated first at a single concentration point of 100 nM for binding toward hCB2 or hCB1. In certain cases, the displacement was tested at compound concentrations ranging from 0.03 nM to 10 μM. Non-specific binding was measured by the addition of 1 μM of unlabelled CP55940 to the tubes. Binding assays were performed in triplicate in a total volume of 500 μl for 60 minutes at 30° C., in a shaking bath. Free and bound radioligands were separated by rapid filtration through GF/C filter plates (PerkinElmer) that had been presoaked with 0.1% Polyethylenimine (Sigma). Filters were shaken for 1 hour in 7 ml scintillation fluid (PerkinElmer) and radioactivity was determined by liquid scintillation counter (Wallac; PerkinElmer).
Measurement: Ki = 357.2 nM (pChEMBL 6.449999809265137)
Descriptors: MW 376.48, ALogP 3.44, HBA 5, HBD 2, PSA 76.66, QED 0.74
Compound (SMILES): COc1ccc(CNc2ccc3ncc(-c4cccc(OC(F)(F)F)c4)n3n2)cc1
InChIKey: JTLUUMDUCUTWEW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Kinase-Glo Assay: These assays are set up in duplicate 50 ul volumes in white, flat bottom 96 well plates. Inhibitors are added to the solution of 1× kinase buffer, 10 uM ATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, and water in serial dilutions ranging from micromolar to nanomolar concentrations. This solution is incubated at 30 degrees Celsius at 360 rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagent is added to each well, including all positive and negative control wells, and incubated at room temperature for 15 minutes. The plate is then read by the Luminoskan Ascent instrument and the results displayed with the Ascent Software version 2.6.
Measurement: IC50 = 523 nM (pChEMBL 6.28000020980835)
Descriptors: MW 414.39, ALogP 4.92, HBA 6, HBD 1, PSA 60.68, QED 0.49
Compound (SMILES): Cc1cc(F)c(NC(=O)c2cc(C#N)cc(C(C)(C)C)c2)cc1-n1ccn2nc(-c3cccnc3)cc12
InChIKey: AUGJTNPFSWOIIC-UHFFFAOYSA-N
Target: Angiopoietin-1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q02763
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: For the assay, 50 nl of a 100-fold concentrated solution of the respective test substance in DMSO were pipetted into a black low-volume 384-well microtitre plate (Greiner Bio-One, Frickenhausen, Germany) 2 μl of a solution of the Tie2 fusion protein in assay buffer [50 mM HEPES/HCl pH 7, mM MgCl2, 2.5 mM MnCl2, 1 mM dithiothreitol, 0.01% (v/v) Nonidet P-40, 0.1% (w/v) bovine serum albumin (BSA), 1× Complete EDTA-free protease inhibitor mixture (Roche)] were added, and the mixture was incubated for 15 min to allow pre-binding of the substance to the enzyme prior to the kinase reaction. The kinase reaction was then started by adding 3 μl of a solution of adenosine triphosphate (ATP, 1.67 mM→final concentration in 5 μl assay volume=1 mM) and substrate (1.67 μM→final concentration in 5 μl assay volume=1 μM) in assay buffer, and the resulting mixture was incubated for a reaction time of 60 min at 22° C. The concentration of the Tie2 fusion protein was adapted to the respective activity of the enzyme and adjusted such that the assay was carried out in the linear range. Typical concentrations were in the range of 30 ng/ml. The reaction was stopped by addition of 5 μl of a solution of TR-FRET detection reagents [200 nM streptavidin-XL665 and 2 nM PT66-Eu chelate, a europium chelate-labelled anti-phosphotyrosine antibody (Perkin-Elmer); alternatively, it is also possible to use PT66-Tb cryptate (Cisbio Bioassays, Codolet, France)] in aqueous EDTA solution [90 mM EDTA, 0.28% (w/v) bovine serum albumin (BSA) in 50 mM HEPES pH 7.5]. The resulting mixture was incubated at 22° C. for 1 h to allow formation of the complex of the biotinylated phosphorylated substrate and the detection reagents. The amount of phosphorylated substrate was then determined by measuring the resonance energy transfer from PT66-Eu chelate to streptavidin-XL665. To this end, the fluorescence emissions at 620 nm and 665 nm after excitation at 350 nm were measured in a TR-FRET measuring instrument (for example Viewlux, Perkin-Elmer).
