lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CNC(=O)O[C@H]1CCC[C@@H]1C(Cn1ccnc1)(c1cccc(F)c1)C1CCN(CC2CN(c3ccc(C#N)cc3)C2)CC1
InChIKey: UDTLYHREMCOXLK-QKZQDKRESA-N
Target: Menin (SINGLE PROTEIN), Homo sapiens
UniProt: O00255
Assay: Fluorescence Polarization (FP) Competitive Binding Assay: A fluorescence polarization (FP) competitive binding assay was used to determine the binding affinities of representative menin inhibitors. A FAM labeled fluorescent probe was designed and synthesized based on a MLL1 peptide (FAM-MM2). Equilibrium dissociation constant (Kd) value of FAM-MM2 to menin protein was determined from protein saturation experiments by monitoring the total fluorescence polarization of mixtures composed with the fluorescent probe at a fixed concentration and the protein with increasing concentrations up to full saturation. Serial dilutions of the protein were mixed with FAM-MM2 to a final volume of 200 μl in the assay buffer (PBS with 0.02% Bovine γ-Globulin and 4% DMSO. 0.01% Triton X-100 was added right before assays). Final FAM-MM2 concentration was 2 nM. Plates were incubated at room temperature for 30 minutes with gentle shaking to assure equilibrium. FP values in millipolarization units (mP) were measured using the Infinite M-1000 plate reader (Tecan U.S., Research Triangle Park, N.C.) in Microfluor 1 96-well, black, v-bottom plates (Thermo Scientific, Waltham, Mash.) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Kd value of FAM-MM2, which was calculated by fitting the sigmoidal dose-dependent FP increases as a function of protein concentrations using Graphpad Prism 6.0 software (Graphpad Software, San Diego, Calif.), is determined as 1.4 nM.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 584.74, ALogP 5.2, HBA 7, HBD 1, PSA 86.42, QED 0.37
Compound (SMILES): Cc1n[nH]c2cc(Nc3ncc(C(N)=O)c(NC4CC4)n3)ccc12
InChIKey: VLNMBPSHYBXUFL-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 323.36, ALogP 2.08, HBA 6, HBD 4, PSA 121.61, QED 0.57
Compound (SMILES): O=C(C[C@H]1CC[C@@H](CNc2cn[nH]c(=O)c2C(F)(F)F)O1)N1CCN(c2ncc(Cl)cn2)CC1
InChIKey: JGOKPLSMIDTDHG-KGLIPLIRSA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay: Displacement of Probe A, a biotinylated probe binding to the TIPARP active site, was measured using a time-resolved fluorescence energy transfer (TR-FRET) assay. 20 nL of a dose response curve of each test compound was spotted in black 384-well polystyrene proxiplates (Perkin Elmer) using a Mosquito (TTP Labtech). Reactions were performed in a 8 μL volume by adding 6 JAL of TIPARP and Probe A in assay buffer (20 mM HEPES pH=8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002% Tween20), incubating with test compound at 25° C. for 30 min, then adding 2 μL of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin (Perkin Elmer). The final concentrations of TIPARP and Probe A were 6 nM and 2 nM, respectively. The final concentration of ULight-anti 6×His and LANCE Eu-W1024 labeled streptavidin were 4 nM and 0.25 nM, respectively. Reactions were incubated at 25° C. for an additional 30 min, then read on an Envision plate reader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 320 nm and emission of 615 nm and 665 nM with a 90 μs delay. The ratio of the 665/615 nm emission were calculated for each well to determine the amount of complex of TIPARP and Probe A in each well.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 501.9, ALogP 1.93, HBA 8, HBD 2, PSA 116.34, QED 0.62
Compound (SMILES): CCn1cnc2cc(-c3ccc4c(c3)N([C@H]3C[C@@H](N5CC(C)(C)C5)C3)C(=O)C4(C)C)nc(Nc3ccncc3F)c21
InChIKey: BJDCEOOTRODPFL-OYRHEFFESA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: Enzymatic Activity Assay: Recombinant HPK1 kinase domain produced via baculovirus infection of insect cells was obtained from Proteros (Proteros Biostructures #PR-0322) and was pre-activated in the presence of 2 mM ATP (Sigma-Aldrich, cat #GE27-2056-01) and 2 mM magnesium chloride for 16 hours at 4° C. The protein reaction mixture was then loaded to a desalting column (Thermo Fisher Scientific, Cat #89889) to remove excess ATP. HPK1 was eluted with buffer containing 20 mM Tris (2-Amino-2-(hydroxymethyl)propane-1,3-diol) pH 8.0, 150 mM NaCl, 2 mM dithiothreitol and 5% glycerol, and was frozen at −80° C. for later use. HPK1 dual phosphorylation was confirmed by mass spectrometry.Ten nanoliters of test compounds dissolved in DMSO at various concentrations were dispensed into a 384-well ProxiPlate (PerkinElmer #6008289). Five microliters of a solution of recombinant HPK1 diluted in HPK1 kinase assay buffer (50 mM BES [N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid], pH 7.0; 10 mM magnesium chloride; 0.01% Triton X-100; 1 mM dithiothreitol; 0.01% bovine serum albumin; 0.1 mM sodium orthovanadate) was added to the compound-containing plate and was incubated for 15 minutes at 25° C. Five microliters of a mixture of ATP (Sigma-Aldrich #A6559) and peptide substrate STK S1 (Cisbio #61ST1BLC) diluted in HPK1 kinase assay buffer was then added to start the reaction. Final concentrations were 0.15 nM for HPK1, 10 μM for ATP, and 1 μM for the STK S1 peptide substrate. The reaction mixture was incubated at 25° C. for 3 hours and was stopped with the addition of 10 μl of an EDTA (Ethylenediaminetetraacetic acid)-containing detection buffer (Cisbio #62SDBRDF) supplemented with Europium cryptate-labeled anti-phospho-serine/threonine antibodies (Cisbio #62ST1PEJ) and XL665-labeled streptavidin (Cisbio #610SAXLG). The mixture was incubated for 16 hours at room temperature and peptide phosphorylation was measured by time-resolved fluorescence energy transfer (665 nm/620 nm) on an Envision plate reader (PerkinElmer).
