lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=P(O)(O)COC[C@H]1O[C@@H](n2ncc3c(N4CC5(CCc6ccc(F)cc65)C4)nc(Cl)nc32)[C@H](O)[C@@H]1O
InChIKey: IGGVNVUROHQPRN-WOCWXWTJSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Enzyme Activity In Vitro Assay: I. Experimental Materials and Equipment1. Malachite green phosphate detection kit (R & D Systems, Cat #, DY996)2. Recombinant human 5′-nucleotidase (CD73) (R & D Systems, Cat #, 5795-EN)3. HEPES buffer (Gibco, Cat #, 15630-080)4. CMP (Sigma, Cat #, C1006)5. DMSO (Fisher Chemical, Cat #, D128-1)6. NaCl 5M (Boston Bioproducts, Cat #, BM-244)7.384-well plate (Fisher, Cat #, 5795-EN)8. TECAN plate reader (TECAN)II. Experimental ProcedureCompounds are first dissolved in DMSO to 10 mM as a stock solution. When determining the IC50 of the compound, prepare 3-fold serial dilutions with a highest concentration of 125 μM for a total of 12 concentration points and ensure each dilution containing equal amount of DMSO. In each well of 384-well plate, 0.34 nM of recombinant human 5′-nucleotidase (CD73) was pre-incubated at 37° C. for 2 hours with the compounds tested in assay buffer containing 20 mM HEPES buffer (pH 7.4), 137 mM NaCl, 0.001% Tween 20. The final reaction volume of the reaction in each well was 12 μL. The highest concentration of compound was 125 μM and the DMSO concentration was 1.25%. After pre-incubation, 3 μL of CMP dissolved in assay buffer was added to each reaction. The final CMP concentration was 45 μM. The reaction was incubated at 37° C. for 15 minutes. Then 3 μL of Malachite Green Reagent A was added to each reaction. Spin the plate briefly in centrifuge for 30 seconds. After incubation for additional 10 minutes at room temperature, 3 μL of malachite green Reagent B was added to each reaction. Spin the plate briefly in centrifuge for 30 seconds. After 20 minutes of incubation at room temperature, the signal was read on a TECAN reader at OD620.
Measurement: IC50 = 0.25 nM (pChEMBL 9.600000381469727)
Descriptors: MW 555.89, ALogP 1.09, HBA 10, HBD 4, PSA 163.29, QED 0.25
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCN(CC4CCCO4)CC3)cc12
InChIKey: WYEPXZBVRBBIHG-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 407.49, ALogP 4.65, HBA 4, HBD 1, PSA 50.38, QED 0.67
Compound (SMILES): COc1ccc(CN(Cc2ccc(OC)cc2)S(=O)(=O)c2c(S(=O)(=O)[C@H]3C[C@@H](CO)N(C(=O)OCc4ccccc4)C3)ccc(I)c2-c2nnn(Cc3ccc(OC)cc3)n2)cc1
InChIKey: TUYZDBKNNCUCAC-UVNLRBHVSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Enzyme Activity: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1; for a review, see: Meine, M.-R.; Llarrull, L. I.; Vila, A. J. Antibiotics, 2014, 3, 285-316) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 μM to 0.00063 μM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 0.148 nM (pChEMBL 9.829999923706055)
Descriptors: MW 1008.91
Compound (SMILES): CN1C(=O)CCc2c1ccc(-c1n[nH]cc1CC1CC3(C1)CN(C(=O)c1cnn(C)c1)C3)c2Cl
InChIKey: PFEXLJYVCIXOLE-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Inhibition Assay: The expression plasmids contains the ORF for either human/cyno CYP11B1 or CYP11B2 under the control of a suitable promoter (CMV-promoter) and a suitable resistance marker (neomycin). Using standard techniques the expression plasmid is transfected into G-402 cells and these cells are then selected for expressing the given resistance markers. Individual cell-clones are then selected and assessed for displaying the desired enzymatic activity using 11-Deoxycorticosterone (Cyp11B2) or 11-Deoxycortisol (Cyp11B1) as a substrate.G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 μg/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 0.038 nM (pChEMBL 10.420000076293945)
Descriptors: MW 478.98, ALogP 3.47, HBA 5, HBD 1, PSA 87.12, QED 0.62
Compound (SMILES): C[C@@H]1CCCN(C(=O)c2cccc(F)c2-c2ncccn2)[C@@H]1CNc1ccc(C(F)(F)F)cn1
