lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(C)(C)CNC(=O)c1ccc(-n2ncc(-c3ccc(C#N)cc3)c2O)nc1
InChIKey: WMLUJJMDWXFQKV-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 63.1 nM (pChEMBL 7.199999809265137)
Descriptors: MW 391.43, ALogP 2.67, HBA 7, HBD 2, PSA 113.06, QED 0.67
Compound (SMILES): COCCCN(C)C1CCN(S(=O)(=O)N2[C@@H]3CC[C@H]2C[C@@H](NC(=O)c2cc(C4CC4)on2)C3)CC1
InChIKey: WCVZZKDAAVJFBL-NUZGMZIFSA-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 = 14.5 nM (pChEMBL 7.840000152587891)
Descriptors: MW 509.67, ALogP 1.95, HBA 7, HBD 1, PSA 108.22, QED 0.48
Compound (SMILES): Cn1nccc1Nc1nccc(-c2cc(F)c3c(c2)CCN3C(=O)Cc2ccncc2Cl)n1
InChIKey: XZVDVXOOEXWNSO-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: In Vitro Enzyme Activity Assay: The substrate Z′-LYTE Ser/Thr 3 Peptide, the phosphorylation substrate Z′-LYTE Ser/Thr 3 Phospho-peptide, 1× kinase buffer (5× kinase buffer was diluted with ultra pure water by five times), ATP, Development Reagent A, Development Buffer, Stop Reagent were balanced to room temperature for use. The screening concentrations for detecting the effect of the compounds of the invention on the ERK kinase activity were seven 3-fold serial dilutions starting from 1 μM (from 0.2 μM for the positive drug control) using 4% DMSO as co-solvent. 5 μL enzyme system (50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 0.01% Brij-35, 4 uM substrate, 0.8 ng/μL enzyme), 2.5 μL Compound, 2.5 μL 400 μM ATP were added into 384 well microplate, followed by incubation at room temperature for 60 min in the dark. After the reaction was completed, 5 μl the developing agent (Development Reagent A) diluted with the developing buffer (Development Buffer) was added to all reaction wells, followed by incubation at the room temperature for 60 min in the dark. 5 μL the terminating agent was added to each well to terminate the reaction. The fluorescence was determined using the multil-mode microplate reader PerkinElmer EnVision® (excitation wavelength 400 nm, emission wavelength 460 nm and 528 nm).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 463.9, ALogP 3.94, HBA 7, HBD 1, PSA 88.83, QED 0.48
Compound (SMILES): C[C@@](O)(c1ccc(-c2nc(C34CCCC(C(=O)O)(CC3)CC4)n3ccnc(N)c23)cc1)c1cccc(C(F)(F)F)c1
InChIKey: AVIZEELGPHTKGW-AYMNYOFFSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay: BTK enzymatic activity was determined with the LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay. In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2) (SEQ.ID.NO.: 1), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity). IC50 values were determined by 4 parameter robust fit of TR-FRET ratio values vs. (Log10) compound concentrations.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 564.61, ALogP 6.32, HBA 6, HBD 3, PSA 113.74, QED 0.26
Compound (SMILES): COCCC1(NC(=O)c2ccc(-n3ncc(-c4ccc(C#N)cc4C)c3O)nc2)CCCC1
InChIKey: VVSVUBZKPXOUKU-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 6.92 nM (pChEMBL 8.15999984741211)
Descriptors: MW 445.52, ALogP 3.9, HBA 7, HBD 2, PSA 113.06, QED 0.57
Compound (SMILES): O=C(Nc1ccc(F)cc1)c1cc(-c2cccn3c(-c4cccnc4)cnc23)ccc1C(F)(F)F