Measurement: IC50 = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 553.69, ALogP 5.89, HBA 7, HBD 1, PSA 79.18, QED 0.32
Compound (SMILES): FC(F)Cn1cc(-c2ccnc(Nc3ccc(N4CCNCC4)nc3)n2)c(-c2cccnc2)n1
InChIKey: BBAJIAMAMQMXMC-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: Enzyme Inhibitory Assay: TABLE 5: An overview of evaluation of an inhibitory effect of a compound of the present invention with respect to ALK2 is as follows. In a buffer solution (20 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2, 1 mM EGTA, 0.02% Brij 35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO), human ALK2 (http://www.expasy.org/uniprot/Q04771) and a substrate (20 μM casein) were incubated at room temperature for 20 minutes together with test compound, 33P-ATP (specific radioactivity: 10 Ci/L) was then added thereto so that the final ATP concentration was 10 μM, and the mixture was additionally incubated at room temperature for 2 hours. The reaction mixture was filtered using a P81 ion exchange filter, and a kinase activity was determined by measuring a radioactivity bound to the filter. An inhibitory effect was determined using test compounds with respective concentrations, and a concentration (IC50) indicating 50% of the maximum inhibitory concentration was calculated.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 463.5, ALogP 3.22, HBA 9, HBD 2, PSA 96.68, QED 0.43
Compound (SMILES): Cn1cc(-c2cc(-c3ccc(N4CCN(C(=O)C5(c6ccccc6)CC5)CC4)nc3)c3c(C#N)cnn3c2)cn1
InChIKey: SKVSKPJVMINBIP-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of General Formula I were screened for their ability to inhibit wild type and V804M mutant RET kinase using CisBio's HTRF® KinEASE -TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Cat. No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Cat. No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Cat. No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 528.62, ALogP 4.05, HBA 8, HBD 0, PSA 95.35, QED 0.34
Compound (SMILES): O=C(CCCc1nc2ccccc2c(=O)[nH]1)N1CC=C(n2c(=O)[nH]c3ccccc32)CC1
InChIKey: HMUTYRXLOPTZOV-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase tankyrase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95271
Assay: Biochemical Assay: The tankyrase 1 biochemical activity of the compounds was assayed in the following assay buffer (50 mM MOPS pH7.5, 100 mM NaCl, 2.5 mM MgCl2, 0.01% Tween-20, 0.05% BSA, and 1 mM DTT) as follows: 0.25 nM of 6×HIS-tankyrase1 (1091-1325) was incubated in the presence of compound (DMSO 1.85% final) in a Perkin Elmer 384 well Proxiplate Plus (cat.no. 6008289) with 400 nM of NAD for 60 minutes at RT. The assay was then stopped with the above assay buffer containing a 0.6 μM inhibitor and the following detection components: 0.05 μg/mL monoclonal anti-PAR antibody (Trevigen cat.no. 4335-MC-01K-AC) prebound for 60 minutes with 0.63 μg/mL protein G AlphaLisa® acceptor bead (Perkin Elmer cat.no. AL102M) and 5 μg/mL AlphaLisa® nickel chelate donor bead (Perkin Elmer cat.no. AS 101M). The assay was incubated for 16 hours at RT in the dark and read on a Perkin Elmer Envision® multi label reader using the default program set with laser excitation at 680 nM and emission at 615 nM.
