lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(CC1CNC1)Nc1ccc(C(F)(F)F)cn1
InChIKey: KBFKFIVPNOIUAB-UHFFFAOYSA-N
Target: Voltage-dependent N-type calcium channel subunit alpha-1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q00975
Assay: High-Throughput Cav2.2/Kir2.3 T-Type Fluorescent Assay: Cells were plated in 384-well, clear-bottom, black-walled, poly-D-lysine coated plates (Becton Dickinson, Franklin Lake, N.J.) 2 days prior to use in the FLIPR assay. 100 μL of cells (1.4×106 cell/mL) containing doxycyline (Sigma-Aldrich, 1.5 μg/mL; to induce channel expression) were added to each well using a Multidrop (Thermo Scientific, Waltham, Mass.) and were maintained in 5% CO2 incubator at 37° C. On the morning of the assay, cells were transferred to a 5% CO2 incubator at 29° C. Cells can then be washed with a wash buffer containing (in mM): 118 NaCl, 18.4 HEPES, 11.7 D-glucose, 2 CaCl2, 0.5 MgSO4, 4.7 KCl, 1.2 KH2PO4, pH adjusted to 7.2 with NaOH. 4.4 μM of the fluorescent indicator dye, Fluo-4 (Invitrogen), prepared in pluronic acid (Sigma-Aldrich), were loaded into the wells and incubated for 45 minutes at 29° C. in 5% CO2. Cells were then rinsed with either a 2 mM KCl closed-state buffer (in mM: 138.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 2 KCl, with the pH adjusted to 7.4 with NaOH) when performing the closed-state assay or 12.5 mM KCl inactivated-state buffer (in mM: 128 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 12.5 mM KCl, with the pH adjusted to 7.4 with NaOH) when performing the inactivated-state assay. Concentration-dependent response curves were generated from 5 mM stock solutions prepared in DMSO (Sigma-Aldrich) and diluted in either the 2 mM KCl buffer or 12.5 mM KCl buffer and incubated for 20 minutes at 29° C. in 5% CO2. Calcium entry was evoked with an addition of 130 mM KCl stimulation buffer (in mM: 10.5 NaCl, 10 HEPES, 10 D-glucose, 1 CaCl2, and 130 KCl, with the pH adjusted to 7.4 with NaOH) for both the closed-state or inactivated-state assay. A change in the Fluo-4 fluorescence signal was assessed using FLIPRTETRA™ instrument (Molecular Devices, Sunnyvale, Calif.) for 3 minutes following the elevation of extracellular KCl using an illumination wavelength of 470-495 nm with emissions recorded at 515-575 nm.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 259.23, ALogP 1.65, HBA 3, HBD 2, PSA 54.02, QED 0.87
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccnc(Cn2ncc3cc(Cl)ccc32)c1
InChIKey: VIIRLZCJMXLFHW-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 420.9, ALogP 3.66, HBA 6, HBD 2, PSA 98.72, QED 0.51
Compound (SMILES): COC1CN(C(=O)C2CCc3c(C4=C[C@H](C)[C@H](C(=O)O)CC4)nn(C(=O)c4c(Cl)cccc4C4(C(F)(F)F)CC4)c3C2)C1
InChIKey: XEDBDFGQMFSCQN-LYNLNPNLSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay for RORgammaT Activity: To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.HIS-tagged RORγ-LBD protein was recombinantly expressed in Escherichia coli. The RORγ-LBD protein was purified by Ni2+-affinity resin. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 50 mM KCl, 1 mM EDTA, 0.1 mM DTT, 100 mg/ml bovine serum albumin, delipidated) to obtain a RORγ-LBD final concentration of 3 nM. Europium tagged anti-HIS antibody was also added to this solution (1.25 nM). Separately, SF9 cells not expressing any recombinant protein were lysed (32,000 cells per ml in 25 mM Tris, 50 mM NaCl) and the previously frozen lysate was added to the diluted RORγ-LBD solution at a ratio of 0.75 ml SF9 lysate per 15 ml of diluted RORγ-LBD.Compounds to be tested were injected to the 384-well assay plate using Acoustic Droplet Ejection technology by Echo 550 liquid handler (Labcyte, CA).A stock of biotinylated-LXXLL peptide from coactivator SRC1 (Biotin-SPSSHSSLTERHKILHRLLQEGSP) (SEQ ID NO: 1) and APC-conjugated streptavidin (final concentrations 100 nM and 8 nM respectively) were also added to each well.The final assay mixture was incubated overnight at 4° C., warmed to room temperature and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 tis, integration time=200 tis). IC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 620.07, ALogP 5.3, HBA 6, HBD 1, PSA 101.73, QED 0.47
Compound (SMILES): O=c1c2cccnc2ncn1Cc1nn(C[C@H](F)c2ccc(OC(F)F)cc2)c(=O)o1