InChIKey: DXJMVIMEVGXSCN-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Cell-Based (HeLa) Assay for Measurement of IDO1 Inhibition: To measure IDO1 inhibition in tissue culture, HeLa cells were treated with a test compound in the presence of IFNγ, which induces IDO1 expression. Following incubation, cell supernatants were assayed for kynurenine levels, an indicator of IDO1 activity.H1-HeLa cells (ATCC #CRL-1958) were seeded in 384-well plates (Greiner #82051-282) at a volume of 50 μL/well in DMEM (Corning #15-018-CM) supplemented with 10% FBS (Corning #35-011-CV) and 1% P/S/G (Corning #30-009-CL) at a density of 1,250 cells/well and incubated overnight at 37° C., 5% CO2/100% humidity. The following day, the test compounds were added in DMSO (0.5% final) at various concentrations, and IDO1 was inducibly expressed by the addition of 50 uL/well of 50 ng/mL of INFγ (Peprotech #300-02) in cell plating media. As a positive control, 50 uL of the cell plating media without IFNγ was added to several wells. Following a 48 hour incubation, the plates were spun down at 1,200 RPM for 5 min at 10° C. 65 μL/well of the supernatant was then transferred to new 384-well plates (Thermo #262160) that contained 10 uL/well of 30% TCA (Sigma #TO699), and the plates were sealed and incubated at 60° C. for 30 min. The plates were then centrifuged for 15 min at 2,000 RPM at 10° C. 40 μL/well of the supernatant was transferred to new 384-well plates (Thermo #262160) and was reacted with 40 μL/well of 2% (w/v) p-dimethlyaminobenzaldehyde (Sigma #156417) in glacial acetic acid (Sigma #A6283). The reaction was incubated at room temperature for 10 min and absorbance at 480 nm was read using a PerkinElmer Envision plate reader.
Measurement: EC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 476.43, ALogP 6.47, HBA 4, HBD 1, PSA 59.29, QED 0.3
Compound (SMILES): Cn1cc(-c2cc(S(C)(=O)=O)cc3c2OC2(CCC2)C(=O)N3)c2cc[nH]c2c1=O
InChIKey: PZOYHUIQLVFINM-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 413.46, ALogP 2.19, HBA 6, HBD 2, PSA 110.26, QED 0.67
Compound (SMILES): O=C(Cc1ccc(F)c(OC(F)(F)F)c1)Nc1ccc(CCCCc2nnc(NC(=O)Cc3ccccn3)s2)nn1
InChIKey: LBUGIVGUIVGPRK-UHFFFAOYSA-N
Target: Glutaminase kidney isoform, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: O94925
Assay: Recombinant Enzyme Assa: Compounds were assessed for their ability to inhibit the enzymatic activity of a recombinant form of Glutaminase 1 (GAC) using a biochemical assay that couples the production of glutamate (liberated by GAC) to glutamate dehydrogenase (GDH) and measuring the change in absorbance for the reduction of NAD+ to NADH. Substrate solution was prepared (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA, 150 mM K2HPO4, 0.1 mg/ml BSA, 1 mM DTT, 20 mM L-glutamine, 2 mM NAD+, and 10 ppm antifoam) and 50 μL added to a 96-well half area clear plate (Corning #3695). Compound (2 μL) was added to give a final DMSO concentration of 2% at 2× the desired concentration of compound. Enzymatic reaction was started with the addition of 50 μL of enzyme solution (50 mM Tris-HCl pH 8.0, 0.2 mM EDTA, 150 mM K2HPO4, 0.1 mg/ml BSA, 1 mM DTT, 10 ppm antifoam, 4 units/ml GDH, 4 mM adenosine diphosphate, and 4 nM GAC) and read in a Molecular Devices M5 plate reader at 20° C. The plate reader was configured to read absorbance (λ=340 nm) in kinetic mode for 15 minutes. Data was recorded as milli-absorbance units per minute and slopes were compared to a control compound and a DMSO-only control on the same plate. Compounds with slopes less than the DMSO control were considered inhibitors and plate variability was assessed using the control compound.