Measurement: IC50 = 17.7 nM (pChEMBL 7.75)
Descriptors: MW 429.48, ALogP 2.66, HBA 5, HBD 2, PSA 103.85, QED 0.51
Compound (SMILES): Cc1c(-c2[nH]c3ccc(N4CC(N(C)CC(=O)N(C)C)C4)nc3c2C(C)C)cn2ncnc2c1C
InChIKey: TUXNFZFAHLZNEZ-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 474.61, ALogP 3.22, HBA 7, HBD 1, PSA 85.66, QED 0.46
Compound (SMILES): CS(=O)(=O)c1cc(C(=O)N(CC#N)c2cnccc2-c2ccccc2Cl)cc(C(F)(F)F)c1
InChIKey: CBVIEVQYYRELSF-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 = 379 nM (pChEMBL 6.420000076293945)
Descriptors: MW 493.89, ALogP 4.99, HBA 5, HBD 0, PSA 91.13, QED 0.47
Compound (SMILES): CN1CCN(c2cc(-c3cnc4[nH]c(=O)n(-c5ccc6[nH]ccc6c5)c4n3)ccn2)CC1
InChIKey: YIUDJPYFKOHYHD-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16584
Assay: Radiometric Filter Plate Assay: 200 ng (130 nM) MLK3 (Dundee, DU8313) was incubated with 1 μM inactive MKK7b (Dundee, DU703) in the presence of 2 μM cold ATP (Km) and 0.5 μCi/assay 33P ATP and appropriate concentrations of compounds. After a twenty minute incubation, the reactions were washed through filter plates and read on a scintillation counter.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 426.48, ALogP 2.4, HBA 7, HBD 2, PSA 98.73, QED 0.46
Compound (SMILES): COc1cc(C#N)ccc1-c1cnn(-c2ccc(C(=O)NCc3ccccc3)cn2)c1O
InChIKey: JJVMMNHCKXEKQA-UHFFFAOYSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: Inhibition Assay: The IC50 values for the PHD2 enzyme (residues 181-417) were determined by mixing increasing amounts of inhibitor with a fixed amount of enzyme (5 nM, final concentration) and Biotin labeled peptide (Biotin-Asp-Leu-Glu-Met-Leu-Ala-Pro-Tyr-Ile-Pro-Met-Asp-Asp-Asp-Phe-Gln-Leu, 1 uM final concentration) and 2-Oxyglutarate (2 uM final concentration) in 50 mM HEPES, 50 mM KCl, 0.5 mM TCEP, 2 uM FeCl2, 0.1 mg/ml BSA, at pH 7.3. The reaction was conducted by pre-incubating the enzyme in the presence of inhibitor for 60 min at room temperature. The activity of the free enzyme was measured by adding the peptide, the 2-Oxoglutarate (see above for final concentrations), and Ascorbic Acid (1 mM final concentration). The enzymatic activity was quenched after 60 min by adding an excess of a tight binding inhibitor to the assay mixture. The amount of product released was measured by using a LC/MS system (Agilent HPLC with Applied Biosystems API3000 Mass Spectrometer).
Measurement: IC50 = 97.7 nM (pChEMBL 7.010000228881836)
Descriptors: MW 425.45, ALogP 3.45, HBA 7, HBD 2, PSA 113.06, QED 0.49
Compound (SMILES): Fc1ccccc1-c1ccccc1-c1nnnn1-c1ccccc1Cl
InChIKey: KWVLAIZVBIWUNV-UHFFFAOYSA-N
Target: Intermediate conductance calcium-activated potassium channel protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O15554
Assay: Inhibition Assay: Human HEK293 cells over-expressing human IK are grown in culture medium (DMEM supplemented with 10% foetal bovine serum), in polystyrene culture flasks (175 mm2) in a humidified atmosphere of 5% CO2 in air, at 37° C. Cell confluence should be 80-90% on day of plating. Cells are rinsed with 4 mL of PBS (phosphate buffered saline) and incubated 2 min with 1 mL of Trypsin-EDTA. After addition of 25 mL of culture medium cells are re-suspended by trituration with a 25 mL pipette.The cells are seeded at a density of 3×106 cells/mL (25 μL/well) in black-walled, clear bottom, 384-well plates pre-treated with 0.01 g/L poly-D-lysin (20 μL/well for ≧30 min). Plated cells were allowed to proliferate for 24 h before loading with dye.BTC-AM (50 mg, Invitrogen) is added 25.5 μl DMSO. The BTC-AM stock solution (2 mM) is diluted to a final concentration of 2 μM in Cl+ free assay buffer (in mM: 140 Na+-gluconate, 2.5 K+-gluconate, 6 Ca2+-gluconate, 1 Mg2+ gluconate, 5 glucose, 10 HEPES, pH 7.3) containing 2 μM ouabain, 2 mM amaranth and 1 mM tartrazine.The culture medium is aspirated from the wells, and 25 μl of the BTC-AM loading solution are added to each well. The cells are incubated at 37° C. for 60 min.After the loading period, the Tl+-sensitive BTC fluorescence signal is measured over time using a FLIPR.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 350.78, ALogP 4.79, HBA 4, HBD 0, PSA 43.6, QED 0.53
Compound (SMILES): Cc1cc(-c2cc(Cn3ccn(C)c3=N)cc(C(=O)NC(C3CC3)C3CC3)c2)ccn1