InChIKey: GUGRIVXZTJXLBP-AWEZNQCLSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay: The IC50 (effective concentration) of compounds on the human and rat TRPA1 channels were determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 15 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 20 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75 uM for human TRPA1 and 45 uM for rat TRPA1) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 433.35, ALogP 2.3, HBA 9, HBD 0, PSA 105.04, QED 0.44
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@@H](Nc3ccc(F)c(Cl)c3)[C@H](C3CCOC3)C2)c1
InChIKey: DOEDZRZQYHKEAM-CRLJJSMGSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 10000 nM (pChEMBL 5)
Descriptors: MW 428.92, ALogP 2.35, HBA 6, HBD 1, PSA 76.46, QED 0.79
Compound (SMILES): O=C(Nc1ccccc1)C(Nc1ncc(-c2nnc(C(F)F)o2)cn1)c1ccc(F)cc1
InChIKey: XOPAONJSFCLMEE-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Activity Assay: All enzymatic reactions were conducted in duplicate at room temperature for 17 hours in a 50 μl mixture containing HDAC assay buffer (50 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, 5 μg BSA), an HDAC substrate, an HDAC enzyme, and a test compound. Compound dilution was prepared ten-fold higher than the final concentration of the compounds with 10% DMSO in HDAC assay buffer and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. After enzymatic reactions, 50 μl of HDAC Developer was added to each well and the plate was incubated at room temperature for an additional 20 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 or Biotek Synergy microplate reader.The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(F−Fb)/(Ft−Fb), where F=the fluorescent intensity in the presence of the compound.
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 440.39, ALogP 4.4, HBA 7, HBD 2, PSA 105.83, QED 0.44
Compound (SMILES): O=C(O)c1ccn(C(=O)N2CCC3(CCCN3Cc3ccc(C(F)(F)F)cc3N3CC4CCC(C3)O4)CC2)n1
InChIKey: JBBUXQKBLBHFMR-UHFFFAOYSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction for mouse assays; human prefrontal cortex membrane fractions for human assays) (50 mL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FP-Rh (1.0 mL, 50 mM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software. IC50 data from this assay is shown in Table 1. All compounds in Table 1 were more potent inhibitors of MAGL than FAAH.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 547.58, ALogP 4.07, HBA 7, HBD 1, PSA 91.14, QED 0.62
Compound (SMILES): COc1ccc(-c2cnc3[nH]c(=O)n(C4CCCC4)c3n2)cc1OC
InChIKey: JYRONAVVWQIHMW-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16584
Assay: Radiometric Filter Plate Assay: 200 ng (130 nM) MLK3 (Dundee, DU8313) was incubated with 1 μM inactive MKK7b (Dundee, DU703) in the presence of 2 μM cold ATP (Km) and 0.5 μCi/assay 33P ATP and appropriate concentrations of compounds. After a twenty minute incubation, the reactions were washed through filter plates and read on a scintillation counter.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 340.38, ALogP 2.92, HBA 6, HBD 1, PSA 82.03, QED 0.79
Compound (SMILES): Cc1nc2sc(N3CCC[C@@H]3C(=O)NCc3ccccc3)nn2c(=O)c1C(C)C
InChIKey: ATRCVPZDWMIPCR-MRXNPFEDSA-N
Target: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q8N5Z0
Assay: KAT-II inhibitory assay: The inhibitory action of the test compound on human recombinant KAT-II was determined by the following method.To a reaction mixture (45 μL) containing 3.0 μmol/L kynurenine, 10 μmol/L pyridoxal phosphate, 2.0 ng/μL human recombinant KAT-II, and 150 mmol/L tris(hydroxymethyl)aminomethane-acetate buffer (pH 8.0) was added a 10% dimethyl sulfoxide solution (5 μL) of each test compound prepared, and the mixture was reacted at 37° C. for 1 hr. After the reaction, 50% trichloroacetic acid (5 μL) was added to terminate the reaction.The resultant kynurenic acid was quantified as follows by high performance liquid chromatography. An enzyme reaction mixture was separated by an octadecylsilane reversed-phase column (SC-50DS, Eicom Corporation; mobile phase: 250 mmol/L zinc acetate, 50 mmol/L sodium acetate, and 5.0% acetonitrile (pH 6.2)) incubated at 30° C., and kynurenic acid was quantified using a fluorescence detector (RF-20Axs, Shimadzu Corporation) at excitation wavelength 354 nm, detection wavelength 460 nm. The analytical curve was drawn every time by an external standard method. Each test compound was tested by dual measurement at each concentration. The kynurenic acid level in the presence of a test compound at each concentration was converted into % relative to kynurenic acid resulting from a reaction with an enzyme alone as 100%, and the obtained values were fitted to S-curve to determine IC50.