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 492.56, ALogP 6.06, HBA 6, HBD 1, PSA 88.01, QED 0.33
Compound (SMILES): Cc1cccc(C(=O)N2CCOC[C@H]2Cc2cc(-c3ncon3)ccc2F)c1-n1nccn1
InChIKey: OQWDRLJUBNCFEY-GOSISDBHSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: OX Inhibition Assay: 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 ug/mL G418, 100 U/mL penicillin, 100 ug/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.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 448.46, ALogP 2.85, HBA 8, HBD 0, PSA 99.17, QED 0.46
Compound (SMILES): COc1c(C(C)Nc2ncnc(N)c2C#N)cc(Cl)c(C)c1-c1cncc(S(C)(=O)=O)c1
InChIKey: XNZMAFVFOMKHPS-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Scintillation Proximity Assay (SPA): The kinase reaction was conducted in polystyrene 384-well matrix white plate from Thermo Fisher Scientific in a final volume of 25 uL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 0.5%. The PI3K assays were carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 uM PIP2, 20 uM ATP, 0.2 uCi [gamma-33P]ATP, 4 nM PI3K delta. Reactions were incubated for 210 min and terminated by the addition of 40 uL SPA beads suspended in quench buffer: 150 mM potassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 uM ATP. The final concentration of SPA beads was 1.0 mg/mL.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 472.96, ALogP 3.54, HBA 9, HBD 2, PSA 143.88, QED 0.55
Compound (SMILES): COc1cc(OC)cc(N(CCCN)c2ccc3ncc(-c4cnn(CCS(C)(=O)=O)c4)nc3c2)c1
InChIKey: DIDLAUMNKKUFSF-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 = 269.15 nM (pChEMBL 6.570000171661377)
Descriptors: MW 510.62, ALogP 3.04, HBA 10, HBD 1, PSA 125.46, QED 0.32
Compound (SMILES): COCCn1cc(-c2ccc3[nH]nc(-c4cc(N5CCOCC5)ncn4)c3c2)cn1
InChIKey: COJPOAVUHSPEHI-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LanthaScreen™ Assay (5 mM ATP): a) 400 nl of a 1:2.15 serial dilution of test compound(98 μM top assay concentration) is spotted via Labcyte Echo to certain wells in a 384 well black, untreated plate. Control wells contain 400 nl of either DMSO or 400 nl of a known inhibitor in DMSO. b) 10 μl of a 2.5 nM LRRK2(G2019S mutation, GST-LRRK2(amino acids 970-2527)) enzyme solution in 1× assay buffer(50 mM Tris pH 8.5, 10 mM MgCl2, 0.01% Brij-35, 1.0 mM EGTA, 2 mM DTT, 0.05 mM NaVO4) is added to all wells. c) A 30 minute room temperature incubation is followed by addition of 10 μl of 800 nM fluorescein labeled LRRKtide peptide substrate and 10 mM ATP solution in 1× assay buffer to all wells. d) After a 35 minute room temperature incubation, 20 μl of TR-FRET Dilution Buffer(Invitrogen PV3756B) containing 4 nM Tb-labeled anti-phospho LRRKtide antibody and 20 mM EDTA is added to all wells. e) Plates are incubated at room temperature for 1 hour and read on an Envision™ multi-mode plate reader with LanthaScreen™ settings. Results are analysed using Assay Data Analyzer.
Measurement: IC50 = 1.5 nM (pChEMBL 8.819999694824219)
Descriptors: MW 405.46, ALogP 2.37, HBA 8, HBD 1, PSA 93.98, QED 0.53
Compound (SMILES): O=C1N[C@@](c2ccc(OCCCCCC(F)(F)F)cc2)(C(F)(F)F)CC(c2ccn(CC3CC3)n2)=C1C(=O)NS(=O)(=O)C1CC1
InChIKey: STPGMXVCTXISQM-MHZLTWQESA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: LCMS Assay: The MGAT enzyme reactions were performed in Corning FALCON® 96-well Polypropylene plates, in a total volume of 60 μL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 μM 2-oleoylglycerol, 15 μM oleoyl-Coenzyme A and 0.0013 μg/μL Human or Mouse MGAT-2 or 0.0026 μg/μL 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 μL of ice cold methanol containing 1 μg/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, 50×2.1 mm column, was used for the chromatography of enzyme products. The buffer system consists of 0.1% formic acid in water with a mobile phase consisting 0.1% formic acid in methanol. The shallow gradient is 90-100% mobile phase in 0.2 min with a total run time of 2.3 min. The first 0.5 minutes of each injection was diverted to waste to eliminate the presence of Phosphate buffer in the enzymatic reaction. The column was run at 0.6 mL/min and a temperature of 65° C.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 662.65, ALogP 5.13, HBA 7, HBD 2, PSA 119.39, QED 0.17