Measurement: IC50 = 33 nM (pChEMBL 7.480000019073486)
Descriptors: MW 589.58, ALogP 4.69, HBA 9, HBD 2, PSA 131.88, QED 0.18
Compound (SMILES): Cc1cc(C)c(CN2CCc3c(c(C)c4c(c3-c3cccs3)O[C@](C)([C@H]3CC[C@H](N(C)C)CC3)O4)C2=O)c(=O)[nH]1
InChIKey: NXSYWBPZACPLLZ-VOSQCMCKSA-N
Target: Histone-lysine N-methyltransferase EZH1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92800
Assay: Inhibitory Activity Assay: The inhibitory activity of the synthesized compounds above against EZH1 or EZH2 methyltransferase was measured. The experiments were consigned to Reaction Biology, and EZH1 or EZH2 activity was measured using a radiometric scintillation proximity assay. To measure the IC50 of the synthesized compounds for EZH1 or EZH2, 2.3 nmol/L EZH1 or EZH2, 1 μmol/L histone H3 (21-44)-lys(biotin), 1.5 μmol/L S-adenyl methionine (SAM), and 500 nmol/L 3H-SAM were added to the compounds or DMSO with a buffer solution and reacted for 90 minutes at room temperature. The buffer solution was comprised of 50 mmol/L Tris-HCl pH 8.0, 50 mmol/L NaCl, 1 mmol/L EDTA, 1 mmoL/L DTT, 1 mmol/L PMSF and 1% DMSO. Trichloroacetic acid was added to end the reaction, and SPA beads coated with PVT streptavidin were added followed by 1 hour reaction at room temperature. A TopCount NXT plate reader was used to measure the methylation value of the substrate peptide. The measured values were converted to percent activity, setting the mean value for wells treated with DMSO as 100% and the background mean value as 0%.
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 561.75, ALogP 5.83, HBA 6, HBD 1, PSA 74.87, QED 0.42
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C(=O)[C@@H]5CC(=O)N(C)C5)CC4)cc3c2C(C)C)cc(C)n1
InChIKey: RXTSZQDKKBHOAC-HSZRJFAPSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 472.63, ALogP 5.15, HBA 3, HBD 1, PSA 69.3, QED 0.57
Compound (SMILES): COC(=O)c1cccc(Nc2nc(=O)c(OC)cn2Cc2ccccc2)c1C
InChIKey: OJDBZMOPWYUZIU-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Evaluation of a Human P2X7 Receptor Inhibitory Activity: Stably expressing cell line (1321N1 cell transfected with the human P2X7 receptor gene (GenBank accession number NM_002562.5 including T606C and G952A SNP)) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (10% fetal bovine serum, 25 mM HEPES, 1% penicillin and streptomycin in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. After replacing with 20 μL of the HBSS buffer (20 mM HEPES, 55.6 mM D-glucose, 1×HBSS(−), pH7.4-7.5), 15 μL of 17.3 μM Yo-Pro solution in the HBSS buffer was added. The plate was placed in high-throughput cellular screening system FLIPR TETRA (Molecullar Devices, LLC.) and 15 μL of 130 μM BzATP solution in the HBSS buffer was added. Measurement of fluorescence intensity by FLIPR TETRA was started. After eight minutes, 15 μL of DMSO solutions containing different concentrations of the compound of the present invention as prepared by dilution with the HBSS buffer were dispensed to each well through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 20 minutes. The maximum fluorescence intensity without the compound of the present invention is calculated as 0% inhibition and the maximum fluorescence intensity when the reference compound was added is calculated as 100% inhibition. Changing values of fluorescence intensity by the compound of the present invention were calculated by difference between maximum and minimum fluorescence intensity for 20 minutes. IC50 was calculated using logistic approximation.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 379.42, ALogP 3.14, HBA 7, HBD 1, PSA 82.45, QED 0.66
Compound (SMILES): Cc1ccc(Cl)cc1-n1cc(-c2ncnc3[nH]ccc23)cc1C#N
InChIKey: DFBJUFDCBMYZJV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Kinase Assay: Assays were performed with the JAK1 kinase domain (residues 861-1152 of the 1154 amino acid long full-length sequence, accession number P23458 of UniProtKB/Swiss-Prot database). The JAK1 kinase assay was run with a final pre activated enzyme concentration of 2.5 nM, in the presence of 100 μM ATP, 2 nM 33P-γ-ATP and 154 μM of substrate BioDBn*333 (Aminoacid sequence: KKHTDDGYMPMSPGVA—SEQ ID NO: 2). The peptidic substrate was purchased from American Peptide Company (Sunnyvale, Calif.).