Measurement: IC50 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 411.53, ALogP 2.87, HBA 7, HBD 1, PSA 79.6, QED 0.7
Compound (SMILES): O=c1[nH]c2ccc(OCCN3CCC(N[C@@H]4C[C@H]4c4ccccc4)CC3)cc2o1
InChIKey: PVOROURTRAYOPB-PZJWPPBQSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 enzymatic activity: Briefly, compounds of the present invention were solubilized in DMSO and a series of 10, three-fold serial dilutions were made for each compound in 15% DMSO. The initial starting concentration for the serial dilutions of each compound was 10 μM. Control samples lacking compound, LSD1 enzyme or various reaction components also were prepared and processed in parallel with compound test samples.An aliquot of each serial dilution of test compound was added to a 96 well plate containing 50 nM purified N-truncated LSD1 enzyme (RBC Cat# PDM-11-350), 50 mM Tris-HCl, pH 7.5, 0.05% CHAPS and 1% DMSO in a 10 microliter reaction volume. The plate was pre-incubated at room temperature for 30 min to which 10 M of histone 3.3 peptide (aa 1-21) was added to initiate the enzymatic reaction. The reaction mixture was incubated at room temperature for one hour. After one hour, 10 μl of a detection mixture of horseradish peroxidase (Sigma Cat #P8375) and Amplex Red (InVitrogen A36006) was added and kinetic measurements were read at 5 minute intervals for a period of 30 minutes using an Envision Multiplate Reader (PerkinElmer; excitation at 535 nM and emmission read at 590 nM). The IC50 value for each compound was determined from each 10-point dose-response curve using GraphPad Prism 4 software with a sigmodial dose response.
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 393.49, ALogP 3.11, HBA 5, HBD 2, PSA 70.5, QED 0.65
Compound (SMILES): Cc1cc(-n2ncc3cc(NC(=O)N[C@H]4CCN(C)C4)ncc32)ccn1
InChIKey: FKQLHVUGRCROJA-AWEZNQCLSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 μM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 μL) was dispensed, followed by the addition of 15 μL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 μL kinase buffer containing 2.45 μM ERK2 IMAP substrate peptides and 75 μM ATP. The final reaction in each well of 25 μL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 μM ATP. Phosphorylation reactions were allowed to proceed for 60 minutes and were immediately quenched by the addition of 60 μL IMAP detection beads (1:1000 dilutions) in IMAP binding buffer (Molecular Devices) with 24 mM NaCl. Plates were read on EnVision reader after 60 minutes binding equilibration using Fluorescence Polarization protocol (Perkin Elmer).
Measurement: IC50 = 767 nM (pChEMBL 6.119999885559082)
Descriptors: MW 351.41, ALogP 1.95, HBA 6, HBD 2, PSA 87.97, QED 0.75
Compound (SMILES): CCn1cc(C(=O)Nc2ccc(Oc3cc(-c4cncn4C)cn4nccc34)c(F)c2)c(=O)n(-c2ccc(F)cc2)c1=O
InChIKey: OIMFYTYTBICGEO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: AXL Kinase Assay: The ability of compounds to inhibit the kinase activity of recombinant human baculovirus-expressed AXL was measured by homogeneous TRF (HTRF) using Cisbio's KinEASE assay system in white 384-well Optiplates. Assay buffer contained 1 mM DTT, 2 mM MnCl2, 2% DMSO, 50 nM supplement enzymatic buffer, and 1× enzymatic buffer. A 2× concentration of tyrosine kinase (TK) substrate-biotin/ATP mixture made in assay buffer was added to plates at 10 μL/well using the Multidrop Combi (Thermo Fisher Scientific, Waltham, Mass.). The final concentrations were 0.3 μM TK substrate-biotin, and 1.3 μM ATP. Compounds (100 nL), diluted in 100% DMSO on the