lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nc([C@](C)(O)C#Cc2ccc3c(c2)-c2nc(C(N)=O)sc2[C@H](F)CO3)no1
InChIKey: VCPRVLNKTAUZOQ-CWTRNNRKSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5'-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5'-diphosphate) assay (BellBrook Labs). Purified NIK (0.2-1 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl.sub.2, 2 mM dithiothreitol, 10 uM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 12 ug/mL ADP antibody and 4 nM ADP-AlexaFluor.RTM. 633 tracer. Reactions were quenched by the addition of 20 mM 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation.
Measurement: Ki = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 414.42, ALogP 2.26, HBA 8, HBD 2, PSA 124.36, QED 0.62
Compound (SMILES): Cc1cccc(C)c1NC(=O)N1CC(CC#N)(n2cc(C(N)=O)c(Nc3ccccc3)n2)C1
InChIKey: YFHNRZFFMXEAJD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 656 nM (pChEMBL 6.179999828338623)
Descriptors: MW 443.51, ALogP 3.5, HBA 6, HBD 3, PSA 129.07, QED 0.54
Compound (SMILES): C[C@@H]1CNCCN1S(=O)(=O)c1ccccc1-c1ccc(-c2cnc(N)cn2)c(F)c1
InChIKey: UJGKCRSGLVSQSQ-CQSZACIVSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: Homogeneous Time Resolved Fluorescence Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBacl (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator [5-[({[2-(2-{3-[3-(tert-Butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid]. The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 ug/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid].
Measurement: Ki = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 427.51, ALogP 2.51, HBA 6, HBD 2, PSA 101.21, QED 0.66
Compound (SMILES): CCOn1c(N[C@H](C)c2ccc(C(F)(F)F)cc2)nc2c(c1=O)CN(C(=O)c1ccc(-c3cnco3)cc1)CC2
InChIKey: ADBZYMOKGJVIQN-QGZVFWFLSA-N
Target: Prolactin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P49683
Assay: Intracellular Calcium Mobilization Assay: HEK293 cells expressing human GPR10 were maintained in Dulbecco's Modified Eagles' medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 20 mM HEPES at 37° C. in a 5% CO2 incubator. GPR10-expressing HEK293 cells were placed in poly-D-lysine coated 96-well culture plates and cultured for 18-24 hr before the test at a density of 3×104 cells/well. The cells were incubated with 2.5 nM Fluo-8 for 1 hr at room temperature in Recording Buffer containing 1% bovine serum albumin, 0.01% pluronic F-127 and 20 mM HEPES, Hanks Balanced Buffered Saline, pH 7.4. The cells were incubated with test compounds for 15 min at room temperature, and then PrRP (the final concentration, 1 nM) was added into the medium. The changes in intracellular calcium-dependent fluorescence were monitored using a fluorescence imaging plate reader (FDSS3000, Hamamatsu Photonics K.K.). Fluo-8 fluorescence was measured with excitation at 490 nm and emission at 520 nm.
