lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(NCc1ccccc1)c1ccc(C2CC2NCc2ccc(-c3cnccn3)cc2)cc1
InChIKey: NCSUOTIPVGKGSZ-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 Inhibition Assay: A test compound dissolved in 2.5% DMSO was added by 4 μL to 3 μL reaction solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 1 mM DTT) containing 7.5 ng of LSD1, and the mixture was reacted at room temperature for 60 min. Biotin-histone H3 mono methylated K4 peptide solution (NH2-ART(me-K)QTARKSTGGKAPRKQLAGGK(Biotin)-CONH2) (3.3 μM) was added by 3 μL to start the reaction. After reaction at room temperature for 5 min, 1 mM 2-PCPA solution was added 5 μL to terminate the reaction. A detection solution (BOO mM potassium fluoride, 0.1% BSA) containing europium-labeled anti-histone H3 antibody (Wako Pure Chemical Industries, Ltd.) and Streptavidin-XL665 (Cisbio) was further added by 5 μL, and the mixture was left standing for 60 min. A time-resolved fluorescence (excitation 320 nm, emission 615 nm, 665 nm) was measured by Envision (PerkinElmer). The LSD1 inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate(%)=(1−(test compound count−blank)÷(control−blank))×100
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 434.54, ALogP 4.72, HBA 4, HBD 2, PSA 66.91, QED 0.42
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(Cc5c[nH]nn5)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: PPSNVVBCVMMFBL-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 454.58, ALogP 4.81, HBA 6, HBD 2, PSA 90.79, QED 0.4
Compound (SMILES): CC1(N)CCN(c2cnc3c(-c4ccccc4Cl)n[nH]c3n2)CC1
InChIKey: JQWPQEJDTUKOCS-UHFFFAOYSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: PTPN11 Enzymatic Assay: Recombinant full-length wild-type and E76K mutant human PTPN11 proteins were cloned, expressed (E. coli system), and isolated via a two-step purification of Ni affinity followed by S75 size exclusion chromatography.Phosphatase activity of full length wild-type PTPN11(PTPN11-WT) or PTPN11-E76K mutant enzyme was measured using the fluorogenic 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP; Molecular Probes) as the substrate. Enzyme (250 pM) was incubated with or without increasing concentrations of compounds in assay buffer (62.5 mM HEPES, 125 mM NaCl, 1 mM EDTA, 1.25 mM TECP, 0.1% BSA) for 30 min at room temperature. Reaction was initiated by addition of DiFMUP (50 μM) at room temperature in 384-well black plate with a final reaction volume of 20 uL in assay buffer. After 1 hour, DiFMUP fluorescence signal was measured (Ex: 340/Em: 460) using Envision plate reader. Dose-response curves were analyzed using IC50 regression curve fitting (GeneData Screener). Curves were normalized to a high controls without inhibitor, and low controls without enzyme. Results are given below in Table 1. Other compounds disclosed herein are expected to have activity similar to the results below, showing activity as PTPN11 inhibitors.
Measurement: IC50 = 326 nM (pChEMBL 6.489999771118164)
Descriptors: MW 342.83, ALogP 2.99, HBA 5, HBD 2, PSA 83.72, QED 0.75
Compound (SMILES): O=C(O)c1ccc(NC(=O)[C@@H](CC2CC2)c2ccc(-c3c(C(F)F)ccc(Cl)c3F)c[n+]2[O-])cc1
InChIKey: YTPAQVBXJDTTMW-SFHVURJKSA-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 half-maximal inhibitory concentrations (IC50), or the inhibitory constant, Ki.Compounds were pre-incubated for 30 minutes at 25° C. with human (0.04 nM) Factor XIa in 50 mM HEPES buffer with 150 mM sodium chloride, 5 mM calcium chloride, 0.1% PEG 8000, pH 7.4. Factor XIa enzymatic activity was determined by addition of the substrate glycine-proline-arginine-7-amido-4-trifluoromethylcoumarin (GPR-AFC) and measurement of the fluorescence at 400/505 nm after a 60 minute incubation at 25° C. The % inhibition for each data point was calculated from the data and analyzed using the log (inhibitor) vs. response four parameters equation to determine the half-maximal inhibitory concentrations (IC50). The IC50 were converted to equilibrium inhibitory constants (Ki) using the Cheng-Prusoff equation.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 504.89, ALogP 5.94, HBA 3, HBD 2, PSA 93.34, QED 0.29
Compound (SMILES): COc1ccc(-n2nc(C(=O)N3CCC[C@@H](N)C3)cc2-c2ccc(C#N)cc2)cc1F
InChIKey: YJLWOYXVHYHONO-QGZVFWFLSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Biochemical Inhibition Activity Assay: The biochemical inhibition activity of the synthesized compounds on LSD1 was measured. The activity measurement was performed using LSD1 fluorescence analysis kit (available from BPS Bioscience Co., Ltd., Catalog No: 50106). This analysis kit is designed to measure activity of LSD1 enzyme. H2O2 generated upon demethylation of Lys4 moiety of histone H3 by LSD1 is reacted with HRP/Amplex Red reagent to form fluorescent Resorufin, which is measured by this kit, thereby confirming demethylation.