Biomek FX, (Beckman Coulter, Inc., Brea, Calif.), were transferred to the assay plates using the Biomek FX pintool (2.5% final DMSO in assay). A 2× concentration (final=12 ng/mL) of GST-AXL (diluted in assay buffer) was added to plates at 10 uL/well using the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 30 minutes. A 4× stock of Streptavidin-XL665 (final=18.8 nM) and a 1:100 diluted stock of TK antibody-cryptate were made in HTRF detection buffer and mixed together just prior to adding 20 μL/well on the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 1 hour. The fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 multi-label plate reader (PerkinElmer, Waltham, Mass.) with an excitation wavelength of 337 nm (laser) and emission wavelengths of 590 and 665 nm. Raw data was expressed as the ratio of 665/590×10,000.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 583.56, ALogP 4.39, HBA 10, HBD 1, PSA 117.45, QED 0.3
Compound (SMILES): CC[C@@H](c1cccc(C#N)c1)n1c(CC2CC2)nc(=O)c(S(=O)(=O)c2ccc(-c3cnccc3C#N)cc2)c1O
InChIKey: IDAOLCHMAZEBPZ-SANMLTNESA-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 ((Pyrl)apelin-13) peptide, which was set to 100%.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 551.63, ALogP 4.54, HBA 9, HBD 1, PSA 149.73, QED 0.34
Compound (SMILES): Cc1ccc([C@H]2C[C@@H](N(C)C(=O)[C@H]3C[C@]4(COC(=O)N4)C3)C2)cc1C
InChIKey: VPUMZFWDTFVZJK-ZQJXWABTSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q99685
Assay: In Vitro Activity Assay: The assay used to measure the in vitro activity of MGL is adapted from the assay used for another serine hydrolase (FAAH) described in Wilson et al., 2003 (A high-throughput-compatible assay for determining the activity of fatty acid amide hydrolase. Wilson S J, Lovenberg T W, Barbier A J. Anal Biochem. 2003 Jul. 15; 318(2):270-5.). The assay consists of combining endogenously expressed MGL from HeLa cells with test compounds, adding [glycerol-1,3-3H]-oleoyl glycerol, incubating for one hour, and then measuring the amount of cleaved [1,3-3H]-glycerol that passes through an activated carbon filter. The amount of cleaved, tritiated glycerol passing through the carbon filter is proportional to the activity of the MGL enzyme in a particular well/test condition.
Measurement: IC50 = 3.6 nM (pChEMBL 8.4399995803833)
Descriptors: MW 342.44, ALogP 2.9, HBA 3, HBD 1, PSA 58.64, QED 0.92
Compound (SMILES): Cc1ccc(CC(CO)n2c(=N)n(Cc3ccsc3)c3ccccc32)cc1
InChIKey: XAZLFWGEBAQWNC-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: IP Accumulation Assay: HEK293 cells stably expressing OX2R are transfected with Gq alpha construct (6.4 ug per 10 cm dish) using Lipofectamine 3000 (ThermoFisher). 24 hours after transfection, cells are plated on poly-lysine-coated 96-well plates at 30,000 cells per well in Inositol-free DMEM+5% FCS with 5 uCi per ml Myo-[2-3H] Inositol (Perkin Elmer). Plates are incubated at 37° C. O/N in 8% C02 tissue culture incubator. Agonists are diluted serially in HBSS containing 10 mM Lithium Chloride. After removal of culture media, agonist solutions are added to the cells (100 ul/well) and incubated at 37° C. 45 min in 8% C02 incubator. Agonist solution is removed and cells are lysed on ice with 10 mM Formic Acid (40 ul/well) for 30 minutes. SPA Poly Lysine YSI beads (Perkin Elmer, cat #RPNQ0010) are added to the wells and mixed on a shaker for 30 minutes before reading on a Microbeta scintillation counter. Positive control curves run with all screens: orexin B and Yanagisawa (J. Med. Chem. 2015) agonist.
