lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cc(OCCCc2cn(CC(=O)O)c3ccccc23)cc(C)c1Cl
InChIKey: BGTSDEKNILHHTD-UHFFFAOYSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Compound affinity was measured using a fluorescence polarization anisotropy competition assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 14 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). For selectivity assays, 40 nM Bcl-2 (residues 1-207A96T,G110R, Δ35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer.Compounds are diluted in DMSO in a 10-point, 3-fold serial dilution scheme. 2.5 uL compound is added to 47.5 uL of assay buffer containing FITC-Bak and protein, for a final DMSO concentration of 5% and a top concentration of 20 uM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 90 minutes at room temperature. Anisotropy is measured at excitation wavelength 480 nm and emission wavelength 535 nm using an EnVision Multi-label plate reader (PerkinElmer, Wellesley, Mass., USA). Fluorescence anisotropy is plotted against compound concentration to generate an IC50 (inhibitor concentration at which 50% of bound peptide is displaced) by fitting the data to a 4-parameter logistic model using XLFit software (Guildford, Surrey, UK). IC50 is converted to a binding dissociation constant (Ki value)
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 371.86, ALogP 5.01, HBA 3, HBD 1, PSA 51.46, QED 0.59
Compound (SMILES): C#Cc1cc(Nc2cc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)cc(NS(C)(=O)=O)c4OC)c4ccccc34)ccn2)cc(C(=O)NCCN2CCN(C)CC2)c1
InChIKey: YMOPDCHJUZNPQB-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: Enzyme Inhibition Assay: Method 2: This method follows the same steps as Method 1 above, but utilises a shorter period of mixing of the test compound (105 minutes instead of 2 hours) with the GSK3-α protein. In addition, the concentrations of test compound employed are either 10 ug/mL, 1 ug/mL, 0.1 ug/mL, or 0.01 ug/mL. Method 1: The inhibitory activities of compounds of the invention against the GSK 3α enzyme isoform (Invitrogen), are evaluated by determining the level of activation I phosphorylation of the target peptide. The GSK3-α protein (500 ng/mL, 2.5 uL) is mixed with the test compound (2.5 uL at either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL) for 2 hr at RT. The FRET peptide (8 uM, 2.5 uL), which is a phosphorylation target for GSK3α, and ATP (40 uM, 2.5 uL) are then added to the enzyme\compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 3565 nM (pChEMBL 5.449999809265137)
Descriptors: MW 819, ALogP 7.05, HBA 10, HBD 5, PSA 166.26, QED 0.08
Compound (SMILES): C[C@H]1CCCN1C(=O)c1nc(C(=O)N2CC(C)(O)C2)sc1-c1cnc(NC(C)(C)C)cc1C(F)(F)F
InChIKey: QVKIGFCIUMSEQW-ZDUSSCGKSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation.Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature. Reference wells contained RORγt without compounds, and the assay conditions were as follows:    0.065 mg/mL RORγt    60 μM 1,8-ANS    100 mM Hepes, pH 7.0    10 mM NaCl    2.5 mM GSH    0.002% Tween-20
Measurement: Kd = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 525.6, ALogP 4.27, HBA 7, HBD 2, PSA 98.66, QED 0.62
Compound (SMILES): C=CC(=O)NC[C@@H]1CCN1C(=O)N(C)[C@H](C(=O)N[C@H]1Cc2nc(cs2)-c2ccc3c(c2)c(c(-c2cccnc2[C@H](C)OC)n3CC)CC(C)(C)COC(=O)[C@@H]2CCCN(N2)C1=O)C(C)C
InChIKey: BRXPXRLWEXNYMQ-MBCZHGPKSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 910.15, ALogP 5.66, HBA 12, HBD 3, PSA 180.33, QED 0.13
Compound (SMILES): Fc1c(-c2cccc(Cl)c2OC(F)(F)F)ncc2c(N3CC4CCC(C3)N4)nc(OCC34CCCN3CCC4)nc12
InChIKey: VIRFQUHGWGWGPR-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Binding Assay: Table 2: The ability of a compound to bind to KRAS G12D was measured using a TR-FRET displacement assay. Biotinylated GDP-loaded recombinant human KRAS G12D (corresponding to amino acids 1:169, produced at Array BioPharma) was incubated with a custom-made Cy5 labelled tracer, europium labelled streptavidin and compound (2% DMSO final) in buffer (50 mM HEPES [pH 7.5], 5mM MgCl2, 0.005% Tween-20 & 1 mM DTT). After a 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using a concentration of control compound that completely inhibits binding of the tracer to KRAS. The POC values were fit to a 4-parameter logistic curve and the IC50 value was determined as the concentration where the curve crosses 50 POC.
