lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(N2CCCNCC2)ccc1-c1cc(Nc2cnc(C#N)cn2)n[nH]1
InChIKey: BLOVTSDJVZJJAR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Kinase Inhibiting Activity Assay: Chk-1 Kinase Inhibiting ActivityThe compounds of the invention were tested for activity against Chk-1 kinase using the materials and protocols set out below.Reaction Buffer:Base Reaction buffer: 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO*Required cofactors are added individually to each kinase reactionReaction Procedure:(i) Prepare indicated substrate in freshly prepared Base Reaction Buffer(ii) Deliver any required cofactors to the substrate solution above(iii) Deliver indicated kinase into the substrate solution and gently mix(iv) Deliver compounds in DMSO into the kinase reaction mixture(v) Deliver 33P-ATP (specific activity 0.01 μCi/μl final) into the reaction mixture to initiate the reaction.(vi) Incubate kinase reaction for 120 minutes at room temperature(vii) Reactions are spotted onto P81 ion exchange paper (Whatman #3698-915)(viii) Wash filters extensively in 0.1% phosphoric acid.(ix) Dry filters and measure counts in scintillation counterKinase Information:CHK-1 Genbank Accession # AF016582Recombinant full length construct, N-terminal GST tagged, purified from insect cells.No special measures were taken to activate this kinase.Final concentration in assay=0.5 nMSubstrate: CHKtidePeptide sequence: [KKKVSRSGLYRSPSMPENLNRPR]Final concentration in assay=20 μM
Measurement: IC50 = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 390.45, ALogP 2.29, HBA 8, HBD 3, PSA 114.78, QED 0.61
Compound (SMILES): CC(C)Oc1nc(N2CC[C@H](O)C2)c2nnn(Cc3ccccc3Cl)c2n1
InChIKey: IKYATIXPQUGMFE-ZDUSSCGKSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1×HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 μl and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 μl detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 μM to 0.13 nM cAMP.
Measurement: EC50 = 14.8 nM (pChEMBL 7.829999923706055)
Descriptors: MW 388.86, ALogP 2.28, HBA 8, HBD 1, PSA 89.19, QED 0.72
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)NCc2cccc(Cl)c2F)c2ccc(C#Cc3ncc(O)cn3)cc12
InChIKey: IQAXUFQJZIEJFY-IBVKSMDESA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 100 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 592, ALogP 3.79, HBA 7, HBD 2, PSA 117.42, QED 0.26
Compound (SMILES): CN1C(=O)[C@@H](NC(=O)c2cc(C3(c4ccccc4)CC3)on2)COc2ccccc21
InChIKey: WQISZFZHWMIKSX-KRWDZBQOSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: Fluorescent Polarization Binding Assay: Fluorescent polarization binding 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.After addition of 5 μL of fluorescent-labeled ligand at twice the final concentrations in assay buffer, the plate was incubated at RT for 30 min. At the end, the binding was measured as FP value with the Envision (PerkinElmer) plate reader using filter for an excitation λ=531 nm FP and an emission λ=595 nm FP (S & P-pol).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 403.44, ALogP 2.91, HBA 5, HBD 1, PSA 84.67, QED 0.72
Compound (SMILES): CCc1nc2ccc(N3CCC(N4CCNC(=O)C4)C3)nn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: HCBYGAWKMFDKNC-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: Compound IC50 values are determined in a fluorescent hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal. 10 uL of a dilution series of compound, starting from 20 uM highest concentration, dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 ug/mL or 0.64 ug/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 uL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 uL of 0.75 uM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 = 5050 nM (pChEMBL 5.300000190734863)
Descriptors: MW 520.64, ALogP 3.33, HBA 9, HBD 1, PSA 81.9, QED 0.42