Compound (SMILES): Cc1c(-c2ccc(N3CCc4cccc(C(=O)Nc5nc6ccccc6s5)c4C3)nc2C(=O)O)cnn1CC1CCCCO1
InChIKey: CXLVLOGFZIAGJS-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The measurement of competition of compounds of Formula (I) with F-Bak for a Bcl-2 family protein (Bcl-xL) binding site using a Time Resolved Fluorescence Resonance Energy Transfer (TR-FRET) binding assay: Test compounds were serially diluted in DMSO starting at 50 M (2x starting concentration; 10% DMSO) and 10 Î¼L transferred into a 384-well plate. Then 10 Î¼L of a protein/probe/antibody mix is added to each well at final concentrations listed in Table 1. Protein: GST-Bcl-xL; 1(nM) Probe: F-Bak (GQVGRQLAIIGDK (6-FAM)INR-amide) SEQ ID NO: 1; 100(nM) Antibody: Tb-anti-GST; 1(nM). The samples are then mixed on a shaker for 1 minute then incubated for an additional 2 hours at room temperature. For each assay plate, a probe/antibody and protein/antibody/probe mixture were included as a negative and a positive control, respectively. Fluorescence was measured on the ENVISION plate reader (Perkin Elmer) using a 340/35 nm excitation filter and 520/525 (F-Bak) and 495/510 nm (Tb-labeled anti-his antibody) emission filters. Dissociation constants (Ki) were determined using Wang's equation (Wang, Z.X. An 20 exact mathematical expression for describing competitive binding of two different ligands to protein molecule. FEBS Lett. 1995 360:111-114). The TR-FRET assay can be performed in the presence of varying concentrations of human serum (HS) or fetal bovine serum (FBS).
Measurement: Ki < 0.1 nM (pChEMBL 10)
Descriptors: MW 608.72, ALogP 5.95, HBA 9, HBD 2, PSA 122.47, QED 0.23
Compound (SMILES): COc1nnc(-c2cc(C(=O)N3CCC(F)(c4ccc(C#N)cc4)CC3)c(C)cc2C2CC2)[nH]1
InChIKey: SPRKROXUGKNHJP-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 459.53, ALogP 4.64, HBA 5, HBD 1, PSA 94.9, QED 0.6
Compound (SMILES): O=S(=O)(c1ccccc1)N(Cc1cccc(OC(F)(F)F)c1)c1cc2ccccc2s1
InChIKey: WSSXMYDFMVLIJE-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 108 nM (pChEMBL 6.96999979019165)
Descriptors: MW 463.5, ALogP 6.2, HBA 4, HBD 0, PSA 46.61, QED 0.34
Compound (SMILES): Cc1nc(N)ccc1CNC(=O)c1cnn(Cc2cc(C(F)(F)F)c3cc(F)ccc3n2)c1
InChIKey: LRSBELBIYXLLQU-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 458.42, ALogP 3.85, HBA 6, HBD 2, PSA 98.72, QED 0.44
Compound (SMILES): CC(C)Oc1ccc(Nc2ncc(NC(=O)C(CO)CO)c(=O)n2Cc2ccc(Cl)cc2)cc1F
InChIKey: ONDCUQVSXINORB-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 in M MgCl2, 5.0 mM CaCl2, 5.6 D-glucose, 2.5 mM probenecid, 1.0% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence inte
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 504.95, ALogP 3.15, HBA 8, HBD 4, PSA 125.71, QED 0.33
Compound (SMILES): C[C@@H]1CN(C(=O)c2ccccc2-n2nccn2)[C@H](Cc2cccc(-c3ncccn3)c2)CO1
InChIKey: GTTJINSRAWFIHD-WIYYLYMNSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: OX Inhibition Assay: 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 ug/mL G418, 100 U/mL penicillin, 100 ug/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.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 440.51, ALogP 3.2, HBA 7, HBD 0, PSA 86.03, QED 0.47
Compound (SMILES): CC(CO)n1cc(C(=O)c2cncc(NC(=O)Cc3ccc(C(F)(F)F)cc3)c2)c2cncc(F)c21
InChIKey: GAGVFTJQVOCNEX-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 264 nM (pChEMBL 6.579999923706055)
Descriptors: MW 500.45, ALogP 4.55, HBA 6, HBD 2, PSA 97.11, QED 0.29
Compound (SMILES): CCCN(Cc1ccc(OCCN2C(=O)CCC2=O)c(OC)c1)C(C(=O)O)c1ccc(Cl)cc1
InChIKey: JLCZBCWSSHXTSH-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 256 nM (pChEMBL 6.590000152587891)
Descriptors: MW 488.97, ALogP 3.91, HBA 6, HBD 1, PSA 96.38, QED 0.45