Measurement: IC50 = 1433 nM (pChEMBL 5.840000152587891)
Descriptors: MW 593.03, ALogP 5.33, HBA 8, HBD 1, PSA 75.64, QED 0.39
Compound (SMILES): CCc1nn(CCCOC(=O)c2ccc(C)cc2OC)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: DOAJUUXUQPLEJK-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: Enzyme Activity Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [5380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 455.56, ALogP 3.09, HBA 7, HBD 1, PSA 91.68, QED 0.51
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(-c3cnc(N4C[C@H]5CC[C@@H](C4)O5)nc3)nn2-c2ccccc2)[C@H](c2ccnc(F)c2)O1
InChIKey: HDTYXQWYEBUUFP-KGWIZABPSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: Seven point five μL per well of human TrkA (PV3144, Lifetechnologies, final concentration: 1 nmol/L) suspended in the assay buffer (100 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20 and 1 mmol/L dithiothreitol (DTT)) was applied in a 384 well plate and the plate was pre-incubated for 15 min at room temperature with 0.4 μL of each compound (final concentration: 200 μmol/L-1 pmol/L) dissolved in DMSO. Then, fluorescent substrate (FL-peptide 27, 760424, PerkinElmer, final concentration: 1.5 μmol/L) and ATP (final concentration 500 μmol/L) dissolved in the assay buffer was added in each well. After the incubation of 120 min at 37° C., fifteen μL of termination buffer (100 mmol/L HEPES, 40 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L magnecium chloride, 0.003 vol % Brij-35, 0.004 vol % Tween20, 1 mmol/L DTT and 0.16 vol % Coating Reagent 3) was added in each well to stop the enzyme reaction. Fluorescent intensities (FI) of phosphorylated and non-phosphorylated fluorescent substrates were measured by LabChip EZReader II (Caliper LifeSciences, Inc.), and conversion ratio (CR) was calculated by the following formula-1. The CR in the well applied with DMSO alone was used as a negative control and the CR in the well without applying TrkA was used as a positive control.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 629.7, ALogP 3.66, HBA 11, HBD 2, PSA 131.79, QED 0.26
Compound (SMILES): N[C@@H](Cc1nc(C2(CN[C@@H]3C[C@H]3c3ccccc3)CC2)no1)C(=O)O
InChIKey: AQTRCOVXWIBMHU-MELADBBJSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Enzyme Activity Assay: The purpose of this test is to test the in vitro inhibitory activities of the compounds against LSD1. The enzyme used in this experiment was human LSD1, and the standard substrate was histone H3(1-21)K4me2 peptide (10 μM). HCI-2509 (SP2509) was used as reference compound, and the fluorescence coupling enzyme assay was employed. The production of FAD-dependent H2O2 as a result of demethylase activity of LSD1 was measured by coupling with HRP and Amplex Red. Compounds as well as control compound HCl-2509 were tested in 10-dose IC50 mode with a 3-fold serial dilution in duplicate starting at 10 μm. All compounds were pre-incubated 30 mins with enzyme before added substrate to start reaction. Fluorescence measurement: Ex/Em=535/590 by EnVision.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 342.4, ALogP 1.2, HBA 6, HBD 3, PSA 114.27, QED 0.66
Compound (SMILES): Cc1cc(Nc2nccc(-c3cc(C#N)c4c(c3)[C@](C)(CO)CN4)n2)n(CC2CCN(CCF)CC2)n1
InChIKey: SUALBZWOPNFWDU-MHZLTWQESA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition of auto-phosphorylation of recombinant human NF-kappaB-inducing kinase (NIK/MAP3K14) activity: NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen® (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 1% (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween® 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision® Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
Measurement: IC50 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 504.61, ALogP 3.62, HBA 9, HBD 3, PSA 114.92, QED 0.43
Compound (SMILES): O=C(Nc1ccc(Oc2ccc(F)cc2)nc1)c1ccc(C(=O)N2CCC(Oc3ccc(F)cc3F)CC2)nc1
InChIKey: GUJSBGWSDRFUAC-UHFFFAOYSA-N
Target: 5'-AMP-activated protein kinase catalytic subunit alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P54646
Assay: Enzyme-Linked Immunosorbent Assay: Compounds were assayed for their ability to activate AMPK using an enzyme-linked immunosorbent assay. Reagents and procedures for measuring AMPK activation are well known and kits for AMPK activation assays are commercially available.
Measurement: EC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 548.52, ALogP 5.62, HBA 6, HBD 1, PSA 93.65, QED 0.32
Compound (SMILES): C=CC(=O)N1CC[C@H](Nc2nc(-c3n[nH]c(=O)[nH]3)cc3ccccc23)C1
InChIKey: MIJKWXXKYTVHGR-LBPRGKRZSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition Assay: The inhibitory properties of compounds relative to BTK is determined using a black 384-well-plate format in a buffer which contains 50 mM Hepes, 10 mM NaCl, 10 mM MgCl2, 0.2 mM EDTA, 0.01% Brij35®, 1 mM DTT, and 0.1 mg/mL BSA at pH 7.3. The test compound is prepared in DMSO using 2-fold serial dilutions for 11 data points, which are added to the buffer so that each dilution contains 3% DMSO. To initiate the assay, 5 μL of 3 μM 5FAM-EEPLYWSFPAKKK-NH2 (in buffer), 5 μL of diluted test compound (3% DMSO in buffer), and 5 μL of 9 nM BTK and 150 μM ATP in buffer are combined in each well. The reaction mixtures are incubated at room temperature for 60 minutes and then quenched by adding 25 μL of 50 mM EDTA. To quantify the fluorescent-labeled substrate and product following reaction, the test plate is loaded on a Caliper LC-3000, which measures percent of conversion by microfluidic-based separation. Corresponding IC50 values are calculated by non-linear curve fitting of the compound concentrations and percent of inhibition to the standard IC50 equation and reported as pIC50, i.e., −log(IC50), where IC50 is molar concentration at 50% inhibition.