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 377.51, ALogP 4.12, HBA 5, HBD 2, PSA 53.94, QED 0.52
Compound (SMILES): O=C(Nc1ccc(Cl)c(S(=O)(=O)[C@H]2CCCNC2)c1O)N[C@@H]1CCCc2cc(C(F)(F)F)oc21
InChIKey: JMULEYOMVPJOPN-SWLSCSKDSA-N
Target: C-X-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25025
Assay: Inhibition Assay: PathHunter CXCR2 β-arrestin cells (DiscoverX) were grown under normal conditions and seeded on a white-walled 384-microwell plate at 20 μL/well. Before testing, the cells were incubated at 37° C. for an appropriate time. The test compounds were serially diluted in DMSO at a three-fold dilution factor to obtain 8 serially diluted test compounds. Shortly before the test, the above serially diluted test compounds were further diluted with test buffer to 5 times the test concentration. 5 μL of the further diluted test compounds was added to the cells and then the cells were incubated at 37° C. for 30 minutes. The vehicle concentration was 1%. 5 μL of 6×EC80 agonist (CXCL8) buffer was added to the cells and then the cells were incubated at 37° C. for 90 minutes. 15 μL (50% v/v) of PathHunter detection reagent mixture was added in one portion, followed by one-hour incubation to generate the test signal. The microplates were read on a PerkinElmer Envision instrument for chemiluminescence signal. The biological activity of the test compounds was analyzed by CBIS data analysis suite (ChemInnovation, CA) and shown as IC50 values.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 521.95, ALogP 4.38, HBA 6, HBD 4, PSA 120.67, QED 0.44
Compound (SMILES): C=CC(=O)N1CC(NC(=O)Cn2c(C)c(-c3ccccc3C3CC3)c3cc(Cl)ccc32)C1
InChIKey: ZSWOTWVAQJMGMS-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (2 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 710 nM (pChEMBL 6.150000095367432)
Descriptors: MW 447.97, ALogP 4.66, HBA 3, HBD 1, PSA 54.34, QED 0.56
Compound (SMILES): O=C(O)c1ccc(-n2cc(C(=O)c3c(Cl)cccc3C(F)(F)F)c3cncnc32)cc1
InChIKey: RWHQBWZULYELHV-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: FRET Assay: The assay was run in black 384 well plates (Greiner cat no: 784900). Various concentrations of test ligands in 0.1 microliters DMSO were dispensed to assay plates using an Labcyte Echo acoustic dispenser. Two pre-mixes were prepared and incubated for 1 hr at room temp in the dark. Pre-mix 1 comprised 100 nM Protein (Biotinylated HN-Avi-MBP-TCS-hRORg (258-518)) and 60 nM Streptavidin APC in assay buffer, 50 mM MOPS pH7.4, 50 mM KF, 0.003% (w/v) CHAPS, 10 mM DTT and 0.01% (w/v) BSA and pre-mix 2 comprised 160 nM biotinylated SRC-1 peptide (NCOA1-677-700) and 20 nM Europium-W8044 labelled Streptavidin in assay buffer.Five microliters of pre-mix 2 was dispensed to assay plates containing test compound and incubated for 15 minutes prior to adding five microliters of pre-mix 1. Plates were incubated at room temperature for 1 hour in the dark, prior to reading in a Pherastar multi-mode plate reader using HTRF filter set (ex 320, em 612 and 665). The FRET signal at 665 nM was divided by the signal at 612 nM and multiplied by 10,000 to generate a signal ratio value for each well. The raw data was transformed to % effect using the equation:Compound % effect=100*[(X−min)/(max−min)],where X represents the normalized value for the compound based on the Min (vehicle) and Max (reference compound) inhibition control.
Measurement: IC50 = 1371 nM (pChEMBL 5.860000133514404)
Descriptors: MW 445.78, ALogP 5.02, HBA 5, HBD 1, PSA 85.08, QED 0.45
Compound (SMILES): OC[C@H]1CC[C@H](Nc2ccc3nnc(-c4cccc(C(F)(F)F)c4)n3c2)CC1
InChIKey: UYOVLDHCOFZASA-SAZUREKKSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Pim-1 Kinase Inhibition Assay: One illustrative manner in which Pim-1 kinase activity can be determined is by quantifying the amount of ATP remaining in solution after an in vitro Pim-1 kinase reaction. The Kinase-Glo Assay Kit (Promega, Inc., Madison, Wis.) allows this. The amount of ATP remaining in the solution after the kinase reaction serves as a substrate for the luciferase to catalyze luciferin to oxyluciferin plus one photon of light. Thus, the luminescent signal read by the Luminoskan Ascent Instrument (Thermo Electron Corp., Milford, Mass.) correlates with the amount of ATP present after the kinase reaction and inversely correlates with the amount of kinase activity. This assay is efficient at determining the IC50 values of kinase inhibitors against the Pim-1 kinase. These assays are set up in duplicate 50 ul volumes in white, flat bottom 96 well plates. Inhibitors are added to the solution of 1× kinase buffer, 10 uM ATP, 100 uM Pim-1-specific substrate, 50 ng of active Pim-1 enzyme, and water in serial dilutions ranging from micromolar to nanomolar concentrations. This solution is incubated at 30 degrees Celsius at 360 rpm for two hours. Following the incubation, 50 ul of Kinase-Glo reagent is added to each well, including all positive and negative control wells, and incubated at room temperature for 15 minutes. The plate is then read by the Luminoskan Ascent instrument and the results displayed with the Ascent Software version 2.6. The IC50 values can then be calculated for each inhibitor tested.