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 333.78, ALogP 4.25, HBA 4, HBD 1, PSA 70.29, QED 0.6
Compound (SMILES): CCc1nn(C)c2c1-c1c(F)ccc3c(CCCOc4ccc(F)c5ccccc45)c(C(=O)O)n(c13)CCCCOC2
InChIKey: JDJKYSNKDLEVMG-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Biological Assay: MCL-1 Assay: In the assay 11 different concentrations of each compound (0.1 nM, 0.33 nM, 1.1 nM, 3.8 nM, 13 nM, 44 nM, 0.15 μM, 0.51 μM, 1.7 μM, 5.9 μM and 20 μM) were typically measured as duplicates in the same microtiter plate. For that, 100-fold concentrated DMSO solutions were prepared by serial dilutions (1:3.4) of a 2 mM stock solution in a clear, 384-well microtiter plate (Greiner Bio-One, Frickenhausen, Germany). From there, 50 nl were transferred in a dark test plate (Greiner Bio-One, Frickenhausen, Germany). The assay was initiated by addition of 2 μl of a 2,5-fold concentrated MBP-MCL-1 solution (usually for a 1 nM end concentration in 5 μl reaction volume) in aqueous assay buffer [50 mM Tris/HCl pH 7, 100 mM sodium chloride (NaCl), 50 mM potassium fluoride (KF), 0.005% Tween-20, 2 mM DTT, 0.1% bovine gamma globulin (BGG)] to the compounds in the assay plate. This was followed by a 10-minute incubation step at 22° C. for pre-equilibration of the putative complex between MBP-MCL-1 and the compounds. After that, 3 μl of a 1.67-fold concentrated solution (in assay buffer) consisting of Noxa BH3-derived peptide (1 nM end concentration) and TR-FRET detection reagents [1.67 nM anti-MBP-Eu cryptate and 1.67 nM streptavidin-XL665 (both from Cisbio Bioassays, Codolet, France)], were added.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 573.64, ALogP 7.06, HBA 6, HBD 1, PSA 78.51, QED 0.21
Compound (SMILES): N#Cc1ccc(N2CCN(c3c(C#N)ccc(COC(=O)NC(=N)N)c3F)CC2)nc1
InChIKey: RTCLRLYCYPJPMC-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Biochemical Assay: The MAO-Glo Assay (commercial available from PROMEGA, #V1402) provides a sensitive method for the measurement of monoamine oxidase (MAO) activity (Valley, M. P. et al., 2006, Anal. Biochem. 359: 238-246) from a variety of tissues, biofluids or recombinant expressed or purified enzymes. As substrate a derivate of the beetle luciferin ((4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazole-carboxylic acid) is used, which is oxidized at a primary amine moiety. After a spontaneous elimination and a catalyzed esterase reaction, the turnover of the luciferine by the luciferase is recorded as a signal of AOC3 activity.For the determination of AOC3 activity or compound inhibition potency, the compound inhibitors are dissolved in DMSO and adjusted to the respective assay concentration with reaction buffer (50 mM HEPES, 5 mM KCl, 2 mM CaCl2), 1.4 mM MgCl2, 120 mM NaCl, 0.001% (v/v) Tween 20, 100 μM TCEP, pH 7.4). An aliquot of 3 μL of the compound dilution is added to a 384 well plate (Optiplate, PS, flat bottom, white, PERKIN ELMER, #6007290) with a final DMSO concentration of 6.6%. Recombinant CHO cells, overexpressing the human (1500 cells/well), mouse (1000 cells/well) or rat (500 cells/well) AOC3 enzyme are diluted in reaction buffer and added in a volume of 15 μL to the wells. After incubation for 20 minutes at 37° C., 2 μL of MAO substrate (dissolved in DMSO at 16 mM, adjusted to assay concentration in reaction buffer to a final assay concentration of 20 μM) is added and further incubated for 60 minutes at 37° C. The turnover of the substrate is determined by the addition of 20 μL of the detection-mix which was generated by the addition of reconstitution buffer with esterase (PROMEGA, #V1402) to the luciferine detection reagent (PROMEGA, #V1402). After an incubation period of 20 minutes, the luminescent signal is measured with Envision 2104 Multilabel Reader (PERKIN ELMER).Alternative assays for the determination of the AOC3 enzymatic activity could be the extraction of 14C-labelled benzylamine reaction product or the Amplex Red Monoamine Oxidase reaction (Molecular Probes, Netherlands) as described in Gella et al. (Gella, A. et al., 2013, J. Neural Transm. 120: 1015-1018).