Measurement: IC50 = 364 nM (pChEMBL 6.440000057220459)
Descriptors: MW 419.46, ALogP 3.12, HBA 6, HBD 1, PSA 97.17, QED 0.7
Compound (SMILES): CN1C(=N)N[C@]2(C)c3cc(NC(=O)c4ccc(C#N)cn4)ccc3CCC[C@H]2S1(=O)=O
InChIKey: DXOCMNBLELQBRX-WIYYLYMNSA-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 3× the final desired concentration in a volume of 10 ul are preincubated with purified human BACE1 catalytic domain (3 nM in 10 μl) 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 μl of 600 nM QSY7-APPswe-Eu substrate (200 nM final) to give a final reaction volume of 30 μl 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 μm value of 8 μM for the QSY7-APPswe-Eu substrate at BACE1.
Measurement: Ki = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 438.51, ALogP 1.93, HBA 6, HBD 3, PSA 139.04, QED 0.65
Compound (SMILES): CC(=O)c1ccc(C(=O)N2C[C@H](NC(=O)[C@@H](C)N)C(=O)N(Cc3c(C)ccc4ccccc34)c3ccccc32)cc1
InChIKey: HDRZXKUDFVCNST-PIKZIKFNSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay for BIR2 and BIR3: Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(c-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations. Percentage of inhibition at each concentration was determined to generate an IC50 value for each test compound.
Measurement: IC50 = 1479 nM (pChEMBL 5.829999923706055)
Descriptors: MW 548.64, ALogP 4.38, HBA 5, HBD 2, PSA 112.81, QED 0.35
Compound (SMILES): FC(F)c1cc(-n2cc(CNc3ncccn3)nn2)ccc1Cl
InChIKey: ZLBVELNRVLGPDW-UHFFFAOYSA-N
Target: Retinoic acid receptor RXR-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P19793
Assay: NR2B standard assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to ¿EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 1260 nM (pChEMBL 5.900000095367432)
Descriptors: MW 336.73, ALogP 3.26, HBA 6, HBD 1, PSA 68.52, QED 0.77
Compound (SMILES): O=C(CN1CN(c2ccccc2)C2(CCN(C(=O)c3ccc4[nH]ncc4c3)CC2)C1=O)N[C@H]1CNC[C@@H]1c1ccccc1
InChIKey: XCVSRUFAZODOLV-PXJZQJOASA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: In Vitro Binding Assay for DDR1: This assay is based on the intracellular domain of the DDR1 protein which contains the kinase active site. The recombinant protein additionally carries a GST-tag that can be recognized by an Eu-labeled anti-GST antibody. A tracer compound binding to the active site is labeled with a dye so that a FRET donor acceptor pair can be formed. Excitation energy absorbed by the Europium complex (350 nm flash light or pulsed laser) is transferred to a suitable fluorescent dye, if it is in close proximity. Compounds binding competitively with the tracer molecule will displace the bound tracer molecules and reduce the FRET signal in a dose dependent manner. Due to the long lifetime of the Eu excited state, the emission of the donor and the acceptor can be measured in time-gated mode such that most of the intrinsic fluorescence contributions have already decayed. This results in high sensitivity, excellent reproducibility and high data quality. This sensitive detection method enables protein concentrations below 20 nM.Protein, tracer and labeled antibody were obtained from commercial sources. The assay was performed in 384 low volume microtiter-plates with a final volume of 15 μl. Dose response curves were generated from 16 compound dilutions in DMSO as solvent compound dilutions, a solution containing protein and labeled antibody, and a solution containing the tracer which is added in the last step. The fluorescence of donor and acceptor were then measured after one hour incubation at room temperature. Every assay run was quality-controlled with dose response curves for two reference compounds.