InChIKey: RYLOAVVOYUGPSB-DNVCBOLYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 473.47, ALogP 5.05, HBA 5, HBD 1, PSA 71.01, QED 0.53
Compound (SMILES): O=C(O)C1CC=C(c2nn(C(=O)c3c(Cl)cccc3C3(C(F)(F)F)CC3)c3c2CCC(C(=O)N[C@@H]2COC[C@@H]2F)C3)CC1
InChIKey: YLUOHJPXIPEXKD-RIBBAFADSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay for RORgammaT Activity: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 mg/ml bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per ml in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 ml SF9 lysate per 15 ml of diluted RORγ-LBD.Compounds to be tested were injected to the 384-well assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, CA).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-SPSSHSSLTERHKILHRLLQEGSP) (SEQ ID NO: 1) and APC-conjugated streptavidin (final concentrations 100 nM and 8 nM respectively) were also added to each well.The final assay mixture was incubated overnight at 4° C., warmed to room temperature and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 tis, integration time=200 tis). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 624.03, ALogP 5.05, HBA 6, HBD 2, PSA 110.52, QED 0.44
Compound (SMILES): CNCC(=O)N1CCC(c2ccc3[nH]c(-c4cc(OC)c5ncnn5c4)c(C(C)C)c3c2)CC1
InChIKey: XWUFQIKVPDASRW-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 460.58, ALogP 3.94, HBA 6, HBD 2, PSA 87.55, QED 0.46
Compound (SMILES): C/C=C/C[C@@H](C)[C@@H](O)[C@H]1C(=O)N[C@@H](CC)C(=O)N(C)[C@H](C)C(=O)N(C)[C@@H]([C@H](C)CCN2CCN(CCOC)CC2)C(=O)N[C@@H](C(C)C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@H](C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N(C)[C@@H](C(C)C)C(=O)N1C
InChIKey: PRSMSTPTQBLJFI-VCWNVUDTSA-N
Target: Peptidyl-prolyl cis-trans isomerase A (SINGLE PROTEIN), Homo sapiens
UniProt: P62937
Assay: Surface Plasmon Resonance (SPR) Assay: Binding of inhibitors to expressed cyclophilins was determined using surface plasmon resonance (SPR) experiments. Briefly, avi-tagged cyclophilin proteins with mono-biotinylation were immobilized onto a Biotin CAPture chip (GE Healthcare, cat. #28920234). SPR experiments were carried out on an upgraded Biacore T200 system using a running buffer containing the inhibitor, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P20, and 3% DMSO. Single-cycle kinetics measurements were used to study the interactions between cyclophilin inhibitors and different cyclophlins. Accordingly, compound at various concentrations was injected to the flow cells with a contact time of 1 min each at a flow rate of 50 μL/min. The final dissociation phase lasts for 10 min following the sample injection. Data was analyzed using Biacore T200 Evaluation Software Version 1.0 and a 1:1 binding model was applied to fit the data to obtain kon, koff, and KD.
Measurement: Kd = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 1358.86
Compound (SMILES): CCOC(=O)c1cn(-c2ccc(-c3nnc(N)o3)cc2)c(=O)n(Cc2cccc(C(F)(F)F)c2C)c1=O
InChIKey: OIZXYDFSZRFTHG-UHFFFAOYSA-N
Target: Chymase (SINGLE PROTEIN), Homo sapiens
UniProt: P23946
Assay: Enzymatic Assay : The enzyme source used is recombinant human chymase (expressed in HEK293 cells) or chymase purified from hamsters' tongues. The substrate used for chymase is Abz-HPFHL-Lys(Dnp)-NH2. For the assay, 1 μl of a 50-fold concentrated solution of test substance in DMSO, 24 al of enzyme solution (dilution 1:80 000 human or 1:4000 hamster) and 25 al of substrate solution (final concentration 10 μM) in assay buffer (Tris 50 mM (pH 7.5), sodium chloride 150 mM, BSA 0.10%, Chaps 0.10%, glutathione 1 mM, EDTA 1 mM) are combined in a white 384-hole microtiter plate (Greiner Bio-One, Frickenhausen, Germany). The reaction is incubated at 32 degrees for 60 min and the fluorescence emission at 465 nm after excitation at 340 nm is measured in a fluorescence reader, for example Tecan Ultra (Tecan, Männedorf, Switzerland).
