lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)(C)C(NC(=O)c1ccc(F)cc1)C(=O)N1CCC[C@H]1C(=O)N1C[C@]2(C[C@H]1C#N)C(=O)Nc1ccccc12
InChIKey: NXJYCMBBFOPRCJ-WWDNEBHZSA-N
Target: Replicase polyprotein 1ab (SINGLE PROTEIN), Severe acute respiratory syndrome coronavirus 2
UniProt: P0DTD1
Assay: Protease Fluorescence Assay (FRET): Recombinant SARS-CoV-2 3CL-protease was expressed and purified. TAMIRA-SITSAVLQSGFRKMK-Dabcyl-OH peptide 3CLpro substrate was synthesized. Black, low volume, round-bottom, 384 well microplates were used. In a typical assay, 0.85 μL of test compound was dissolved in DMSO then incubated with SARS-CoV-2 3CL-protease (10 nM) in 10 μL assay buffer (50 mM HEPES [pH 7.5], 1 mM DTT, 0.0100 BSA, 0.01% Triton-X 100) for 30 min at RT. Next, 10 μL of 3CL-protease substrate (40 μM) in assay buffer was added and the assays were monitored continuously for 1 h in an Envision multimode plate reader operating in fluorescence kinetics mode with excitation at 540 nm and emission at 580 nm at RT. No compound (DMSO only) and no enzyme controls were routinely included in each plate. All experiments were run in duplicate. Data Analysis: SARS-CoV-2 3CL-protease enzyme activity was measured as initial velocity of the linear phase (RFU/s) and normalized to controlled samples DMSO (10000 activity) and no enzyme (000 activity) to determine percent residual activity at various concentrations of test compounds (0-10 μM).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 545.62, ALogP 2.98, HBA 5, HBD 2, PSA 122.61, QED 0.61
Compound (SMILES): COc1cc(F)ccc1-c1ccnc(Cl)c1N(C)C(=O)c1cc(C(F)(F)F)cc(S(C)(=O)=O)c1
InChIKey: VCZJLOHYWLJQJM-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 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), lx 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. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), 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 measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. 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 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 516.9, ALogP 5.25, HBA 5, HBD 0, PSA 76.57, QED 0.34
Compound (SMILES): CC(C)[C@H]1c2ncc(C(=O)NC[C@@H]3CCN(C(=O)OC(C)(C)C)C[C@H]3O)cc2CN1Cc1ccc(C(F)(F)F)cc1
InChIKey: ROYKINDILJIIEK-KHMOSXFSSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay: Compounds of the present invention were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 μg of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader.
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 576.66, ALogP 5.16, HBA 6, HBD 2, PSA 95, QED 0.49
Compound (SMILES): COc1cc(CN2CCCC(c3cc(C)nc4ncnn34)C2)ccc1C
InChIKey: OXBHINOHTRUFBQ-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Enzymatic Assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.PDE2
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 351.45, ALogP 3.13, HBA 6, HBD 0, PSA 55.55, QED 0.72
Compound (SMILES): COC(=O)Nc1ccc2c(c1)N[C@H](C1=NN(C)CC1Cl)CCCC[C@H](NC(=O)/C=C/c1cc(Cl)ccc1-n1cnnn1)c1nc-2c[nH]1
InChIKey: KCSPAARWGBCQGA-QBDOVHSJSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa assay : 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 = 3851 nM (pChEMBL 5.409999847412109)
Descriptors: MW 678.58, ALogP 5.02, HBA 11, HBD 4, PSA 167.34, QED 0.16
Compound (SMILES): CCc1cccc(CC)c1-n1nc2c(c1-c1cc(F)c(NC(N)=O)cc1F)CN(C(=O)C1CCOCC1)C(C)(C)C2
InChIKey: CGWJRMZBGNRDIW-UHFFFAOYSA-N
Target: C5a anaphylatoxin chemotactic receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21730
Assay: chemotaxis assay: Optionally, compounds may be further assayed for their ability to inhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μm pore polycarbonate, polyvinylpyrrolidone-coated filters in 96-well chemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxis buffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands (i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluate compound mediated inhibition of C5aR mediated migration. Other chemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used as specificity controls. The lower chamber is loaded with 29 μl of chemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the top chamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambers are incubated 1.5 hours at 37° C., and the number of cells in the lower chamber quantified either by direct cell counts in five high powered fields per well or by the CyQuant assay (Molecular Probes), a fluorescent dye method that measures nucleic acid content and microscopic observation.