Measurement: IC50 = 518 nM (pChEMBL 6.289999961853027)
Descriptors: MW 390.41, ALogP 4.38, HBA 5, HBD 2, PSA 62.45, QED 0.69
Compound (SMILES): CN(C(=O)CCc1ccccc1)[C@H]1C[C@@H](C(=O)O)N(C(=O)C(c2ccccc2)c2ccccc2)C1
InChIKey: YAHHFSHBEGPKKO-DQEYMECFSA-N
Target: Type-2 angiotensin II receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P50052
Assay: Receptor Binding Assay:     120 μL membrane (5 mg protein/well) was incubated with 15 μL of [125I]-CGP42112A and 15 μL of compound at RT for 1.5 hrs.    The binding reaction was stopped by rapid filtration through Unifilter GF/C plates (presoaked in 0.3% (v:v) BSA).    Plate was washed three times with ice cold wash buffer.    The filtration plates were dried at 37° C. overnight.    50 μL of scintillation cocktail was added to each well.    Radioactivity was determined using MicroBetaTriluxmicroplate scintillation counter.
Measurement: IC50 = 1664 nM (pChEMBL 5.78000020980835)
Descriptors: MW 470.57, ALogP 3.96, HBA 3, HBD 1, PSA 77.92, QED 0.54
Compound (SMILES): Cc1ccc2nc(N3CCC(NC4CCCCC4)C(CO)C3)c(-c3nnc(C)o3)cc2c1
InChIKey: YJRLNSQUCPKJIT-UHFFFAOYSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: OPRK1 antagonist assay: The cell line for the OPRK1 antagonist assay stably expresses the following elements. The carboxy terminus of the OPRK1 receptor has a 7 amino acid linker, followed by the TEV protease cleavage site and a GAL4-VP16 fusion protein. The cell line also expresses a b-arrestin-2-TEV protease fusion protein and contains a reporter construct consisting of the UAS response element and the b-lactamase (bla) reporter gene. Upon activation of the receptor, g-protein receptor kinase (GRK) phosphorylates specific intracellular residues of OPKR1 and this induces recruitment of B-arrestin2-TEV protease fusion protein. The TEV protease recognizes and cleaves the TEV site, releasing the GAL4-VP16 fusion protein, which then translocates to the nucleus. The GAL4-V16 binds to the UAS element, driving expressing of the b-lactamase gene. B-lactamase expression is detected with the cell permeable, fluorescent substrate, CCF4-AM. This substrate consists of coumarin tethered to fluoroscein via a b-lactam ring. In the absence of b-lactamase, excitation of the dye with 405 nm light results in FRET from the coumarin to fluoroscein and emission of green (525 nm maximum) light. B-lactamase cleavage of the substrate separates the courmarin fluorophore from the fluorscein, and 405 nm excitation results in blue (460 nm maximum) emission. The assay is monitored by the blue/green emission ratio. The antagonist assay is performed by seeding the cells into 384 well plates and incubating them 16-24 hours at 37° C. Test antagonist compounds are added and incubated for 30 minutes at 37° C. Next an EC80 challenge of U-50488 (OKRK1 agonist) is added and the samples are incubated for 4 hours at 37° C., followed by addition of CCF4-AM substrate. The plates are then incubated 2 hours at room temperature in the dark and the blue/green ratio determined on a fluorescent plate reader. See J Biomol Screen April 2009, vol. 14 no. 4, pp 381-394.