Measurement: IC50 = 580 nM (pChEMBL 6.239999771118164)
Descriptors: MW 515.45, ALogP 3.18, HBA 10, HBD 1, PSA 135.24, QED 0.39
Compound (SMILES): CCOC(=O)N1CC/C(=C\C#Cc2cc(F)ccc2F)C(C)(C)C1
InChIKey: UEYFUTHRQLEZHM-VIZOYTHASA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Radioligand Binding Assay at Cloned Human mGluR5 Receptor Subtype: Affinity at transmembrane glutamate metabotropic cloned human mGluR5 receptor was evaluated according to the methods of Anderson (Anderson et al., J Pharmacol. Exp. Ther., (2002), Vol. 303(3), pp. 1044-51), with some modifications. Human cloned mGluR5 was obtained by re-suspending CHO T-REx h-mGluR5 cells (50 μg/well) in 20 mM HEPES, 2 mM MgCl2, 2 mM CaCl2, pH 7.4, that then were incubated in a final volume of 1 ml for 60 min at 25° C. with 4 nM [3H]MPEP in the absence or presence of competing drugs. Non-specific binding was determined in the presence of 10 μM MPEP. The incubation was stopped by addition of cold Tris buffer pH 7.4 and rapid filtration through 0.2% polyethyleneimine pretreated Filtermat 1204-401 (Perkin Elmer) filters. The filters were then washed with cold buffer and the radioactivity retained on the filters was counted by liquid scintillation spectrometry (Betaplate 1204 BS-Wallac).
Measurement: Ki = 0.89 nM (pChEMBL 9.050000190734863)
Descriptors: MW 333.38, ALogP 4.13, HBA 2, HBD 0, PSA 29.54, QED 0.72
Compound (SMILES): COCc1ccc(NC(=O)c2c(C#Cc3cncc(C#N)c3)n(C3(C)CC3)c3ncnc(N)c23)cc1
InChIKey: OQPFUWAMXGSTNL-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Measurement of Inhibitory Activity Against RET (In Vitro): Regarding the conditions for measurement of in vitro inhibitory activity of compounds against RET kinase activity, the website of AnaSpec states that Srctide (GEEPLYWSFPAKKK) corresponds to the substrate peptide for reaction to measure RET kinase activity. Thus, the amino acid sequence was partly modified and biotinylated to prepare biotinylated peptides (biotin-EEPLYWSFPAKKK). The purified recombinant human RET protein used in the test was purchased from Carna Biosciences, Inc.To measure the inhibitory activity, first, the compounds of the present invention were individually diluted with dimethyl sulfoxide (DMSO) stepwise. Subsequently, RET protein, the substrate peptide (the final concentration: 250 nM), magnesium chloride (the final concentration: 10 mM), ATP (the final concentration: 10 μM), and a solution of the compound of the present invention in DMSO (the final concentration of DMSO: 2.5%) were added to a buffer for kinase reaction (13.5 mM Tris, pH of 7.5, 2 mM dithiothreitol, 0.009% Tween-20). Each of the mixtures was incubated at 25° C. for 100 minutes to perform a kinase reaction. EDTA was then added thereto to give a final concentration of 24 mM so that the reaction was terminated. A detection solution containing Eu-labeled antiphosphotyrosine antibody PT66 (PerkinElmer) and SureLight APC-SA (PerkinElmer) was added thereto, and each mixture was allowed to stand at room temperature for 2 hours or more. Finally, the intensity of fluorescence under the excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at the two wavelengths of 620 nm and 665 nm. The phosphorylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which phosphorylation was inhibited by 50% was defined as the IC50 value (nM).