Measurement: IC50 = 158 nM (pChEMBL 6.800000190734863)
Descriptors: MW 553.54, ALogP 4.74, HBA 8, HBD 1, PSA 102.49, QED 0.35
Compound (SMILES): C[C@@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1ncc(-c2ccc(Cl)c(Cl)c2)cn1
InChIKey: FZJHBXOYJXRIDM-PSTGCABASA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 524 nM (pChEMBL 6.28000020980835)
Descriptors: MW 475.34, ALogP 4.12, HBA 8, HBD 2, PSA 113.36, QED 0.55
Compound (SMILES): Cc1c([C@H](O)CN2CC3CCN(C[C@@H](O)c4ccc5c(c4C)COC5=O)C3C2)ccc2c1COC2=O
InChIKey: AYKTVDFUHYNDJO-VGIKYBBCSA-N
Target: ATP-sensitive inward rectifier potassium channel 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P48048
Assay: Electrophysiology Assay: Block of Kir1.1 (ROMK1) currents was examined by whole cell voltage clamp (Hamill et. al. Pfluegers Archives 391:85-100 (1981)) using the IonWorks Quattro automated electrophysiology platform (Molecular Devices, Sunnyvale, Calif.). Chinese hamster ovary cells stably expressing Kir1.1 channels were maintained in T-75 flasks in cell culture media in a humidified 10% CO2 incubator at 37° C. Prior to an experiment, Kir1.1 expression was induced by overnight incubation with 1 mM sodium butyrate. On the day of the experiment, cells were dissociated with 2.5 ml of Versene (Invitrogen 15040-066) for approximately 6 minutes at 37° C. and suspended in 10 ml of bath solution containing (in mM): 150 NaCl, 10 KCl, 2.7 CaCl2, 0.5 MgCl2, 5 HEPES, pH 7.4. After centrifugation, the cell pellet was resuspended in approximately 4.0 ml of bath solution and placed in the IonWorks instrument. The intracellular solution consisted of (in mM): 80 K gluconate, 40 KCl, 20 KF, 3.2 MgCl2, 3 EGTA, 5 Hepes, pH 7.4. Electrical access to the cytoplasm was achieved by perforation in 0.13 mg/ml amphotericin B for 4 minutes. Amphotericin B (Sigma A-4888) was prepared as a 40 mg/ml solution in DMSO.
Measurement: IC50 = 117.3 nM (pChEMBL 6.929999828338623)
Descriptors: MW 492.57, ALogP 2.42, HBA 8, HBD 2, PSA 99.54, QED 0.59
Compound (SMILES): CC(CCO)Nc1cc(-n2ccc3cc(C#N)cnc32)ncc1C(=O)NC[C@@H](F)C(C)(C)O
InChIKey: NSCFZAOLQWGDEU-JANGERMGSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 L prepared from 15 L additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij 35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction mixture was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 454.51, ALogP 2.31, HBA 8, HBD 4, PSA 136.09, QED 0.39
Compound (SMILES): O=C(Nc1ccc(OC(F)(F)F)cc1)N1CCC(F)(c2ncc([C@H](O)CO)cc2Cl)CC1
InChIKey: WSPUTZHMMTZHDI-MRXNPFEDSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 ("wash buffer"), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 ("assay buffer"). Plates are then transferred to a FLIPR for assay. The test compound is diluted in assay buffer, and 50 μL of the resultant solution is added to the cell plates and the solution is monitored for two minutes. The final concentration of the test compound is adjusted to range from about 50 picoM to about 3 μM. Agonist buffer (wash buffer titrated with 1N HCl to provide a solution having a pH of 5.5 when mixed 1:1 with assay buffer) (0.1 mL) is then added to each well, and the plates are incubated for 1 additional minute.
