lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCCC(=O)N1CCC(c2cccc([C@H](Cc3ccccc3)c3ccc(-c4cc(Cl)ccc4-n4cnnn4)cn3)c2)CC1
InChIKey: BLTANBDUBMLEEC-HKBQPEDESA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Inhibition Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, 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 40 pM (Sekisui Diagnostics) and the synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 100 μM.
Measurement: IC50 = 11.2 nM (pChEMBL 7.949999809265137)
Descriptors: MW 607.16, ALogP 6.49, HBA 7, HBD 0, PSA 86.03, QED 0.18
Compound (SMILES): C/C=C/C[C@@H](C)[C@@H](O)[C@H]1C(=O)N[C@@H](CC)C(=O)N(C)[C@H](C)C(=O)N(C)[C@@H]([C@H](CN2CCN(CCOC)CC2)OC)C(=O)N[C@@H](C(C)C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@H](C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N(C)[C@@H](CC(C)C)C(=O)N(C)[C@@H](C(C)C)C(=O)N1C
InChIKey: MWUVAGZAJWQVEP-VKBFEWJASA-N
Target: Peptidyl-prolyl cis-trans isomerase A (SINGLE PROTEIN), Homo sapiens
UniProt: P62937
Assay: Surface Plasmon Resonance (SPR) Assay: Binding of inhibitors to expressed cyclophilins was determined using surface plasmon resonance (SPR) experiments. Briefly, avi-tagged cyclophilin proteins with mono-biotinylation were immobilized onto a Biotin CAPture chip (GE Healthcare, cat. #28920234). SPR experiments were carried out on an upgraded Biacore T200 system using a running buffer containing the inhibitor, 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% P20, and 3% DMSO. Single-cycle kinetics measurements were used to study the interactions between cyclophilin inhibitors and different cyclophlins. Accordingly, compound at various concentrations was injected to the flow cells with a contact time of 1 min each at a flow rate of 50 μL/min. The final dissociation phase lasts for 10 min following the sample injection. Data was analyzed using Biacore T200 Evaluation Software Version 1.0 and a 1:1 binding model was applied to fit the data to obtain kon, koff, and KD.
Measurement: Kd = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 1360.84
Compound (SMILES): CN1CC[C@@](O)(C#Cc2cccc(-n3nc(C(N)=O)c4cnc(Cl)cc43)c2)C1=O
InChIKey: DJEIIOBJWIKKRM-FQEVSTJZSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: NIK Enzyme Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified NIK (0.5 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl2, 2 mM dithiothreitol, 10 μM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 7 μg/mLADP antibody and 5 nM ADP-MR121 633 tracer. Reactions were quenched by the addition of 20 mM 2,2′,2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation at 633 nm with a Fluorescence Correlation Spectroscopy Plus reader (Evotec AG). Equilibrium dissociation constant (Ki) values for NIK inhibitors are calculated from plots of activity vs. inhibitor concentration using Morrison's quadratic equation that accounts for the potential of tight binding, and by also applying the conversion factor that accounted for competitive inhibition and the concentration of substrate used in the assay relative to its Michaelis constant (Km).
Measurement: Ki = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 409.83, ALogP 1.12, HBA 6, HBD 2, PSA 114.34, QED 0.49
Compound (SMILES): CCn1ccc(C2=C(c3nnn[nH]3)C(=O)N[C@@](c3ccc(CCCCC(F)(F)F)cc3F)(C(F)(F)F)C2)n1
InChIKey: YJFKXWQHQUGEKN-NRFANRHFSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in Corning FALCON® 96-well polypropylene plates, in a total volume of 60 μL of 50 mM potassium phosphate buffer pH 7.4, containing a final concentration of 100 μM 2-oleoylglycerol, 15 μM oleoyl-coenzyme A and 0.0013 μg/μL human or mouse MGAT-2 or 0.0026 μg/μL rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 μL of ice cold methanol containing 1 μg/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters SYMMETRY C8, 50×2.1 mm column, was used for the chromatography of enzyme products. The buffer system consists of 0.1% formic acid in water with a mobile phase consisting 0.1% formic acid in methanol. The shallow gradient is 90-100% mobile phase in 0.2 min with a total run time of 2.3 min. The first 0.5 minutes of each injection was diverted to waste to eliminate the presence of phosphate buffer in the enzymatic reaction. The column was run at 0.6 mL/min and a temperature of 65° C. Mass spectrometry analysis of the samples was performed on a ThermoFisher Quantum Triple quad utilizing APCI (+) as the mode of ionization. Data was acquired in Single Ion Monitoring (SIM) mode analyzing Diolein=m/z 603.6 (PRODUCT) and 1,2-distearoyl-rac-glycerol (IS)=m/z 607.6. The ratio of Diolein to internal standard (Peak Area Ratio) is utilized to calculate IC50 values.