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(C#CC5CC5)n4c3)ccc2o1
InChIKey: ARPYIUHHHXVAHV-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: PI 3-Kinase HTRF Assay: Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K. The kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate). The total reaction volume is approximately 20 ul per well. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, approximately 14.5 ul of a kinase/PIP2 mixture (diluted in 1x reaction buffer) is added per well for a final concentration of 0.25-0.3 ug/ml kinase and 10 uM PIP2. The plate is sealed and incubated for 15 minutes at room temperature. To start the reaction, 3.5 ul of ATP (diluted in 1x reaction buffer) is added per well for a final concentration of 10 uM ATP. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 5 ul of Stop Solution per well and then 5 ul of Detection Mix is added per well. The plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 314.35, ALogP 3.49, HBA 5, HBD 1, PSA 69.35, QED 0.55
Compound (SMILES): C=CC(=O)N1CC(N(C)c2ccc(C(N)=O)c3[nH]c(-c4ccc5c(c4)CCN(CC)C5)cc23)C1
InChIKey: OUAPHHDAERUUPW-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) : The in-house BTK corresponds to recombinant human catalytic domain (aa 393-659), which was expressed in SF9 cells with an N-terminal his tag and purified by immobilized metal affinity chromatography. BTK was mixed with peptide substrate (biotin-TYR1, Sequence: Biotin-(Ahx)-GAEEEIYAAFFA-COOH, 0.2 μM final) at varying inhibitor concentrations in reaction buffer: 50 mM MOPSO pH 6.5, 10 mM MgCl2, 2 mM MnCl2, 2.5 mM DTT, 0.01% BSA, 0.1 mM Na3VO4 and 0.001 mM ATP. After about 60 mM incubation at room temperature, the reaction was quenched by addition of EDTA (final concentration: 100 mM) and developed by addition of detection reagents (final approximate concentrations: 30 mM HEPES pH 7.0, 0.06% BSA, 0.006% Tween-20, 0.24 M KF, 80 ng/mL PT66K (europium labeled anti-phosphotyrosine antibody cat #61T66KLB Cisbio, Bedford, Mass.) and 0.6 μg/mL SAXL (Phycolink streptavidin-allophycocyanin acceptor, cat #PJ25S, Prozyme, San Leandro, Calif.). The developed reaction was incubated in the dark for about 60 mM at room temperature, then read via a time-resolved fluorescence detector (Rubystar, BMG) using a 337 nm laser for excitation and monitoring emission wavelength at 665 nm. Within the linear range of the assay, the observed signal at 665 nm was directly related to phosphorylated product and can be used to calculate the IC50 values.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 457.58, ALogP 3.14, HBA 4, HBD 2, PSA 85.67, QED 0.56
Compound (SMILES): COC(=O)Nc1ccc2c(c1)NC(=O)CCCCCC(C(=O)NCc1csc3ccc(Cl)cc13)NC2=O
InChIKey: LNNXLPPTNQWRKB-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: In Vitro Assay for FXIa: The effectiveness of compounds of the present invention as inhibitors of the coagulation Factors XIa, VIIa, IXa, Xa, XIIa, plasma kallikrein or thrombin, can be determined using a relevant purified serine protease, respectively, 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. Hydrolysis of the substrate resulted in the release of pNA (para nitroaniline), which was monitored spectrophotometrically by measuring the increase in absorbance at 405 nm, or the release of AMC (amino methylcoumarin), which was monitored spectrofluorometrically by measuring the increase in emission at 460 nm with excitation at 380 nm. A decrease in the rate of absorbance or fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 1337 nM (pChEMBL 5.869999885559082)
Descriptors: MW 543.05, ALogP 5.05, HBA 6, HBD 4, PSA 125.63, QED 0.37
Compound (SMILES): Cc1cccc([C@@H](CO)NC(=O)[C@@H](C)N2Cc3ncc(-c4nc(NC5CCC(O)CC5)ncc4Cl)cc3C2=O)c1
InChIKey: VFCBVYHOBLCJTO-RZBJAIAPSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 7.4 nM (pChEMBL 8.130000114440918)
