lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1ccc(F)c(Nc2nc(=O)c(F)cn2Cc2cc(F)c(F)c(F)c2)c1
InChIKey: SRTITJZGFIOWSA-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 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 397.3, ALogP 3.74, HBA 5, HBD 1, PSA 56.15, QED 0.53
Compound (SMILES): NC/C(=C\F)COc1ccc2c(c1)CCN(C1CCOCC1)C2=O
InChIKey: FRNVWLVHOPPEPZ-JLHYYAGUSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Biological Assay: 1. Determination of Inhibitory Activity of Human Recombinant SSAO/VAP-1Test purpose: The following methods can be used to determine the inhibitory activity of the compounds described in the examples of the invention for human recombinant SSAO/VAP-1.Test Materials:Human recombinant SSAO/VAP-1 (VAP-1, human) purchased from Sigma, Cat. No. SRP6241;Amplex® Red Monoamine Oxidase Assay Kit purchased from Invitrogen, Cat. No. A12214;384-Well plate purchased from Corning, Cat. No. 6005174;Amplex® Red Hydrogen PeroxidePeroxidase Assay Kit purchased from Invitrogen, Cat. No. A22188.Benzylamine hydrochloride purchased from Sigma, Cat. No. B5136-25G;DMSO (Dimethyl Sulfoxide) purchased from Sigma, Cat. No. D2650-100ML;Test Method:The test compound was dissolved in DMSO and diluted to 10 concentrations in 4-fold serial dilution. In 384 well plates, 25 μL of human recombinant SSAO/VAP-1 (1.6 μg/ml) was added to each well. 100 nL of test compounds with different concentrations were added to each well containing human recombinant SSAO/VAP-1 and incubated at room temperature for 30 min. After 30 min incubation, 25 μl of Amplex® red monoamine oxidase assay kit (containing a reaction mixture of 200 μm Amplex Red reagent, 1 U/ml HRP and 1 mm benzylamine hydrochloride) was added to the corresponding well, and incubated at room temperature and dark for 60 min. After 60 min, the relative fluorescence unit (RFU) was read by using PerkinElmer's Envision at 530-560 nm excitation and 590 nm emission. The curve was drawn by using the graph pad prism 5 software, and the IC50 value was calculated. The results were shown in table 1, wherein the compound listed in table 1 is the compound in the preparation examples with the same compound number:
Measurement: IC50 = 0.22 nM (pChEMBL 9.65999984741211)
Descriptors: MW 334.39, ALogP 2.05, HBA 4, HBD 1, PSA 64.79, QED 0.9
Compound (SMILES): c1nc2c(Nc3cnn(C4CCNCC4)c3)nc(C3CCCC3)cn2n1
InChIKey: HXTYAORDNOENER-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: In Vitro CSF-1R Kinase Activity: The CSF-1R construct corresponds to recombinant human catalytic domain (aa 538-910), which was purchased from Invitrogen (cat #PV4092). CSF-1R was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 4 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTI, 0.01% BSA, 0.1 mM Na3VO4 and 0.1 mM ATP. After about 60 min incubation at rt, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 min at rt, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 352.45, ALogP 2.65, HBA 8, HBD 2, PSA 84.96, QED 0.75
Compound (SMILES): CNCC1(c2ccc(Cl)c(Cl)c2)CCC(C)(O)CC1
InChIKey: IEEXXKGDHWBIJX-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Functional Uptake Assay (hSERT): Inhibition of human serotonin reuptake transporter was assayed using the recombinant human serotonin transporter expressed in HEK-293 cells. HEK-293 cells expressing human serotonin transporter were plated before the assay. Test compound and/or vehicle was preincubated with cells in modified HEPES buffer pH 7.1 or pH 7.4 for 20 minutes at 18 to 25° C. and 65 nM [3H]serotonin was then added for an additional timed incubation period (ten to thirty minutes). Cells with internalized [3H]serotonin were washed and the amount of tritium taken into cells is counted using a liquid scintillation counter to determine [3H]serotonin uptake. Non-specific binding of tritium was measured in a control reaction containing 10 uM fluoxetine, and was subtracted from the counts for assays to correct for non-specific binding of tritium. Reduction of [3H]serotonin uptake by 50 percent or more (50%) relative to an uninhibited control reaction indicates significant inhibitory activity. Compounds were screened at 10, 1, 0.1, 0.01 and 0.001 uM. The reference compound for the assay was fluoxetine, for which the IC50 value of 7.1 nM was obtained in a typical experiment.