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 422.42, ALogP 1.41, HBA 8, HBD 3, PSA 155.15, QED 0.49
Compound (SMILES): CO[C@@H]1C[C@H](C(=O)Cc2cc(C(CCC3CC3)n3ccccc3=O)ccc2F)N(C(=O)Nc2ccc(Cl)cn2)C1
InChIKey: PZNMKTKIJGLXDD-UVAYFRITSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Activity Assay: The effect of compounds of the invention on human plasma kallikrein activity was determined using the chromogenic substrates (DiaPharma Group, Inc., West Chester, Ohio, USA). In these experiments, 2 nM kallikrein (Enzyme Research Laboratories, South Bend, Ind., USA) was incubated with 80 μM S2302 (H-D-Pro-Phe-Arg-p-nitroaniline) in the absence or presence of increasing concentrations of compounds of the invention in a final volume of 200 μL Tris-HCl buffer (200 mM NaCl; 2.5 mM CaCl2; 50 mM Tris-HCl, pH 7.8).After incubation at 30° C., the activity of kallikrein was measured as a change in absorbance at OD 405 nm using BioTek PowerWave X340 Microplate Reader.
Measurement: Ki < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 567.06, ALogP 5.25, HBA 6, HBD 1, PSA 93.53, QED 0.36
Compound (SMILES): CCOC1=CN2N=CC(C#N)C2C(c2ccc(N3CCC(C)(NC(=O)OC(C)C)CC3)nc2)=C1
InChIKey: BESRGAFKDRVEHW-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1× TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve. The IC50.
Measurement: IC50 = 9.8 nM (pChEMBL 8.010000228881836)
Descriptors: MW 464.57, ALogP 3.66, HBA 8, HBD 1, PSA 103.08, QED 0.69
Compound (SMILES): CNc1cccc(-c2ccc(-n3ccc4ccncc43)nc2)c1
InChIKey: UJVPNGCHONKGFS-UHFFFAOYSA-N
Target: Microtubule-associated protein tau (SINGLE PROTEIN), Homo sapiens
UniProt: P10636
Assay: Tau binding assay: For displacement tau binding assay, unlabeled test compounds were dissolved in DMSO at 1 mM. Dilutions of tests compounds to various concentrations were made in 100% DMSO at 1000× final assay concentration and 0.225 μl aliquots were dispensed into assay plates. Brain homogenates were diluted to 0.5 mg/mL from original 30 mg/mL volume in Assay Buffer, and 200 μl were added to the assay plate for a final concentration of 100 μg wet weight/assay tube. [3H]-6 was prepared at 100× final concentration in Assay Buffer plus 20% DMSO and 25 μl was added to the assay plate for final assay concentration of 0.25 nM. The assay plate was incubated at room temperature (25° C.) for 90 minutes. Unbound and bound ligand were separated by filtration of bound onto GF/C filter plates (pre-treated for 30 min with 0.2% polyethyleninamine) using a Packard UniFilter Harvester and washing away unbound with 2.5 ml per well ice cold 5 mM Tris at pH 7.4. Filter plates were dried for 1 hour in a vacuum oven and 50 μl/well MicroScint-20 were added. Plates were counted 1 min per well by Packard Topcount. Total amount of radioligand used in the assay was determined by counting 25 μl of 10× ligand stock. Data was analyzed using Activity Base software to generate 4 p fit of dose responses; and, Ki values were calculated.