Descriptors: MW 565.07, ALogP 3.41, HBA 8, HBD 4, PSA 140.57, QED 0.33
Compound (SMILES): C[C@H]1C[C@@H](NC(=O)c2cc(C3CC3)on2)CCN1S(=O)(=O)CCCNCc1ccccc1
InChIKey: SOSTZMGCOMSPAE-PXNSSMCTSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 8.13 nM (pChEMBL 8.09000015258789)
Descriptors: MW 460.6, ALogP 2.64, HBA 6, HBD 2, PSA 104.54, QED 0.53
Compound (SMILES): C/C=C/Cn1cc(-c2ccc(C(=O)N3CCOCC3)c(F)c2F)c2cc(C)[nH]c2c1=O
InChIKey: QHSUUTWPKDIQCI-ONEGZZNKSA-N
Target: Bromodomain-containing protein 9 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H8M2
Assay: IC50 measurements for inhibitors using BRD9 AlphaLisa Binding Assay: His/Flag epitope tagged BRD9134-239 was cloned, expressed, and purified to homogeneity. BRD9 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-11/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD9 (50 nM final) was combined with peptide (3 nM final) in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.8% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature AlphaLisa Streptavidin Acceptor Beads (Perkin-AL125C) and AlphaLisa Nickel donor beads (Perkin AS 10 ID) were added to a final concentration of 15 ug/mL each. After ninety minutes of equilibration in the dark, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 427.45, ALogP 3.63, HBA 4, HBD 1, PSA 67.33, QED 0.65
Compound (SMILES): COc1ccc([C@H]2C[C@@H]2COc2cccc([C@H](C3CC3)[C@H](C)C(=O)O)c2)cn1
InChIKey: WADQKXMIUJWXDX-AVYAGANSSA-N
Target: Free fatty acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14842
Assay: Inositol Phosphate Turnover (IP1) Assay 1: The assay was performed in 96-well format. HEK cells stably expressing human GPR40 were plated to be 60-80% confluent within 72 h. After 72 h, the plates were aspirated and the cells washed with inositol-free DMEM (ICN). The wash media was replaced with 150 μL of 3H-inositol labeling media (inositol-free media containing 0.4% human albumin or 0.4% mouse albumin, 1× pen/strep antibiotics, glutamine, 25 mM HEPES to which was added 3H-myo-inositol NEN #NET114A 1 mCi/mL, 25 Ci/mmol diluted 1:150 in loading media with a final specific radioactivity of 1 μCi/150 μL). Alternatively, the human and mouse albumin can be added after the overnight labeling step before the addition of LiCl.The assay was typically run the next day after 18 h labeling. On the day of the assay, 5 μL of 300 mM LiCl was added to all wells and incubated at 37 degrees for 20 min. 0.75 μL of each compound was added and incubated with the cells for 60 min at 37 degrees. The media was then aspirated off and the assay terminated with the addition of 60 μL 10 mM formic acid. The cells were lysed for 60 min at room temperature. 15-30 μL of lysate was mixed with 70 μL/1 mg YSi SPA beads (Amersham) in clear bottom Isoplates. The plates were shaken for 2 h at room temperature. Beads were allowed to settle and the plates were counted in the Wallac Microbeta.
Measurement: EC50 = 1159 nM (pChEMBL 5.940000057220459)
Descriptors: MW 381.47, ALogP 4.49, HBA 4, HBD 1, PSA 68.65, QED 0.69
Compound (SMILES): N[C@H]1CC[C@H](NC(=O)c2ccc3c(c2)OC(F)(F)O3)CC1
InChIKey: OSAQGTAWWUGSGQ-MGCOHNPYSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
Measurement: IC50 = 69120 nM (pChEMBL 4.159999847412109)
Descriptors: MW 298.29, ALogP 2.01, HBA 4, HBD 2, PSA 73.58, QED 0.88
Compound (SMILES): C[C@@]1(C(F)(F)F)Cc2cc(NC(=O)c3cnn4cccnc34)c(N3CCOCC3)cc2O1
InChIKey: JHLAHBHKNQPFJZ-FQEVSTJZSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 447.42, ALogP 3.07, HBA 7, HBD 1, PSA 80.99, QED 0.66
Compound (SMILES): O=C1CCCc2ccc(-c3ccc4cnn(-c5cccc(C(F)(F)F)n5)c4c3)nc21