Measurement: IC50 = 830 nM (pChEMBL 6.079999923706055)
Descriptors: MW 302.25, ALogP 3.78, HBA 2, HBD 2, PSA 32.26, QED 0.89
Compound (SMILES): Cc1nn(C(C)C)cc1Nc1ncc(F)c(OC[C@H]2CC[C@H](O)CC2)n1
InChIKey: BJOMCWNDDDYKAV-HDJSIYSDSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Enzymatic Assay: LRRKtide substrate (peptide sequence RLGRDKYKTLRQIRQ, derived from human ezrin [amino acids 561-573], moesin [amino acids 539-553] and radixin [amino acids 558-570], obtained from SignalChem, catalogue #L10-58, reconstituted in 20 mM Tris-HCl at pH 7.5 to a final concentration of 1 mg/mL, assay concentration 20 μM) and recombinant human LRRK2 (catalytic domain only [amino acids 970-2527], GST-tagged, expressed in insect cells, obtained from ThermoFisher Scientific, catalogue #PV4874, 0.35 mg/mL, assay concentration 30 nM) were mixed in assay buffer (20 mM Hepes pH 7.5, 10 mM MgCl, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM NaVO, 2 mM DTT, 1% DMSO). Compounds of interest (in DMSO, serial 3-fold dilution from 10 μM to 0.5 nM) or control (1% DMSO) were dispensed into the kinase reaction mixture by Acoustic technology (Echo550; nanoliter range). After incubation at room temperature for 20 minutes, the kinase reaction was initiated by addition of [P]-ATP (Specific activity 10 μCi/μl) and the mixture was incubated at room temperature for 2 hours. The reaction was then stopped by spotting the reaction mixture on strips of phosphocellulose P81 paper. Following washing, the radioactivity of the P81 paper was measured and kinase activity data were expressed as the percent remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 = 125 nM (pChEMBL 6.900000095367432)
Descriptors: MW 363.44, ALogP 3.38, HBA 7, HBD 2, PSA 85.09, QED 0.82
Compound (SMILES): O=C(NC1CC2CC3(S(=O)(=O)CCN4CCOCC4)CC(C1)N23)c1cc(C2CC2)on1
InChIKey: VQGXSUCSGKXZPA-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 450.56, ALogP 0.73, HBA 8, HBD 1, PSA 104.98, QED 0.65
Compound (SMILES): Cc1nc2c(-c3ccncc3)cc(N3CCOCC3)cc2n1-c1ccnc2c(Cl)cccc12
InChIKey: ADPUOVFQQBPWRT-UHFFFAOYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O00459
Assay: TR-FRET PI3K Beta Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 μM, and PI3Kγ at 2 nM. The compounds were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean. As shown in Table 1, the compounds provided herein are inhibitors of PI3Kβ.