Measurement: IC50 = 577 nM (pChEMBL 6.239999771118164)
Descriptors: MW 577.69, ALogP 2.72, HBA 6, HBD 3, PSA 113.67, QED 0.32
Compound (SMILES): N#Cc1ccc(CN2CCC(NC(=O)c3ccc(C(=O)N4CCNCC4)cn3)CC2)cc1
InChIKey: XJFFGYUNDZFZOG-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 > 10000 nM (pChEMBL 5)
Descriptors: MW 432.53, ALogP 1.39, HBA 6, HBD 2, PSA 101.36, QED 0.74
Compound (SMILES): COc1ccc(C(O)CCc2cc(OC)c(OC)c(OC)c2)c(O)c1
InChIKey: QSAWBQYXISOLBK-UHFFFAOYSA-N
Target: Polyphenol oxidase 1 (SINGLE PROTEIN), Agaricus bisporus
UniProt: Q00024
Assay: Tyrosinase Inhibition Assay: A tyrosinase inhibition assay was carried out using the method reported by Jones et al. (2002) Pigment. Cell Res. 15:335. Using this method, the conversion of L-Dopa, a substrate of tyrosinase, into dopachrome is followed by monitoring absorption at 450 nm. Tyrosinase was prepared in 50 mM potassium phosphate buffer, pH 6.8 (assay buffer) at 2000 U/ml and stored at −20° C. in 1 ml aliquots prior to use. For use in assays, stock enzyme solutions were thawed and diluted to 200 U/ml with assay buffer. A 2 mM working solution of substrate, L-DOPA, was prepared in assay buffer for each assay. Samples were dissolved in 10% DMSO (0.5 ml) and diluted to 5 ml with assay buffer. The reaction mixture consisted of 0.050 ml 2 mM L-DOPA, 0.050 ml 200 U/ml mushroom tyrosinase and 0.050 ml inhibitor. Reaction volume was adjusted to 200 μl with assay buffer. Assays were performed in 96 well Falcon 3097 flat-bottom microtiter plates (Beckton Dickinson, N.J.). Appearance of dopachrome was measured with a WALLAC 1420 Multilable Counter (Turku, Finland). Average velocity was determined from linear enzyme rate as measured by change in absorbance (ΔA450) at 450 nm per minute.
Measurement: IC50 = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 348.4, ALogP 3.09, HBA 6, HBD 2, PSA 77.38, QED 0.76
Compound (SMILES): O=S(=O)(Nc1nccs1)c1cc(Cl)c(N[C@@H]2CCCC[C@H]2N2CC3CC3C2)cc1F
InChIKey: ZXTJOCUXAIBHEZ-ZBIUXTHTSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Electrophysiological Assay (EP) (In Vitro Assay): Patch voltage clamp electrophysiology allows for the direct measurement and quantification of block of voltage-gated sodium channels (NaV's), and allows the determination of the time- and voltage-dependence of block which has been interpreted as differential binding to the resting, open, and inactivated states of the sodium channel (Hille, B., Journal of General Physiology (1977), 69: 497-515).The following voltage clamp electrophysiology studies were performed on representative compounds using cells heterologously expressing Nav1.7 or Nav1.5 channels. cDNAs for Nav1.7 (NM_002977) and Nav1.5 (AC137587) were stably expressed in Chinese Hamstr Ovary (CHO) cells and CHL (Chinese Hamster Lung) cells respectively. Sodium currents were measured in the whole-cell configuration using Syncropatch 384PE (NanIon Technologies, Germany). 1NPC®-384 chips with custom medium resistance and single hole mode are used. Internal solution consists of (in mM): 110 CsCl, 10 CsCl, 20 EGTA, and 10 Hepes (pH adjusted to 7.2); and external solution contains (in mM): 60 NMDG, 80 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 2 D-Glucose monohydrate, 10 Hepes (pH adjusted to 7.4 with NaOH).After system flushing, testing compounds are dissolved in external solution containing 0.1% Pluronic F-127. The chip is moved into the measuring head and the instrument primes the chip with external and internal solutions. 10l cells are added to the chip from a cell hotel, and a negative pressure of −50 mBar is applied to form a seal. Following treatment with seal enhancer solution and wash-off with external solution, negative pressure of −250 mbar is applied for 1 second to achieve the whole-cell configuration, followed by three washing steps in external solution. 20 μl of compounds is added to 40 μl in each well (1:3 dilution of compounds), and after mixing, 20 μl is removed so the volume is retained at 40 μl. After approximately 13 minutes recordings, 20 μl/well of 2 uM TTX, or 333 uM Tetracaine (for Nav1.5) is added to achieve full block.For voltage protocol, an holding potential of −50 mV is applied during the whole experiment. A depolarizing step is applied to −10 mV for 10 ms, followed by a hyperpolarization step to −150 mV for 20 ms to allow channel recovery from inactivation. A second depolarizing step is applied from −150 mV to −10 mV for 10 ms, where currents were measured to derive blocking effects of compounds. Inhibition is determined based on 7.5 min of compound incubation.