InChIKey: CRSLRHNJAYCANC-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biochemical Assay: The biochemical assay is in a AlphaScreen format. The kinase reaction is based on the IRAK-4 phosphorylation of a biotin labeled peptide. The phosphopeptide is incubated with anti-phosphothreonine antibody as well as streptavidin- and protein A-coated beads. Binding of the protein-A coated beads to the antibody and the streptavidin beads to the peptide, leads to an energy transfer from one bead to the other, ultimately producing a luminescent/fluorescent signal.Generally, the kinase reaction is carried out at 1 nM IRAK4, 1.6 μM peptide, 1 mM ATP in reaction buffer 50 mM Hepes, 60 mM NaCl, 5 mM MgCl2, 0.25 mM MnCl2, 2 mM DTT, 0.01% BSA, 0.01% Tween-20) for 3.5 h at RT.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 408.38, ALogP 5.02, HBA 5, HBD 0, PSA 60.67, QED 0.47
Compound (SMILES): COc1cc(C)c(C2CC2NS(=O)(=O)N(C)C)cc1C(=O)Nc1cccc(-c2nncn2C(C)C)n1
InChIKey: GIWWKXJGZIWSTE-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: HTRF KinEASE Assay: ASK1 was purchased from Thermofisher (Catalogue #PV4011), ATP was purchased from Sigma (Catalogue #A7699), HTRF® KinEASE Assay System was obtained from Cisbio (Bedford, Mass.). ½ Area plate was purchased from Perkin Elmer (Catalogue # #6005560). HTRF® KinEASE-STK is a generic method for measuring serine/threonine kinase activities using a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. The IC50 value for each compound was determined in the presence of compound (various concentration from 0 to 10 μM) and a fixed amount of ATP and peptide substrates. The test compound, 1 uM STK3 peptide substrate, and 5 nM of ASK1 kinase are incubated with kinase reaction buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, and 1 mM EGTA for 30 minutes. 100 uM ATP is added to start kinase reaction and incubated for 3 hours. The STK3-antibody labeled with Eu3+-Cryptate and 125 nM streptavidin-XL665 are mixed in a single addition with stop reagents provided by the Cisbio kit used to stop the kinase reaction. Fluorescence is detected using an Envision Multilabeled 2014 reader from PerkinElmer. The Fluorescence is measured at 615 nm (Cryptate) and 665 nm (XL665) and a ratio of 665 nm/615 nm is calculated for each well. The resulting TR-FRET is proportional to the phosphorylation level. Staurosporine was used as the positive control.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 513.62, ALogP 2.74, HBA 8, HBD 2, PSA 131.34, QED 0.45
Compound (SMILES): C[C@H]1CCCN1C(=O)c1nc(C(=O)NCC(C)(C)O)sc1-c1cnc(C(O)(C(F)(F)F)C(F)(F)F)cc1C(F)(F)F
InChIKey: YUDBWJYFGQQUOM-JTQLQIEISA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: Project compounds were arranged in a pre-dosed mother plate (Greiner Bio-one) wherein compounds are serially diluted in 100% DMSO by 1:2 from a high concentration of 10 mM over 12 columns within a series (column 12 is a reference well containing DMSO, no compound). The compounds were robotically dispensed directly into assay plates (1×=46 nL) using a Hummingbird capillary liquid handling instrument (Digilab). Following compound dispense, protein and dye in buffer was added to achieve the final assay volume of 3 μL, followed by 1 μL of silicone oil.
Measurement: Kd = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 622.51, ALogP 4.66, HBA 7, HBD 3, PSA 115.65, QED 0.4
Compound (SMILES): CCn1c(=O)[nH]/c(=N\c2ccc(Oc3ccc(CO)nc3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: MLXSNVIJYASMNZ-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1.% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 50 nM ATP solution (50 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 3 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 479.92, ALogP 2.97, HBA 8, HBD 2, PSA 114.5, QED 0.42