Measurement: IC50 = 27.5 nM (pChEMBL 7.559999942779541)
Descriptors: MW 565.67, ALogP 5.52, HBA 5, HBD 2, PSA 102.48, QED 0.41
Compound (SMILES): Cc1ccc(-n2nc3c(c2NC(=O)CS(C)(=O)=O)C(=O)N[C@@]2(CCc4cc(OCC(C)(C)F)ccc42)C3)nc1
InChIKey: IRHBUQVCQWMXHG-MHZLTWQESA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: Inhibitory Activity: Solutions of the compounds of the present invention in DMSO were each aliquoted into 0.2-μL portions in a 384-well polystyrene microplate produced by Corning Incorporated, and 5 μL of an enzyme solution prepared with an assay buffer (100 mmol/L phosphate buffer (pH 7.4) containing 2 mmol/L DTT) and 5 μL of a substrate solution (100 mmol/L phosphate buffer (pH 7.4), 30 μmol/L 2-Oleoylglycerol, 10 μmol/L Oleoyl-CoA) were added thereto, and the resultant was stirred and centrifuged, and incubated in a moist chamber at room temperature for 1 hour. After enzymatic reaction, 50 μL of a quenching solution (containing 0.2 μmol/L Diolein-d5, 0.4% formic acid, and 50% isopropanol) containing Internal Standard (IS) was added to terminate the reaction, and the resultant was sealed in a plate produced by Shimadzu GLC Ltd., and then stirred and centrifuged, and measurement was performed by using an electrospray ionization method with a RapidFire360 and Agilent 6550 Q-TOF mass spectrometer. Diolein as a reaction product (P) of 2-Oleoylglycerol as the substrate and an ammonium adduct ion of the IS were detected, and the peak intensity ratio, P/IS, was calculated from the peak heights to evaluate the inhibitory activity. Inhibitory activities with/without addition of enzyme were defined as Control (+)/Control (−), respectively, and the respective % inhibitions were defined as 0% inhibition and 100% inhibition. The inhibitory activity was calculated from formula below with TIBCO Spotfire (produced by TIBCO Software Inc.):Inhibitory activity (%)=[1−{Sample−Control(−)}/{Control(+)−Control(−)}]*100where Sample indicates a peak intensity ratio: P/IS, when the compound of the present invention was added.
Measurement: IC50 = 4.67 nM (pChEMBL 8.329999923706055)
Descriptors: MW 555.63, ALogP 2.81, HBA 8, HBD 2, PSA 132.28, QED 0.46
Compound (SMILES): C[C@]1(O)CC[C@@](F)(C(=O)NCc2c(F)cc(F)cc2Cl)c2cccnc21
InChIKey: CRDYDEYVXIRAPR-ROUUACIJSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Pharmacological Assay: 1. Wash each well with 80 microL of assay buffer (20 mM HEPES, 1×HBSS, pH 7.4 adjusted with NaOH) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).2. Add 20 microL of assay buffer containing 2.5 mM probenecid, 0.5 microM Fluo-4-AM (Molecular Probes) and 0.1% Pluronic F-127 to each well.3. Incubate the plate at 37° C. in 5% CO2 for 1 h.4. Wash each well with 80 microL of assay buffer (see below) three times and leave 20 microL using plate washer, ELx-405 Select CW (BIO-TEK).5. Test compounds were prepared at 100× the test concentration in DMSO by serial dilution with Biomek-FX liquid handling instrument. 33× diluted compound solutions in assay buffer were prepared in intermediate compound plate with Biomek-NX liquid handling instrument. A further 3× dilution occurred in below steps 6 and 7.6. Add 20 microL of 33× diluted compound solutions into each well and leave the plate for 10 min under the dark at room temperature.7. Measure activity by FDSS as follows:Set the assay plate on the stacker of FDSS.Start the detection of fluorescence intensity at 540 nm by 480 nm exicitation.After 30 seconds, add 20 microL of assay buffer containing 240 microM BzATP (final concentration 80 microM).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 384.79, ALogP 3.5, HBA 3, HBD 2, PSA 62.22, QED 0.85
Compound (SMILES): CC(C)c1c(-c2ccc(N3CCN(C(C)C)CC3)cc2)[nH]c2ncnn2c1=O
InChIKey: YMQBXGHSTCRWML-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGL1