InChIKey: NDIPAWRSXYHFKR-UHFFFAOYSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: TR-FRET Binding Assay : A Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay that measures the displacement of the 10mer-Thr-FAM probe in response to compound treatment was performed for compounds wherein the IC50 from FPA assay using 10mer-Thr-FAM was below the lower assay IC50 limit 1 nM. Excess 10mer-Thr-FAM probe was utilized with His-tagged WDR5 in conjunction with a commercial anti-His antibody containing a Terbium label. The LanthaScreen Elite Tb-anti-HIS Antibody from ThermoFisher Scientific was used for this purpose. This Th-anti-HIS has an excitation/emission of 340 nm and 490 nm, respectively. The 10mer-Thr-FAM probe when bound to WDR5 will undergo a FRET interaction with the Tb-anti-HIS and emit at 520 nm. The ratio of the 520 and 495 signals are then utilized to generate a dose-response curve to calculate an IC50 value. By virtue of FRET there is little to no background fluorescence interference from 10mer-Thr-FAM probe allowing an excess of the probe to be used permitting an increase in the lower limit of the calculated Ki when testing against highly potent inhibitors with Ki<<1 nM. WDR5-His Tag (A23, residues 24-334) is expressed and purified in our lab in sufficient quantities for screening. 10mer-Thr-FAM peptide is used anywhere from 15 to 150 nM depending on the window of sensitivity required. WDR5-His tag protein is used at 2 nM. A source plate is prepared using an Echo Liquid Handler, which distributes the compounds to the assay plate (white, flat-bottom; OptiPlate) in a 10-point, 3-fold dilution schemes with a top concentration of either 5 or 20 μM depending on the anticipated potency of the compounds, in a final volume of 20 μL. A final target (WDR5)/Tb-Ab concentration of 2 nM/1 nM is dispensed from appropriate stock solutions, respectively. The final DMSO concentration in each well of the assay plate is 1% or lower. As before the plate is covered, shielded from light, and incubated for 60 minutes at room temperature with rocking. Anisotropy is then measured on a Biotek Cytation 3 at excitation wavelength of 340 nm, and emission wavelengths of 495 nm and 520 nm. Working buffer conditions (pH 7.0) are similar to that in FPA above. TR-FRET signal is plotted and IC50 and Ki values are calculated in the same manner as the fluorescence polarization anisotropy based competition assays.
Measurement: Ki = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 415.54, ALogP 3.64, HBA 5, HBD 2, PSA 75.7, QED 0.62
Compound (SMILES): Cn1c(=O)sc2cc(-c3ccc(C(=O)N4CC[C@H](N)C4)cc3-c3ccc(C#N)c(F)c3)c(F)cc21
InChIKey: LXTAASQWJGJAQY-KRWDZBQOSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: Inhibitory Activity Assay: The conditions for measuring inhibitory activity of compounds against LSD1 activity were determined with reference to a document available from the website of PerkinElmer (U-TRF #38) and a patent of GlaxoSmithKline (WO2012135113).To measure the inhibitory activity, first, the compound of the present invention was serially diluted in dimethylsulfoxide (DMSO). Sequentially, the solution of the compound of the present invention in DMSO (final concentration of DMSO: 5%) and human LSD1 protein (Abcam, ab80379) were added to a reaction buffer (25 mM Tris-HCl (pH 7.5), 50 mM KCl, 2 mM CHAPS, 1 mM DTT, 0.02% BSA). The mixture was preincubated at 25° C. for 30 minutes. Thereafter, a H3K4 (Me1)-biotin-labeled peptide (Anaspec #64355) (final concentration: 200 nM) was added thereto and reacted for 60 minutes. Tranylcypromine (final concentration: 3 mM) was then added thereto to terminate the reaction. Thereafter, a detection solution containing an Eu-labeled anti-H3K4 antibody (PerkinElmer, TRF0404) and Streptavidin Alexa Fluor 647 (Thermo Fisher Scientific, 521374) was added thereto, and the mixture was allowed to stand at room temperature for 1 hour. Finally, the intensity of fluorescence under the excitation light with a wavelength of 337 nm was measured with a PHERAstar FS (BMG Labtech) at two wavelengths: 620 nm and 665 nm. The demethylation level was calculated from the ratio of the fluorescence intensity at the two wavelengths, and the compound concentration at which demethylation was inhibited by 50% was defined as IC50 (nM).