Measurement: IC50 = 0.3 nM (pChEMBL 9.520000457763672)
Descriptors: MW 477.53, ALogP 3.59, HBA 8, HBD 2, PSA 131.74, QED 0.42
Compound (SMILES): CC(Nc1ccc(C#N)n(C)c1=O)c1cc2cc(Cl)c(OCc3ccccn3)cc2[nH]c1=O
InChIKey: PJBUFTGEHWYEER-UHFFFAOYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: In Vitro Biochemical Assay: Please see paper.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 461.91, ALogP 3.9, HBA 7, HBD 2, PSA 112.8, QED 0.45
Compound (SMILES): Cc1nn(C(C)(C)C#N)cc1Nc1nc(C2CC2)c2cc[nH]c2n1
InChIKey: CLIYBEGBFHJLFI-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Biochemical Assay : 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT, 5 nM LRRK2, 134 μM ATP, 60 minute reaction time, 23° C. reaction temperature, and 10 μL total reaction volume.Detection Reaction Conditions:1×TR-FRET dilution buffer, 10 mM EDTA, 2 nM antibody, 23° C. reaction temperature, and 10 μL total reaction volume.Compound solutions were prepared by initially diluting to 1 mM with DMSO. 35 μL of reference compound solution, 35 μL of test compound solution, and 35 μL HPE were successively added to the source plate (384-well assay plate, Labcyte). The plates were centrifuged at 2500 rpm for 1 minute and sealed in foil. POD was used to perform a 3.162 fold serial dilution and 100 nL of reference compound solution, test compound solution, HPE and ZPE were transferred to assay plates. The assay plate was centrifuged at 2500 rpm for 1 minute, and sealed with foil.
Measurement: IC50 > 60 nM (pChEMBL 7.2220001220703125)
Descriptors: MW 321.39, ALogP 3.34, HBA 6, HBD 2, PSA 95.21, QED 0.77
Compound (SMILES): COc1ncccc1-c1nc2c(n1C(C)C)C(c1ccc(Cl)cc1)N(c1cc(C)c3nnc(CF)n3c1)C2=O
InChIKey: NEOVBISHUWMCQM-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In-Vitro Binding Assay: All assays were performed in 384 well microtiter plates. Each assay plate contained 8-point serial dilutions for 40 test compounds, plus 16 high- and 16 low controls. Liquid handling and incubation steps were done on an Innovadyne Nanodrop Express equipped with a robotic arm (Thermo CatX, Perkin Elmer/Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO HummingBird nanodispenser (Zinsser Analytic). The assay was started by stepwise addition of 4.5 ul per well of bromo domain protein (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 45 nM His-Brd2(60-472) or 45 nM His-Brd3(20-477) or 45 nM His-Brd4(44-477) all proteins produced in-house) and 4.5ul per well of peptide solution (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 60 nM acetyl-histone H4 (AcK 5, 8, 12, 16) (Biosyntan GmbH)). Reactions were incubated at 30° C. for 35 minutes. Subsequently 4.5 ul per well detection mix (50 mM HEPES, pH 7.5, 0.005% Tween20, 0.1% BSA, 50 mM NaCl, 3 nM Eu-labeled anti-His6 antibody, 21 nM streptavidin-allophycocyanin) were added. After 35 minutes incubation at 30° C., plates were measured in a Perkin Elmer EnVision multilabel reader. Concentrations causing 50% inhibition (IC50 values) were determined from percent inhibition values at different compound concentrations by non-linear regression analysis.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 546.01, ALogP 5.76, HBA 8, HBD 0, PSA 90.44, QED 0.27
Compound (SMILES): Cc1nc2nonc2c(N)c1Cc1ccc(Br)c(NCCC(F)(F)F)n1
InChIKey: NYLZEQKDDLTFMM-UHFFFAOYSA-N
Target: Membrane-bound ghrelin O-acyltransferase MBOAT4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96T53