Measurement: IC50 = 8600 nM (pChEMBL 5.070000171661377)
Descriptors: MW 455.95, ALogP 5.43, HBA 6, HBD 0, PSA 56.07, QED 0.36
Compound (SMILES): CC(C)Cc1cccc2cc(Cn3cccc(NC(=O)[C@H](CC/C=C/C(=O)N(C)C)NC(=O)OCCO)c3=O)[nH]c12
InChIKey: NDWORGPTGGPLLL-FCHBTLIASA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 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 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 565.67, ALogP 3.03, HBA 7, HBD 4, PSA 145.76, QED 0.23
Compound (SMILES): COc1ccc(Oc2cc(C(F)(F)F)cc(C(F)(F)F)c2C(=O)Nc2cccc(S(N)(=O)=O)c2)cc1F
InChIKey: KAYRZLWFCJSCJU-UHFFFAOYSA-N
Target: Sodium channel protein type 10 subunit alpha (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q62968
Assay: Electrophysiology Assay: Patch clamp electrophysiology was used to assess the efficacy and selectivity of sodium channel blockers in dorsal root ganglion neurons. Rat neurons were isolated from the dorsal root ganglions and maintained in culture for 2 to 10 days in the presence of NGF (50 ng/ml) (culture media consisted of NeurobasalA supplemented with B27, glutamine and antibiotics). Small diameter neurons (nociceptors, 8-12 μm in diameter) were visually identified and probed with fine tip glass electrodes connected to an amplifier (Axon Instruments). The voltage clamp mode was used to assess the compound's IC50 holding the cells at −60 mV. In addition, the current clamp mode was employed to test the efficacy of the compounds in blocking action potential generation in response to current injections.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 552.42, ALogP 5.56, HBA 5, HBD 2, PSA 107.72, QED 0.39
Compound (SMILES): COc1ncc(-c2nc(N3CCOCC3)nc3c(CN4CCC(S(C)(=O)=O)CC4)c(C)sc23)cc1NS(C)(=O)=O
InChIKey: PPEOHBNJYLIUCA-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Enzyme Activity Inhibition 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, and added to a 384-well plate to a final DMSO concentration of 1%.3. PI3Kα, PI3Kδ, PI3Kβ and PI3Kγ enzymes were diluted to an 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, and transferred to a 384-well plate and incubated with the compound for a certain time.4. Substrate was diluted to an 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, 50 μM PIP2, 25 μM ATP; then added to a 384-well plate to initiate the reaction, and for PI3Kα, PI3Kβ and PI3Kγ at room temperature reacted for 1 hr, for PI3Kδ at room temperature reacted for 2 hrs. For PI3Kβ and PI3Kγ, it was still necessary to further add 10 μL ADP-Glo Detection Reagent, and then equilibrated at room temperature for 30 minutes.
Measurement: IC50 = 3.2 nM (pChEMBL 8.489999771118164)
Descriptors: MW 610.78, ALogP 2.29, HBA 12, HBD 1, PSA 143.92, QED 0.4
Compound (SMILES): Cc1c(C(=O)O)ccc2c1nc(Nc1ccc(OC(F)(F)F)cc1)n2[C@H]1C[C@H](C)CC(C)(C)C1
InChIKey: UZOSCNKBPBEHBO-YOEHRIQHSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: mIDH1 catalyzes the NADPH-dependent reduction of alpha-ketoglutarate (α-KG) to (2R)-2-hydroxyglutarate (2-HG). NADPH consumption was measured by luminescent readout.The biochemical reactions were performed at 32° C. in 384-well plates using a reaction volume of 41 μl and the following assay buffer conditions: 50 mM Tris pH 7.5, 100 mM NaCl, 20 mM MgCl2, 0.05% BSA, 0.01% Brij, 1 μM NADPH, and 250 μM α-KG. The IDH1 R132H enzyme was used in a final concentration of 1.5 nM. Test compounds were used in a concentration range between 0.002 and 10 μM. The final DMSO concentration was 2.4%.The reaction was incubated for 30 minutes, then 40 μl of detection mix (0.75 μg/ml Luciferase, 0.02 U/ml Oxidoreductase, 4 μg/ml FMN, 2 μl/ml Decanal/Ethanol, 50 mM Tris pH 7.5, 0.5% Glycerin, 0.01% Tween-20, 0.05% BSA) was added. Luminescence was measured on a luminescent reader (10 seconds measuring time, 1 second integration period, 30% sensitivity). The decrease in luminescence is proportional to mIDH1 activity. IC50 values are determined by interpolation from plots of relative luminescence versus inhibitor concentration.