Measurement: IC50 = 0.25 nM (pChEMBL 9.600000381469727)
Descriptors: MW 490.54, ALogP 4.26, HBA 6, HBD 1, PSA 92.12, QED 0.46
Compound (SMILES): CCOC(=O)N1CC=C(c2cccn3nc(Nc4ccc(C(=O)N5CCCCC56CCCNC6)cc4)nc23)CC1
InChIKey: VORFXOKACUBSES-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 4.37 nM (pChEMBL 8.359999656677246)
Descriptors: MW 543.67, ALogP 4.47, HBA 8, HBD 2, PSA 104.1, QED 0.49
Compound (SMILES): CC(C)(C)C(=O)NCc1ccc(F)c(-c2nc(-c3ccc(OC(F)F)nc3)cc(=O)[nH]2)c1
InChIKey: ADTVBBVXUVQMPK-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibitory Activity Assay: Microsomes were prepared from COS-1 cells transiently transfected with a plasmid containing human mPGES-1 cDNA, and used as mPGES-1 enzyme. The mPGES-1 enzyme was diluted with a sodium phosphate buffer (pH 7.2) containing reduced glutathione (2.5 mM) and EDTA (1 mM), DMSO or a DMSO solution of a test compound (final concentration of DMSO was 1%) was added to the enzyme, and the mixture was preincubated at 4° C. for 15 minutes. Then, PGH2 as the substrate was added at a final concentration of 1 μM to start the enzymatic reaction, and after incubation at 4° C. for 4 minutes, a solution of ferric chloride (25 mM) and citric acid (50 mM) was added to terminate the enzymatic reaction. Generated PGE2 was measured by using Prostaglandin E2 Express EIA Kit (Cayman Chemical).
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 446.43, ALogP 3.9, HBA 5, HBD 2, PSA 96.97, QED 0.6
Compound (SMILES): CCOc1cccc(CN2[C@H]3Cn4c(nc5cc(C(=O)NO)ccc54)[C@H]3[C@H](c3cccc(Cl)c3F)[C@]23C(=O)Nc2cc(Cl)ccc23)c1
InChIKey: UEFJJQGFIIYIDV-VLWKUZPGSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: AlphaScreen Assay: 15 μL of compound in 20% DMSO (serial pre-dilutions of compound are done in 100% DMSO) is pipetted to the wells of a white OptiPlate-96 (Perkin Elmer). A mix consisting of 20 nM GST-MDM2 protein (aa 23-117) and 20 nM biotinylated p53 wt peptide (encompassing aa 16-27 of wt human p53, amino acid sequence QETFSDLWKLLP-Ttds-Lys-Biotin, molecular weight 2132.56 g/mol) is prepared in assay buffer (50 mM Tris/HCl pH 7.2; 120 mM NaCl; 0.1% bovine serum albumin (BSA); 5 mM dithiothreitol (DTT); 1 mM ethylenediaminetetraacetic acid (EDTA); 0.01% Tween 20). 30 μL of the mix is added to the compound dilutions and incubated for 15 min at rt while gently shaking the plate at 300 rounds per minute (rpm). Subsequently, 15 μL of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/mL each) are added and the samples are incubated for 30 min at rt in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HIS Multilabel Reader using the AlphaScreen protocol from Perkin Elmer.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 672.54, ALogP 6.61, HBA 7, HBD 3, PSA 108.72, QED 0.14
Compound (SMILES): C=CC(=O)N[C@H]1CCCC[C@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1cnc(Oc2ccccc2)nc1
InChIKey: XVLIKCUEMWVBPD-RBUKOAKNSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 5.89 nM (pChEMBL 8.229999542236328)
Descriptors: MW 555.62, ALogP 4.91, HBA 8, HBD 3, PSA 138.44, QED 0.28
Compound (SMILES): C[C@](C(=O)N[C@H]1COC[C@H]1O)(c1ccccc1)n1ncc2c1nc(N)n1nc(-c3ccco3)nc21
InChIKey: WAVQOFHBBOPGGM-NXHTTZLHSA-N
Target: Adenosine deaminase (SINGLE PROTEIN), Homo sapiens
UniProt: P00813
Assay: Luciferase Reporter Assay: HEK293-A2AR-luc2p/CRE/Hygro cells were seeded at a density of 5,000 cells/well in DMEM with 1% FBS and 1 U/mL adenosine deaminase (ADA) (Sigma). After 18 h, the cells were treated with 3 nM CGS21680 plus a series dilution of A2AR antagonist, the compounds disclosed herein at the concentration of 0.110000 nM, prepared in DMEM with 1% FBS. After 5 h incubation, the luciferase activity in cells were measured using the Bright-Glo Luciferase Assay System (Promega) according to manufacturer's instructions. The luminescence signal was measured using a PHERAstar FS plate reader (BMG Labtech). Luminescence intensity from 10 μM preladenant treatment was set as 0%. Maximal luminescence intensity was determined in the presence of 3 nM CGS21680 and was set as 100%. The IC50 value was calculated from a dose dependent inhibition curve across the range of compound concentrations.