Compound (SMILES): CCc1nn(C[C@@H](C)COC(=O)c2ccc(C(=O)N3CCCC3)cc2)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: HPIWSEFPRVYVRO-HXUWFJFHSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: Enzyme Activity Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [5380-L740]) was incubated with a mixture of non-labelled cAMP (cyclic adenosine monophosphate) and fluorescein amidite (FAM) conjugated cAMP and titrated test or reference compound. Following brief incubation the enzymatic reaction was stopped by addition of binding buffer containing nanoparticles with immobilized trivalent metal ions capable of binding 1) AMP phospho groups and 2) terbium (Tb) donor fluorophores. Subsequent excitation of the Tb donor triggers time-resolved FRET to adjacent FAM acceptor molecules resulting in light emission. In the presence of a PDE4 inhibitor, AMP generation was reduced resulting in a lower fluorescence signal. The cAMP phosphodiester is not bound by the detection system.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 522.65, ALogP 3.26, HBA 7, HBD 1, PSA 102.76, QED 0.56
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1ccc2c(c1)C1OC2c2ccc(S(=O)(=O)c3ccccc3)cc21
InChIKey: AMKDZYLEYYHWBL-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Plasma Kallikrein Inhibition Assay: The chromogenic substrate D-Pro-Phe-Arg-pNa, 2HCl (BIOPHEN CS-31(02) from Hyphen BioMed, Neuville-Sur-oise, France) was dissolved in 5 mL deionized water and stored at 4° C. Concentration was determined in the spectrophotometer at 342 nm using an extinction coefficient of 8270. All other chemicals were of analytical grade.Human plasma kallikrein was purchased from Enzyme Research Labs (South Bend, Ind., USA, batch HPKa 2830). A stock solution of 7 μM in 50% glycerol was stored at −20° C.Enzyme reactions were conducted in assay buffer comprised of 20 mM HEPES at pH 7.4, 150 mM NaCl, 0.1% PEG-8000 and 0.01% Triton X-100.Both enzyme and substrate were diluted in assay buffer.The compound solutions as well as the enzyme and the substrate solutions were transferred to 96-well plates (Clear, UV-Star, Flat-bottom, Half-Area plates; cat. No. 675801 Greiner Bio-one, purchased from VWR International, Arlington Heights, Ill., USA) using a Rainin LTS 96-channel pipettor (Rainin, Columbus, Ohio, USA). Plate measurements were conducted using a SPECTROStar Nano reader (BMG Labtech, San Francisco, Calif., USA). The SPECTROStar Nano is a spectrophotometer and absorbance was measured at 405 nm. We used discrete wavelength, precise, kinetic reads of 15 cycles with a 60 sec cycle time.Determination of IC50 valuesFor the determination of IC50 values, the assays were performed at room temperature in 96-well plates with a total assay volume of 85 μL per well. The test compound was dissolved in 100% DMSO. The compounds were serially diluted in DMSO in a 7 point dose response. For the assays, 66.5 μL of protease solution (protease in assay buffer) was added per well followed by the addition of 8.5 μL of compound in 100% DMSO. The final assay concentration of the human plasma kallikrein was 250 μM. After 30 min. incubation at room temperature on an orbital shaker, the reactions were started by the addition of 10 μL substrate solution (in assay buffer, final assay concentration was 600 uM). After the addition of the substrate solution the final DMSO concentration was 10%. The plate was placed again on the shaker for 5 sec, spun at 2000 rpm for 5 sec and read on the spectrophotometer. The effect of the compound on the enzymatic activity was obtained from the linear part of the progress curves and determined after 15 minutes.
Measurement: IC50 = 1023 nM (pChEMBL 5.989999771118164)
Descriptors: MW 511.6, ALogP 4.57, HBA 6, HBD 2, PSA 111.38, QED 0.41
Compound (SMILES): Cc1[nH]nc2c1CCC2NC(=O)c1cn(Cc2ccc(N3CC4CC4C3)nc2C#N)cn1
InChIKey: CZKPZBKFDVJHIP-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibition Assay: Human PKK (1.78 nM or 0.025 U/mL; Enzyme Research Laboratories) was incubated at 24° C. with 0.25 mM fluorogenic substrate H-Pro-Phe-Arg-AMC (11295 from Bachem) and various concentrations of the test compound in assay buffer. Measurements were performed in a kinetic interval every 2nd minute for 16 min using a Spectramax M5 (Molecular Devices) with the following settings of the wavelength excitation of 350 nm and wavelength emission of 450 nm. Ki values for compounds according to the invention are shown in the following table