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 475.51, ALogP 7.07, HBA 5, HBD 2, PSA 76.38, QED 0.41
Compound (SMILES): CC(C)C1Oc2c(-c3cn(C)c(=O)c4[nH]ccc34)cc(S(=O)(=O)C(C)C)cc2N(C)C1=O
InChIKey: ACIXQIIPFSJIFH-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 40 μL for BD1 and 60 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature in the assay buffer (50 mM Tris-HCl, pH 7.5, 0.01% Tween-20, 0.01% BSA, 5 mM DTT), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4) and BRD4-BD1 or BRD4-BD2 protein at concentration less than 1 nM. The incubation for 75 min. was followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at final concentration 2-4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech). IC50 determination was performed by fitting the curve of percent control activity versus the log of the inhibitor concentration using the GraphPad Prism 5.0 software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 457.55, ALogP 3.1, HBA 6, HBD 1, PSA 101.47, QED 0.65
Compound (SMILES): CC(=O)N1CC2(C1)CN(c1ccc(CNC(=O)C3(c4ccc(OCc5ccc(C(F)F)nc5)nn4)CC3)cc1F)C2
InChIKey: DXVYUGPXFQIDBO-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical ATX Assay: 5 nM recombinant ATX (Cayman Chemicals) was supplemented to 50 mM Tris buffer (pH 8.0) containing 3 mM KCl, 1 mM CaCl2, 1 mM MgCl2 0.14 mM NaCl, and 0.1% bovine serum albumin. Test compounds were dissolved in DMSO and tested in the range of 0.1 nM to 10 μM. The enzymatic reaction (22.5 μL) was started by addition of 2.5 μL 10 μM 18:1 LPC (Avanti Lipids, Alabaster, Ala., USA). After 2-h incubation at room temperature, the reaction was stopped by addition of 20 μL water containing 500 nM 20:4 LPA as internal standard and 100 μL 1-butanol for extracting LPA. Subsequently, the plates were centrifuged at 4000 rpm, 4° C., for 2 min. The resultant upper butanol phase was directly used for injection at a RapidFire system (Agilent).
Measurement: IC50 = 4.1 nM (pChEMBL 8.390000343322754)
Descriptors: MW 566.58, ALogP 3.54, HBA 7, HBD 1, PSA 100.55, QED 0.42
Compound (SMILES): CC(=O)N(C)c1cnn(C(=O)N2CCC3(CCN(Cc4ccccc4C(F)(F)F)C3)C2)c1
InChIKey: CPCDKGPZNKATJS-UHFFFAOYSA-N
Target: Fatty-acid amide hydrolase 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08914
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction or cell lysates for mouse assays; human prefrontal cortex or cell membrane fractions for human assays) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FPRh or HT-01 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice.Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 449.48, ALogP 3.45, HBA 5, HBD 0, PSA 61.68, QED 0.72
Compound (SMILES): CC(C)COc1ccc(/N=c2\[nH]c(=O)n([C@@H](C)[C@@H](C)C(=O)O)c(=O)n2Cc2ccc(Cl)cc2)cc1
InChIKey: WXXLOAIDIOWQJO-SJORKVTESA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 500.98, ALogP 3.59, HBA 7, HBD 2, PSA 118.68, QED 0.47
Compound (SMILES): Cc1nc(SC[C@H]2CN(S(=O)(=O)c3cccc(C(F)(F)F)c3)c3cc(NC(=O)OC(C)(C)C)ccc3O2)[nH]c1C
InChIKey: KVWWBLWSFMHATH-LJQANCHMSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).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 (SEQ ID NO:1) (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.