Assay: hGOAT Activity in HEK293 Cells after Incubation: Cells are plated with a density of 5000 cells/well in 384-well poly-D-lysin plates and incubated for 1 day at 37° C., 5% CO2 in DMEM medium, 10% FCS, 1×NEAA, Puromycin (0.5 μg/ml) and G418 (1 mg/ml). Then the medium is changed to a identical medium without FCS and containing Octanoate-BSA (final concentration 100 μM each) and compound in DMSO (final DMSO concentration 0.3%). After incubation for 5 hours acylghrelin in the medium is measured by ELISAThe medium sample is diluted 1:25 in Elisa buffer, A 25 ul aliquot is transferred to a 384-well ELISA plate previously washed 4 times with 100 μL wash buffer, and 25 μl tracer-solution is added. After incubation overnight (20 h) at 4° C. temperature the plate is washed 4 times with 100 μl wash-buffer per well. Finally 50 μl Ellman's reagent is added to each well and the plate is incubated in the dark for 20 minutes. The absorbance is measured at 405 nm in an Envision multilabel reader and the amount of acylated ghrelin is calculated according to a acylated ghrelin standard curve provided in the same plate.Each assay plate contains wells with vehicle controls (1% DMSO) for the measurement of non-inhibited transfer reaction (=100% Ctl) and wells with 10 μM ([Dap3]-Ghrelin) as controls for fully inhibited GOAT enzymeThe analysis of the data is performed by calculation of the percentage of acyl-ghrelin produced in the presence of test compound compared to the amount of acyl-ghrelin produced in the vehicle control samples. An inhibitor of the GOAT enzyme will give values between 100% CTL (no inhibition) and 0% CTL (complete inhibition). IC50 values are calculated with Assay Explorer or other suited software based on curve fitting of results of 8 different compound concentrations.
Measurement: IC50 = 0.24 nM (pChEMBL 9.619999885559082)
Descriptors: MW 431.22, ALogP 3.62, HBA 7, HBD 2, PSA 102.75, QED 0.64
Compound (SMILES): Cc1nc2c(c(C3CC3)nn2[C@@H](C)c2ccc(C(F)(F)F)nc2)c(=O)[nH]1
InChIKey: ICUBKVAGXAKIKJ-QMMMGPOBSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: IMAP TR-FRET PDE2A assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A 1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 120 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 363.34, ALogP 3.33, HBA 5, HBD 1, PSA 76.46, QED 0.77
Compound (SMILES): O=C(NS(=O)(=O)c1ccc(NCC2CCOCC2)c([N+](=O)[O-])c1)c1ccc(C2CCC(N3CCC[C@H]3c3cccc(Cl)c3)CC2)cc1Oc1cnc2[nH]ccc2c1
InChIKey: SFRKJBCLZUCXAC-FMKFJFLSSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X Fluorescence Polarization Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human 2.7 nM Bcl-2/1.3 nM Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 1 μM for Bcl-2 assay, and 10 μM or 1 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 ml potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK (FITC)INR-amide (1 nM for Bcl-2, 0.82 nM for Bcl-xl) was added to plate and further incubated at room temperature for 0.5 h. The FP signals (485 nm-520 nm-520 nm) were read on BMG PHERAstar FS or BMG PHERAstar FSX instalment. The inhibition percentage of Bcl-2/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 813.38, ALogP 8.74, HBA 10, HBD 3, PSA 168.79, QED 0.08
Compound (SMILES): CC(C)[C@H](N[C@H]1CC[C@H](C(=O)N2Cc3cccnc3Nc3ccc(N4CCOCC4)cc32)CC1)C(F)(F)F