Measurement: IC50 = 4.5 nM (pChEMBL 8.350000381469727)
Descriptors: MW 545.46, ALogP 4.72, HBA 6, HBD 2, PSA 101.38, QED 0.32
Compound (SMILES): COc1ccccc1-c1cc(C(=O)/N=c2\[nH]c3cc(C4CCCN(C(C)=O)C4)ccc3n2CC(C)(C)O)ccn1
InChIKey: AZORWFVYYSMUNT-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 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 541.65, ALogP 4.28, HBA 6, HBD 2, PSA 112.81, QED 0.38
Compound (SMILES): FC(F)(F)c1nc(-c2ccc(N3CCc4nc5ncccc5cc4C3)nc2)no1
InChIKey: XWHTZTXCVQYOAH-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Assay: HDAC1 and 6 reagents: FLAG-tagged HDACs 1 and 6 were prepared in-house by protein expression in HEK293F cells followed by anti-FLAG affinity purification. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. In addition to the above buffer ingredients, TCEP [CalBiochem, catalog #580561, 500 mM stock] was added at a final concentration of 0.5 mM to the buffer for the HDAC6 assays. HDAC1, 2, 3, and 6 enzymes were run at the final concentrations of 0.3 nM, 1.5 nM, 0.3 nM, and 1.333 nM, respectively. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-104], used to evaluate enzyme activity, was added at the final concentrations of 20 uM, 40 uM, 20 uM, and 2.5 uM for HDACs 1, 2, 3, and 6. To enable detection of the signal, Developer [BioMol Research Laboratories, catalog #KI-105] was added at a 1:250 dilution to the stop solution, which also included 10 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 398.35, ALogP 3.66, HBA 7, HBD 0, PSA 80.83, QED 0.51
Compound (SMILES): CC(C)n1cc(C(=O)Nc2ccc(Oc3cc(Nc4ncccn4)cn4nccc34)c(F)c2)c(=O)n(-c2ccc(F)cc2)c1=O
InChIKey: GEZJPGSEQPDBRG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: AXL Kinase Assay: The ability of compounds to inhibit the kinase activity of recombinant human baculovirus-expressed AXL was measured by homogeneous TRF (HTRF) using Cisbio's KinEASE assay system in white 384-well Optiplates. Assay buffer contained 1 mM DTT, 2 mM MnCl2, 2% DMSO, 50 nM supplement enzymatic buffer, and 1× enzymatic buffer. A 2× concentration of tyrosine kinase (TK) substrate-biotin/ATP mixture made in assay buffer was added to plates at 10 μL/well using the Multidrop Combi (Thermo Fisher Scientific, Waltham, Mass.). The final concentrations were 0.3 μM TK substrate-biotin, and 1.3 μM ATP. Compounds (100 nL), diluted in 100% DMSO on the Biomek FX, (Beckman Coulter, Inc., Brea, Calif.), were transferred to the assay plates using the Biomek FX pintool (2.5% final DMSO in assay). A 2× concentration (final=12 ng/mL) of GST-AXL (diluted in assay buffer) was added to plates at 10 uL/well using the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 30 minutes. A 4× stock of Streptavidin-XL665 (final=18.8 nM) and a 1:100 diluted stock of TK antibody-cryptate were made in HTRF detection buffer and mixed together just prior to adding 20 μL/well on the Multidrop Combi. Plates were sealed, briefly shaken and incubated at 25° C. for 1 hour. The fluorescence of the resulting solution was measured using the PerkinElmer EnVision 2102 multi-label plate reader (PerkinElmer, Waltham, Mass.) with an excitation wavelength of 337 nm (laser) and emission wavelengths of 590 and 665 nm. Raw data was expressed as the ratio of 665/590×10,000.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 610.58, ALogP 5.08, HBA 11, HBD 2, PSA 137.44, QED 0.24
Compound (SMILES): Cc1nc(-c2ccccc2-c2ccc3ncc(C(N)=O)n3c2)cs1
InChIKey: FCJHFBFYQVBFLD-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Inhibitory Activity Toward ALK5 Enzyme: 1. Preparation of kinase buffer: 40 mM Tris (pH 7.5), 20 mM MgCl2, 0.10% BSA, 1 mM DTT.2. Preparation of the compound: the final concentration of the compound was 10 μM, and the compound was prepared to a 100-fold concentration, i.e, 1 mM. 100 μL compound of the 100-fold concentration was added to the second well of a 384-well plate, and 60 μL of 100% DMSO was added to other wells. 30 μL compound in the second well was transferred to the third well, and the compound was 3-fold diluted in sequence to give 10 different concentrations. 50 nL compound was transferred to a reaction plate by echo.3. Kinase reaction: the kinase was added to 1× kinase buffer to prepare 2× kinase solution. The final concentration of kinase solution was 25 nM of ALK5. Peptide TGFbR1 (purchased from Signal Chem, catalog number T36-58) and ATP were added to 1× kinase buffer to prepare 2× substrate solution. The final concentration of substrate solution was 0.1 mg/mL of polypeptide TGFbR1 and 7 μM of ATP. 2.5 μL of 2× kinase solution was added to a 384-well reaction plate (containing 50 nL compound dissolved in 100% DMSO), 1× kinase buffer was added to the negative control well. The reaction plated was incubated at room temperature for 10 minutes. 2.5 μL of 2× substrate solution was added to the 384-well reaction plate. The 384-well plate was covered with a lid and incubate at 30° C. for 1 hour. ADP-Glo reagent (purchased from Promege, catalog number v9102) was equilibrated to room temperature. 5 μL of ADP-Glo reagent was transferred to the reaction well of the 384-well plate to terminate the reaction.4. Determination of reaction results: 10 μL of kinase detection reagent was added to each reaction well, the 384-well plate was shaken for 1 minute, and allowed to stand for 30 minutes at room temperature. The luminescence value of the sample was read by Synegy.5. Curve Fitting: the data of the luminescence reading was copied from the Synegy program.