Assay: TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) Assay: In this assay, the potency (IC50) of each compound was determined from a ten point (1:3 serial dilution; final compound concentration range in assay from 100000 nM to 5.08 nM) titration curve using the following outlined procedure. To each well of a white Greiner 1536 Lumitrac 1536 well-plate, 50 nL of compound (100 fold dilution in final assay volume of 5 μL) was dispensed, followed by the addition of 4 μL of 1× assay buffer (50 mM Hepes 7.3, 0.5 mM TCEP, 0.005% Brij-35, 0.02% BSA, 50 μM Na-L-Ascorbate and 2 μM AmFe(II)Sulfate) containing 5 nM of Full-length KDM5B enzyme (recombinant protein from baculovirus-transfected Sf21 cells: full-length KDM5B; MW=176.825 kDa). Following a 30 minutes compound and enzyme incubation in a humidified chamber, each reaction was initiated by the addition of 1 μL 1× assay buffer containing 50 nM biotinylated H3K4Me3 peptide, and 500 nM α-ketoglutarate. The final reaction in each well of 5 μL consists of 5 nM KDM5B, 500 nM biotinylated-peptide, and 500 nM α-ketoglutarate. De-methylation reactions were allowed to proceed for 60 minutes. Reactions were immediately quenched by the addition of 5 uL of 2× Lance Detection Buffer (PerkinElmer) with 0.5 mM EDTA and 2 nM of Eu-anti-H3K4Me1-2 antibody, and 30 nM of Streptavidin-conjugated Dylight 650 detection reagents. 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-H3K4Me1-2-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 = 15.5 nM (pChEMBL 7.809999942779541)
Descriptors: MW 380.5, ALogP 2.74, HBA 6, HBD 1, PSA 69.53, QED 0.75
Compound (SMILES): CC(C)N(CC(=O)NCc1cccc(Cl)c1F)C(=O)Cn1nc(C(N)=O)c2cc(NC(=O)N3CCC(F)(F)CC3)ccc21
InChIKey: JAOGKGHWYVWLEC-UHFFFAOYSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: An established esterolytic assay for the measurement of Factor D activity and inhibition of Factor D activity was used (Kam, C. M.; McRae, B. J.; Harper, J. W.; Niemann, M. A.; Volanakis, J. E.; Powers, J. C. Human complement proteins D, C2, and B Active site mapping with peptide thioester substrates. J Biol. Chem. 1987, 262, 3444-3451). For this assay Z-Lys-SBzl, 1.29 mM (Kim, S.; Narayana, S. V. L; Volanakis, J. E. Mutational analysis of the substrate binding site of human complement Factor D. Biochemistry. 1994, 33, 14393-14399) was used as the substrate for Factor D (104 mM). Hydrolysis of this compound by Factor D liberated a free sulfhydryl group which is then reacted with 5,5′-dithiobis(2nitrobenzoic acid) producing an intense yellow color (Habeeb, A. F. S. A. Reaction of protein sulfhydryl groups with Ellman's Reagent. Methods in Enzymol. 1976, 25, 457-464.). The assays were performed in 96 well microtiter plates and rates of hydrolysis were monitored at 405 nm on a Biotek Synergy H1 plate reader. Hydrolysis rates were reported as change in mOD/min. The assay was conducted in 100 mM HEPES, 500 mM NaCl, pH 7.5 containing 10% DMSO in a final volume of 50 μL per well.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 622.05, ALogP 3.74, HBA 6, HBD 3, PSA 142.66, QED 0.33
Compound (SMILES): Oc1cc(CC(F)(F)F)c(-c2nc(NCCC3CC3)c3cn[nH]c3n2)cc1F
InChIKey: FTSYURCNSVPOMF-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Caliper Enzyme Assay at 1 mM ATP: Test article was solubilized in dimethyl sulfoxide (DMSO) to a stock concentration of 30 mM. An 11-point half log dilution series was created in DMSO with a top concentration of 600 μM. The test compound plate also contained positive control wells containing a known inhibitor to define 100% inhibition and negative control wells containing DMSO to define no inhibition. The compound plates were diluted 1 to 60 resulting in a top final assay compound concentration of 10 μM and a 2% DMSO concentration.Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 41.6 nM (pChEMBL 7.380000114440918)