InChIKey: UVRKFQPHSDRJQZ-OWHZGTRUSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Enzymatic Assay: Each test compound (10 mM stock in DMSO) is diluted in DMSO to make a 10-point, 3-fold dilution series. 125 nL of each dilution or DMSO alone is dispensed to a 384-well Greiner Lumitrac 200 assay plate using an Echo® Liquid Handler. To each well of the plate is added 20 uL of enzyme in assay buffer or assay buffer alone. Assay buffer consists of 50 mM sodium phosphate, pH 7.0, 50 mM magnesium chloride, 50 mM sodium chloride, and 0.01% (w/v) bovine serum albumin. When present, the R132H mutant IDH1 enzyme is at a working concentration of 1.875 nM (final concentration in assay of 1.5 nM). The assay plate is allowed to incubate for 30 minutes at room temperature and 5 uL of 5× substrate mixture (2.5 uM nicotinamide adenine dinucleotide phosphate, 100 uM adenosine diphosphate, 7.5 mM glyceraldehyde-3-phosphate, 7.5 ug/mL of spinach glyceraldehyde-3-phosphate dehydrogenase, 25 nM phosphoglycerate kinase, and 5 mM alpha-ketoglutarate in assay buffer) is added to all wells. The reaction plate is incubated for 60 minutes followed by addition of 25 uL of Promega Kinase-GLO reagent to all wells and 10-minute incubation.Luminescence is measured using a PerkinElmer Envision plate reader. The percent activity of each dilution is determined as the ratio of background corrected signal to the background corrected signal of wells receiving only DMSO. IC50 values are determined by fitting percent activity data to a four-parameter logistic dose response equation.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 531.62, ALogP 5.24, HBA 6, HBD 2, PSA 69.73, QED 0.55
Compound (SMILES): C[C@H]1CN(C(=O)CCC2(c3cccnc3)NC(=O)NC2=O)CCN1c1ccc(Cl)c(F)c1
InChIKey: JVYZFADRQWLBNU-XLEXHMCLSA-N
Target: A disintegrin and metalloproteinase with thrombospondin motifs 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UNA0
Assay: In Vitro Assay: ADAMTS-5:Protocol 2: The basis for the assay is the cleavage of the substrate TBIS-1 (5 FAM-TEGEARGSVILLK (5TAMRA)K-NH2) (SEQ ID No 2) by human ADAMTS-5.For the dose response (10 point), 4 μL of a dilution series of compound (2 mM highest concentration, 1/5 dilution in DMSO further diluted 1 in 10 in water, corresponding to a final highest concentration of 20 μM), is transferred to 384 well Fluotrac 200 plate (Greiner, cat #781076) and incubated at room temperature for 30 min with a 26 μL buffer solution (50 mM Hepes pH7.5, 100 mM NaCl, 5 mM CaCl2, 0.1% CHAPS 1) containing hADAMTS-5 (1 ng/μL, affinity purified, followed by overnight digestion of 6His tag by thrombin and dialysis) (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration).The reaction is initiated by adding to the assay plate TBIS-1 (10 μL, 4.5 μM, Anaspec) in the same buffer.Finally, the fluorescence is read on the Envision (Perkin Elmer) after an incubation of 45 min at 37° C. (Excitation 485 nm, emission 530).
Measurement: IC50 = 42 nM (pChEMBL 7.380000114440918)
Descriptors: MW 459.91, ALogP 2.43, HBA 5, HBD 2, PSA 94.64, QED 0.67
Compound (SMILES): COCC(O)Cn1cc(C(=O)Nc2ccc(-c3n[nH]c(=O)c4ccccc34)cc2)cn1
InChIKey: FMXFIMYBYJPBOM-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 419.44, ALogP 2.05, HBA 7, HBD 3, PSA 122.13, QED 0.42
Compound (SMILES): CN(C)Cc1nc(Nc2ccc(-c3cnc4cc(F)ccn34)c3c2C(=O)NC3)ccc1N1CCOC[C@H](O)C1
InChIKey: KLAVGPJWKWRHDG-GOSISDBHSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 l/well termination buffer (Carna Biosciences) and signal determination using aCaliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 531.59, ALogP 2.78, HBA 9, HBD 3, PSA 107.26, QED 0.35
Compound (SMILES): COC(=O)c1ccc2c(c1)c(-c1cccc(S(=O)(=O)N(C)C)c1)c(C)n2C/C(F)=C/CN
InChIKey: NRSYNKVPOCKDIH-ZDLGFXPLSA-N
Target: Lysyl oxidase homolog 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y4K0