Measurement: IC50 = 32.2 nM (pChEMBL 7.489999771118164)
Descriptors: MW 474.48, ALogP 0.96, HBA 11, HBD 3, PSA 158.62, QED 0.34
Compound (SMILES): N#CCC1(CNC2CC2c2ccccc2)CCN(Cc2cccnc2)CC1
InChIKey: PSHWGBHTCRJQSL-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 Histone Demethylase Biochemical Assay: LANCE LSD1/KDM1A demethylase assay10 μL of 1 nM LSD-1 enzyme (ENZO BML-SE544-0050) in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 25 mM NaCl, 5 mM DTT) were preincubated for 1 hour at 25° C. with 0.8 μL compound/DMSO dotted in black 384 well polystyrene plates. Reactions were started by addition of 10 μL of assay buffer containing 0.4 μM Biotin-labeled Histone H3 peptide substrate: ART-K(Me1)-QTARKSTGGKAPRKQLA-GGK(Biotin) SEQ ID NO: 1 (AnaSpec 64355) and incubated for 1 hour at 25° C. Reactions were stopped by addition of 10 μL 1× LANCE Detection Buffer (PerkinElmer CR97-100) supplemented with 1.5 nM Eu-anti-unmodified H3K4 Antibody (PerkinElmer TRF0404), and 225 nM LANCE Ultra Streptavidin (PerkinElmer TRF102) along with 0.9 mM Tranylcypromine-HCl (Millipore 616431). After stopping the reactions plates were incubated for 30 minutes and read on a PHERAstar FS plate reader (BMG Labtech). Compounds having an IC50 of 1 μM or less were considered active.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 360.51, ALogP 3.72, HBA 4, HBD 1, PSA 51.95, QED 0.82
Compound (SMILES): N#Cc1ccc(C(=O)N[C@H](c2ccc(Cl)cc2)c2ncc(Cl)cc2F)cc1F
InChIKey: OQKYKTVLUYGAQX-GOSISDBHSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: TBD: TBD
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 418.23, ALogP 5.06, HBA 3, HBD 1, PSA 65.78, QED 0.64
Compound (SMILES): CNc1cc(Nc2cccn(-c3ccc(C)nc3)c2=O)nc2c(C(=O)N[C@H]3CC[C@]3(C)OC)cnn12
InChIKey: YCAAYSHRRACSIP-DFBJGRDBSA-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 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 488.55, ALogP 2.67, HBA 10, HBD 3, PSA 127.47, QED 0.36
Compound (SMILES): CC#CC(=O)N1CC[C@@H](c2nc(-c3ccc(C(=O)Nc4ccccn4)cc3)c3cnccn23)C1
InChIKey: QSKBYRCFAPFUKO-HXUWFJFHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Assay: Btk and Other Kinases: Btk kinase activity was determined using a homogenous time resolved fluorescence (HTRF) methodology. Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 μM peptide substrate were incubated in a reaction buffer compose of Hepes50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of Eμ-labeled antibody and XL-665 in 1×Detection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVision® Multilabel Reader, Perkin Elmer) with an excitation wavelength (2Ex) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls were used to determine baseline fluorescence levels.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 450.5, ALogP 3.38, HBA 6, HBD 1, PSA 92.49, QED 0.48
Compound (SMILES): Cc1nc(C(=O)N2CC[C@H](C)[C@H]2c2nc3cc(C)c(C)cc3[nH]2)c(-c2ccc(F)cc2)s1
InChIKey: HFVBVYUFLNJLFO-XMHCIUCPSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 448.57, ALogP 5.97, HBA 4, HBD 1, PSA 61.88, QED 0.42
Compound (SMILES): CC(C)C=C(C#N)C(=O)N1CCCC(n2c(=NC(=O)c3ccc(-c4cn[nH]c4)s3)[nH]c3ccccc32)C1
InChIKey: OBTJDABPRHPKBP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Enzymatic Activity Assay: The ability of the compounds of the present disclosure to inhibit ITK was measured using the Caliper assay format, which is an electrophoretic separation of a phosphorylated peptide substrate from unphosphorylated peptide. The enzymatic reaction occurred in a buffer of 100 mM HEPES pH 7.5, 5 mM MgCl2, 0.01% Triton-X 100, 0.1% Bovine Serum Albumin, and 1% DMSO. Three-fold dilutions of compounds were prepared in DMSO. Compounds were added to enzyme and pre-incubated for 15 minutes prior to reaction. The enzymatic reaction was initiated by addition of phospho-acceptor peptide FAM-GEEPLYWSFPAKKK-NH2 (also known as SRCtide) to 1 uM and ATP to its Km value (ITK: 10 uM). The reaction proceeded for 3 hours and was terminated by addition of EDTA. The assay employed an enzyme concentration of 1 nM. The top compound concentration was 5 uM. The amount of phosphorylated substrate was determined by the Caliper instrumentation, and dose-response curves were fit using standard methods to determine the IC50 values.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 513.63, ALogP 4.43, HBA 6, HBD 2, PSA 122.93, QED 0.3