Measurement: IC50 < 2.51 nM (pChEMBL 8.600000381469727)
Descriptors: MW 350.38, ALogP 1.51, HBA 5, HBD 3, PSA 106.77, QED 0.62
Compound (SMILES): COc1cc2ncnc(-n3nc(N)nc3Nc3ccc(N4CCN(C5CCCCC5)CC4)cc3)c2cc1OC
InChIKey: KDDFPHVERPRXCY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: PHOSPHO-AKT IN-CELL WESTERN ASSAY: The resulting chemiluminescence was read with a Luminomitor within 10 minutes to minimize changes in signal intensity. After reading the chemiluminescence, the cells were washed 1× with wash buffer and 1× with PBS by plate washer. The plate was tapped onto paper towels to remove excess liquid from wells and air-dried at ambient temperature for 5 minutes.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 529.65, ALogP 4.01, HBA 11, HBD 2, PSA 119.48, QED 0.36
Compound (SMILES): COC(=O)Nc1ccc(Nc2cc(-c3ccc(S(C)(=O)=O)cc3)[nH]n2)c(C)c1
InChIKey: OECYWQZZLCQYFD-UHFFFAOYSA-N
Target: TRAF2 and NCK-interacting protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UKE5
Assay: ADP-Glo Kinase Assay: The inhibitory properties of compounds were evaluated with TNIK kinase enzyme system and luminescent ADP-GloFigure US11485711-20221101-P00001 Kinase Assay from Promega Corporation according to the manufacturer's protocol. The tested compounds were incubated with TNIK kinases. Reactions were performed in 10 μl kinase buffer supplemented with reaction mixture containing 2 μl ATP, 2 μl MBP protein and 2 μl TNIK at 37° C. for 30 min. The reactions' conditions are provided in Table 5. After kinase reaction, 5 Figure US11485711-20221101-P00002 reaction mixtures were transferred to 384 well assay plate (Greiner, solid white low-binding plates). Next, 5 μl of ADP-Glo Reagent was added to each well and incubated at 37° C. for 30 min. After 45 min, 10 μl Kinase Detection Reagent was added to each well and luminescence signal was detected with the SpectraMax M5e Microplate Reader (Molecular Devices, Menlo Park, Calif.) after 30 min at 37° C. incubation. The IC50 values were calculated using GraphPad Prism software (GraphPad Software Inc., La Jolla, Calif., USA) to determine kit performance. To the cut-off assay, the inhibitory activity of the compounds against TNIK kinase was expressed as the percentage of the kinase inhibitory activity for an indicated concentration of inhibitor.
Measurement: IC50 = 623 nM (pChEMBL 6.210000038146973)
Descriptors: MW 400.46, ALogP 3.71, HBA 6, HBD 3, PSA 113.18, QED 0.6
Compound (SMILES): C=CC(=O)N1C[C@@H](n2nc(C#Cc3ccc4nc(C)cnc4c3)c(C(N)=O)c2NC)C[C@@H]1COC
InChIKey: WQTFRFULRFIUTD-ZWKOTPCHSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: FGFR2 Kinase Assay: Small molecule inhibition of FGFR2 kinase activity was evaluated using a fluorescence-based microfluidic mobility shift assay. FGFR2 catalyzes the production of ADP from ATP during phosphoryl transfer to the substrate peptide, FLPeptide30 (5-FAM-KKKKEEIYFFFCONH2) (Perkin Elmer, 760430). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product were measured and the ratio of these values used to generate % conversion of substrate to product by the LabChip EZ reader (Perkin Elmer). Wild type FGFR2 (Carna Bioscience, 08-134) at 0.06 nM was prepared with 1.5 μM substrate, 10 mM MgCl and 100 μM ATP in a butler containing 50 nM HEPES, 1 mM EGTA, 0.01% Brij-35, 0.05% BSA, and 2 mM DTT prior to addition of compounds in DMSO and incubation for 80 minutes at room temperature. The reaction was terminated by addition of 0.5 MEDIA. IC50 values were calculated using the inhibition of conversion ratio using Dotniatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 473.54, ALogP 1.65, HBA 8, HBD 2, PSA 128.26, QED 0.41
Compound (SMILES): O=C(NC(Cc1ccc(-c2ccccc2)cc1)C[C@@H](O)C(=O)O)c1ccc2[nH]nnc2c1
InChIKey: MQJDBDUFOAMADZ-AVKWCDSFSA-N
Target: Neprilysin (SINGLE PROTEIN), Homo sapiens
UniProt: P08473
Assay: Enzyme Inhibition Assay: The assays were performed in 384-well white opaque plates at 37° C. using the fluorogenic peptide substrates at a concentration of 10 μM in Assay Buffer (NEP: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% polyethylene glycol sorbitan monolaurate (Tween-20), 10 μM ZnSO4; ACE: 50 mM HEPES, pH 7.5, 100 mM NaCl, 0.01% Tween-20, 1 μM ZnSO4). The respective enzymes were used at concentrations that resulted in quantitative proteolysis of 1 μM of substrate after 20 minutes at 37° C.Test compounds were assayed over the range of concentrations from 10 μM to pM. Test compounds were added to the enzymes and incubated for 30 minute at 37° C. prior to initiating the reaction by the addition of substrate. Reactions were terminated after 20 minutes of incubation at 37° C. by the addition of glacial acetic acid to a final concentration of 3.6% (v/v).