Measurement: IC50 = 193 nM (pChEMBL 6.710000038146973)
Descriptors: MW 334.4, ALogP 3.53, HBA 5, HBD 1, PSA 73.28, QED 0.62
Compound (SMILES): CNc1cc(-c2cnc3n([C@@H]4COC[C@H]4O)cccc2-3)nc2c(C(=O)NC3CC[C@@H]3OC)cnn12
InChIKey: FJYKVWWNTBYVMJ-NLBBHCLQSA-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 477.53, ALogP 1.58, HBA 10, HBD 3, PSA 127.83, QED 0.38
Compound (SMILES): O=C1CCc2cc(Nc3ncc(Br)c(NC4CC4)n3)ccc2N1
InChIKey: UVQDXQYIDNQTLH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ULK1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75385
Assay: ULK1 inhibition assay: Gamma-32P assays to measure ULK1 kinase activity were performed as previously described. Briefly, Flag ULK1 was transfected into HEK293T cells and 20 hours later treated as indicated. The immmunoprecipitate was washed in IP buffer 3 times, and washed in kinase buffer (25 mM MOPS, pH 7.5, 1 mM EGTA, 0.1 mM Na3VO4, 15 mM MgCl2,). Hot and cold ATP were added at a 100 μM final concentration. As substrates, GST or the recombinant protein GST-Atg101 purified from E. coli were used at 1 μg for each reaction. Reactions were boiled, run out on SDS page gel. The gel was dried, and imaged using PhosphoImager software. For cold assays to asses ULK1, Flag ULK1 which was transiently overexpressed and immunoprecipited from HEK293T cells. Reactions were then run out on SDS page gel, transferred to PVDF membrane and blotted for total levelsFluorescence MicroscopyVps34flox/flox MEFs were reconstituted with Flag-VPS34 and either p40FX or GFP-DFCP1. 48 hours post infection with adenovirus expressing Cre recombinase (MOI of 100), cells were plated on glass coverslips at a density of 3×105 cells per well in 6-well tissue culture plates. 18 h later, cells were fixed in 4% PFA in PBS for 10 minutes and permeabilized in 0.2% Triton in PBS for 10 minutes. The following primary antibodies were used: mouse anti-Myc epitope and LC3B XP antibody (2276 and 3868 respectively, Cell Signaling Technologies). Secondary antibodies were anti-rabbit Alexa488 and anti-mouse Alexa594 (Molecular Probes, 1:1000. Cells were then fixed and counter stained with DAPI. Coverslips were mounted in FluoromountG (SouthernBiothech). Images were acquired on a Zeiss Axioplan2 epifluorescence microscope coupled to the Openlab software. Confocal images of mitotracker were taken on Zeiss LSM 710 laser scanning confocal microscope. 10 random fields per condition were acquired using the 100× objective and representative images shown. Glass coverslips were mounted directly on plate with FluoromountG and images taken on Zeiss Axioplan2 epifluorescence microscope.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 374.24, ALogP 3.44, HBA 5, HBD 3, PSA 78.94, QED 0.76
Compound (SMILES): O=C(N[C@H]1CC[C@H](NCC(F)F)CC1)c1nc(-n2ccnc2)nc2ccsc12
InChIKey: ZHOCLDVUBUAZOH-HAQNSBGRSA-N
Target: ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28907
Assay: Enzyme Assay: The CD38 enzyme assay was performed as described previously (Becherer, J D, et al. J. Med. Chem. 2015, 58, 7021-7056). Briefly, 200 nL of a dose response titration of each test compound dissolved in 100% DMSO was spotted in clear polystyrene 384-well plate (Thermo #264704) using a Mosquito (TTP Labtech). A 10 μL solution of 2 nM CD38 (BPS Biosciences #71227) suspended in 100 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH=7.5), 4 mM EDTA (2,2′2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid) and 1 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) was incubated with test compound at 25° C. for 30 min. The enzyme reaction was initiated by adding 10 μL of 400 μM nicotinamide adenine dinucleotide (NAD+), 1000 μM (E)-2-(2-(pyridin-4-ylmethylene)hydrazinyl)pyridine in buffer containing 5 mM sodium acetate (pH=5.2) and 1 mM CHAPS. The reactions were incubated at 25° C. and the absorbance at 405 nm was measured after 15 minutes and 60 min on an Envision plate reader (Perkin Elmer) The absorbance values from the 15 min timepoint were subtracted from the absorbance value from the 60 min timepoint.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 406.46, ALogP 2.77, HBA 7, HBD 2, PSA 84.73, QED 0.66
Compound (SMILES): Cn1nc(C2CC2)c(F)c1CSc1cc(N)nc2[nH]nnc12
InChIKey: REKXNERHFWGQLF-UHFFFAOYSA-N
Target: Myeloperoxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P05164