Measurement: Ki = 1 nM (pChEMBL 9)
Descriptors: MW 428.5, ALogP 2.1, HBA 7, HBD 2, PSA 115.52, QED 0.64
Compound (SMILES): CC(C)(C)OC(=O)NC(C(=O)NC(C(=O)NC(=N)c1ccccc1)c1cccc(Cl)c1)c1ccc(Cl)cc1
InChIKey: ICNIKUFUZRASPF-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: TBD: TBD
Measurement: IC50 = 20100 nM (pChEMBL 4.699999809265137)
Descriptors: MW 555.46, ALogP 5.56, HBA 5, HBD 4, PSA 120.38, QED 0.23
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(-c4nc(C(=O)NCCCN5CCCCC5)co4)cc3c2C(C)C)cc(C)n1
InChIKey: SKEQTTAOIRBHMY-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: 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-κc Band 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 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-Bue 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 = 18 nM (pChEMBL 7.75)
Descriptors: MW 499.66, ALogP 6.23, HBA 5, HBD 2, PSA 87.05, QED 0.28
Compound (SMILES): COc1cc(N2CCC(Oc3ccc(-c4ccncc4Cl)cc3)C2=O)ccc1OCC(C)(C)O
InChIKey: QPDVUVACXQOVOT-UHFFFAOYSA-N
Target: Melanin-concentrating hormone receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99705
Assay: Radioligand Binding Assay: An in vitro binding assay was used to determine the compound Ki value or ability to antagonize binding of a peptide agonist to the human melanin concentrating hormone receptor (MCHR1). Membranes from stably transfected HEK-293 cells expressing a mutated (E4Q, A5T) hMCHR1 receptor were prepared by dounce homogenization and differential centrifugation. Binding experiments were carried out with 0.5-1.0 μg of membrane protein incubated in a total of 0.2 mL in 25 mM HEPES (pH 7.4) with 10 mM MgCl2, 2 mM EGTA, and 0.1% BSA (Binding Buffer) for 90 min. For competition binding assays, reactions were carried out in the presence of with 0.06-0.1 nM [Phe13, [125I]Tyr19]-MCH and increasing concentrations of unlabeled test molecules. Reactions were terminated by rapid vacuum filtration over 96 well-GFC UNIFILTER® plates pre-coated with 0.075 mL binding buffer containing 1% BSA, and washed 3 times with 0.4 mL of Phospho-buffered Saline (pH 7.4) containing 0.01% TX-100. Filters were dried, 0.05 mL MicroScint 20 was added to each well and radioactivity was subsequently quantified by scintillation counting on a TOPCOUNT® microplate scintillation counter (Packard).
Measurement: Ki = 634 nM (pChEMBL 6.199999809265137)
Descriptors: MW 482.96, ALogP 4.74, HBA 6, HBD 1, PSA 81.12, QED 0.5
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2cc(-c3ccccc3)nn2-c2ccccc2)[C@H](c2ccccc2)C1
InChIKey: NKQYPBUMCGKFST-AHKZPQOWSA-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 481.6, ALogP 4.78, HBA 5, HBD 2, PSA 71.42, QED 0.38
Compound (SMILES): Cn1cnc(S(=O)(=O)N2C[C@@H](CNc3cccc(Cl)c3)[C@H](c3ccc(F)cc3)C2)c1
InChIKey: AXYHGEAZNDEHRN-UZLBHIALSA-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 < 1000 nM (pChEMBL 6)
Descriptors: MW 448.95, ALogP 3.73, HBA 5, HBD 1, PSA 67.23, QED 0.62
Compound (SMILES): CC(=O)N1CCC2(CC1)NC(=O)c1c(Cl)cc(Nc3ccncn3)c(=O)n12
InChIKey: FDUKGUHPTGOCIY-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: Mnk Biochemical Enzymatic Assay: Compounds are screened for Mnk inhibition using the ADP-Glo kinase assay kit (Promega, catalogue No. V9101). All kinase reactions are performed in Reaction Buffer E (15 mM HEPES pH7.4, 20 mM NaCl, 1 mM EGTA, 10 mM MgCl2, 0.1 mg/ml BGG, and 0.02% Tween-20). Final Mnk1 reactions contained 10 nM recombinant Mnk1 (Life Technologies, PR9138A), 100 μM Mnk substrate peptide Ac-TATKSGSTTKNR-NH2 (American Peptide Company), 300 μM ATP, and varying concentrations of the inhibitory