Descriptors: MW 395.36, ALogP 4.18, HBA 5, HBD 3, PSA 86.72, QED 0.55
Compound (SMILES): CS(C)(=O)=Nc1cc(N2CCOC[C@H]2CF)nc(-c2ccnc3[nH]ccc23)n1
InChIKey: YBGPHBWOMSWCRF-GFCCVEGCSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: pCHK1 Cellular Assay: Inhibitors of ATR kinase are effective at inhibiting the ATR-driven phosphorylation of the downstream target Chk1 kinase at Serine 345, following the addition of 4-nitroquinoline N-oxide, a chemical used to induce DNA damage. Cellular IC50 for the inhibitors of ATR described herein were measured in HT-29 colorectal adenocarcinoma cells. HT-29 cells were routinely maintained in McCoy's 5A media (ATCC Catalog #30-2007) supplemented with 10% fetal bovine serum (Sigma Catalog # F2442) and 1λ Penicillin-Streptomycin (Gibco Catalog #15140-122) using a humidified incubator (37° C., 5% CO2, and ambient O2). In preparation for the CHK1 (p-Ser345) ALPHASCREEN® SUREFIRE® assay, cells were harvested and resuspended in McCoy's 5A media supplemented with 10% fetal bovine serum and 1× Penicillin-Streptomycin. Cells were seeded onto a 384-well black CELLSTAR® Tissue Culture Plate (VWR Catalog #89085-314) at a density of 13,000 cells/well in a volume of 40 μL. The microplate was incubated overnight (approximately 20 hours) at 37° C. with 5% CO2 and ambient O2. Stock solutions of the test compounds were prepared in 100% DMSO (Sigma, Catalog # D2650) and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:33 in culture medium, and 10 μL/well were transferred to the tissue culture plate. Following the compound addition the microplate was incubated at 37° C. for 90 minutes. 10 μL of 4-nitroquinoline N-oxide (Sigma Aldrich Catalog # N8141-1G) diluted in media (final concentration 12 uM) were added to the tissue culture plate followed by a 120 minute incubation at 37° C. The cells were then washed with PBS and lysed using 10 μL/well SUREFIRE® Kit lysis buffer diluted to 1× in water (PerkinElmer Catalog # TGRCHK1S50K), with mixing on an orbital shaker at 500 rpm for 20 min at RT. Lysates were frozen at −20° C. overnight.4 μL/well of lysate was then transferred from the tissue culture plate to a 384-well, white, low volume, PROXIPLATE (PerkinElmer Catalog #600828). 5 μL/well of the acceptor bead solution, prepared by diluting SUREFIRE® Kit activation buffer (PerkinElmer Catalog # TGRCHK1S50K) and ALPHASCREEN® Protein A acceptor beads (PerkinElmer Catalog #6760617R) in SUREFIRE® Kit reaction buffer (PerkinElmer Catalog # TGRCHK1S50K), were added to the lysates under subdued light and incubated at room temperature for 120 min. 2 uL/well of the donor bead solution, prepared by diluting ALPHASCREEN® Streptavidin donor beads (PerkinElmer Catalog #6760617R) in SUREFIRE® Kit dilution buffer (PerkinElmer Catalog # TGRCHK1S50K), were added under subdued light and incubated at room temperature for an addition 120 minutes. The pCHK1 ALPHASCREEN® signal was measured using an ENVISION® plate reader (PerkinElmer). IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software.
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 404.47, ALogP 2.55, HBA 7, HBD 1, PSA 96.36, QED 0.72
Compound (SMILES): CN(C)S(=O)(=O)c1c(Cl)ccc(Nc2c(NC(c3ccccc3)C(F)(F)F)c(=O)c2=O)c1O
InChIKey: DKOUVWBSBUVOPA-UHFFFAOYSA-N
Target: C-C chemokine receptor type 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P51684
Assay: Biological Assay: HEK-Gqi5 cells stably expressing human CCR6 were cultured in DMEM high glucose, 10% FBS, 1% PSA, 400 ug/ml geneticin and 50 ug/ml hygromycin. Appropriate positive control chemokine (MIP3α) was used as the positive control agonist for screening compound-induced calcium activity assayed on the FLIPRTetra. The drug plates were prepared in 384-well microplates using the EP3 and the MultiPROBE robotic liquid handling systems. Compounds disclosed herein were tested for CCR6 activity.