Measurement: IC50 = 11.3 nM (pChEMBL 7.949999809265137)
Descriptors: MW 477.84, ALogP 4.15, HBA 5, HBD 3, PSA 94.92, QED 0.56
Compound (SMILES): O=C1CCc2c(Oc3ccc4c(c3)[C@@H]3C(c5nc6ccc(C(F)(F)F)cc6[nH]5)[C@@H]3O4)ccnc2N1
InChIKey: NGFFVZQXSRKHBM-NHXNGRAPSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 8.8 nM (pChEMBL 8.0600004196167)
Descriptors: MW 478.43, ALogP 5.3, HBA 5, HBD 2, PSA 89.13, QED 0.42
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)[C@H](C)CCC[C@H](N1CCC(c3nccc(Cl)c3F)=CC1=O)c1cc-2ccn1
InChIKey: XXPYQZDZFMYLHR-NSYGIPOTSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 578.04, ALogP 6.23, HBA 6, HBD 2, PSA 113.52, QED 0.38
Compound (SMILES): C[C@@H]1CCC[C@H](N2CCC(c3c(F)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-n2nc(C(=O)O)cc2NC1=O
InChIKey: NPHHNMJIOHQPCH-SZNDQCEHSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 501.2 nM (pChEMBL 6.300000190734863)
Descriptors: MW 555.97, ALogP 5.01, HBA 6, HBD 2, PSA 117.42, QED 0.44
Compound (SMILES): Cc1nc(N)ncc1-c1nc(N2CCOCC2)nc2c(CN3CCN(C(=O)[C@@H](C)O)CC3)csc12
InChIKey: QVYPYOVXCSHCCB-OAHLLOKOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzymatic Inhibitory Activity Assay:   1. Buffer preparation: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS.  2. Compound was formulated in 100% DMSO in a concentration gradient, deposited to a 384-well plate to make final DMSO concentration of 1%.  3. PI3Kα and PI3Kδ enzymes were diluted to be the optimum concentration with the following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT. Transferred to a 384-well plate and incubated with the compound for a certain time.  4. Substrate was diluted to an optimum concentration with following buffer: 50 mM HEPES, pH 7.5, 3 mM MgCl2, 1 mM EGTA, 100 mM NaCl, 0.03% CHAPS, 2 mM DTT, 50 μM PIP2, Km ATP. The reaction was performed in a 384-well plate for 1 h at room temperature for PI3Kα and 2 hrs at room temperature for PI3Kδ.  5. Read the conversion rate using Caliper Reader, and calculate the inhibition rate as the average of two tests.
Measurement: IC50 = 4.7 nM (pChEMBL 8.329999923706055)
Descriptors: MW 498.61, ALogP 0.9, HBA 11, HBD 2, PSA 133.83, QED 0.52
Compound (SMILES): C[C@@H]1c2nnn(-c3ccccn3)c2CCN1C(=O)c1ccc(F)c(Cl)c1Cl
InChIKey: MDWQVJGLGUXAKF-SNVBAGLBSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: FLIPR Assay: 1321 N1 cells expressing the recombinant human, rat or mouse P2X7 channel was cultured in HyQ DME/(HyClone/Dulbecco's Modified Eagle Medium) high glucose supplemented with 10% Fetal Bovine Serum (FBS) and appropriate selection marker. Cells were seeded at a density of 25000 cells/well (96-well clear bottom black walled plates) in 100 ul volume/well. On the day of the experiment, cell plates were washed with assay buffer, containing (in mM): 130 NaCl, 2 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, 5 glucose; pH 7.40 and 300 mOs. After the wash, cells were loaded with the Calcium-4 dye (Molecular Device) and incubated in the dark for 60 minutes. Test compounds were prepared at 250Ã¿ the test concentration in neat DMSO. Intermediate 96-well compound plates were prepared by transferring 1.2 uL of the compound into 300 uL of assay buffer. A further 3x dilution occurred when transferring 50 uL/well of the compound plate to 100 uL/well in the cell plate. Cells were incubateÂ  with test compounds and dye for 30 minutes. Calcium dye fluorescence was monitored in FLIPR as the cells were challenged by adding 50 uL/well of BzATP (final concentration is 250 uM BzATP (human and rat) or 600 (mouse)). The fluorescence change was measured 180 seconds after adding the agonist.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 406.25, ALogP 3.87, HBA 5, HBD 0, PSA 63.91, QED 0.61
Compound (SMILES): CC(=O)N1CC2[C@](c3cc(NC(=O)c4ccc(F)cn4)ccc3F)(C1)NC(=N)N(C)S2(=O)=O
InChIKey: JDCNHYZNJNRUDX-UUSAFJCLSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 HTRF FRET Assay: A homogeneous time-resolved FRET assay was used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitored the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contained an N-terminal QSY7 moiety that served as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence was low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme. Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors was manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3x the final desired concentration in a volume of 10 ul were preincubated with purified human BACE1 catalytic domain (3 nM in 10 ul) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0.