Measurement: IC50 = 202 nM (pChEMBL 6.699999809265137)
Descriptors: MW 471.02, ALogP 4.41, HBA 6, HBD 2, PSA 74.33, QED 0.65
Compound (SMILES): C=CC(=O)N1CCC(c2cc3cn[nH]c3c(-c3ccc(C(=O)Nc4ccccn4)cc3)n2)C1
InChIKey: LHEAIJGUVZUPFF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTK Enzyme Activity Testing: In vitro activity of BTK is measured by detecting ADP produced in a kinase reaction with an ADP-Glo kinase assay kit from Promega Company. In the kinase assay, the kinase consumes ATP to phosphorylate the substrate, while producing ADP. ADP-Glo reagent then terminates the kinase reaction and completely consumes the remaining ATP. Finally, a kinase detection reagent is added to convert generated ADP into new ATP. Luciferase in the detection reagent is capable of catalyzing fluorescein with the participation of ATP and O2 to produce oxidized fluorescein, AMP, and generate light quantum, thereby converting a chemical signal into an optical signal (Luminecence). The intensity of optical signal is positively correlated with the production of ADP in the kinase reaction, so that the activity of kinase BTK can be thereby quantitatively determined.All assays are conducted at a constant temperature of 23° C., using a Corning 3674 Type white 384-well plate; the BTK kinase (full length with His-Tag) is expressed and purified internally by the company; the substrate of kinase is polypeptide (4:1 Glu,Tyr) (from SignalChem) and ATP (from Sigma); and a microplate reader EnVision (Perkin Elmer) is used for reading an optical signal. The assay buffer includes 40 mM Tris-HCl (pH 7.5), 10 mM MgCl2 (Sigma), 2 mM MnCl2 (Sigma), 0.05 mM DTT (Sigma), and 0.01% BSA (Sigma); the BTK kinase is diluted with the assay buffer to a concentration of 1.6 ng/uL as a kinase reaction solution; and the substrate reaction solution comprises 0.2 mg/mL polypeptide substrate and 50 uM ATP.Compound's IC50 is calculated from 10 concentration points by the following method. The compound is dissolved and diluted in 100% DMSO to a concentration of 1 mM, followed by a serial 3× dilution with DMSO to a minimum concentration of 0.05 uM. Each concentration stock is further diluted 40× with the assay buffer. To a 384-well assay plate are added 1 uL of a series of compound solutions and 2 uL of the kinase reaction solution, followed by mixing homogeneously, and incubating in the dark at room temperature. After in the dark for 30 min, 2 uL of the substrate reaction solution is added to allowed the total reaction volume to 5 uL. The reaction mixture is incubated in the dark at room temperature in the dark for another 60 min. An equal volume of 5 uL ADP-Glo reagent is then added to terminate the reaction. The resulting mixture is homogeneously mixed, and stands at room temperature for 40 min. Finally, 10 uL of the kinase detection reagent is added, stands at room temperature for 30 min, and then a value is read on Envision.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 438.49, ALogP 3.77, HBA 5, HBD 2, PSA 103.87, QED 0.46
Compound (SMILES): Nc1nc2c3c(ccc2c2nc(CN4CCC(Oc5ccc(F)cc5)CC4)nn12)OC(F)(F)O3
InChIKey: DOIPPUIIOMEOSU-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: competition binding assay: The following table shows representative data for the compounds of the Examples as A2a receptor antagonists as determined by a competition binding assay using Scintillation Proximity technology. Thus, 1.0 μg of membranes from CHO-K1 cells expressing the human A2a receptor were incubated with a compound of the invention at concentrations ranging from 3000 nM to 0.15 nM in a reaction mixture also containing 2.0 nM of a tritiated form of 5-amino-7-[2-phenethyl]-2-(furan-2-yl)-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidine (the tritiated compound) and 100 μg of wheatgerm agglutin-coated yttrium silicate SPA beads for one hour at room temperature with agitation. The beads were then allowed to settle to the bottom of the wells for 1 hr, after which the membrane-associated radioactivity was determined by scintillation counting in a TopCount microplate reader. Ki values were determined using the Cheng-Prusoff equation.