Measurement: Ki = 3.55 nM (pChEMBL 8.449999809265137)
Descriptors: MW 430.46, ALogP 2.8, HBA 5, HBD 4, PSA 128.2, QED 0.34
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(OC[C@H](C)O)nn2-c2ccccc2)[C@H](c2cc(F)cc(F)c2)C1
InChIKey: RIJOZBDVFAOVFM-YRUKQIKQSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 529.59, ALogP 3.45, HBA 7, HBD 3, PSA 100.88, QED 0.37
Compound (SMILES): CN(C)C/C=C/C(=O)N1CCOc2c1ccc1ncnc(Nc3ccc(OCc4cccc(F)c4)cc3)c21
InChIKey: KPEKPAFTRUCZDF-QPJJXVBHSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Inhibition of EGFR and HER2 Kinase Activity Assay: The assay for the inhibition of EGFR and HER2 kinase activity by small molecular compounds was carried out using the method as follows:1) Dilution of the compoundsIn a 96-well plate a, the compounds were diluted with DMSO using a 3-fold gradient dilution to form 11 concentrations, the 12th concentration is pure DMSO (as a positive control); and in a new 96-well plate b the above solutions were diluted 25 times with ultrapure water (DMSO concentration is 4%).2) Transferring the compounds to 384-well plateThe compound solutions diluted with ultrapure water in the 96-well plate b above was transferred to the corresponding wells of a 384-well plate in duplicate.3) Addition of 4×kinase solution: 2.5 μl of the above 4× kinase solution was taken using multichannel pipette and added to the corresponding reaction wells of the 384-well plate, mixed well and pre-reacted at room temperature for 5 minutes.4) Addition of 2×substrate/ATP mixed solution: 5 μl of the above 2×substrate/ATP mixed solution was taken using multichannel pipette and added to the corresponding reaction wells of the 384-well plate.5) Negative control: negative control wells were set in the 384-well plate, and 2.5 μl 4×substrate, 2.5 μl 4×enzyme solution, 2.5 μl 1×Kinase Assay Buffer and 2.5 μl ultrapure water containing 4% DMSO were added to each well.6) Mixed by centrifugation and kept at room temperature for 2 hours in the dark.7) Termination of the enzymatic reaction:5 μl of the above 4× stop solution was pipetted to the corresponding wells of the 384-well plate, centrifuged and mixed, and reacted at room temperature for 5 minutes.8) Development reaction:5 μl of the above 4× detection solution was pipetted into the corresponding wells of the 384-well plate, centrifuged and mixed, and reacted at room temperature for 1 hour.9) The 384-well plate was placed into a microplate reader and the signal was detected using the corresponding program.10) IC50 analysis:Well reading value=10000*EU665 value/EU615 valueInhibition rate=(reading value of positive control well−reading value of experimental well)/(reading value of positive control well−reading value of negative control well)*100%Corresponding IC50s can be calculated by entering the drug concentrations and the corresponding inhibition rates into GraphPad Prism 5.
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 513.57, ALogP 4.93, HBA 7, HBD 1, PSA 79.82, QED 0.33
Compound (SMILES): CC(=O)N[C@H]1CC[C@@H](COc2nc(Nc3ccc(N4CCOCC4)cc3)ncc2F)C1
InChIKey: DWXCAMAUXBJZOH-AEFFLSMTSA-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 429.5, ALogP 2.88, HBA 7, HBD 2, PSA 88.61, QED 0.7
Compound (SMILES): CNc1cc(Nc2cccn([C@H]3C[C@@H]3F)c2=O)nc2c(C(=O)N[C@H]3CC[C@@H](O)C3)cnn12
InChIKey: FVVAPARGJLGJEV-LMWOUKEDSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 441.47, ALogP 1.6, HBA 9, HBD 4, PSA 125.58, QED 0.46
Compound (SMILES): Cc1cnc2c(C(=O)NC3(c4ccc(C(F)(F)F)cc4F)CCCOC3)cnn2c1
InChIKey: PVIHIXVNTDNMAQ-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Phosphodiesterase (PDE) 2A and 10 Assay: All reactions were performed in 384 well plates, Perkin Elmer black optiplates and IMAP reaction buffer with 0.1% Tween20 (kit component)Compounds were serial diluted in DMSO. With an intermediate dilution step with reaction buffer DMSO concentration was reduced to achieve 1% DMSO in the assay reaction. Setup of the assay started with 10 μl enzyme ( 10 ng/well, depending on prep. batch), 5 μl compound, reaction was started by addition of 5 μl labeled cAMP (30 nM, final concentration), immediately mixed for 15 seconds on a Eppendorf mixmate (2000 rpm) followed by an incubation at room temperature for 90 minutes in the dark. Reaction is stopped by adding of 60 μl binding buffer for FP/cAMP (kit component). After at least 90 min of further incubation (room temperature, dark) the assay was measured at 485 nm excitation/525 nm emission in an Envision multilabel reader (PerkinElmer).Each assay plate contained wells with vehicle controls (1% DMSO) for the measurement of non-inhibited reaction (=100% control) and wells without enzyme as 0% controls.The analysis of the data was performed by calculation of the percentage of inhibition in the presence of test compound compared to the vehicle control samples (100% control, no inhibition) and a low control (0% control, no enzyme).