Measurement: Ki = 32 nM (pChEMBL 7.5)
Descriptors: MW 478.48, ALogP 0.84, HBA 6, HBD 3, PSA 135.56, QED 0.6
Compound (SMILES): CNC(=O)[C@@H](NC(=O)c1cccc(-c2cccc(F)c2)n1)C(C)(C)C
InChIKey: YJOVMDYYBGIRNV-MRXNPFEDSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 = 361.1 nM (pChEMBL 6.440000057220459)
Descriptors: MW 343.4, ALogP 2.78, HBA 3, HBD 2, PSA 71.09, QED 0.9
Compound (SMILES): CCC(NC(=O)c1cc(C(=O)N[C@H](CC)c2ccccc2)c2n1CCOC2)c1cnc(OC)nc1
InChIKey: JHFADEAAFZIUAA-VQCQRNETSA-N
Target: Potassium channel subfamily K member 3 (SINGLE PROTEIN), Homo sapiens
UniProt: O14649
Assay: TASK-1 Inhibiition Assay: Human TASK-1 channels were expressed in Xenopus oocytes. For this purpose, oocytes were isolated from Xenopus laevis and defoliculated. Subsequently, TASK-1-encoding RNA synthesized in vitro was injected into the oocytes. After two days of TASK-1 protein expression, TASK-1 currents were measured by two-microelectrode voltage clamp. Data were acquired and analyzed using a TEC-10cx amplifier (NPI Electronic, Tamm, Germany) connected to an ITC-16 interface (Instrutech Corp., Long Island, USA) and Pulse software (HEKA Elektronik, Lambrecht, Germany). Oocytes were clamped to -90 mV and TASK-1 mediated currents were measured during 500 ms voltage pulses to 40 mV. Oocytes were continuously superfused with ND96 buffer containing 96 mM sodium chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM 4-(2-hydroxyethyl)-piperazine-1-ethanesulfonic acid (HEPES; pH adjusted to 7.4 with sodium hydroxide). All experiments were performed at room temperature.
Measurement: IC50 = 760 nM (pChEMBL 6.119999885559082)
Descriptors: MW 477.57, ALogP 3.58, HBA 7, HBD 2, PSA 107.37, QED 0.49
Compound (SMILES): CC(Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1nc(CCc2ccccc2)no1
InChIKey: LXDWPRMFMLFXHK-IGLHTZBQSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 1587 nM (pChEMBL 5.800000190734863)
Descriptors: MW 424.46, ALogP 2.52, HBA 9, HBD 2, PSA 126.5, QED 0.56
Compound (SMILES): COc1ccc2nc(N[C@@H](c3ccc[nH]3)P(=O)(OC)OC)sc2c1
InChIKey: OSYYITDVMDIJJZ-CQSZACIVSA-N
Target: 3-hydroxyacyl-CoA dehydrogenase type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99714
Assay: ABAD Enzymatic Activity Assay: The assay for the inhibition of reduction of S-acetoacetyl-CoA (SAAC) by ABAD was carried out with ABAD (418 ng/ml), SAAC (172 μM), NADH (102 μM), and different concentrations of inhibitors (from 0 to 1000 μM) in 93 mM potassium phosphate buffer (pH 7.3). Before the assay, all the assay components except SAAC were pre-incubated for 5 minutes, and the reaction started with the addition of SAAC into the reaction mix. The reaction was carried out for a total 6 minutes at room temperature under steady-state conditions, and the decrease of NADH absorbance at 340 nm was determined every 10 seconds.