Measurement: IC50 = 10.3 nM (pChEMBL 7.989999771118164)
Descriptors: MW 598.67, ALogP 6.14, HBA 7, HBD 2, PSA 113.62, QED 0.33
Compound (SMILES): C[C@@H]1CN(c2nc3ccc(C(=O)NCCCc4ncc[nH]4)cc3nc2-c2ccc(F)cc2)C[C@H](C)O1
InChIKey: OGESYMUJKALXAV-HDICACEKSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: NAMPT Enzyme Assay: To measure the inhibition of NAMPT activity hNAMPT protein stock and anti 6His-Tb (Cisbio; Cat. No. 61HISTLB) is diluted to 3× final concentration with assay buffer (50 mM Tris-HCl (pH7.5), 1 mM DTT, 100 mM NaCl, 10 mM MgCl2, 0.005% Tween 20). To this solution is added test compounds or vehicle control (DMSO) and BodiPY ligand (structure below). The plate is shaken for 1-2 min, sealed and incubated for 1 hour at rt in the dark. The TR-FRET signal is measured using BMG Pherastar (Lanthascreen protocol on BMG Pherastar). Excitation was carried out at 320 nm, and the ratio of emission of BodiPY (520 nm) to terbium (486 nm) was determined. Concentration response curves are generated by calculating the excitation increase in test compound-treated samples relative to DMSO-treated controls. For the assay method described above, test compound percent inhibition values at a single concentration are calculated relative to control (DMSO) treated samples. Compound concentration response curves are fitted to generate IC50 values from those curves. One skilled in the art will appreciate that these values generated either as percentage inhibition at a single concentration or IC50 values are subject to experimental variation.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 488.57, ALogP 4.14, HBA 6, HBD 2, PSA 96.03, QED 0.38
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](OC)c2ncc(Cl)cn2)nnc1[C@H]1C[C@H](C(F)(F)F)C1
InChIKey: RASYLDYWESUZJL-MNUAXYBXSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.). All the results presented are means of several independent experiments and analyzed by non-linear regression methods using the commercially available program Prism (GraphPad, San Diego, Calif.) providing the EC50 values.
Measurement: EC50 = 4.2 nM (pChEMBL 8.380000114440918)
Descriptors: MW 591.01, ALogP 4.3, HBA 10, HBD 1, PSA 130.35, QED 0.37
Compound (SMILES): CN(C)CCC1CCN(c2ccnc(S(N)(=O)=O)c2-c2nnn[nH]2)CC1
InChIKey: JNZPXLJEZSLFRW-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: 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) 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 uM to 0.00063 uM. 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 = 1061 nM (pChEMBL 5.96999979019165)
Descriptors: MW 380.48, ALogP 0.08, HBA 8, HBD 2, PSA 133.99, QED 0.72
Compound (SMILES): COc1cnc(C(=O)Nc2ccc(F)c([C@@]3(C)N=C(N)[C@](C)(F)C[C@@H]3F)c2)cn1
InChIKey: LMPPAZCPPBTWFD-GDIGMMSISA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Binding Assay: The binding assay was performed as SPA-based assay using a biotinylated form of human BACE1 recombinantly expressed and subsequently purified from Freestyle HEK293 cells. The binding assay was run in a 50 mM sodium acetate buffer, pH 4.5 containing 50 mM NaCl and 0.03% Tween-20 in white clear bottom 384 plates (Corning #3653). 10 nM (final concentration) radioligand ([3H]¿N-((1S,2R)-1-benzyl-3-cyclopropylamino-2-hydroxy-propyl)-5-(methanesulfonyl-methyl-amino)-N¿((R)-1-phenyl-ethyl)-isophthalamide) (TRQ11569 purchased from GE Healthcare) was mixed with test compound at a given concentration, 6 nM (final concentration) human BACE1 and 25 μg Streptavidin coated PVT core SPA beads (RPNQ0007, GE Healthcare Life Sciences) in a total volume of 40 μl. Several concentrations of each test compound were tested in the assay for IC50 determination. The plates were incubated for one hour at room temperature and counted in a Wallac Trilux counter. Total and non-specific binding were determined using buffer and 1 μM (final concentration) of the high affinity BACE1 reference inhibitor (S)-6-[3-chloro-5-(5-prop-1-ynyl-pyridin-3-yl)-thiophen-2-yl]-2-imino-3,6-dimethyl-tetrahydro-pyrimidin-4-one, respectively. For each test compound, a IC50 value (the concentration mediating 50% inhibition of the specific binding of the radioligand) was determined from concentration-response curve and used to calculate the Ki from the equation Ki=IC50/(1+L/Kd), where L and Kd are the final concentration of the radioligand used in the assay and the dissociation constant of the radioligand, respectively.