Assay: MPO Chlorination (APF) Assay : MPO chlorination activity was measured in 100 mM KPi (pH 7.4) by utilizing the non-fluorescent reagent Aminophenyl fluorescein (APF, Invitrogen catalog #A36003). APF is cleaved by (OCl) to yield the fluorescent compound fluorescein. Reactions were carried out in 50 μL total volume by adding a 25 μL mixture of 200 pM myeloperoxidase and 120 mM NaCl to 100 nL inhibitor in 100% DMSO in a 384 well, non-binding surface clear bottom plate (CORNING® #3655). Enzyme, inhibitor, and chloride were preincubated for ten minutes at rt.After the ten minute preincubation, 25 μL of an APF mixture containing 10 mM APF, 120 mM NaCl and 10 μM H2O2 was added to the plate using the internal dispensing system of a Hammatsu FDSS 6000. Kinetic determinations were carried out immediately on the FDSS 6000 (3 minute kinetic read, 1 read every second, ex: 485 nm, em: 535 nm). IC50 values for inhibitors were calculated by taking the slope of the linear portion of the kinetic measurement (20 seconds to 80-120 secs).IC50 values were calculated by determining the slope of the linear portion of the kinetic trace (180-540 secs), and using that calculated slope to determine % inhibition occurring at each concentration of inhibitor using the following equation:Y = A + B - A 1 + ( C / x ) Dwhere A=minimal Y value (activity level of inhibited sample), B=maximal Y value (activity level of uninhibited sample), C=Log IC50, D=Hill Slope, x=concentration of inhibitor.
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 319.37, ALogP 1.98, HBA 7, HBD 2, PSA 98.3, QED 0.71
Compound (SMILES): Cc1ccc(C(CC(=O)O)c2ccc(C#N)cc2C)cc1CN1CC(C)Cc2ccccc2S1(=O)=O
InChIKey: QXXYHMOQLMDKMF-UHFFFAOYSA-N
Target: Nuclear factor erythroid 2-related factor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16236
Assay: BEAS-2B NQO1 MTT Assay: NAD(P)H:quinone oxidoreductase 1 (NQO1), also called DT diaphorase, is a homodimeric FAD-containing enzyme that catalyzes obligatory NAD(P)H-dependent two-electron reductions of quinones and protects cells against the toxic and neoplastic effects of free radicals and reactive oxygen species arising from one-electron reductions. The transcription of NQO1 is finely regulated by Nrf2, and thus NQO1 activity is a good marker for Nrf2 activation. On day one, frozen BEAS-2B cells (ATCC) are thawed in a water bath, counted, and re-suspended at a concentration of 250,000 cells/mL. Fifty microliters of cells are plated in 384 well black clear-bottomed plates. Plates are incubated at 37° C., 5% CO2 overnight. On day two, plates are centrifuged and 50 nL of compound or controls are added to the cells. Plates are then incubated at 37° C., 5% CO2 for 48 h. On day four, medium is aspirated from the plate and crude cell lysates are made by adding 13 μL of 1× Cell Signaling Technologies lysis buffer with 1 Complete, Mini, EDTA-free Protease Inhibitor Tablet (Roche) for each 10 mL of lysis buffer. After lysis plates are incubated for 20 min at room temperature. Two microliters of lysate are removed for use in Cell Titer Glo assay (Promega) and MTT cocktail is prepared (Prochaska et. al. 1998) for measurement of NQO1 activity. Fifty microliters of MTT cocktail is added to each well, plate is centrifuged, and analyzed on an Envision plate reader (Perkin Elmer) using Absorbance 570 nm label for 30 min. Product formation is measured kinetically and the pEC50 of NQO1 specific activity induction is calculated by plotting the change in absorbance (Delta OD/min) versus the log of compound concentration followed by 3-parameter fitting.
Measurement: EC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 502.64, ALogP 5.16, HBA 4, HBD 1, PSA 98.47, QED 0.5
Compound (SMILES): CC1CCN(S(=O)(=O)NC[C@H]2CN(S(=O)(=O)c3ccc(F)c(Cl)c3)c3cc(NC(=O)OC(C)(C)C(F)(F)F)ccc3O2)C1
InChIKey: RPQBRQBORRYQFD-LWKPJOBUSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Biological Assay: The compounds of the invention inhibit RORgammaT activity. Activation of RORgammaT activity can be measured using, e.g., a biochemical TR-FRET assay. In such an assay, interaction of cofactor-derived peptides with human RORgammaT-Ligand Binding Domain (LBD) can be measured. The TR-FRET technique is a sensitive biochemical proximity assay that will give information concerning the interaction of a ligand with the LBD, in the presence of cofactor-derived peptides (Zhou et al., Methods 25:54-61, 2001).To identify novel antagonists of RORgammaT, an assay was developed which employs the interaction of RORgammaT with its co-activator peptide SRC1_2. This peptide mimics the recruitment of co-activators to RORgammaT through its interaction with the LXXLL (SEQ ID NO:1) (e.g., NR box) motifs (Xie et al., J. Immunol. 175: 3800-09, 2005; Kurebayashi et al., Biochem. Biophys. Res. Commun. 315: 919-27, 2004; Jin et al., Mol. Endocrinology 24:923-29, 2010). The RORγ-Ligand Binding Domain TR-FRET Assay was run according to the following protocol.