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 435.57, ALogP 4.01, HBA 7, HBD 2, PSA 87.31, QED 0.63
Compound (SMILES): N=C(NC(=O)C(NC(=O)c1ccc(Cl)cc1)c1ccc(Cl)c(Cl)c1)c1ccccc1
InChIKey: RKYTYQAMWFBKKY-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: TBD: TBD
Measurement: IC50 = 1200 nM (pChEMBL 5.920000076293945)
Descriptors: MW 460.75, ALogP 5.26, HBA 3, HBD 3, PSA 82.05, QED 0.36
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2nnc(Cc3ccccc3)[nH]2)COc2c(C#N)cccc21
InChIKey: ZLGZXEYUQKINOD-HNNXBMFYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding (FP Binding) assay: Assay was performed in polystyrene low volume 384-well black plate, at Room Temperature (RT) in a final volume of 10.1 μl/well using 10 nM of GST-hRIPK1 (8-327) enzyme and 5 nM of fluorescent labeled ligand (14-(2-{[3-({2-{[4-(cyanomethyl)phenyl]amino}-6-[(5-cyclopropyl-1H-pyrazol-3-yl)amino]-4-pyrimidinyl}amino) propyl]amino}-2-oxoethyl)-16,16,18,18-tetramethyl-6,7,7a,8a,9,10,16,18-octahydrobenzo[2″,3″]indolizino[8″,7″:5′,6]pyrano[3′,2′:3,4]pyrido[1,2-a]indol-5-ium-2-sulfonate.Test Compounds were serially diluted in DMSO at 100 fold final concentrations in the assay (1% DMSO final). In each well of a 384-well Plate were dispensed 0.1 μL of compound solution (or DMSO for controls) followed by 5 μL of GST-hRIPK1 (8-327) at twice the final concentrations in assay buffer (50 mM HEPES pH 7.5, 10 mM NaCl, 50 mM MgCl2, 0.02% CHAPS, 0.5 mM DTT and 0.01% Pluronic F127). For negative control the enzyme addition was replaced by assay buffer only.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 402.41, ALogP 1.42, HBA 6, HBD 2, PSA 124, QED 0.68
Compound (SMILES): C[C@@H]1[C@H](F)CCN1C(=O)[C@]12C[C@H]1[C@@](C)(c1cc(/C=C(\F)c3ccc(C#N)cn3)ccc1F)N=C(N)S2
InChIKey: DFYAUWDSHXLSJC-OJNSPYEBSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05M acetate, pH 4.5, 8% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 511.57, ALogP 4.55, HBA 6, HBD 1, PSA 95.37, QED 0.65
Compound (SMILES): CCn1c2ccccc2c2cc(-c3nc4cc(C(=O)O)ccc4n3CCOC(F)(F)F)ccc21
InChIKey: VTALIBBCVSUPQG-UHFFFAOYSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Test for Antagonism: 4 μl of a cAMP-d2/cell suspension (625000 cells/ml) were added to a test plate containing the substance solutions already initially introduced (0.05 μl; 100% DMSO, concentration range from 0.8 nM-16.5 μM). After a 20-minute preincubation at room temperature (RT), 2 μl of a 3xPGD2 solution (6 nM, in PBS-IBMX) were added and incubated in the presence of the agonist for a further 30 min at RT (volume: ¿6 μl). Subsequently, the reaction was stopped by addition of 2 μl of lysis buffer and incubated for a further 20 min at RT before the actual measurement (volume: ¿8 μl).
Measurement: IC50 = 14.3 nM (pChEMBL 7.840000152587891)
Descriptors: MW 467.45, ALogP 6.07, HBA 5, HBD 1, PSA 69.28, QED 0.33
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(C)c(-c3ccc(=O)n(C(C)C)c3)nn2-c2ccccc2)[C@H](c2ccc(F)cc2)C1
InChIKey: YFSJOZAPCLPFPG-WUFINQPMSA-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 572.69, ALogP 4.97, HBA 7, HBD 2, PSA 93.42, QED 0.3
Compound (SMILES): N#Cc1cc(-c2ncnc(Nc3ccc(C(=O)C(F)(F)F)cc3)n2)ccc1OC1CCOCC1
InChIKey: OOYRAPOOQHGEBB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: TBK1 and IKKE Biological Assays: Enzymatic activity of IKKε and TBK1 was measured using a homogeneous time resolved fluorescence resonance energy transfer (TR-FRET) assay that monitors enzyme dependent phosphorylation of a biotinylated serine/threonine peptide substrate. An increase in the amount of phosphorylated peptide results in an increase in TR-FRET signal. TBK1 and IKKε were expressed and purified as full length recombinant proteins. Detection reagents for the assay were purchased from Cisbio. TBK1 and IKKε enzymes were assayed under initial rate conditions in the presence of 2×Km ATP (40-80 μM) and 1 μM peptide, hepes (pH 7), 0.1 mM orthovanadate, 0.02% NaN3, 0.01% BSA, 10 mM MgCl2, 0.01% (v/v) tritonX, 1 mM dithiothreitol, 0.5% (v/v) DMSO at the following concentrations for each enzyme: TBK1 at 2.5 nM and IKKε at 0.3 nM. After an assay reaction time of 240 minutes at 25° C., reactions were terminated with EDTA.Amount of phosphorylated peptide was determined by the addition of 125 nM streptavidin XL665 and europium cryptate labeled anti-phospho monoclonal antibody and the resulting TR-FRET signal was recorded on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 200 μs read window). Data was normalized based on a positive (1 μM Staurosporine) and negative (DMSO) controls and IC50 values calculated from the fit of the dose-response curves to a four-parameter equation. All IC50 values represent geometric mean values of a minimum of four determinations.