Measurement: IC50 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 422.38, ALogP 3.63, HBA 5, HBD 1, PSA 68.52, QED 0.65
Compound (SMILES): COc1cc(CCc2cnc3[nH]c(C(=O)N(C)C)cc3n2)cc(OC)c1
InChIKey: HCOXTFKFUWXMBG-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzymatic Assay: The inhibitor potency of compounds was measured in an enzyme assay that measures peptide phosphorylation using FRET measurements to detect product formation. Inhibitors were serially diluted in DMSO and a volume of 0.5 μL was transferred to the wells of a 384-well plate. For FGFR3, a 10 μL volume of FGFR3 enzyme (Millipore) diluted in assay buffer (50 mM HEPES, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween-20, 5 mM DTT, pH 7.5) was added to the plate and pre-incubated for 5-10 minutes. Appropriate controls (enzyme blank and enzyme with no inhibitor) were included on the plate. The assay was initiated by the addition of a 10 μL solution containing biotinylated EQEDEPEGDYFEWLE peptide substrate (SEQ ID NO: 1) and ATP (final concentrations of 500 nM and 140 μM respectively) in assay buffer to the wells. The plate was incubated at 25° C. for 1 hr. The reactions were ended with the addition of 10 μL/well of quench solution (50 mM Tris, 150 mM NaCl, 0.5 mg/mL BSA, pH 7.8; added fresh 30 mM EDTA and Perkin Elmer Lance Reagents for HTRF at 3.75 nM Eu-antibody PY20 and 180 nM APC-Streptavidin). The plate was allowed to equilibrate for 1 hr before scanning the wells on a PheraStar plate reader.FGFR1 and FGFR2 were measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 μM, respectively and FGFR2, 0.01 nM and 100 μM, respectively.
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 354.41, ALogP 2.46, HBA 5, HBD 1, PSA 80.34, QED 0.74
Compound (SMILES): C[C@H]1C[C@@H](n2c(Nc3ccc(OC(F)(F)F)cc3)nc3cc(OCC(=O)Nc4cccc(C(F)(F)F)c4)ccc32)CC(C)(C)C1
InChIKey: QSXHXLCRBCVIQX-WMZHIEFXSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: Biochemical Assay: 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.
Measurement: IC50 = 4100 nM (pChEMBL 5.389999866485596)
Descriptors: MW 634.62, ALogP 9.1, HBA 6, HBD 2, PSA 77.41, QED 0.19
Compound (SMILES): COc1cnc(C(=O)Nc2ccc(F)c([C@]3(C)CS(=O)(=O)[C@@](C)(CC4CCOCC4)C(=N)N3)c2)cn1
InChIKey: XSWUKTIPUCIOSI-ZEQRLZLVSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 HTRF FRET Assay: A homogeneous time-resolved FRET assay can be used to determine IC50 values for inhibitors of the soluble human BACE1 catalytic domain. This assay monitors the increase of 620 nm fluorescence that resulted from BACE1 cleavage of an APPswedish APPswe mutant peptide FRET substrate (QSY7-EISEVNLDAEFC-Europium-amide). This substrate contains an N-terminal QSY7 moiety that serves as a quencher of the C-terminal Europium fluorophore (620 nm Em). In the absence of enzyme activity, 620 nm fluorescence is low in the assay and increased linearly over 3 hours in the presence of uninhibited BACE1 enzyme. Inhibition of BACE1 cleavage of the QSY7-APPswe-Eu substrate by inhibitors is manifested as a suppression of 620 nm fluorescence.Varying concentrations of inhibitors at 3Ã¿ the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 ul) for 30 minutes at 30° C. in reaction buffer containing 20 mM Na-Acetate pH 5.0, 10% glycerol, 0.1% Brij-35 and 7.5% DSMO. Reactions are initiated by addition of 10 ul of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 ul in a 384 well Nunc HTRF plate. The reactions are incubated at 30° C. for 1.5 hours. The 620 nm fluorescence is then read on a Rubystar HTRF plate reader (BMG Labtechnologies) using a 50 millisecond delay followed by a 400 millisecond acquisition time window. Inhibitor IC50 values are derived from non-linear regression analysis of concentration response curves. Ki values are then calculated from IC50 values using the Cheng-Prusoff equation using a previously determined um value of 8 uM for the QSY7-APPswe-Eu substrate at BACE1. The compounds of Examples 1, 2, and 3 were measured in this assay and each exhibited a Ki value of less than about 5 nM.