Measurement: IC50 = 14.6 nM (pChEMBL 7.840000152587891)
Descriptors: MW 673.11, ALogP 4.5, HBA 7, HBD 2, PSA 134.35, QED 0.39
Compound (SMILES): NC(=O)c1cnc(Nc2ccc(N3CCCC(F)(F)C3)cc2)nc1NC1CC1
InChIKey: KNALDSQTJPPEKT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: In Vitro Assay: SYK tyrosine phosphorylation activity is measured using the LANCE Technology developed by Perkin Elmer Life and Analytical Sciences (Boston, Mass.). LANCE refers to homogeneous time resolved fluorometry applications using techniques such as time-resolved fluorescence resonance energy transfer assay (TR-FRET) (see generally for procedures in Perkin Elmer Application NoteHow to Optimize a Tyrosine Kinase Assay Using Time Resolved Fluorescence-Based LANCE Detection, wwww.perkinelmer.com/lifesciences). The assay principle involves detection of a phosphorylated substrate using energy transfer from a phosphospecific europium-labeled antibody to streptavidin-allophycocyanin as an acceptor.To test the ability of candidate molecules to inhibit SYK tyrosine phosphorylation activity, molecules are reconstituted in 30% DMSO and serially diluted 1:3 with the final dilution containing DMSO in the absence of the candidate molecule. The final DMSO concentration in the assay is 3%. Kinase assays are performed as a two part reaction. The first reaction is a kinase reaction and which comprises of a candidate molecule, full length active recombinant SYK enzyme (Millipore, CA) and biotin-labeled SYK-specific substrate biotin-DEEDYESP-OH. The second reaction involves termination of the kinase reaction and the simultaneous addition of the detection reagents-europium-labeled anti-phosphotyrosine reagent (Eu-W1024-PY100, Perkin Elmer, Boston, Mass.) and Streptavidin-Allophycocyanin detection reagent (SA-APC, Prozyme, CA). The kinase reaction is performed in a black U-bottom 96-well microtitre plate. The final reaction volume is 50 μL and contains a final concentration of 1 nM active SYK enzyme, 550 nM SYK-substrate, and 100 μM ATP diluted in a buffer containing 50 mM Tris pH 7.5, 5 mM MgCl2, and 1 mM DTT. The reaction is allowed to proceed for 1 hour at room temperature. The quench buffer contains 100 mM Tris pH 7.5, 300 mM NaCl2, 20 mM EDTA, 0.02% Brij35, and 0.5% BSA. The detection reagents are added to the reaction mixture at the following dilutions1:500 for Eu-W1024-PY100 and 1:250 for SA-APC. The kinase reaction is terminated by the addition of 50 μL quench buffer containing the detection reagents. The detection is allowed to proceed for 1 hr at room temperature.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 388.42, ALogP 3.13, HBA 6, HBD 3, PSA 96.17, QED 0.7
Compound (SMILES): CC1CN(c2ccnc3c2ccn3CC(=O)Nc2ccc(-c3cnccn3)cn2)CC(C)N1
InChIKey: DNRIVPKXAFXIIJ-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Mus musculus
UniProt: P27467
Assay: Dual-Cell beta-Catenin Reporter Assay: Mouse L cells transfected to constitutively produce biologically active murine Wnt-3a, referred to as L-Wnt cells, were purchased from the American Type Culture Collection, ATCC, Manassas, Va. (ATCC). These cells were cultured in DMEM supplemented with 10% FCS (Gibco/Invitrogen, Carlsbad, Calif.), 1% geneticin and 1% sodium pyruvate (Sigma) at 37° C. with 5% CO2. The cells were seeded into 96 well plates and treated with serial dilutions of compound diluted to 0.1% DMSO concentration. After 24 hours, cell supernatants were transferred to a 96 well plate previously seeded with Leading Light® Wnt Reporter Cells, stably transfected with a luciferase gene under control of Wnt pathway response elements. After a further 24 hours, cells are treated with One-glo luciferase assay system (Promega, Madison, Wis.) and the luminescent signal read by envision. The IC50 of the compound is determined as the concentration that reduces the induced luciferase signal to 50% of the DMSO control.