Measurement: IC50 = 2000 nM (pChEMBL 5.699999809265137)
Descriptors: MW 469.42, ALogP 4.46, HBA 8, HBD 1, PSA 110.02, QED 0.53
Compound (SMILES): CC(C)(C)c1ccc([C@]2(O)C[C@H]3CN(C(=O)Nc4cccc(C#N)c4)C[C@H]3C2)cc1
InChIKey: AGNSNCDHOXNOOE-HBNMSFMJSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: FDSS Assay: To each well of the plate, 10 μL test compound, control (MK801) or HHnoCa buffer was added to a final concentration of 10 μM with a final concentration of DMSO of 0.1%. Following 10 minutes pre-incubation in the dark, plates are loaded onto the Hamamatsu FDSS 6000. After collecting baseline fluorescence images, 3 μM glutamate, 3 glycine, and 1 mM Ca2+ in HHnoCa buffer is added to each well, and Ca2+ is recorded for 3 minutes. Data were processed by computing ratio of fluorescence at the end of data collection to baseline fluorescence to assess degree of Ca′ influx inhibition relative to that observed in MK801.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 403.53, ALogP 4.62, HBA 3, HBD 2, PSA 76.36, QED 0.77
Compound (SMILES): COC1CCC2(CC1)Cc1ccc(Br)cc1C21N=C(SCC2CCO2)N(CC2CCO2)C1=O
InChIKey: AIYXFVKSPTWDCO-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Assay: For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 μl OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 μl water containing the desired concentration of test compound and 1% DMSO, 1 μM substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 μl in a 384 well plate. In general, 25 μl of compound dilution were given to the plate followed by the addition of 10 μl of BACE containing OptiMEM diluted 1:2 in water with 0.2% Triton X-100. The reaction was started with the addition of 15 μl substrate in NaOAc buffer. The reaction was incubated at 30° C. in a fluorimeter and the cleavage of the substrate was recorded as kinetic for 60 min. at ex: 530 nm, em: 590 nm. Blank wells containing either no inhibitor or no enzyme were included on each plate.The intensity of fluorescence was regressed against time in order to derive velocities of reaction in all 384 wells. These velocities were used for calculating percent inhibition using an uninhibited control containing 1% DMSO and a fully inhibited control incubations performed in the absence of enzyme. IC50 values were calculated by fitting percent inhibition vs. inhibitor concentration using standard software like GraphPadPrism.
Measurement: IC50 = 13.7 nM (pChEMBL 7.860000133514404)
Descriptors: MW 535.5, ALogP 4.29, HBA 6, HBD 0, PSA 60.36, QED 0.56
Compound (SMILES): O=c1[nH]c2ncc(-c3cccc(C(F)(F)F)c3)cc2n1CC1CCO1
InChIKey: LYAQGBFZDHGDNN-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 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 349.31, ALogP 3.2, HBA 4, HBD 1, PSA 59.91, QED 0.79
Compound (SMILES): CN(C)Cc1nc(Nc2ccc(-c3cnc4cc(F)ccn34)c3c2C(=O)NC3)ccc1N1CCC(C)(O)CC1
InChIKey: JEHLHKCMEROVJY-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Biochemical Enzyme Assay: HPK1 biochemical enzyme assay: HPK1 enzyme inhibition was measured using a microfluidic mobility shift assay. Reactions were performed in a 384-well plate, containing 1.5 nM HPK1 (Invitrogen), in assay buffer (Carna Biosciences; pH 7.4). Test compounds were titrated in ten point curves (top final assay concentration 3 μM), and preincubated with enzyme/substrate mix for 30 min prior to initiation of the reaction by addition of ATP (1 mM final concentration) and substrate (1 μM final concentration; Carna Biosciences) diluted in assay buffer supplemented by MgCl2 (final assay concentration of 5 mM). Following 60 min incubation at RT, the reaction was terminated by addition of 60 μl/well termination buffer (Carna Biosciences) and signal determination using a Caliper EZ Reader (Perkin Elmer, UK).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 529.62, ALogP 3.94, HBA 8, HBD 3, PSA 98.03, QED 0.35