Measurement: Ki = 4.9 nM (pChEMBL 8.3100004196167)
Descriptors: MW 472.43, ALogP 3.36, HBA 9, HBD 1, PSA 100.03, QED 0.48
Compound (SMILES): CNC1CCN(c2nc(-c3ccc(C#N)cc3)c(-c3ccc(C)cc3)n(C)c2=O)CC1
InChIKey: JCGNLVXIPOIVDN-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: In Vitro Enzyme Inhibition Assay: The enzymatic assay of LSD1 activity is based on Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) detection. The inhibitory properties of compounds to LSD1 were determined in 384-well plate format under the following reaction conditions: 0.1-0.5 nM LSD1, 50 nM H3K4me1-biotin labeled peptide (Anaspec cat #64355), 2 uM FAD in assay buffer of 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA. Reaction product was determined quantitatively by TR-FRET after the addition of detection reagent Phycolink Streptavidin-allophycocyanin (Prozyme) and Europium-anti-unmodified histone H3 lysine 4 (H3K4) antibody (PerkinElmer) in the presence of LSD1 inhibitor such as 1.8 mM of Tranylcypromine hydrochloride (2-PCPA) in LANCE detection buffer (PerkinElmer) to final concentration of 12.5 nM and 0.25 nM respectively. The assay reaction was performed according to the following procedure: 2 uL of the mixture of 150 nM H3K4me1-biotin labeled peptide with 2 uL of 11-point serial diluted test compound in 3% DMSO were added to each well of plate, followed by the addition of 2 uL of 0.3 nM LSD1 and 6 LM of FAD to initiate the reaction. The reaction mixture was then incubated at room temperature for one hour, and terminated by the addition of 6 uL of 1.8 mM 2-PCPA in LANCE detection buffer containing 25 nM Phycolink Streptavidin-allophycocyanin and 0.5 nM Europium-anti-unmodified H3K4 antibody. Enzymatic reaction is terminated within 15 minutes if 0.5 LSD1 enzyme is used in the plate. Plates were read by EnVision Multilabel Reader in TR-FRET mode (excitation at 320 nm, emission at 615 nm and 665 nm) after 1 hour incubation at room temperature.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 413.53, ALogP 3.48, HBA 6, HBD 1, PSA 73.95, QED 0.71
Compound (SMILES): Cc1cccc2nc([C@@H](Nc3nc(N)nc(N)c3C#N)C3CC3)n(-c3cc(F)cc(F)c3)c(=O)c12
InChIKey: HTNPZVQEQLIDFG-IBGZPJMESA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: TR-FRET assay : TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site. Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. The compounds of Table 1, Compound X ((S)-2-(1-((9H-purin-6-yl)amino)ethyl)-5-chloro-3-phenylquinazolin-4(3H)-one) and Compound Y ((S)-4-amino-6-((1-(5-chloro-4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile), were added to the assay solution and incubated for 30 minutes at 25° C. Additionally, compounds 19 to 116 were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 474.48, ALogP 3.36, HBA 9, HBD 3, PSA 148.53, QED 0.4
Compound (SMILES): C=CC(=O)N1CCN(CCn2c(=O)ccc3cnc(Nc4ccn(C(C)C)n4)nc32)[C@@H](CC)C1
InChIKey: HUGFHSWFIHCEQV-IBGZPJMESA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK and Tyk2 Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies. Serially or discretely diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1 h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.