Measurement: Ki = 3.9 nM (pChEMBL 8.40999984741211)
Descriptors: MW 519.6, ALogP 2.66, HBA 8, HBD 3, PSA 143.36, QED 0.53
Compound (SMILES): Cc1cnc2c(C(N)=O)cc(Cc3cc(C(=O)NCc4cc5c(Cl)c[nH]c5cc4F)ccn3)cc2c1
InChIKey: WETFPOPVXHIHFG-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Enzyme Inhibition Assay: The ability of the compounds disclosed herein to inhibit human plasma kallikrein activity was quantified according to the procedures below.A 10 mM solution of the test compound was made in DMSO. This solution was serially diluted 1:5 in DMSO to yield 2000, 400, 80, 16, 3.2, 0.64, 0.128, 0.0256 and 0.00512 μM compound test solutions. A control tube containing only DMSO is included. 16 μL of each compound test solution was combined with 384 μL of assay buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% Triton X-100) to yield a 4× test compound buffer stock.Separately, a 40 nM solution of human Plasma Kallikrein (Abcam) and a 93.6 μM solution Pro-Phe-Arg-AMC (Bachem) were made using assay buffer. These solutions are hereby referred to as 4×hPK and 2×PFR-AMC, respectively.60 μL of each 4× test compound buffer stock was combined with 60 μL of 4×hPK to yield 120 μL of 2× test compound buffer stock/2×hPK. 50 μL was removed from this mixture and placed into duplicate wells on a Microfluor 1Black U-bottom microtiter plate (Thermo Scientific). This plate was incubated for 5 minutes at 37° C. To each well, 50 μL of pre-warmed 2×PFR-AMC was added to start the enzymatic reaction. Cleavage of PFR-AMC was monitored in a Biotek Synergy H4 reader set at 37° C. Readings are taken every 43 seconds for 1 hour. The highest mean velocity over 20 reads (15 minutes) is used to calculate the IC50.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 501.95, ALogP 4.83, HBA 4, HBD 3, PSA 113.76, QED 0.31
Compound (SMILES): C/C=C/c1cc(Cl)ccc1N(Cc1cocn1)c1ccnc(N)n1
InChIKey: CAVMYJHIDNAPTM-NSCUHMNNSA-N
Target: Histamine H4 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H3N8
Assay: Binding Assay on Recombinant Human Histamine H4 Receptor: Receptor binding was assessed using isolated plasma membranes from SKNMC neuroblastoma cell lines stably expressing recombinant human H4 receptor. To isolate plasma membrane enriched in recombinant human H4 receptor, cells were collected from 100 culture dishes (150 mm) in PBS-EDTA. The cells were pellet by centrifugation (800 g) and resuspended in 5×TE buffer (50 mM Tris/5 mM EDTA). The cells were then homogenized and centrifuged at 1000 g for 10 min. The supernatant was collected and centrifuged for an additional 26000 g for 30 min. The supernatant was removed and the pellet was gently resuspended in 30 ml of 5×TE buffer. Protein concentration was then determined using Bradford protein assay (Bradford, M. M., Anal. Biochem., 1976, 72: 248-254). Isolated cell membranes were incubated with 2×KD (10 nM), [3H] histamine (Specific activity: 23 Ci/mmol), with or without test compounds for 45 min at 25° C. Nonspecific binding was defined with 100 μM cold histamine. Ki values were calculated based on an experimentally determined appropriate KD values according to Cheng and Prusoff (Biochem. Pharmacol. 1973, 22(23):3099-3108). Membranes were harvested by rapid filtration using the 96 well Brandel system or a cell harvester using a Whatman GF/C filter or filter plates treated with 0.5% polyethylenimine (PEI), and washed 4 times with ice-cold 50 mM Tris/5 mM EDTA buffer. Filters were then dried, mixed with scintillant and radioactive counts were determined.
Measurement: Ki = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 341.8, ALogP 4.07, HBA 6, HBD 1, PSA 81.07, QED 0.75
Compound (SMILES): C=C(C)c1c(Nc2ccncc2C(=O)OC)cc(-c2cc(Cl)ccc2F)nc1NC(=O)OC(C)(C)C
InChIKey: UXFTVPGMWIESDP-UHFFFAOYSA-N
Target: Activin receptor type-1-like (SINGLE PROTEIN), Homo sapiens
UniProt: P37023
Assay: In Vitro Kinase Assay Inhibition of ALK1/2/3/4/5/6 Kinases: Compound of the invention were screened in an in vitro kinase assay against several members of the TGFβ family of Ser/Thr kinases. The kinases tested were ALK1 (ACVRL1), ALK2 (ACVR1), ALK3 (BMPR1A), ALK4 (ACVR1B), ALK5 (TGFBR1), and ALK6 (BMPR1B). Standard kinase testing conditions and techniques were employed. For each case, specific kinase/substrate pairs along with required cofactors were prepared in reaction buffer. Compound of the invention were delivered into the reaction, followed 15-20 min later by addition of a mixture of ATP and 33P ATP to a final concentration of 10 μM. Reactions were carried out at RT for 120 min, followed by spotting of the reactions onto P81 ion exchange filter paper. Unbound phosphate was removed by extensive washing of filters in 0.75% phosphoric acid. Kinase activity data was expressed as the percent of remaining kinase activity in test samples compared to vehicle. IC50 values were generated from activity values performed at multiple concentrations.