Compound (SMILES): O=C(NCc1ccc(F)c(F)c1)[C@]1(F)CC[C@H](O)c2ncccc21
InChIKey: FXQMIOGNSKMYQY-YOEHRIQHSA-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 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 336.31, ALogP 2.67, HBA 3, HBD 2, PSA 62.22, QED 0.91
Compound (SMILES): CNCC(Cc1cccnc1)c1ccc(Cl)c(Cl)c1
InChIKey: OOCQXYIWPXQLSE-UHFFFAOYSA-N
Target: Sodium-dependent noradrenaline transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P23975
Assay: Uptake Assay Protocol: NET: This protocol was designed to measure inhibition of uptake by the human norepinephrine transporter. The reagents were human NET (HEK293F) cells, desipramine (Sigma), nomifensine, neurotransmitter transporter uptake assay kit (Molecular Devices), freestyle 293 expression medium (Invitrogen), 10× Hank's Balanced Salt Solution (HBSS; Invitrogen), 1 M HEPES (Mediatech), Biocoat poly-D-lysine 96-well, black, clear plates (Becton, Dickinson), and 500 μL polypropylene U-bottom 96-well plates (Fisher). The Assay Buffer (AB) was 1×HBSS and 0.02 M HEPES.The HEK293F cells were transfected with the human norepinephrine transporter and frozen in 1 mL aliquots at about 1E+07 cells/mL. On the day of the experiment, the cells were removed from −80° C. or liquid nitrogen and thawed in a room temperature water bath. The cells were dilute to about 1-2E+06 with Freestyle medium. A 1 mL sample (1:2 dilution) was prepared, and the cells were counted. The cells were spun at 1100 rpm for 5 minutes, and the medium was aspirated off. The cells were resuspend in medium at 1.5E+06 cells/mL for about 60,000 cells per well. 40 μL of cells were dispensed per well in the Biocoat plates. The plates were spun at 1100 rpm for 1 minute to improve homogeneity of the cell layer and were incubated at 37° C. for a minimum of 3 hours.
Measurement: Ki = 14.5 nM (pChEMBL 7.840000152587891)
Descriptors: MW 295.21, ALogP 3.93, HBA 2, HBD 1, PSA 24.92, QED 0.9
Compound (SMILES): CCCc1nc(Nc2ccc(C(=O)O)cc2)nc(-c2ccc(Cl)s2)n1
InChIKey: BFZMHXGHLXTZLU-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Kinetic Assay: Assays are performed in 96-well plates in a total volume of 200 μl/well. Compounds are dissolved in dimethylsulfoxide (DMSO) and added to plates in a volume of 10 μl followed by addition of 165 μl of assay mix. Plates are pre-incubated at 25° C. for 15 min and the reactions are initiated by the addition of 25 μl of cAMP followed by thorough mixing. Reaction rates are measured by monitoring the decrease in fluorescence using excitation at 355 nm and emission at 460 nm for a period of 10 min in a fluorescence plate reader. Initial rates (slopes) are determined from linear portions of the progress curves. Final concentrations of assay components are as follows: 50 mM Tris, pH 8, 10 mM MgCl2, 50 mM KCl, 2% DMSO, 5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.4 mM phosphenolpyruvate (PEP), 0.01 mM NADH, 0.04 mM adenosine triphosphate (ATP), 0.004 mM cAMP, 7.5 units myokinase from yeast, 1.6 units pyruvate kinase, 2 units lactate dehydrogenase, and either 0.5 nM human PDE4D7 or 10 nM human PDE4B1. All data are percent normalized relative to controls and are presented as percent inhibition. An inhibitory concentration 50% (IC50) value is calculated by fitting of a sigmoidal dose response curve. Human PDE4D7 contained a mutation of serine 54 to aspartic acid to mimic activation by cAMP-dependent protein kinase A (PKA). Human PDE4B1 contained a corresponding mutation of serine 133 to aspartic acid to also mimic PKA activation.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 374.85, ALogP 4.65, HBA 6, HBD 2, PSA 88, QED 0.65
Compound (SMILES): COc1ccc(-n2ccc3nc(Nc4cc(C)ns4)nc(OC4CCOCC4)c32)cc1