Measurement: Ki > 100 nM (pChEMBL 7)
Descriptors: MW 464.57, ALogP 2.42, HBA 9, HBD 1, PSA 101.18, QED 0.51
Compound (SMILES): Cc1ccc(-c2noc(C)c2COc2cc3c(cn2)C(=O)N(C2CCSCC2)C3)cn1
InChIKey: STIAETBDCMTLLC-UHFFFAOYSA-N
Target: Gamma-aminobutyric acid receptor subunit alpha-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14867
Assay: Electrophysiology: Electrophysiological experiments were performed on days 3 to 5 after the micro-injection of mRNA. During the experiment the oocytes were constantly superfused by a solution containing (in mM) NaCl 90, KCl 1, HEPES 5, MgCl2 1, CaCl2 1 (pH 7.4). Oocytes were impaled by two glass microelectrodes (resistance: 0.4 MΩ) which were filled with a solution containing KCl 1M+K-acetate 1.5 M and voltage-clamped to −80 mV. The recordings were performed at room temperature using the Roboocyte two-electrode voltage clamp system (Multichannelsystem). After an initial equilibration period of 1.5 min GABA was added for 1.5 min at a concentration evoking approximately 20% of a maximal current response (EC20). After another rest interval of 2.5 min GABA was again added evoking a response of similar amplitude and shape. 0.5 min after the onset of this second GABA application the test compound, at a concentration corresponding to approximatively 30 fold its Ki, was added while GABA was still present. Current traces were recorded at a digitization rate of 10 Hz during and shortly before and after the GABA application.
Measurement: Ki = 9.6 nM (pChEMBL 8.020000457763672)
Descriptors: MW 436.54, ALogP 4.18, HBA 7, HBD 0, PSA 81.35, QED 0.59
Compound (SMILES): C[C@@H]1OCC2(CCN(c3nc4[nH]nc(-c5cc(F)ccc5F)c4c(=O)n3C)CC2)[C@@H]1N
InChIKey: ZVWVAOSFFYJEDW-APPDUMDISA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Allosteric Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phosphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.More specifically, the phosphatase reactions were performed at room temperature in 384-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat #3575) using a final reaction volume of 25 μL and the following assay buffer conditions: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT.The inhibition of SHP2 by compounds of the invention (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.5 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) (SEQ ID NO:1). After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567) was added to the reaction and incubated at 25° C. for 30 minutes. The reaction was then quenched by the addition of 5 μl of a 160 μM solution of bpV(Phen) (Enzo Life Sciences cat #ALX-270-204). The fluorescence signal was monitored using a microplate reader (Envision, Perki-Elmer) using excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 430.46, ALogP 1.93, HBA 7, HBD 2, PSA 102.06, QED 0.64
Compound (SMILES): Cc1ncoc1-c1nnc(SCCCN2C[C@H]3CCN(c4ccc(C(F)(F)F)cc4F)[C@H]3C2)n1C
InChIKey: YTXILLIRPDSLHZ-BEFAXECRSA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Inhibition Assay: 3H]-Spiperone Binding Assay at hD3 and hD4 recombinant receptors CHO cells transiently transfected with human dopamine type 3 or 4 receptors (CHO-hD3 or CHO-hD4, respectively), were re-suspended in 20 mM HEPES, 2 mM EDTA (pH 7.4), homogenised and centrifuged at 40,000 g (20 min, 4° C.). After re-suspension, homogenization and centrifugation as above, the final pellet was re-suspended in 20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 1 mM EDTA (pH 7.4) and aliquots were kept at −80° C. [3H]-Spiperone Binding experiments were performed in 96 deep-well polypropylene plates in 50 mM Tris/HCl, 120 mM NaCl, 5 mM KCl, 5 mM MgCl2 (pH 7.4). Compounds of invention were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final in the assay). Displacement was performed in the presence of 0.3 nM [3H]-Spiperone. The reaction was initiated by the addition of membrane suspension (4 μg and 12 μg of protein for CHO-hD3- and CHO-hD4 membranes, respectively) and lasted for 90 or 100 min (for hD3 or hD4 membranes, respectively) at 23° C. in a final volume of 500 μl. Non specific binding (NSB) was determined in the presence of 1 μM Spiperone. The binding reaction was stopped by rapid filtration through GF/B filterplates pre-soaked in 0.5% polyetylenimmine (PEI) using a Packard cell harvester. After washing with ice-cold 0.9% NaCl, the plate was left to dry before the addition of Microscint 20 (50 μl/well, PerkinElmer). Radioactivity was counted with a TopCount (PerkinElmer). Data were analysed by non-linear regression analysis using GraphPad Prism 5.0 (GraphPad Software). Saturation binding experiments were performed similar to the competition binding experiments using a radioligand concentrations ranging from 0.015 to 4.0 nM. Ref: Mackenzie R. G. et al. (1994). Characterization of the human dopamine D3 receptor expressed in transfected cell lines. Eur. J. Pharmacol. 266: 79-85.