Measurement: Ki = 0.76 nM (pChEMBL 9.119999885559082)
Descriptors: MW 300.37, ALogP 4.13, HBA 4, HBD 1, PSA 42.74, QED 0.62
Compound (SMILES): OC[C@@H]1CCCN1c1nc(Nc2cn(-c3cn(C4CC4)cn3)cn2)c2cccn2n1
InChIKey: AHXCQPHEWTXGSK-HNNXBMFYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Biochemical assay: Biochemical assays to measure the inhibitory effects of the compounds were performed by ThermoFisher Scientific (Life Technologies). ALK2 inhibition was tested using LanthaScreen Eu Kinase Binding Assay screening protocol.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 405.47, ALogP 2.15, HBA 10, HBD 2, PSA 101.33, QED 0.51
Compound (SMILES): COC(=O)Nc1ccc(NC(=O)N[C@@H](Cc2ccccc2)C(=O)N2CCC3(C2)OC(=O)Nc2ccc(Cl)cc23)cc1
InChIKey: IGUKVCNEIQUPGE-CTLOQAHHSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 40 pM (Sekisui Diagnostics) and he synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration≤0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. Assuming competitive inhibition, and that unity is negligible compared Km/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in the following equation. V o /V i=1+[I]/K iThe activities shown by this assay indicate that the compounds of the invention may be therapeutically useful for treating or preventing various cardiovascular and/or cerebrovascular thromboembolic conditions in patients suffering from unstable angina, acute coronary syndrome, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, stroke such as thrombotic stroke or embolic stroke, venous thrombosis, coronary and cerebral arterial thrombosis, cerebral and pulmonary embolism, atherosclerosis, deep vein thrombosis, disseminated intravascular coagulation, and reocclusion or restenosis of recanalized vessels.
Measurement: Ki = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 578.03, ALogP 4.94, HBA 6, HBD 4, PSA 138.1, QED 0.33
Compound (SMILES): C=CCn1c(=O)c2cnc(Nc3cnn(C4CCCCC4)c3)nc2n1-c1cccc(N=S(C)(C)=O)n1
InChIKey: UNNHKHDPDWSSRW-UHFFFAOYSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: Kinase Activity Assay: All reactions took place in 60 μL volumes in reaction buffer containing 40 mM Tris-HCl and 20 mM magnesium chloride, supplemented with 0.1 mg/mL bovine serum albumin and 2 mM DTT. Compounds were serially diluted in buffer and 5 μL of each concentration pipetted into a white 384 well plate (Sigma Aldrich M6186). A 5 μL aliquot of the Wee-1 enzyme was added to each well and the plate centrifuged for 1 min to ensure mixing of the enzyme and inhibitor. The plate was incubated at room temperature for 30 minutes before the addition of 2.0 μg/mL of substrate and 30 μM ATP in a 5 μL aliquot. The plate was centrifuged for one minute and incubated for 1 h at RT. 15 μL of ADP-Glo stop reagent was added to each well to quench the reaction and deplete unconverted ATP. The plate was incubated for a further 40 min in the dark at RT. 30 μL of ADP-Glo kinase detection reagent was added to each well, converting ADP to ATP, catalysing the generation of luciferin by luciferase. The plate was shaken for 1 min, and incubated in the dark for an additional hour.
Measurement: IC50 = 0.4 nM (pChEMBL 9.399999618530273)
Descriptors: MW 507.62, ALogP 3.97, HBA 11, HBD 1, PSA 124.88, QED 0.38
Compound (SMILES): CC(C)c1cc(=O)n2nc([C@H]3CCCN3C(=O)Oc3ccc(F)cc3)sc2n1
InChIKey: IAWBLQZNXDMCGW-OAHLLOKOSA-N
Target: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q8N5Z0
Assay: KAT-II inhibitory assay: The inhibitory action of the test compound on human recombinant KAT-II was determined by the following method.To a reaction mixture (45 μL) containing 3.0 μmol/L kynurenine, 10 μmol/L pyridoxal phosphate, 2.0 ng/μL human recombinant KAT-II, and 150 mmol/L tris(hydroxymethyl)aminomethane-acetate buffer (pH 8.0) was added a 10% dimethyl sulfoxide solution (5 μL) of each test compound prepared, and the mixture was reacted at 37° C. for 1 hr. After the reaction, 50% trichloroacetic acid (5 μL) was added to terminate the reaction.The resultant kynurenic acid was quantified as follows by high performance liquid chromatography. An enzyme reaction mixture was separated by an octadecylsilane reversed-phase column (SC-50DS, Eicom Corporation; mobile phase: 250 mmol/L zinc acetate, 50 mmol/L sodium acetate, and 5.0% acetonitrile (pH 6.2)) incubated at 30° C., and kynurenic acid was quantified using a fluorescence detector (RF-20Axs, Shimadzu Corporation) at excitation wavelength 354 nm, detection wavelength 460 nm. The analytical curve was drawn every time by an external standard method. Each test compound was tested by dual measurement at each concentration. The kynurenic acid level in the presence of a test compound at each concentration was converted into % relative to kynurenic acid resulting from a reaction with an enzyme alone as 100%, and the obtained values were fitted to S-curve to determine IC50.