Measurement: IC50 = 293 nM (pChEMBL 6.53000020980835)
Descriptors: MW 503.89, ALogP 3.35, HBA 7, HBD 3, PSA 115.81, QED 0.33
Compound (SMILES): COC(=O)NC(C(=O)NC(C(=O)NC(=N)c1ccccc1)c1cccc(Cl)c1)c1cccc(Cl)c1
InChIKey: WBTSGSPPSRSUTC-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: TBD: TBD
Measurement: IC50 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 513.38, ALogP 4.39, HBA 5, HBD 4, PSA 120.38, QED 0.28
Compound (SMILES): C[C@H](c1ccccc1)N1CC[C@H](N(C)c2cc(F)c(S(=O)(=O)Nc3cccc(F)n3)cc2Cl)C1
InChIKey: OYELAONRLOYCOX-AEFFLSMTSA-N
Target: Sodium channel protein type 8 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQD0
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5 h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS ρCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaCl2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 1980 nM (pChEMBL 5.699999809265137)
Descriptors: MW 507.01, ALogP 5.09, HBA 5, HBD 1, PSA 65.54, QED 0.45
Compound (SMILES): CNc1cc(-c2cn([C@@H]3CCOC[C@H]3OC)c3ncccc23)nc2c(C(=O)NC3CC[C@@H]3OC)cnn12
InChIKey: KLMHVSFAZXFYHP-ZXJUBJKJSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 JH2 Domain Binding Assay: The binding reaction was assembled by combining 16 μl of DNA-tagged kinase extract, 3.8 μl liganded affinity beads, and 0.18 μl test compound (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA)]. Extracts were used directly in binding assays without any enzyme purification steps at a ≥10,000-fold overall stock dilution (final DNA-tagged enzyme concentration <0.1 nM). Extracts were loaded with DNA-tag and diluted into the binding reaction in a two step process. First extracts were diluted 1:100 in 1× binding buffer (PBS/0.05% Tween 20/10 mM DTT/0.1% BSA/2 μg/ml sonicated salmon sperm DNA) containing 10 nM DNA-tag. This dilution was allowed to equilibrate at room temperature for 15 minutes and then subsequently diluted 1:100 in 1× binding buffer. Test compounds were prepared as 111× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing) in 100% DMSO
Measurement: Kd < 0.2 nM (pChEMBL 9.699000358581543)
Descriptors: MW 505.58, ALogP 2.67, HBA 10, HBD 2, PSA 116.83, QED 0.39
Compound (SMILES): NC(=O)c1nnc(N2CCC[C@@H](NC(=O)N3CCCCC3)C2)nc1Nc1ccc2cccnc2c1
InChIKey: JHOQHRMLDARZGN-GOSISDBHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk In Vitro Inhibitory Assay: Human Btk kinase (Genbank accession # NP_000052) was purified from insect cells as a full-length construct containing a N-terminal 6×-His tag. Btk kinase activity was determined using a radiometric filter binding assay. Measurements are performed in a low μL reaction volume 384-well assay plates. BTK enzyme (8 nM final in reaction), inhibitor (at requested doses), and 0.2 mg/mL peptide substrate (Poly-Glu-Tyr, 4:1 ratio) are incubated in a reaction buffer composed of 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 for 15 min. followed by addition of 1 μM ATP to start the assay. Kinase reactions are carried out for 120 min. at room temperature. The reaction was stopped by spotting of reaction sample onto P81 cationic exchange paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% Phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme (via addition of saturating EDTA), kinase activity data for each dose of compound tested was expressed as the percent of remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 475.56, ALogP 2.43, HBA 8, HBD 3, PSA 142.26, QED 0.51
Compound (SMILES): Cc1cc(-n2ncc3cc(NC(=O)N[C@H]4CN(C)C[C@@H]4c4ccccc4C)ncc32)ccn1
InChIKey: PIKZAOBNOWWJRU-YADHBBJMSA-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 = 0.83 nM (pChEMBL 9.079999923706055)
Descriptors: MW 441.54, ALogP 3.65, HBA 6, HBD 2, PSA 87.97, QED 0.5
Compound (SMILES): COc1cc(C(=O)Nc2n[nH]c3cc(-c4c(F)c(OC)cc(OC)c4F)ccc23)ccc1N1CCNC(C)(C)C1