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 510.56, ALogP 4.63, HBA 8, HBD 0, PSA 63.22, QED 0.26
Compound (SMILES): COCCN1CC2CCCC(C1)C2(OC)c1cccc(C(N)=O)c1
InChIKey: DXGHLKRYVCIZLD-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Competitive Displacement Assay: Membrane protein from CHO (Chinese Hamster Ovarian) cells that stably expressed one type of the cloned human opioid receptor were incubated with 12 different concentrations of the compound in the presence of 0.25 nM [3H]DAMGO, 0.2 nM [3H]naltrindole or 1 nM [3H]U69,593 in a final volume of 1 mL of 50 mM Tris-HCl, pH 7.5 at 25° C. Incubation times of 60 min were used for [3H]DAMGO and [3H]U69,593. Because of a slower association of [3H]naltrindole with the receptor, a 3 h incubation was used with this radioligand. Samples incubated with [3H]naltrindole also contained 10 mM MgCl2 and 0.5 mM phenylmethylsulfonyl fluoride. Nonspecific binding was measured by inclusion of 10 μM naloxone. The binding was terminated by filtering the samples through Schleicher & Schuell No. 32 glass fiber filters using a Brandel 48-well cell harvester. The filters were subsequently washed three times with 3 mL of cold 50 mM Tris-HCl, pH 7.5, and were counted in 2 mL Ecoscint A scintillation fluid. For [3H]naltrindole and [3H]U69,593 binding, the filters were soaked in 0.1% polyethylenimine for at least 60 min before use.
Measurement: Ki < 1 nM (pChEMBL 9)
Descriptors: MW 332.44, ALogP 2.01, HBA 4, HBD 1, PSA 64.79, QED 0.87
Compound (SMILES): CCCCOc1nc(N)c2[nH]cc(Cc3ccc(CN4CCN(C)CC4)cc3)c2n1
InChIKey: MBWXIDSZYILEKR-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: In Vitro Receptor Binding Activity Screening Assay: Reagents:HEK-blue hTLR7 cell and HEK-blue hTLR8 cell (available from InvivoGen)DMEM mediumheat inactivated fetal bovine serumAnti Mycoplasma reagent Normocin bleomycinblasticidinScheme:1. Preparation of 96-well compound plate:The compounds were gradient diluted with DMSO in 3-fold using liquid work station POD starting at a concentration of 10 mmol/L and 10 points were diluted (2nd column to 11th column, and each point was duplicated). At 12th column, 1 μL of 5 mg/mL positive compound R848 was added as positive control; and at 1st column, 1 μL of DMSO was added as negative control. Each well contained 1 μL of DMSO.2. The cells in culture flask were collected and the cell density was diluted to 250,000 cells/mL.3. 200 μL (50,000 cells/well) of cell suspension was added into prepared compound plate and the final concentration of DMSO in each well was 0.5%.4. The culture plates containing cells and the compounds were incubated in CO2 incubator for 24 h at 37° C., 5% CO2.5. After 24 h incubation, 20 μL of supernatant was removed from each well to a 96-well transparent assay plate. To each well of the assay plate was added 180 μL of Quanti-Blue reagent and the plate was incubated in an incubator at 37° C., 5% CO2 for 1 h.6. After 1 h, the content of alkaline phosphatase in 20 μL of supernatant was determined using Microplate Reader OD650.7. EC50 of each compound was obtained with Prism software.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 408.55, ALogP 3.06, HBA 6, HBD 2, PSA 83.3, QED 0.56
Compound (SMILES): CCCCc1nc(O)c(-c2nnc(Cc3n[nH]c(=O)c4ccccc34)o2)c(O)c1-c1c(OC)cccc1OC
InChIKey: MXOLKZIXNWZDDT-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyr1)apelin-13) peptide, which was set to 100%.The examples disclosed below were tested in the APJ in vitro assays described above and were found having human APJ cyclic AMP (hcAMP) activity. The EC50 value of each compound is presented at the end of the example description.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 529.55, ALogP 4.4, HBA 10, HBD 3, PSA 156.48, QED 0.25
Compound (SMILES): N#Cc1ccc(N2CCc3c(ncnc3Nc3ccc(C(=O)NCC(F)F)nc3)C2)cc1C(F)(F)F