Measurement: IC50 = 77 nM (pChEMBL 7.110000133514404)
Descriptors: MW 402.45, ALogP 3.75, HBA 7, HBD 0, PSA 76.8, QED 0.67
Compound (SMILES): C[C@H]1CC[C@H](Cn2c(N3CCOC[C@H]3c3ccncc3)nc3nc(-c4n[nH]c(=O)o4)nc(-c4cccc(Cl)c4)c32)CC1
InChIKey: HPKDSOXQEGISMF-YDHSSHFGSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 3.807 nM (pChEMBL 8.420000076293945)
Descriptors: MW 587.08, ALogP 5.29, HBA 10, HBD 1, PSA 127.85, QED 0.28
Compound (SMILES): CC(C)(C=C(C#N)C(=O)N1CCC[C@@H](n2nc(-c3ccc(C(=O)Nc4ccccn4)cc3)c3c(N)nccc32)C1)N1CCN(C2COC2)CC1
InChIKey: ZYQGPDFPQNIGTA-MUUNZHRXSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzymatic Activity Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of BTK kinase activity of a compound of the present disclosure. Serial dilutions of test compounds were incubated with human recombinant BTK (0.5 nM), ATP (16 μM) and a phosphoacceptor peptide substrate FAM-GEEPLYWSFPAKKK-NH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper Desktop Profiler (Caliper LabChip 3000). Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 674.81, ALogP 3.74, HBA 11, HBD 2, PSA 158.53, QED 0.21
Compound (SMILES): Cn1ccc2c(-c3ncc(Nc4ccc([C@@H]5CCOC5)c(CN5CC[C@@H](O)C5)n4)c4c3CNC4=O)ccnc21
InChIKey: CGIWBBUBKUCHKG-QZTJIDSGSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 M), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 l/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 525.61, ALogP 3.09, HBA 9, HBD 3, PSA 117.43, QED 0.35
Compound (SMILES): Cc1cc(-c2c(CF)cc3nc(C)n(CCOc4ccc(Cl)cc4-c4cc(C)nc5c(C(=O)O)csc45)c(=O)c3c2C#N)cc(C)n1
InChIKey: JETJJVBSVXGDIY-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 122 nM (pChEMBL 6.909999847412109)
Descriptors: MW 668.15, ALogP 7.74, HBA 9, HBD 1, PSA 130.99, QED 0.17
Compound (SMILES): COc1ncc(-c2cc(CN3CCC(F)(F)CC3)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)OCC(F)(F)F)C[C@@H]1F
InChIKey: QCVYVXJJEQQYEJ-ZWKOTPCHSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 6600 nM (pChEMBL 5.179999828338623)
Descriptors: MW 630.55, ALogP 3.06, HBA 10, HBD 2, PSA 140.21, QED 0.38
Compound (SMILES): CCn1c(=O)[nH]/c(=N\c2ccc(Oc3cc(C(=O)OC)ncn3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: IPGXPPKZGWGBGV-UHFFFAOYSA-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 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 508.92, ALogP 2.66, HBA 10, HBD 1, PSA 133.46, QED 0.38
Compound (SMILES): C=CCc1cn(-c2cnc(-n3ncc4cc(C#N)cnc43)cc2NC(C)C)nn1
InChIKey: UVTKYUPIONLULP-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Inhibition Assay: The assays 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 7.2, 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 = 298 nM (pChEMBL 6.53000020980835)
Descriptors: MW 385.44, ALogP 2.82, HBA 9, HBD 1, PSA 110.13, QED 0.51