Measurement: IC50 = 5670 nM (pChEMBL 5.25)
Descriptors: MW 512.97, ALogP 6.85, HBA 7, HBD 2, PSA 102.44, QED 0.35
Compound (SMILES): C[C@H](NC(=O)[C@H](N)CCc1ccccc1)C(=O)NCc1cnc(N)c(Cl)c1
InChIKey: JSBVRBAPCFFDNY-BLLLJJGKSA-N
Target: Mannan-binding lectin serine protease 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00187
Assay: Enzymatic Assay for MASP-2: The assay was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50 values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed., John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 389.89, ALogP 1.4, HBA 5, HBD 4, PSA 123.13, QED 0.54
Compound (SMILES): O=C(NCCONc1nonc1/C(=N/O)Nc1ccc(F)c(Br)c1)NC1CCCC1
InChIKey: ROQFHRLCNNYIJH-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Inhibitory Activity Assay: 1. Materials, Kits and EquipmentsSodium L-ascorbate (Cat: A4034-100G, SIGMA)4-(dimethylamino)benzaldehyde (Cat: 156477-25g, SIGMA)Trichloroacetic acid (Cat: T0699-100ML, SIGMA)L-Tryptophan (Cat: T8941-25G, SIGMA)Methylene blue (Cat: M9140-25G, SIGMA)Potassium dihydrogen phosphate (Cat: 10017618, Sinopharm Chemical Reagent)Disodium hydrogen phosphate (Cat: 20040618, Sinopharm Chemical Reagent)Constant temperature water tank (Cat: DK-8D, Shanghai Jinghong Experimental Equipment)Multifunctional microplate reader (Cat: M5, Molecular Devices)96-well reaction plate (Cat: 3590, costar)IDO1 protease (commercially available)Desktop Microplate Reader: SpectraMax M5 Microplate Reader (Molecular Devices)Test compounds: self-madePositive control agent: INCB024360 (commercially available)2. Reagent Preparation100 mM PBS:100 mM disodium hydrogen phosphate and 100 mM potassium dihydrogen phosphate mixed in a ratio of 3:5, pH 6.5IDO1 assay buffer:100 mM PBS containing 400 μM L-tryptophan, 20 mM ascorbate, 20 μM methylene blue and 1000 U/ml catalase, pH 6.530% trichloroacetic acidddH2 O solution of 30% trichloroacetic acidEhrlich reagent1% (w/v) diluted solution of 4-(dimethylamino) benzaldehyde compoundAll compounds were dissolved with DMSO. During the assay, each compound was diluted to a concentration as needed. The compound of each concentration was added to multi-wells, and the final concentration of DMSO was controlled at 1%.3. Test Methoda.) the reaction mixture was prepared by adding 50 nM IDO1 and the desired concentration of the test compound to 100 μL of IDO1 assay buffer. IDO1 and assay buffer need to be preheated to 37° C.b.) The mixture was reacted in a constant temperature water tank at 37° C. for 30 minutes.c.) 50 μL of 30% trichloroacetic acid was added.d.) The above mixture was reacted in a constant temperature water tank at 52° C. for 30 minutes.e.) The reaction mixture was centrifuged at 12000 g for 10 minutes at room temperature.f.) 100 μL of the obtained supernatant and 100 μL of Ehrlich reagent were mixed.g.) the absorbance at 480 nm was measured using an M5 microplate reader.4. Data AnalysisInhibition rate=(ODpostive−ODsample)/(ODpositive−ODnegative)*100%5. Results and Discussion
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 486.3, ALogP 2.8, HBA 8, HBD 5, PSA 145.93, QED 0.13
Compound (SMILES): Cc1ccc(CNC(=O)[C@]2(F)CC[C@@H](O)c3ncccc32)c(Cl)c1
InChIKey: YYNFXPNCORZADY-QAPCUYQASA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pharmacological Assay: 1. Wash each well with 80 microL of assay buffer (20 mM HEPES, 1×HBSS, pH 7.4 adjusted with NaOH) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).2. Add 20 microL of assay buffer containing 2.5 mM probenecid, 0.5 microM Fluo-4-AM (Molecular Probes) and 0.1% Pluronic F-127 to each well.3. Incubate the plate at 37° C. in 5% CO2 for 1 h.4. Wash each well with 80 microL of assay buffer (see below) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).5. Test compounds were prepared at 100× the test concentration in DMSO by serial dilution with Biomek-FX liquid handling instrument. 33× diluted compound solutions in assay buffer were prepared in intermediate compound plate with Biomek-NX liquid handling instrument. A further 3× dilution occurred in below steps 6 and 7.6. Add 20 microL of 33× diluted compound solutions into each well and leave the plate for 10 min under the dark at room temperature.7. Measure activity by FDSS as follows:Set the assay plate on the stacker of FDSS.Start the detection of fluorescence intensity at 540 nm by 480 nm exicitation.After 30 seconds, add 20 microL of assay buffer containing 240 microM BzATP (final concentration 80 microM).