InChIKey: YKLABHRBKMZJRH-UHFFFAOYSA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Antagonist Activity for AR: Antagonist activity for AR was evaluated according to the following method. COS-7 cells (ATCC) were transfected with pMMTV-luc vector (reporter plasmid having, as an androgen response element, murine mouse mammary virus long terminal repeat) and pEX-hAR vector (human androgen receptor expression vector: which expresses human AR gene under control of CMV promoter) by using Nucleofector (registered trademark) Kit R (Lonza) as a transfection reagent and Amaxa (Lonza). The COS-7 cells obtained after transfection were seeded in a clear bottom 96 well microplate (BD) at 1.5×104/well with phenol red free RPMI1640 containing 10% charcoal-treated fetal bovine serum (hereinbelow, DCC-FBS) (hereinbelow, the medium is referred to as an evaluation medium), and then cultured overnight. The culture was added with the evaluation medium containing dihydrotestosterone (DHT) (final concentration of DHT: 1 nmol/L) or the evaluation medium containing the compound of Examples or the compound of Comparative Examples (final concentration of the compound of Examples or the compound of Comparative Examples: 5, 14, 41, 123, 370, 1111, 3333, or 10000 nmol/L), followed by culture for 24 hours. Then, the transcription activity value was measured. The transcription activity was measured by using Bright-Glo Luciferase Assay System (Promega). From the measured transcription activity, 50% transcription activity inhibition concentration (IC50 value) was calculated by logistic regression when the transcription activity value obtained by using 1 nmol/L DHT was 100% and the transcription activity value obtained by using the evaluation medium only was 0%.
Measurement: IC50 = 490 nM (pChEMBL 6.309999942779541)
Descriptors: MW 503.44, ALogP 4.06, HBA 7, HBD 2, PSA 106.83, QED 0.49
Compound (SMILES): N=C(NC(=O)C(NC(=O)c1cc(Br)co1)c1ccc(Cl)c(Cl)c1)c1ccccc1
InChIKey: XXPWBHCDEBIDSO-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O95136
Assay: TBD: TBD
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 495.16, ALogP 4.96, HBA 4, HBD 3, PSA 95.19, QED 0.35
Compound (SMILES): CC#C[C@@H](CC(=O)O)c1ccc(OCc2ccc3scc(-c4ccc(CC5CCNCC5)cc4C)c3c2)cc1
InChIKey: FKURDDHSZLVEKL-NDEPHWFRSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Calcium Flux Assay: Compounds were tested in a calcium flux assay using transfected HEK293 cells stably expressing either human GPR40 or rat GPR40. Human GPR40 expressing cells were cultured in DMEM-High Glucose media supplemented with 10% fetal bovine serum, 1×L-Glutamine, 1× Penicillin/Streptomycin and 500 μg/mL G418. Rat GPR40 expressing cells were cultured in DMEM-High Glucose media supplemented with 10% fetal bovine serum and 1 μg/mL puromycin. Cells were plated into poly-D-lysine coated 384-well plates and cultured overnight in a 37° C. humidified tissue culture incubator under 5% CO2/90% O2 atmosphere. On the day of the experiment, the culture media was replaced with assay buffer (HBSS, 20 mM HEPES, 0.1% BSA) and the cells incubated at 37° C. for 1 h. Calcium-sensitive fluorescent dye (Fluo 8 No-Wash Calcium Dye, ABD Bioquest) was then added and the cells incubated for another 30 min at 37° C. followed by 15 min at room temperature while protected from the light. The cell plate and a plate of diluted compounds of Formula (I) were loaded into a fluorescent plate reader that added compounds onto the cells while measuring the fluorescence intensity of each well.
Measurement: EC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 537.73, ALogP 7.58, HBA 4, HBD 2, PSA 58.56, QED 0.22
Compound (SMILES): Cn1c(=O)n(Cc2cccnn2)c2cc(-c3ccc(F)c(Cl)c3)cnc21
InChIKey: FMIAMQMNCXUHOE-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: Standard cell-based calcium flux assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 369.79, ALogP 3.03, HBA 6, HBD 0, PSA 65.6, QED 0.56