Measurement: IC50 > 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 442.53, ALogP 2.71, HBA 8, HBD 2, PSA 100.86, QED 0.49
Compound (SMILES): O=C(Nc1cn2nc(-c3c(-c4ccc(F)cc4)nc(C4CC4)n3C3CCN(c4ccncc4)CC3)ccc2n1)c1ccnc(F)c1
InChIKey: QXQRGPHTEGOOFA-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 16.7 nM (pChEMBL 7.78000020980835)
Descriptors: MW 617.66, ALogP 6.3, HBA 9, HBD 1, PSA 106.13, QED 0.21
Compound (SMILES): Cn1nc(NC(=O)c2csc3cnc(N[C@@H]4CCCC[C@@H]4N)nc23)c2ccc(F)cc21.O=C(O)C(F)(F)F
InChIKey: XLAZCYODDDRHNT-LDXVYITESA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: SYK kinase assay: Determination of IC50 of Spleen Tyrosine Kinase (SYK) inhibition: SYK kinase assay is a standard kinase assay adapted to a 96 well plate format. This assay is performed in 96-well format for IC50 determination with 8 samples which represented 10 half log dilutions and a 40 μL reaction volume. The assay measures the incorporation of radiolabeled 33P gATP into an N-terminally biotinylated peptide substrate, derived from naturally occurring phosphoacceptor consensus sequence (Biotin-11aa DY*E). Phosphorylated products were detected upon termination of reactions with EDTA and the addition of Streptavidin coated beads.
Measurement: IC50 = 5977 nM (pChEMBL 5.21999979019165)
Descriptors: MW 439.52, ALogP 3.65, HBA 8, HBD 3, PSA 110.75, QED 0.45
Compound (SMILES): Cn1nccc1-c1cc2ncccc2c(-c2ccn(C3(CC#N)CN(CC(F)(F)F)C3)n2)n1
InChIKey: AMGVZSBEJWQCPE-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: JAK Caliper Enzyme Assay at 1mM ATP: Compounds were added to a 384-well plate. Reaction mixtures contained 10 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% 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 JAK 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% to 30% phosphorylation. The assays were stopped with 15 μL of 180 mM HEPES, pH 7.4, 20 mM EDTA, and 0.2% Coating Reagent 3. The assay plates were placed on a Caliper Life Science LC3000 instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 214 nM (pChEMBL 6.670000076293945)
Descriptors: MW 452.44, ALogP 3.38, HBA 8, HBD 0, PSA 88.45, QED 0.46
Compound (SMILES): CCOc1ccc(S(=O)(=O)N2C[C@@H](C)[C@@H](n3nc(CC(=O)O)c4ccccc43)C2)c(N)c1
InChIKey: KMWVGWOFNWFALC-VLIAUNLRSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Receptor Binding Inhibition Assay: Cell membrane was prepared from CRTH2-expressing K562 cell. The cell membrane (20 μg/well) was mixed with reaction buffer (50 mM Tris/HCl, pH 7.4, 10 mM MgCl2). Then, [3H]-PGD2 was added to the mixture and incubated for 60 min at room temperature. The concentration of [3H]-PGD2 was determined as equal to Kd calculated from Scatchard plot. Then, the mixture was filtrated with glass paper immediately. The filter paper was washed several times and radio activity on the filter was measured. Specific binding was defined as difference between total and non-specific binding (radio activity with 10 μM PGD2). Ki values for each compound were calculated from displacement curve.
Measurement: Ki = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 458.54, ALogP 2.53, HBA 7, HBD 2, PSA 127.75, QED 0.52
Compound (SMILES): CCn1nc(CCCOC(=O)c2cnn(C)c2)c2c1C(=O)NCC1(CCOCC1)C2
InChIKey: XPXXLIMDMGAHED-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4D (SINGLE PROTEIN), Homo sapiens
UniProt: Q08499
Assay: PDE4 Assay: The human PDE4D catalytic domain (UniProt no. Q08499 [S380-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.The results were calculated as the molar concentrations resulting in 50% inhibition of the substrate cleavage compared to controls samples, and are expressed as a range of IC50 (nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 415.49, ALogP 1.51, HBA 8, HBD 1, PSA 100.27, QED 0.57
Compound (SMILES): C=CC(=O)N1CC2(CC(n3nc(-c4ccc(N5CCN(C)C5=O)cc4)c(-c4c(Cl)c(C)cc5[nH]ncc45)c3C)C2)C1
InChIKey: FHRQAHZULFOPKY-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Scintillation Proximity Assay (SPA): Assays were run using 384-well plates (781207/Greiner) in which one column was designated as the high signal (no inhibition) control, and contained DMSO with no test compound, and another column was designated as the low signal control (maximal inhibition), and contained no protein. Serial dilutions of compounds to be tested were added to the assay plate (resulting in duplicate 11-point dose response with semi-log compound dilutions from 50 μM to 0.5 nM or from 5 μM to 0.05 nM for the most potent compounds). A 1/20 isotopic dilution of labeled (Compound B) and non-labeled covalent (Compound A) probe was prepared and added to all wells on the plate. The reaction was started by addition of KRasG12C (M1-K169, biotinylated on the N-terminus) to the compounds and incubated for 2 hours with continuous agitation allowing for full modification of KRasG12C with probe or test compounds. Final concentrations in an assay volume of 40 μL were 10 nM KRasG12C, 25 nM radio-ligand and 475 nM unlabelled ligand. The assay buffer contained 20 mM Tris-HCl pH 7.5 (Invitrogen), 150 mM NaCl (Sigma Aldrich), 0.1 mM MgCl2 (Sigma Aldrich), and 0.01% Tween-20 (Sigma Aldrich). Following addition of 50 μL of a 400 μg/ml. suspension of streptavidin-coated YSi beads (Perkin Elmer), plates were incubated for a further 30 min with continuous agitation before reading the plates on a scintillation counter (Topcount NXT 384 (Packard).