Measurement: IC50 = 34100 nM (pChEMBL 4.46999979019165)
Descriptors: MW 367.37, ALogP 4.23, HBA 7, HBD 2, PSA 85.47, QED 0.61
Compound (SMILES): Cn1c(=O)n([C@@H]2CCCOC2)c2c3cc(-c4ccc(OCCCN5CCCC5)nc4)ccc3ncc21
InChIKey: YXJGKECEJNCEIH-JOCHJYFZSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Cellular Assay: Assay b) HT29 cells (ECACC #85061109) were seeded into 384 well assay plates (Costar #3712) at a density of 6000 cells/well in 40 μl EMEM medium containing 1% L glutamine and 10% FBS and allowed to adhere overnight. The following morning compound of Formula (I) in 100% DMSO was added to assay plates by acoustic dispensing. After 1 h incubation at 37° C. and 5% CO2, 40 nl of 3 mM 4NQO in 100% DMSO was added to all wells by acoustic dispensing, except minimum control wells which were left untreated with 4NQO to generate a null response control. Plates were returned to the incubator for a further 1 h. Then cells were fixed by adding 20 μl of 3.7% formaldehyde in PBS solution and incubating for 20 mins at r.t. Then 20 μl of 0.1% Triton X100 in PBS was added and incubated for 10 minutes at r.t., to permeabalise cells. Then the plates were washed once with 50 μl/well PBS, using a Biotek EL405 plate washer. Phospho-Chk1 Ser 345 antibody (Cell Signalling Technology #2348) was diluted 150 fold in PBS containing 0.05% polysorbate/Tween and 15 μl was added to each well and incubated over night at r.t. The next morning plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and then 20 μl of secondary Ab solution, containing 500 fold diluted Alexa Fluor 488 Goat anti-rabbit IgG (Molecular Probes #A-11008) and 0.002 mg/ml Hoeschst dye (Molecular Probes #H-3570), in PBST, was added. After 2 h incubation at r.t., the plates were washed three times with 50 μl/well PBS, using a Biotek EL405 plate washer, and plates were then sealed with black plate seals until read. Plates were read using an ArrayScan VTI instrument, using an XF53 filter with 10× objective. A two laser set up was used to analyse nuclear staining with Hoeschst (405 nm) and secondary antibody staining of pChk1 (488 nm).
Measurement: IC50 > 19100 nM (pChEMBL 4.718999862670898)
Descriptors: MW 487.6, ALogP 4.17, HBA 8, HBD 0, PSA 74.41, QED 0.36
Compound (SMILES): CCCc1nn(C)c(C)c1NS(=O)(=O)c1ccc(-c2cccc(CN(C)C)c2)cc1
InChIKey: SDTSVFNDBKQQQK-UHFFFAOYSA-N
Target: Glycylpeptide N-tetradecanoyltransferase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30419
Assay: Enzyme Inhibition Assay: Measurements were performed using a modification of the scintillation proximity assay platform described previously by Georgopapadakou, N. H. et al. (22nd International Congress on Chemotherapy, 2001, Abstract P16.001). Compounds were solubilised in DMSO at a top concentration of 10 mM and serially diluted in half log steps to achieve a range of final assay concentrations of 100 uM to 1 nM. Compound at each concentration (100-fold final) was added to white 384 well plates in a volume of 0.5 ml. Human, A. fumigatus, T. brucei or L. major N-myristoyl transferase enzyme, dissolved to a working concentration of 10 nM in assay buffer (30 mM Tris/HCl pH 7.4, 0.5 mM EGTA, 0.5 mM EDTA, 1.25 mM DTT, 0.1% Triton X-100), was then added to columns 1 to 11 and 13 to 23 of the plates in a volume of 20 ml. To columns 12 and 24, 20 ml assay buffer was added to provide a no enzyme control. Following a 5 minute incubation at room temperature the substrates (GCGGSKVKPQPPQAK(Biotin)-Amide and myristoyl coenzyme A), dissolved in assay buffer, were added to all wells in a volume of 20 ml to start the reaction. The final concentrations of peptide and 3H-myristoyl coenzyme A were 0.5 mM and 125 nM respectively and the specific activity of the radiolabel was 8 Ci/mmol. Plates were then incubated at room temperature for up to 50 minutes (dependant upon the period of linearity for the different enzyme species) before SPA beads, suspended to 1 mg/ml in a stop solution (200 mM Phosphoric Acid/NaOH pH 4, 750 mM MgCl2), were added in a volume of 40 ml.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 426.59, ALogP 4.21, HBA 5, HBD 1, PSA 67.23, QED 0.59