InChIKey: YJVPQFIFRWLZOB-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Enzyme Kinase Activity Assay: The enzyme reaction substrate Poly(Glu,Tyr) 4:1 was diluted with PBS without potassium ion (10 mM sodium phosphate buffer, 150 mM NaCl, pH7.2-7.4) to 20 μg/mL, 125 μL/well to coat the enzyme plate, and reacted at 37° C. for 12-16 hours. After the liquid was removed from the wells, the plate was washed three times with 200 μL/well of T-PBS (PBS containing 0.1% Tween-20) for 5 minutes each. The enzyme plate was dried in a 37° C. oven for 1-2 hours.50 μL of the ATP solution diluted with the reaction buffer (50 mM HEPES pH 7.4, 50 mM MgCl2, 0.5 mM MnCl2, 0.2 mM Na3VO4, 1 mM DTT) was added into each well at a 5 μM final concentration. The compounds were diluted to the appropriate concentration in DMSO, 1 μL/well or containing the corresponding concentrations of DMSO (negative control wells). The reaction was initiated by addition of each kinase domain recombinant protein diluted with 49 μL of reaction buffer. Two control wells without ATP were set in each experiment. The reaction mixtures were placed on a shaker (100 rpm) to react at 37° C. for 1 hour. The plates were washed with T-PBS for three times. 100 μL/well of the primary antibody PY99 dilution was added, and reacted on a shaker (100 rpm) at 37° C. for 0.5 hour. The plates were washed with T-PBS for three times. 100 μL/well of the secondary anti-horseradish peroxidase-labeled goat anti-mouse IgG dilution was added, and reacted on a shaker at 37° C. for 0.5 hour. The plates were washed with T-PBS for three times. 100 μL/well of 2 mg/mL OPD developing solution (diluted with 0.1M citric acid-sodium citrate buffer containing 0.03% H2O2 (pH=5.4)), and reacted in dark for 1-10 minutes at 25° C. (Ultrasound is needed in OPD dissolution, and the developing solution should be prepared on the site). The reaction was quenched with 50 μL/well of 2M H2SO4, and read out at 490 nm using a tunable microplate microplate reader SPECTRA MAX 190.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 551.59, ALogP 4.97, HBA 7, HBD 3, PSA 100.74, QED 0.3
Compound (SMILES): Cc1nc(-c2ccc(OC(C)C)c(C#N)c2)sc1C(=O)O
InChIKey: UBKMOFWTLCOKFO-UHFFFAOYSA-N
Target: Xanthine dehydrogenase/oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P47989
Assay: Inhibition Assay: The inhibition of the activity of xanthine oxidase (XO) is tested through a coupled enzymatic reaction of xanthine oxidase, horseradish peroxidase (HRP) and a substrate thereof. First, xanthine oxidase oxidizes hypoxanthine to produce xanthine and hydrogen peroxide, and further oxidizes xanthine to produce uric acid and hydrogen peroxide. Then, hydrogen peroxide reacts with 10-acetyl-3,7-dihydroxyphenoxazine (Ampliflu Red) under catalytic action of horseradish peroxidase so as to produce resorufin, a compound with strong fluorescence. The fluorescence intensity of resorufin is determined by using a fluorescence microplate, which is in direct proportion to the activity of xanthine oxidase.
Measurement: IC50 = 2.78 nM (pChEMBL 8.5600004196167)
Descriptors: MW 302.36, ALogP 3.48, HBA 5, HBD 1, PSA 83.21, QED 0.93
Compound (SMILES): Cc1c(C#N)ccnc1O[C@@H]1CC[C@@H](C)N(C(=O)c2ccsc2-n2nccn2)C1
InChIKey: VNPQSNXVFVZPTG-CZUORRHYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Radioligand binding assay: Radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: Ki = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 408.49, ALogP 2.98, HBA 8, HBD 0, PSA 96.93, QED 0.66
Compound (SMILES): C[C@@H]1CNc2c(sc3ccc4nc(Nc5nccc(Cl)n5)ccc4c23)C(=O)N1
InChIKey: QFWPZRYSEPLDPE-SECBINFHSA-N
Target: MAP kinase-activated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P49137
Assay: Mass Spectrometry Assay: Compounds I-1 through I-50 of the invention were assayed in a mass spectrometric assay to measure their ability to covalently modify MK2 protein. The procedure for this assay follows. Intact MK2 protein (Invitrogen, Cat. No. PR5320A) was incubated for 60 minutes at room temperature with a 10-fold excess of test compound to protein. Aliquots of the resulting mixture (6 μL each) were diluted with 0.2% trifluoroacetic acid (TFA, 10 μL) prior to micro C4 ZipTipping directly onto the MALDI target using sinapinic acid as the desorption matrix (10 mg/ml in 0.1% TFA:acetonitrile 50:50, v/v). The centroid mass of the target protein in the control sample was compared with the centroid mass of the target protein incubated with compound. A shift in the centroid mass of the treated protein compared to the untreated protein was divided by the molecular weight of the compound. This number corresponds to the percentage of modified protein (a measure of the proportion of total target protein covalently bound to the test compound) after one hour incubation. Results from this assay are reported in Table A under the column ¿Mass Modification¿.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 410.89, ALogP 4.18, HBA 7, HBD 3, PSA 91.83, QED 0.43