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 570.1, ALogP 5.58, HBA 5, HBD 1, PSA 90.36, QED 0.32
Compound (SMILES): O=C(Nc1nc2cccc(-c3ccc(OCCc4cccnc4)cc3)n2n1)C1CC1
InChIKey: IRSZZTQMGBSCPB-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK1 Inhibition Assay: Recombinant human JAK1 catalytic domain (amino acids 850-1154; catalog number 08-144) was purchased from Carna Biosciences. 10 ng of JAK1 was incubated with 12.5 μg polyGT substrate (Sigma catalog number P0275) in kinase reaction buffer (15 mM Tris-HCl pH 7.5, 1 mM DTT, 0.01% Tween-20, 10 mM MgCl2, 2 μM non-radioactive ATP, 0.25 μCi 33P-gamma-ATP (GE Healthcare, catalog number AH9968) final concentrations) with or without 5 μL containing test compound or vehicle (DMSO, 1% final concentration), in a total volume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom). After 45 min at 30° C., reactions were stopped by adding of 25 μL/well of 150 mM phosphoric acid. All of the terminated kinase reaction was transferred to prewashed (75 mM phosphoric acid) 96 well filter plates (Perkin Elmer catalog number 6005177) using a cell harvester (Perkin Elmer). Plates were washed 6 times with 300 μL per well of a 75 mM phosphoric acid solution and the bottom of the plates was sealed. 40 μL/well of Microscint-20 was added, the top of the plates was sealed and readout was performed using the Topcount (Perkin Elmer). Kinase activity was calculated by subtracting counts per minute (cpm) obtained in the presence of a positive control inhibitor (10 μM staurosporine) from cpm obtained in the presence of vehicle. The ability of a test compound to inhibit this activity was determined as:Percentage inhibition=((cpm determined for sample with test compound present−cpm determined for sample with positive control inhibitor) divided by (cpm determined in the presence of vehicle−cpm determined for sample with positive control inhibitor))*100%.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 399.45, ALogP 3.76, HBA 6, HBD 1, PSA 81.41, QED 0.51
Compound (SMILES): O=C(Nc1cc(-c2ccc3ncn(Cc4cccc(F)c4)c3c2)c(Cl)cn1)C1CCC(O)(C(F)(F)F)CC1
InChIKey: XTTXIBHOQTWGCE-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615665 nm).
Measurement: IC50 = 130 nM (pChEMBL 6.889999866485596)
Descriptors: MW 546.95, ALogP 6.36, HBA 5, HBD 2, PSA 80.04, QED 0.29
Compound (SMILES): Cn1nccc1Nc1nccc(-c2cc3n4c(nnc4c2)C(Oc2cncc(F)c2)CCO3)n1
InChIKey: YVCBASICINFAPL-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Enzymatic Assay: Compounds were tested in an enzymatic assay using human ERK-2 (Mitogen Activated Kinase 1), recombinantly expressed as an n-terminal 6-His fusion protein in E. coli and corresponding to aa 8-360. The substrate used was the fluorescent Omnia peptide S/T17 (Invitrogen of Carlsbad, Calif.; Cat. KNZ1171C). Test compounds were diluted in DMSO in 3-fold serial dilutions at 100× final concentrations. In addition to compound, the assay contained 50 mM HEPES [pH 7.3], 10 mM MgCl2, 2 mM DTT, 0.005% Triton-X100, 5 nM ERK-2 enzyme, 6.25 μM S/T17 peptide substrate and 25 μM ATP (corresponding to the observed Km) for a total reaction volume of 25 μL. The assay was run at ambient temperature in a white 384-well polypropylene plate (Nunc, Inc of Naperville, Ill.; Cat. 267462) collecting data every 50 seconds for approximately 30 minutes on an Envision plate reader (PerkinElmer, Inc. of Waltham, Mass.); Excitation 340 nm/Emission 495 nm.
Measurement: IC50 = 0.035 nM (pChEMBL 10.460000038146973)
Descriptors: MW 459.45, ALogP 3.1, HBA 11, HBD 1, PSA 117.17, QED 0.42
Compound (SMILES): COc1ncc(-c2ccc(C(=O)N3[C@@H](c4cccc(F)c4F)CC[C@H]3C(=O)O)cc2)c(OC)n1
InChIKey: BMFYCRZTAZXVGC-MOPGFXCFSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: BINDING ASSAY: The following assay can be used for determination of GPR43 activation. When a GPCR is in its active state, either as a result of ligand binding or constitutive activation, the receptor couples to a G protein and stimulates the release of GDP and subsequent binding of GTP to the G protein. The alpha subunit of the G protein-receptor complex acts as a GTPase and slowly hydrolyses the GTP to GDP, at which point the receptor normally is deactivated. Activated receptors continue to exchange GDP for GTP. The non-hydrolysable GTP analog, [35S]GTPγS, was used to demonstrate enhance binding of [35S]GTPγS to membranes expressing receptors. The assay uses the ability of GPCR to stimulate [35S]GTPγS binding to membranes expressing the relevant receptors. The assay can, therefore, be used in the direct identification method to screen candidate compounds to endogenous or not endogenous GPCR.