Compound (SMILES): CN(C)[C@@H]1CN(c2nc3ccccc3n([C@H]3C[C@H]4CCC[C@@H](C3)N4[C@@H]3C[C@@H]4CCCC[C@@H](C4)C3)c2=O)[C@@H]1C(=O)O
InChIKey: DWHVTNXMIARYFX-IGSKVMNOSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: In Vitro Mu-Opioid Receptor Binding Assay: Radioligand binding assays were conducted using freshly thawed membranes expressing human μ-receptors (Perkin Elmer, Shelton, Conn.). Radioligand dose-displacement binding assays for human μ-opioid receptors used 0.2 nM [3H]-diprenorphine (NEN, Boston, Mass.), with 5-20 mg membrane protein/well in a final volume of 500 μL binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Reactions were carried out in the absence or presence of increasing concentrations of unlabeled naloxone. All reactions were conducted in 96-deep well polypropylene plates for 1-2 hr at about 25 °C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard, Meriden, Conn.) presoaked in 0.5% polyethylenimine using a 96-well tissue harvester (Brandel, Gaithersburg, Md.) followed by performing three filtration washes with 500 μL of ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 2-3 hours. BetaScint scintillation cocktail (Wallac, Turku, Finland) was added (50 μL/well), and plates were counted using a Packard Top-Count for 1 min/well.
Measurement: Ki = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 547.74, ALogP 4.52, HBA 7, HBD 1, PSA 81.91, QED 0.59
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(c5ccccc5)CC[C@@]5(C[C@@H]4COCc4ccccc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: JKXDZBOIGSVIIM-BGMHAWJCSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Radioligand Dose-Displacement Binding Assay: Radioligand dose displacement assays used 0.4 nM [3H]-U69,593 (GE Healthcare, Piscataway, N.J.; 40 Ci/mmole) with 15 μg membrane protein (recombinant κ opioid receptor expressed in HEK 293 cells; in-house prep) in a final volume of 200 μl binding buffer (5% DMSO, 50 mM Trizma base, pH 7.4). Non-specific binding was determined in the presence of 10 μM unlabeled naloxone or U69,593. All reactions were performed in 96-well polypropylene plates for 1 hr at a temperature of about 25° C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Perkin Elmer, Shelton, Conn.) presoaked in 0.5% polyethylenimine (Sigma). Harvesting was performed using a 96-well tissue harvester (Perkin Elmer, Shelton, Conn.) followed by five filtration washes with 2001 ice-cold binding buffer. Filter plates were subsequently dried at 50° C. for 1-2 hours.
Measurement: Ki = 1.07 nM (pChEMBL 8.970000267028809)
Descriptors: MW 547.74, ALogP 6.69, HBA 4, HBD 0, PSA 30.93, QED 0.31
Compound (SMILES): N=C(Nc1cccc(OC[C@H]2CCN[C@@H]2COCc2cccc(F)c2)c1)c1cccs1
InChIKey: HEEDPPMMSPLXOE-XMSQKQJNSA-N
Target: Nitric oxide synthase 3 (SINGLE PROTEIN), Bos taurus
UniProt: P29473
Assay: Inhibition Assay: The compounds were evaluated for in vitro inhibition against three NOS isozymes: rat nNOS, bovine eNOS and marine iNOS using known literature methods. (Hevel, J. M.; Marietta, M. A. Nitric-oxide synthase assays. Method Enzymol. 1994, 233, 250-258).