Measurement: Ki = 51 nM (pChEMBL 7.289999961853027)
Descriptors: MW 407.4, ALogP 2.92, HBA 6, HBD 2, PSA 102.49, QED 0.81
Compound (SMILES): Cc1cc(C(=O)NC[C@@H]2C[C@H]3CC[C@@H](C2)N3CC(=O)NC(C)(C)C)n(C)n1
InChIKey: SQIJXUHUGXXASR-PHZGNYQRSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Activity Assay: The HEK293 cells having human Cav3.2 stably expressed therein were cultured at 37° C. in Alpha-MEM, to which 10% (v/v) fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), and G418 (250 g/mL) had been added. The cells were suspended in the culture liquid and were inoculated onto a 96-well plate. Subsequently, the cells were cultured for 48 hours. The culture liquid was removed, and the liquid was changed to S-MEM, to which 5% (v/v) FBS, calcium chloride (0.5 mmol/L), L-glutamine (2 mmol/L), L-alanine (8.9 ng/mL), L-asparagine (13.2 ng/mL), L-aspartic acid (1.33 ng/mL), L-glutamic acid (14.7 ng/mL), glycine (7.5 ng/mL), L-proline (11.5 ng/mL, L-serine (10.5 ng/mL), penicillin (100 U/mL), and streptomycin (100 μg/mL) had been added. The cells were cultured for another 24 hours. The culture liquid was removed again, and the cells were washed with an assay buffer (140 mmol/L sodium chloride, 5 mmol/L potassium chloride, 0.5 mmol/L magnesium chloride, 0.5 mmol/L calcium chloride, 10 mmol/L glucose, 0.4 mmol/L magnesium sulfate, 10 mmol/L HEPES, and 250 mol/L sulfinpyrazone, pH 7.4) that had been kept warm at 37° C. Subsequently, an assay buffer prepared by dissolving Fura2-AM, which is a fluorescent Ca2+ indicator, to a concentration of 5 μM, was added to the cells, and the system was incubated for 30 minutes at 37° C. The assay buffer having Fura2 dissolved therein was removed, and the cells were washed with the assay buffer. Subsequently, an assay buffer prepared by adding a test compound was added to the cells, and the system was incubated for 15 minutes. The plate was mounted on a fluorescence analyzer (FLEX STATION II, Molecular Devices, LLC), and the baseline was measured for 20 seconds. Subsequently, any change in the intracellular calcium concentration that was induced when an assay buffer prepared by adding 100 mmol/L calcium chloride was added to the cells, was measured (excited at 340 nm or 380 nm, and detected at 510 nm).
Measurement: IC50 = 203 nM (pChEMBL 6.690000057220459)
Descriptors: MW 375.52, ALogP 1.62, HBA 5, HBD 2, PSA 79.26, QED 0.82
Compound (SMILES): C[C@@H]1C[C@H](N)CN(c2ccncc2NCc2ccc(F)c(-c3ccccc3F)n2)C1
InChIKey: BGNZFPGHJLJRBF-CVEARBPZSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05M Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light. Pim-2 kinase assay20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05M Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 409.48, ALogP 4.21, HBA 5, HBD 2, PSA 67.07, QED 0.66
Compound (SMILES): CC(=O)N1CCN(C(=O)[C@@H]2CC[C@@]3(S(=O)(=O)c4ccc(F)cc4)c4ccc(C(F)(C(F)(F)F)C(F)(F)F)cc4CC[C@@H]23)CC1
InChIKey: SEKBIOTUGGJROQ-KTIWRFAQSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: General RORgamma Gal4 Reporter Assay: Inverse agonist activity of potential ligands to RORγ was measured by inhibition of luminescence in a Gal4-luciferase reporter assay in Jurkat cells.Jurkat cells stably over-expressing the RORγ receptor, Jurkat pEx/Gal/hRORγ CLBD/HYG pG5luc/blast, were plated at a concentration of 10,000 cells/well in a 384-well solid white cell culture plate (Perkin Elmer #6007899) in assay buffer RPMI 1640 (Gibco 11875-085 1L) containing 0.1% BSA, 100×HEPES (Gibco 15360-080), 100 mM sodium pyruvate (Gibco 11360-040), 50 mg/mL Hygromycin B (Invitrogen 10687-010) and 10 mg/mL blasticidin (Invitrogen R210-01). 100 nL of test compound in a 3-fold serial dilution, with final concentrations ranging from 40 μM to 0.67 nM, were added to the cells which were then incubated overnight.The following day, cells were lysed with 10 μL of Steady-Glo Luciferase Assay System (Promega Cat. No. EZ550), and analyzed immediately. IC50 values were determined. The IC50 value is defined as the concentration of test compound needed to reduce luciferase activity by 50% and is calculated using the four parameter logistic equation to fit the normalized data.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 652.6, ALogP 5.45, HBA 4, HBD 0, PSA 74.76, QED 0.33