Measurement: EC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 469.44, ALogP 3.87, HBA 6, HBD 1, PSA 101.85, QED 0.58
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(Cl)c(-c3ccncc3)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)C1
InChIKey: AMSDVSGDBOTSNA-XUZZJYLKSA-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 553.01, ALogP 5.1, HBA 6, HBD 2, PSA 84.31, QED 0.32
Compound (SMILES): CCC1(n2nc(Nc3ccc(C(=O)N4CCCOC(C)C4)c(C)c3)c3c(=O)[nH]ccc32)CCN(C(=O)OC(C)(C)C)CC1
InChIKey: WKNIADKWGSNCEG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Jak Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (Xex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated. The plot of percent inhibition versus the log of compound concentration was fit with a 4-parameter dose response equation to calculate IC50 values.Final reaction conditions were:[E] [S] [ATP] [Eu-pY20] [SA-Dylight]Enzyme (nM) (μM) (μM) (nM) (nM)JAK1 1.405 0.75 31.8 9 312.5JAK2 0.052 0.75 8.5 9 312.5JAK3 0.031 0.75 2.9 9 312.5Tyk2 2.612 0.75 6.9 9 312.5Compound concentrations tested were 1496, 499, 175, 49.9, 18.7, 6.2, 2.1, 0.75, 0.24, 0.075, and 0.0125 nM, with 1.25% residual DMSO.
Measurement: IC50 = 1.3 nM (pChEMBL 8.890000343322754)
Descriptors: MW 592.74, ALogP 5.16, HBA 8, HBD 2, PSA 121.79, QED 0.42
Compound (SMILES): S=C(NCc1ccccc1Cl)Nc1ccc2[nH]cnc2c1
InChIKey: PGOQNHPKVGBXFR-UHFFFAOYSA-N
Target: Glutaminyl-peptide cyclotransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16769
Assay: Spectrophotometric Assay: This novel assay was used to determine the kinetic parameters for most of the QC substrates. QC activity was analyzed spectrophotometrically using a continuous method, that was derived by adapting a previous discontinuous assay (Bateman, R. C. J. 1989 J Neurosci Methods 30, 23-28) utilizing glutamate dehydrogenase as auxiliary enzyme. Samples consisted of the respective QC substrate, 0.3 mM NADH, 14 mM α-Ketoglutaric acid and 30 U/ml glutamate dehydrogenase in a final volume of 250 μl. Reactions were started by addition of QC and persued by monitoring of the decrease in absorbance at 340 nm for 8-15 min.The initial velocities were evaluated and the enzymatic activity was determined from a standard curve of ammonia under assay conditions. All samples were measured at 30° C., using either the SPECTRAFluor Plus or the Sunrise (both from TECAN) reader for microplates. Kinetic data was evaluated using GraFit software.
Measurement: Ki = 1028 nM (pChEMBL 5.989999771118164)
Descriptors: MW 316.82, ALogP 3.7, HBA 2, HBD 3, PSA 52.74, QED 0.64
Compound (SMILES): O=C(Nc1cc(C2=CCC(NC3CN(C(=O)c4ccnc(F)c4)C3)CC2)c2cc[nH]c2n1)C1CC1
InChIKey: RGFRSUJPTGBZRK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: ADP-Glo Kinase Assay: A control material and a test material were prepared for each concentration, in such a way that they were diluted by means of DMSO. At the same time, ATP (250 uM) and JAK substrate (JAK1, IRS-itide 40 ng/mL) were prepared, in such a way that they were diluted by means of kinase buffer (40 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.5 mg/mL BSA, 50 uM DTT).A test drug for each concentration, substrate, ATP and JAK enzyme were mixed in an eppendorf tube, and reacted in a 30° C. incubator for 40 minutes.ADP-Glo reagent contained in ADP-Glo Kinase Enzyme System (Promega, USA, V9571) was added into each eppendorf tube, and reacted in a 30° C. incubator for 40 minutes. A kinase detection reagent contained in ADP-Go Kinase Enzyme System was inserted into the eppendorf tube, then an integration time was set to 1 second by using Wallac Victor 2, then luminescence was measured, and then JAKs phosphorylation inhibition ability of the test material was analyzed. The concentration of the compound, which causes 50% of the JAK enzyme activity inhibition in comparison With the control group, was determined as IC50 (nM) of an inhibitor, wherein the results thereof are shown in a following table 1.
Measurement: IC50 > 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 474.54, ALogP 3.5, HBA 5, HBD 3, PSA 103.01, QED 0.47