Measurement: Ki = 5890 nM (pChEMBL 5.230000019073486)
Descriptors: MW 439.56, ALogP 4.9, HBA 5, HBD 3, PSA 66.37, QED 0.33
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccccc3)n2)CN(C(=O)OCCc2ccccn2)C1
InChIKey: WEHGSQQTGDJRGL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 849 nM (pChEMBL 6.070000171661377)
Descriptors: MW 445.48, ALogP 2.42, HBA 8, HBD 2, PSA 139.16, QED 0.54
Compound (SMILES): C[C@@H]1CN(C(=O)C2(C#N)COC2)C[C@@H]1Nc1c(C(N)=O)cnn2cc(-c3cnn(C)c3)nc12
InChIKey: JFVFQHPIFSEZMB-DOMZBBRYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Kinase Assay: The assay reactions were performed in U-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 25 mM beta-glycerol phosphate, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of GST-JAK1 enzyme with substrates and test compounds. The reactions were incubated at room temperature for 3 hours and terminated by adding 60 μl of 35 mM EDTA to each sample. Each reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product Inhibition data were calculated by comparison with no enzyme control reactions for 100% inhibition, and vehicle-only treated reactions for 0% inhibition. The final concentration of reagents in the assays was: ATP, 100 μM; fluoresceinated peptide, 1.5 μM; GST-JAK1, 12.5 nM; and DMSO, 1.6%.
Measurement: IC50 = 12.9 nM (pChEMBL 7.889999866485596)
Descriptors: MW 449.48, ALogP 0.03, HBA 10, HBD 2, PSA 156.46, QED 0.55
Compound (SMILES): CC(C)C[C@](C)(CO)NC(=O)c1cc(OCC2CC2)c(Cl)cn1
InChIKey: QSYHRAPPRQEOJJ-QGZVFWFLSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 340.85, ALogP 3.05, HBA 4, HBD 2, PSA 71.45, QED 0.76
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccc(N(S(C)(=O)=O)S(C)(=O)=O)cc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: FHUJYYXUEXGFOF-SVMMDPMYSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Functional Assay: Functional [35S]GTPgammaS binding assays were conducted as follows. kippa opioid receptor membrane solution was prepared by sequentially adding final concentrations of 0.026 ug/ul kippa membrane protein (in-house), 10 ug/mL saponin, 3 uM GDP and 0.20 nM [35S]GTPgammaS to binding buffer (100 mM NaCl, 10 mM MgCl2, 20 mM HEPES, pH 7.4) on ice. The prepared membrane solution (190 ul/well) was transferred to 96-shallow well polypropylene plates containing 10 ul of 20x concentrated stock solutions of agonist prepared in DMSO. Plates were incubated for 30 min at a temperature of about 25° C. with shaking. Reactions were terminated by rapid filtration onto 96-well Unifilter GF/B filter plates (Perkin Elmer, Shelton, Conn.) using a 96-well tissue harvester (Packard) and followed by three filtration washes with 2001 ice-cold binding buffer (10 mM NaH2PO4, 10 mM Na2HPO4, pH 7.4). Filter plates were subsequently dried at 50° C. for 2-3 hours.
Measurement: EC50 = 276.71 nM (pChEMBL 6.559999942779541)
Descriptors: MW 672.87, ALogP 3.86, HBA 9, HBD 0, PSA 111.68, QED 0.35
Compound (SMILES): CC(C)(C#N)COC(=O)N1CC[C@@H](n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)[C@H](C#N)C1
InChIKey: BPRJZYBIBILSRC-RDTXWAMCSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 411 nM (pChEMBL 6.389999866485596)
Descriptors: MW 453.48, ALogP 2.94, HBA 8, HBD 2, PSA 150.06, QED 0.68
Compound (SMILES): CC(=O)NC1CCC(N[C@H]2C[C@@H]2c2ccc(-c3cccc(C(F)(F)F)c3)nc2)CC1
InChIKey: MAMLPUSGTQONJN-PELUFGRGSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Fluorescence-Based (Inhibitor)-Screening Assay: In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescence-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAO-A and MAO-B activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 uL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in duplicate within the same experiment.Briefly, a fixed amount of MAO (0.25 ug for MAO-A and 0.5 ug for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of at least eight 3-fold serial dilutions each. Clorgyline and Deprenyl (Sigma Aldrich) was used as a control for specific inhibition of MAO-A and MAO-B respectively. After leaving the enzyme(s) interacting with the inhibitor, KM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. in the dark. The oxidative deamination of the substrate was stopped by adding 50 uL of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor. The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes. The IC50 values of each inhibitor were calculated with GraphPad Prism Software.