Compound (SMILES): Nc1nc(OCc2cn(-c3ccc(Cl)c(OC(F)F)c3)nn2)ncc1F
InChIKey: IUIDXVARBCARLT-UHFFFAOYSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NMDA receptor FLIPR assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 149 mM NaCl, 4 mM KCl, 2 mM CaCl2, and 1.5 mM MgCl2, 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (149 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC80 (standard assay) or EC40 (HTS assay) is used to maximize the assay's signal window or the ability to detect NMDA receptor antagonists and negative allosteric modulators, respectively. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 386.72, ALogP 2.61, HBA 8, HBD 1, PSA 100.97, QED 0.69
Compound (SMILES): Cc1cccc(-c2nc3n(c2-c2cc4cnccc4s2)C2CCC3C2)n1
InChIKey: RRSBIRUCCSICSI-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Kinase Activity Assay: TGFβRI kinase assay kit (V4093, Promega) was used to assay enzyme activity. 2 μl of enzyme solution (the final concentration of enzyme in the reaction system was 2 ng/μL) formulated with reaction buffer (40 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA), 1 μl of a 3-fold gradient dilution of the compounds dissolved in 5% DMSO, and 2 μl of a mixed solution of ATP and TGFβRI substrate peptide (the final concentration of ATP was 50 μM, and the final concentration of substrate was 0.2 μg/μL) were added successively to a 384-well plate (4514, Corning). After reaction at 27° C. for 2.5 hours, 5 μl of ADP-Glo solution in the kit was added to each well, then the plate was placed at 27° C. for 40 minutes. 10 μl of kinase assay reagent was then added to each well, then the plate was placed at 27° C. for 30 minutes. The chemiluminescence signal values were measured with a Victor 3 (PerkinElmer) multi-function microplate reader. The IC50 values of the compounds for enzyme inhibition were calculated using Graphpad prism software based on each concentration of the compound and the corresponding signal value thereof.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 358.47, ALogP 5.35, HBA 5, HBD 0, PSA 43.6, QED 0.48
Compound (SMILES): N#Cc1ccncc1-c1ccc(NC(=O)c2ccnc(-c3c(F)cccc3F)n2)c(N2C[C@@H](N)C[C@H]2CO)c1
InChIKey: LDCDYMPNMGJUDB-OALUTQOASA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: HPK1 Kinase Binding Assay: A stock solution of 1 mM test compound was prepared in DMSO. The compound plate was prepared by 3-fold and 11-point serial dilutions. 0.1 μL of the compound in DMSO was transferred from the compound plate to the white 384 well polystyrene plates. The assay buffer contained 50 mM HEPES, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, and 5 mM DTT. 5 μL of 4 nM active HPK1 (SignalChem M23-11G) prepared in the buffer was added to the plate. The enzyme concentration given was based on the given stock concentration reported by the vender. 5 μl of 18 nM tracer 222 (ThermoFisher PV6121) and 4 nM LanthaScreen Eu-Anti GST antibody (ThermoFisher PV5595) were added. After one hour incubation at 25° C., the plates were read on a PHERAstar FS plate reader (BMG Labtech). Ki values were determined.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 527.54, ALogP 3.51, HBA 8, HBD 3, PSA 141.05, QED 0.35
Compound (SMILES): CC[C@@H](CO)Nc1ccc(C(=O)N2CCC(N3CCC[C@@H](C)C3)CC2)cn1
InChIKey: AANPPHBCLOSKSY-AEFFLSMTSA-N
Target: Alpha-1A adrenergic receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35348
Assay: Binding Assay: To prepare cell membranes with human α1- and α2-adrenergic receptors, CHO cells stably overexpressing α1- and α2-adrenergic receptors are lysed and then subjected to differential centrifugation. After lysis in binding buffer (50 mM tris(hydroxymethyl)aminomethane/1 N hydrochloric acid, 5 mM magnesium chloride, pH 7.4) using an Ultra Turrax (Jahnke & Kunkel, Werk), the homogenate is centrifuged at 1000 g and at 4° C. for 10 min. The resulting sediment is discarded and the supernatant is centrifuged at 20 000 g and at 4° C. for 30 min. The supernatant is discarded and the sediment is resuspended in binding buffer and stored at −70° C. until the binding test. For the binding test the radioligands 3H-MK-912 (2.2-3.2 TBq/mmol, PerkinElmer) (0.4 nM for α2C-adrRez and 1 nM for α2A-adrRez), 0.25 nM 3H-prazosin (α1AC-adrRez; 2.6-3.3 TBq/mmol, PerkinElmer), 0.25 nM 3H-rauwolscine (α2B-adrRez, 2.6-3.2 TBq/mmol, PerkinElmer) are incubated for 60 minutes with 5-20 μg cell membranes in binding buffer (total test volume 0.2 ml) in the presence of the test substances at 30° C. in 96-well filter plates (FC/B glass fibre, Multiscreen Millipore). The incubating is terminated by aspiration of the unbound radioactivity and the plates are then washed with binding buffer and subsequently dried at 40° C. for 1 hour. Liquid scintillator (Ultima Gold, PerkinElmer) is then added and the radioactivity that remained on the plates is measured in a liquid scintillation counter (Microbeta, Wallac). Non-specific binding is defined as radioactivity in the presence of 1-10 μM WB-4101 (α2C-adrRez and α2A-adrRez), prazosin (α2B-adrRez and α1AC-adrRez) (all from Sigma) and is generally <25% of the bound total radioactivity.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 374.53, ALogP 2.6, HBA 5, HBD 2, PSA 68.7, QED 0.8
Compound (SMILES): Cc1cc(C(=O)/N=c2\[nH]c3ccc(CN4CCOCC4)cc3n2CC(C)(C)O)cc(-c2cnn(C)c2)n1
InChIKey: ZTZMTFFPFILZLL-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Biochemical EGFR Inhibition Assays (5 mM ATP): 5 μL of EGFR enzyme form in assay buffer (50 mM HEPES pH 7.3, 10 mM MgCl2, 1 mM EGTA, 0.01% Tween 20, 2 mM DTT) are dispensed into columns 1-23, than 5 μL of ATP and ULight-poly-GT substrate (PerkinElmer; TRF0100-M) mix in assay buffer is added to all wells (final assay concentration of the ULight-poly-GT substrate 200 nM). Each of the different EGFR enzyme form assays is available at low ATP (final assay concentration 5 μM). After 90 minutes incubation at room temperature 5 μL EDTA (final assay concentration 50 mM) and LANCE Eu-anti-P-Tyr (PT66) antibody (PerkinElmer, AD0069) (final assay concentration 6 nM) mix are added to stop the reaction and start the binding of the antibody. After additional 60 minutes incubation at room temperature the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the TR-FRET LANCE Ultra specs of PerkinElmer (used wavelengths: excitation 320 nm, emission 1 665 nm, emission 2 615 nm). Each plate contains 16 wells of a negative control (diluted DMSO instead of test compound; w EGFR enzyme form; column 23) and 16 wells of a positive control (diluted DMSO instead of test compound; w/o EGFR enzyme form; column 24). Negative and positive control values are used for normalization and IC50 values are calculated and analysed using a 4 parametric logistic model.
Measurement: IC50 = 54.1 nM (pChEMBL 7.269999980926514)
Descriptors: MW 503.61, ALogP 2.42, HBA 8, HBD 2, PSA 113.56, QED 0.42
Compound (SMILES): Cc1cc(F)c(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)C2CCC(F)(F)CC2)C[C@@H]1F
InChIKey: PRSWVLILAUOBMJ-RBUKOAKNSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 7100 nM (pChEMBL 5.150000095367432)
Descriptors: MW 586.51, ALogP 4.55, HBA 6, HBD 2, PSA 105.62, QED 0.44
Compound (SMILES): CCN1CCN(Cc2ccc(Nc3ncc(F)c(-c4ccc5ncc(C#N)n5c4)n3)nc2)CC1
InChIKey: OSNAHLHLNKIDPE-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P11802
Assay: Caliper Mobility Shift Assay: CDK4: The test compound was dissolved in DMSO and diluted with a kinase buffer solution (20 mM HEPES-pH 7.5, 0.01% Triton X-100, 10 mM MgCl2, 2 mM DTT) and 5 μL of compound at 5-fold final concentration of reaction dissolved in 10% DMSO was added in a 384-well plate. The compound-free control well was 5 μL of 10% DMSO, and the no-activity control well was 5 μL of kinase buffer. 10 μL of a 2.5-fold diluted CDK4 enzyme solution (GST-CDK4(1-303 end)) was added and incubated at room temperature for 10 min, and then 10 μL of the 2.5-fold diluted substrate solution Peptide FAM-P8 was added. The reaction was stopped by adding 25 μL of stop solution after incubation at 28° C. for 3 h, and the conversion rate data was read on a Caliper EZ Reader II (Caliper Life Sciences) and the conversion rate was converted to inhibition rate data according to above method.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 457.52, ALogP 3.08, HBA 9, HBD 1, PSA 98.27, QED 0.47