InChIKey: OOQIGKHPPZZNHX-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Activity Inhibition Assay: IRAK4 kinase (purchased from Life Technologies, Cat. No.: PR5612U) was diluted to 2 folds of the final concentration (the final concentration is 0.76 ng/4) with reaction buffer (40 mM Tris-HC1, pH 7.5; 20 mM MgCl2; 0.1 mg/mL BSA; 1 mM DTT), and added to a 384-well plate at 5 μL/well. The test drugs were 10-fold serial diluted from 10 μM to set six concentration points and added to the test wells of the 384-well plate at 2.5 μL/well. After incubation for 10 min at 25° C., 10 μM of ATP and 0.1 μg/μL of an enzyme reaction substrate, myelin basic protein MBP (purchased from Signal Chem, Cat. No.: M42-51N) were added at 2.5 μL/well, and reacted at 25° C. for 60 minutes. After the reaction was completed, the kinase activity assay was performed using the ADP-Glo kinase assay kit (purchased from Promega, Cat. No.: V9102) according to the manufacture's instruction, i.e., 10 μL of ADP-Glo reaction reagent was first added and reacted at 25° C. for 40 minutes, 10 μL of the reaction mixture was taken and mixed with 10 μL of ADP-Glo detection reagent, and reacted at 25° C. for 30 minutes.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 437.53, ALogP 4.5, HBA 9, HBD 1, PSA 83.32, QED 0.48
Compound (SMILES): CN1CCC(CNc2nn3c(-c4cccc(OC(F)(F)F)c4)nnc3c3c2OCC(C)(C)N3C)CC1
InChIKey: HJGKZOCLHWKVJH-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM-1 Biochemical Assay: The biochemical assay to measure PIM-1 activity relies on the ADP Hunter assay kit (DiscoveRx Corp., Cat. #90-0077), that determines the amount of ADP as direct product of the kinase enzyme activity.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 505.55, ALogP 4.05, HBA 9, HBD 1, PSA 80.05, QED 0.56
Compound (SMILES): C[C@H](C(=O)N[C@H](C)c1cc(F)cc(N2CCN(C)CC2=O)c1)N1Cc2ccc(-c3nc(NC4CCOCC4)ncc3Cl)cc2C1=O
InChIKey: ILHSXXYFGFRZAA-WOJBJXKFSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: ERK2 In Vitro Inhibition Assay: Activity of ERK2 enzyme (Life Technologies) was determined using a time-resolved fluorescence format measuring the phosphorylation of a truncated version of Activating transcription factor 2 labelled with green fluorescent protein (ATF2-GFP) (Life Technologies). Assay reactions containing 50 mM Tris pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Triton X-100, 1 mM DTT, 2.5% DMSO, 0.4 mM ATF2-GFP, 20 μM ATP and 0.25 nM ERK2 were set up in the presence of compound and allowed to proceed for 30 min at room temperature. Reactions were then stopped using TR-FRET dilution buffer (Life Technologies), 25 mM EDTA and 2 nM Tb-Anti-pATF2 (Thr71) (Life Technologies). After a further incubation period of at least 30 minutes, fluorescence was read on a Pherastar reader (Lanthascreen optic module; excitation 340 nm, emission 520 nm (channel A), 495 nm (channel B)). The ratio between A and B counts was used to calculate signal. IC50 values were calculated using a sigmoidal dose response equation (Prism GraphPad software, La Jolla, Calif., USA).
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 650.15, ALogP 4.03, HBA 8, HBD 2, PSA 120, QED 0.38
Compound (SMILES): CCS(=O)(=O)Nc1cc(-c2cc(C)c(=O)n(C)c2)cc2c1cnn2Cc1ccc(F)cc1
InChIKey: UPRXDQXRQLZTBQ-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: In Vitro Enzyme Inhibition Assay: Determination of the IC50 for the heterocyclic derivative BRD4 inhibitors disclosed herein was performed as follows. His-tagged BREM was cloned, expressed and purified to homogeneity. Filipakopoulos et al., 468 Nature 1067 (2010). BRD4 binding and inhibition was assessed by monitoring the interaction of biotinylated H4-tetraacetyl peptide (AnaSpec, H4K5/8/12/16(Ac), biotinlabeled) with the target using the AlphaScreen technology (Life Technologies). In a 384-well ProxiPlate BRD4(BD1) (2 nM final) was combined with peptide (15 nM final) in 50 mM HEPES (pH 7.3), 10 mM NaCl, 0.25 mM TCEP, 0.1% (w/v) BSA, and 0.005% (w/v) Brij-35 either in the presence of DMSO (final 0.4% DMSO) or compound dilution series in DMSO. After 20 min incubation at RT, Alpha streptavidin donor beads and Nickel Chelate acceptor beads were added to a final concentration of 5 μg/mL. After 2 hr of equilibration, plates were read on an Envision instrument and the IC50 was calculated using a four parameter non-linear curve fit.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 454.53, ALogP 3.66, HBA 6, HBD 1, PSA 85.99, QED 0.48