Measurement: IC50 = 2450 nM (pChEMBL 5.610000133514404)
Descriptors: MW 417.48, ALogP 4.66, HBA 3, HBD 2, PSA 54.02, QED 0.74
Compound (SMILES): C[C@]1(c2nc(NC(=O)c3ncc(OCF)nc3N)ccc2F)N=C(N)OCC1(F)F
InChIKey: FMYPBFZNJZIDKT-OAHLLOKOSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Inhibition Assay: Recombinant BACE-2 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 μM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals. The fluorescence/time slopes were calculated from duplicate wells and from wells without inhibitor and the IC50 values were calculated using a logistic 4-parameter model.
Measurement: IC50 = 730 nM (pChEMBL 6.139999866485596)
Descriptors: MW 429.33, ALogP 1.35, HBA 9, HBD 3, PSA 150.63, QED 0.6
Compound (SMILES): Cc1cc(C#N)cnc1C(=O)Nc1ccc(F)c([C@]23COC[C@H]2S(=O)(=O)C(C)(C)C(=N)N3)c1
InChIKey: WPFPYTKQXHYXQI-VGOFRKELSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3× the desired final concentration in 1×BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1×BACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 μM for 4 μM for autoBACE-2) prepared in 1×BACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% fin
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 471.51, ALogP 2.02, HBA 7, HBD 3, PSA 145.03, QED 0.62
Compound (SMILES): Cc1nc(-c2nnc3n2CCN(C(=O)c2ccc(-c4cccs4)cc2)[C@@H]3C)c(C)s1
InChIKey: LWCJWXWXIZQQQQ-CYBMUJFWSA-N
Target: Neuromedin-K receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P29371
Assay: Binding Competition Assay: The ability of compounds of the invention to inhibit the binding of the NK-3 receptor selective antagonist 3H-SB222200 was assessed by an in vitro radioligand binding assay. Membranes were prepared from Chinese hamster ovary recombinant cells stably expressing the human NK3 receptor. The membranes were incubated with 5 nM 3H-SB222200(ARC) in a HEPES 25 mM/NaCl 0.1M/CaCl2 1 mM/MgCl2 5 Mm/BSA 0.5%/Saponin 10 μg/ml buffer at pH 7.4 and various concentrations of compounds of the invention. The amount of 3H-SB222200 bound to the receptor was determined after filtration by the quantification of membrane associated radioactivity using the TopCount-NXT reader (Packard).
Measurement: Ki = 32 nM (pChEMBL 7.5)
Descriptors: MW 435.58, ALogP 4.96, HBA 7, HBD 0, PSA 63.91, QED 0.46
Compound (SMILES): O=C(Nc1ccc(C(F)(F)F)c(F)c1)N1CC=C(c2ncc([C@H](O)CO)cc2F)CC1
InChIKey: HTAIIDLOWOVQQD-QGZVFWFLSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 ("wash buffer"), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 ("assay buffer"). Plates are then transferred to a FLIPR for assay. The test compound is diluted in assay buffer, and 50 μL of the resultant solution is added to the cell plates and the solution is monitored for two minutes. The final concentration of the test compound is adjusted to range from about 50 picoM to about 3 μM. Agonist buffer (wash buffer titrated with 1N HCl to provide a solution having a pH of 5.5 when mixed 1:1 with assay buffer) (0.1 mL) is then added to each well, and the plates are incubated for 1 additional minute.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 443.37, ALogP 3.73, HBA 4, HBD 3, PSA 85.69, QED 0.63