Assay: Inhibition Assay: LOX and LOXL2: Lysyl oxidase (LOX) is an extracellular copper dependent enzyme which oxidizes peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. This catalytic reaction can be irreversibly inhibited by β-aminopropionitrile (BAPN) that binds to the active site of LOX (Tang S. S., Trackman P. C. and Kagan H. M., Reaction of aortic lysyl oxidase with beta-aminoproprionitrile. J Biol Chem 1983; 258: 4331-4338). There are five LOX family members; these are LOX, LOXL1, LOXL2, LOXL3 and LOXL4. LOX and LOXL family members can be acquired as recombinant active proteins from commercial sources, or extracted from animal tissues like bovine aorta, tendons, pig skin; or prepared from cell cultures. The inhibitory effects of the compounds of the present invention were tested against the given LOX-LOXL preparation using a high-throughput coupled colorimetric method (Holt A. and Palcic M., A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat. Protoc. 2006; 1: 2498-2505). The assay was developed using either 384 or 96 well format. Briefly, in a standard 384 well plate assay 25 μL of a dilution of any of the isoenzymes and orthologues in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were added into each well in the presence of 1 μM mofegiline and 0.5 mM pargyline (to inhibit SSAO and MAO-B and MAO-A, respectively). Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 11 data points, typically in the micromolar or nanomolar range after incubation with the enzyme for 30 min at 37° C. Twenty five L of a reaction mixture containing twice the KM concentration of putrescine (Sigma Aldrich, e.g. 20 mM for LOX, or 10 mM for LOXL2 and LOXL3), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were then added to the corresponding wells. The above volumes were doubled in the case of 96 wells plate.
Measurement: IC50 < 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 459.54, ALogP 3.47, HBA 6, HBD 1, PSA 94.63, QED 0.55
Compound (SMILES): Nc1cc(Br)c(-c2ccnc(F)c2)c(-c2ccncc2)n1
InChIKey: GURBAXXWOCKMDW-UHFFFAOYSA-N
Target: Adenosine receptor A2b (SINGLE PROTEIN), Homo sapiens
UniProt: P29275
Assay: Adenosine A2B Receptor Subtype Competition Radioligand Binding Assay: The binding assay for adenosine A2B receptor subtype was carried out on human recombinant source (HEK-293 cells) and [3H]DPCPX as radioligand, according to assay disclosed by Fredholm et al. (International Union of Pharmacology. XXV. Nomenclature and classification of adenosine receptors, Pharmacol Rev. 2001December; 53(4):527-52).
Measurement: Ki = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 345.18, ALogP 3.69, HBA 4, HBD 1, PSA 64.69, QED 0.72
Compound (SMILES): Cn1cc(-c2cnc(N)c(C(=O)N[C@H]3CCC[C@@H]3OCc3cccc(C4CCCC4)c3)c2)cn1
InChIKey: RPHAHGRSQTZHAE-DQEYMECFSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: TBD: TBD
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 459.59, ALogP 4.59, HBA 6, HBD 2, PSA 95.06, QED 0.54
Compound (SMILES): Cc1c(Cl)cccc1Nc1nc(=O)c(CC(=O)NC2COC2)cn1Cc1ccccc1
InChIKey: XYWNTQFBWAVGQJ-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 438.92, ALogP 3.05, HBA 6, HBD 2, PSA 85.25, QED 0.59
Compound (SMILES): CCc1cc(C(N)=O)ccc1-c1ccc2cc(-c3nc4cc(C(=O)N5CC6CCC5[C@@H]6N)cc(OC)c4n3C)n(CC3CC3)c2n1
InChIKey: ORALVNWJRJMLLQ-SGAJGPLNSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 148 nM (pChEMBL 6.829999923706055)
Descriptors: MW 617.75, ALogP 4.9, HBA 8, HBD 2, PSA 134.29, QED 0.25
Compound (SMILES): Cn1cc(Nc2ncnc(-c3ccc4c(c3)CCCC[C@H]4NC(=O)c3nc(C(C)(C)C)no3)n2)cn1