Compound (SMILES): COC[C@H](Cc1ccc(C)cc1)n1c(=N)n(Cc2ccc(C(C)(C)C)cc2)c2ccccc21
InChIKey: ZZMFIGUVLYLINV-VWLOTQADSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: IP Accumulation Assay: HEK293 cells stably expressing OX2R are transfected with Gq alpha construct (6.4 ug per 10 cm dish) using Lipofectamine 3000 (ThermoFisher). 24 hours after transfection, cells are plated on poly-lysine-coated 96-well plates at 30,000 cells per well in Inositol-free DMEM+5% FCS with 5 uCi per ml Myo-[2-3H] Inositol (Perkin Elmer). Plates are incubated at 37° C. O/N in 8% C02 tissue culture incubator. Agonists are diluted serially in HBSS containing 10 mM Lithium Chloride. After removal of culture media, agonist solutions are added to the cells (100 ul/well) and incubated at 37° C. 45 min in 8% C02 incubator. Agonist solution is removed and cells are lysed on ice with 10 mM Formic Acid (40 ul/well) for 30 minutes. SPA Poly Lysine YSI beads (Perkin Elmer, cat #RPNQ0010) are added to the wells and mixed on a shaker for 30 minutes before reading on a Microbeta scintillation counter. Positive control curves run with all screens: orexin B and Yanagisawa (J. Med. Chem. 2015) agonist.
Measurement: EC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 441.62, ALogP 6.01, HBA 4, HBD 1, PSA 42.94, QED 0.38
Compound (SMILES): C[C@H](O)c1nc2c(NC(=O)c3ccc(C(F)(F)F)c(-c4ccn(C)n4)c3)c(-c3ccccc3)ccc2s1
InChIKey: GTFCBMUQTGKVCN-HNNXBMFYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Biological Assay: TrkA functional activity was measured using a DiscoverX PathHunter assay. In this assay, U2OS cells express the human TrkA receptor as a fusion with the weakly complementing fragment of B-galactosidase, which DiscoverX calls Prolink (PK); additionally, Shc1 is fused with a larger fragment, which is called Enzyme Acceptor (EA). Activation of the TrkA receptor, upon NGF addition, results in the kinase domain being phosphorylated, resulting in subsequent recruitment of Shc1-EA protein. That recruitment results in an active B-galactosidase enzyme that is detected by addition of a chemiluminescent substrate. The human p75NTR protein was also expressed as a co-receptor for NGF.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 522.55, ALogP 6.69, HBA 6, HBD 2, PSA 80.04, QED 0.27
Compound (SMILES): CC(C)Oc1ccc(-c2n[nH]c3ccc(N4CC[C@]5(CCN(CC(=O)N6CCC(F)(c7ccc(-c8ncn(C)n8)cc7)CC6)C5)C4=O)cc23)cn1
InChIKey: YFOMHXSYGKYYAL-QNGWXLTQSA-N
Target: Mitogen-activated protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P27361
Assay: Kinase Assay: Activated ERK1 and ERK2 activity was determined in a Mobility Shift Assay (MSA) format as follows: Compound and kinase solution were prepared with assay buffer (20 mM HEPES, 0.01% Triton X-100, 2 mM DTT, pH7.5) and mixed and incubated in for 30 mins at rt. ERK1 & ERK2 were then activated by the addition of Fl-Substrate, ATP and metal solution and incubated for 1 h at rt. After 1 h, the reaction was terminated by the addition of 70 mL of Termination Buffer (QuickScout Screening Assist MSA; Carna Biosciences) to the well. The reaction mixture was applied to LabChip system (PerkinElmer), and the product and substrate peptide peaks were separated, analyzed and quantitated.