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 348.81, ALogP 3.35, HBA 3, HBD 2, PSA 62.22, QED 0.89
Compound (SMILES): Nc1ccc(-c2ccc(C(F)(F)F)cc2)cc1NC(=O)N1Cc2ccncc2C1
InChIKey: JRCJUDMTSGYLDK-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Reporter Displacement Assay: HDAC2 containing a C-terminal HIS-Tag (Proteros) and fluorescently-labeled anti-HIS-antibody are diluted in one vial in assay buffer 50 mM Tris, pH 8.0, 1 mM DTT, 150 mM NaCl, and 0.01% Tween20. Components are pre-incubated for 30 min, then a small volume of reporter probe (Proteros) is added from a highly concentrated stock and incubation is continued for another 30 min. Final concentrations after adding the reporter probe amount to 20 nM HDAC2, 4 nM antibody, and 180 nM probe.
Measurement: Kd = 2750 nM (pChEMBL 5.559999942779541)
Descriptors: MW 398.39, ALogP 4.9, HBA 3, HBD 2, PSA 71.25, QED 0.6
Compound (SMILES): C[C@@H](Oc1ccc2c(n1)CCN(CC[C@H]1CC[C@H](NC(=O)c3cccc(=O)n3C)CC1)CC2)C(F)(F)F
InChIKey: GFYPLRKHLRVZID-SFHLNBCPSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Binding Inhibition for Dopamine D3 Receptor: Buffer solution: 50 mM Tris-HCl (35409-45, Nacalai Tesque) (pH 7.4) containing 120 mM NaCl (31320-05, Nacalai Tesque), 1 mM MgCl.6HO (20909-55, Nacalai Tesque), 5 mM KCl (28514-75, Nacalai Tesque) and 2 mM CaCl) (067-31, NAKARAI CHEMICALS, LTD.) Radioligand: (final concentration) 2 nM [H]-Methylspiperone ([H���N-methyl-]-Methylspiperone, NET-856, 83.8 Ci/mmol, PerkinElmer)Non-specific ligand: (final concentration) 10 uM Butaclamol [(+)-Butaclamol Hydrochloride, D033, Sigma] SPA beads solution: SPA beads [WGA PVT SPA Scintillation Beads, RPNQ0001 (500 mg), RPNQ0060 (2 g), PerkinElmer] (0.2 mg/well) Incubation time and temperature: 120 min at 25° C.
Measurement: Ki = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 520.6, ALogP 3.89, HBA 6, HBD 1, PSA 76.46, QED 0.6
Compound (SMILES): NC/C(=C\F)COc1ccc(S(=O)(=O)CC2CCN(C(=O)C3CCCC3)CC2)c(F)c1
InChIKey: UZQQFMBJPBAQJR-SFQUDFHCSA-N
Target: Amine oxidase [copper-containing] 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75106
Assay: SSAO Activity Assay: Amine oxidase activity of recombinant hSSAO, hMAO-A, and hMAO-B isoforms are measured using the MAO-Glo assay kit (Promega, V1402). Ten nL test compounds (with DMSO as vehicle, 0.1% v/v for hSSAO, hMAO-A and hMAO-B) were added into assay plates by Echo, and pre-incubated with 5 μL of enzyme for 30 mins at room temperature before the addition of 5 μL of luminogenic substrate. The substrate concentration is 40 μM for human recombinant SSAO, 40 μM for human recombinant MAO-A, and 4 μM for human recombinant MAO-B. The hSSAO and hMAO-A assays were conducted in the MAO-A reaction buffer in the kit, and the MAO-B assay was conducted in the MAO-B reaction buffer. Oxidation of the substrate was conducted for 1 hr, before the addition of detecting reagent according the manufacture's protocol. The IC50 value of the tested compounds was calculated by fitting the dose response curve using a 4-parameter non-linear regression routine.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 456.56, ALogP 3.22, HBA 5, HBD 1, PSA 89.7, QED 0.65
Compound (SMILES): CC#CC(=O)N1CC[C@]2(C1)C[C@H](n1nc(-c3cn(Cc4cccc(C(F)(F)F)c4)nc3C)c(C(N)=O)c1N)C2
InChIKey: NFLKTKKJFVNGLH-KQODVJPSSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Lanthscreen Eu Kinase Binding Assay : Lanthscreen Eu Kinase Binding assay for BMX is performed as described above for BTK except that 1 nM human recombinant full length TEC (His-tagged) kinase and 1 nM Alexa Fluor647-labeled Kinase Tracer #178 were used instead.Representative compounds of the present invention are assessed for inhibition of BTK, BMX, and TXK measuring phosphorylation of a substrate (Z′-LYTE assay, Life Technologies) and TEC measuring displacement of a tracer (Lanthscreen® Eu Kinase Binding assay, Life Technologies).
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 539.56, ALogP 3.38, HBA 7, HBD 2, PSA 125.06, QED 0.48