InChIKey: KFHCKKGBZAIHBI-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: In Vitro Kinase Assay: The purpose of the BTK in vitro assay is to determine compound potency against BTK through the measurement of IC50. Compound inhibition is measured after monitoring the amount of phosphorylation of a fluorescein-labeled polyGAT peptide (Invitrogen PV3611) in the presence of active BTK enzyme (Upstate 14-552), ATP, and inhibitor. The BTK kinase reaction was done in a black 96 well plate (costar 3694). For a typical assay, a 24 pL aliquot of a ATP/peptide master mix (final concentration; ATP 10 kM, polyGAT 100 nM) in kinase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 200 μM Na3PO4, 5 mM DTT, 0.01% Triton X-100, and 0.2 mg/ml casein) is added to each well. Next, I pL of a 4-fold, 40× compound titration in 100% DMSO solvent is added, followed by adding 15 μL of BTK enzyme mix in 1× kinase buffer (with a final concentration of 0.25 nM). The assay is incubated for 30 minutes before being stopped with 28 μL of a 50 mM EDTA solution. Aliquots (5 μL) of the kinase reaction are transferred to a low volume white 384 well plate (Corning 3674), and 5 μL of a 2× detection buffer (Invitrogen PV3574, with 4 nM Tb-PY20 antibody, Invitrogen PV3552) is added. The plate is covered and incubated for 45 minutes at room temperature. Time resolved fluorescence (TRF) on Molecular Devices M5 (332 nm excitation; 488 nm emission; 518 nm fluorescein emission) is measured. IC50 values are calculated using a four parameter fit with 100% enzyme activity determined from the DMSO control and 0% activity from the EDTA control.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 487.57, ALogP 3.89, HBA 10, HBD 2, PSA 136.54, QED 0.4
Compound (SMILES): O=C(O)c1ccc(-c2noc(C34CCC(OCc5c(-c6ccccc6C(F)(F)F)noc5C5CC5)(CC3)CC4)n2)cn1
InChIKey: ULQXKUPCZCEAAN-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/ml Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 2700 nM (pChEMBL 5.570000171661377)
Descriptors: MW 580.56, ALogP 6.94, HBA 8, HBD 1, PSA 124.37, QED 0.24
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2cc(C3CC3)nn2-c2ccccc2)[C@H](c2ccccc2)C1
InChIKey: RJDHMHQMANTTFA-LADGPHEKSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 445.57, ALogP 3.99, HBA 5, HBD 2, PSA 71.42, QED 0.55
Compound (SMILES): Cc1cc(N2C[C@@H](C)n3c(c(CCCOc4cc(C)c(Cl)c(C)c4)c4ccc(Cl)c(-c5c(C)nn(C)c5C)c43)C2=O)c2c(c1)cc(C(=O)O)n2CC(=O)N1CCOCC1
InChIKey: QWPGTIXCDBCSLN-HHHXNRCGSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: TR-FRET Binding Assay: Measurements were carried out in OptiPlate-384 White Opaque 384-well plates (Perkin Elmer. Shelton, Conn.). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification, 300 nM FITC-Bak peptide, 1 nM recombinant Maltose Binding Protein (MBP) tagged Mcl-1 (residues 172-327) and 1 nM terbium conjugated-MBP antibody were added to assay buffer (4.5 mM Monobasic potassium phosphate, 15.5 mM Dibasic potassium phosphate, 1 mM Sodium EDTA, 50 mM NaCl, 1 mM DTT, 0.05% Pluronic F-68, pH 7.5). Compounds are diluted in DMSO in a 16-point, semilog serial dilution scheme. For the BAK FITC TR-FRET assay 50 nL compound is added to 20 μL of assay buffer containing Bak-FITC. MBP tagged Mcl-1 protein and antibody, for a final DMSO concentration of 0.25% and a top concentration of 10 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 3 hours at room temperature. TR-FRET signal (Delta F) is measured on a Biotek Cytation 3 multimode plate reader equipped with a filter cube containing an Ex 340/30 Em 620/10 nm filter and an Ex 340/30 Em 520 filter.
Measurement: Ki < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 839.82, ALogP 8.64, HBA 9, HBD 1, PSA 124.06, QED 0.14