Measurement: IC50 > 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 691.81, ALogP 5.12, HBA 9, HBD 1, PSA 125.37, QED 0.24
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1CC[C@H](Oc2ccc3nc(N4CCC(F)(F)C4)sc3c2F)CC1
InChIKey: KKJHKMVNBHWUEG-BPUTZDHNSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 27 nM (pChEMBL 7.570000171661377)
Descriptors: MW 471.55, ALogP 4.51, HBA 6, HBD 1, PSA 63.69, QED 0.64
Compound (SMILES): Cc1cccc(C#N)c1Oc1nc(NS(=O)(=O)CCCF)cc(-c2cc(C)c(=O)n(C)c2)n1
InChIKey: BYJDWWUCDVYZMX-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: In Vitro Enzyme Inhibition Assay-CREBBP Inhibition: Determination of the IC50 for the CREBBP inhibitors disclosed herein was performed as follows: CREBBP was cloned and expressed in E. Coli as His-tag protein and purified by Nickel affinity and gel-filtration chromatography. The protein was further characterized as a single band with the correct molecular weight by SDS-PAGE. CREBBP binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotin-labeled) with the target using the AlphaScreen technology (Perkin Elmer). In a 384-well ProxiPlate CREBBP (50 nM final) was combined with peptide (20 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20-minute incubation at room temperature, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After two hours of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.The ability of the compounds disclosed herein to inhibit CBP activity was quantified and the respective IC50 value was determined.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 471.51, ALogP 3.22, HBA 8, HBD 1, PSA 126.97, QED 0.53
Compound (SMILES): N[C@@]1(C(=O)O)CCNC[C@H]1CCCB(O)O
InChIKey: QGCXHYHXXBYKHC-APPZFPTMSA-N
Target: Arginase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P05089
Assay: Inhibition Assay of Arginase: Inhibition of arginase I (ARG I) and arginase II (ARG II) by Formula I or Formula II compounds is followed spectrophotometrically at 530 nm. The compound to be tested was dissolved in DMSO at an initial concentration 50-fold greater than its final concentration in the cuvette. 10 μl of the stock solution was diluted in 90 μl of the assay buffer that comprises 0.1M sodium phosphate buffer containing 130 mM NaCl, pH 7.4, to which is added ovalbumin (OVA) at a concentration of 1 mg/ml. Solutions of arginase I and II were prepared in 100 mM sodium phosphate buffer, pH 7.4 containing 1 mg/ml of OVA to give an arginase stock solution at a final concentration of 100 ng/ml. To each well of a 96-well microtiter plate was added 40 μl of enzyme, 10 μl of an inventive compound and 10 μl of enzyme substrate (L-arginine+manganese sulfate). For wells that were used as positive controls, only the enzyme and its substrate were added, while wells used as negative controls contained only manganese sulfate. After incubating the microtiter plate at 37° C. for 60 minutes, 150 μl of a urea reagent obtained by combining equal proportions (1:1) of reagents A and B is added to each well of the microtiter plate to stop the reaction. The urea reagent is made just before use by combining Reagent A (10 mM o-phthaldialdehyde, and 0.4% polyoxyethylene (23) lauryl ether (w/v) in 1.8 M sulfuric acid) with Reagent B (1.3 mM primaquine diphosphate, 0.4% polyoxyethylene (23) lauryl ether (w/v), 130 mM boric acid in 3.6 mM sulfuric acid). After quenching the reaction mixture, the microtiter plate is allowed to stand for an additional 10 minutes at room temperature to allow the color to develop. The inhibition of arginase was computed by measuring the optical density (OD) of the reaction mixture at 530 nm and normalizing the OD value to percent inhibition observed in the control. The normalized OD is then used to generate a dose-response curve by plotting the normalized OD values against log [concentration] and using regression analysis to compute the IC50 values.
Measurement: IC50 = 625 nM (pChEMBL 6.199999809265137)
Descriptors: MW 230.07