compound of interest. Final Mnk2 reactions contained 3 nM recombinant Mnk2 (Life Technologies, PV5607), 50 μM Mnk substrate peptide Ac-TATKSGSTTKNR-NH2 (American Peptide Company), 10 μM ATP, and varying concentrations of the inhibitory compound of interest. Final DMSO concentration in each reaction is 1%. Kinase reactions are carried out in 96-well half-area white flat-bottom polystyrene plates in a final volume of 25 μl. Mnk1/2 enzymes are pre-incubated with compound and peptide substrate for 5 minutes prior to the addition of ATP. After the addition of ATP, kinase reactions are incubated at room temperature for 40 minutes. Reactions are subsequently stopped by the addition of 25 μl of ADP-Glo Reagent and incubating for an additional 40 minutes. The final luminescent signal used for kinase activity readout is produced by the addition of 45 μl of Kinase Detection Reagent (ADP-Glo kit, Promega) and incubating for 40 minutes. The luminescent signal is detected using a Victor 2 multilabel counter (Perkin Elmer) and the concentration of compound necessary to achieve inhibition of enzyme activity by 50% (IC50) is calculated using signals from an 8-point compound dilution series.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 388.82, ALogP 1.07, HBA 7, HBD 2, PSA 109.22, QED 0.8
Compound (SMILES): Cc1ccc2c(c1)nc1n2C[C@@](NC(=O)c2ccc(-n3cc(CCO)nn3)cc2Cl)(c2ccccc2)CC1
InChIKey: WUECUDXWOJCSJF-GDLZYMKVSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: TBD: TBD
Measurement: IC50 = 8.83 nM (pChEMBL 8.050000190734863)
Descriptors: MW 527.03, ALogP 4.39, HBA 7, HBD 2, PSA 97.86, QED 0.34
Compound (SMILES): Cc1ccc([C@H]2C[C@@H]2COc2nc(C)ncc2-c2ccc(C(O)C(C)C)nc2)nc1
InChIKey: PLIZCBLCKNZOJC-BEWYTNLXSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60;
Measurement: Ki = 0.37 nM (pChEMBL 9.430000305175781)
Descriptors: MW 404.51, ALogP 4.42, HBA 6, HBD 1, PSA 81.02, QED 0.63
Compound (SMILES): CC(C)C(=O)N1CCC[C@@H](c2nn(-c3ccc(Oc4ccccc4)cc3)c3c(N)nccc23)C1
InChIKey: MVRZGXNZKPEINC-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: LANCE (Lanthanide Chelate Excite) TR-FRET (Time-resolved fluorescence resonance energy transfer) assay: In this assay, the potency (IC50) of each compound was determined from an eleven point (1:3 serial dilution; final compound concentration range in assay from 1 μM to 0.017 nM) titration curve using the following outlined procedure. To each well of a black non-binding surface Corning 384-well microplate (Corning Catalog #3820), 5 nL of compound (2000 fold dilution in final assay volume of 10 μL) was dispensed, followed by the addition of 7.5 μL of 1× kinase buffer (50 mM Hepes 7.5, 10 mM MgCl2, 0.01% Brij-35, 1 mM EGTA, 0.05% BSA & 1 mM DTT) containing 5.09 pg/μL (66.67 pM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 2.5 μL 1× kinase buffer containing 8 μM biotinylated A5 peptide (Biotin-EQEDEPEGDYFEWLE-NH2), and 100 μM ATP. The final reaction in each well of 10 μL consists of 50 pM hBTK, 2 μM biotin-A5-peptide, and 25 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes. Reactions were immediately quenched by the addition of 20 uL of 1× quench buffer (15 mM EDTA, 25 mM Hepes 7.3, and 0.1% Triton X-100) containing detection reagents (0.626 nM of LANCE-Eu-W1024-anti-phosphoTyrosine antibody, PerkinElmer and 86.8 nM of Streptavidin-conjugated Dylight 650, Dyomics/ThermoFisher Scientific). After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate:anti-phospho-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 455.56, ALogP 5.16, HBA 6, HBD 1, PSA 86.27, QED 0.45
Compound (SMILES): CC(C)(C)N1CCC(N2C=C([C@@H](Nc3cc(Cl)c4ncc(C#N)c(Nc5ccc(Cl)c(Cl)c5)c4c3)c3cscn3)NN2)CC1
InChIKey: DGFWKOGKCFGFKO-SSEXGKCCSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 26 nM (pChEMBL 7.579999923706055)
Descriptors: MW 669.09, ALogP 7.85, HBA 10, HBD 4, PSA 104.17, QED 0.16