lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc2nn3c(c2cc1C#N)OC[C@H]1[C@@H]3[C@H](c2cccc(Cl)c2F)[C@]2(C(=O)Nc3cc(Cl)ccc32)N1CC1CC1
InChIKey: DBQQVGHPOOQHRX-XYKPQQNDSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: Mdm2-p53 Inhibition AlphaScreen: This assay is used to determine whether the compounds inhibit the p53-MDM2 interaction and thus restore p53 function.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 (PerkinElmer). 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 HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer.Each plate contains negative controls where biotinylated p53-peptide and GST-MDM2 are left out and replaced by assay buffer. Negative control values are entered as low basis value when using the software GraphPad Prism for calculations. Furthermore, a positive control (5% DMSO instead of test compound; with protein/peptide mix) is pipetted. Determination of IC50 values are carried out using GraphPad Prism 3.03 software (or updates thereof).
Measurement: IC50 = 331 nM (pChEMBL 6.480000019073486)
Descriptors: MW 604.47, ALogP 6.02, HBA 7, HBD 1, PSA 92.41, QED 0.31
Compound (SMILES): N#Cc1cc2c(C#N)ccc(-c3cnccc3SC3(C(=O)O)CCC3)n2c1
InChIKey: FPLWKQCRUYIRPQ-UHFFFAOYSA-N
Target: Solute carrier family 22 member 12 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96S37
Assay: URAT1 In Vitro Inhibition Activity-Method B: HEK293-URAT1 cell Lines were donated by Japan Fuji Biomedical Research Institute. Negative control cell of HEK293 (MOCK cells) which was transfected with pcDNA3.1 empty vector. HEK293-URAT1 cell lines and MOCK cell lines were cultured in complete growth medium consisting of DMEM supplemented with 10% FBS, penicillin and streptomycin.Preparation of working solution: Each stock solutions was diluted to different concentrations (6, 20, 60, 200 and 600 μmol/L) with DMSO as 200× working solution, which was then diluted to 2×compound working solution with HBSS (Cl− free) buffer. Radiolabeled substrate 14C-Uric acid solution was diluted with HBSS (Cl− free) buffer to obtain 2× working solution which was mixed with an equal volume of 2× compound working solution to obtain the mixture of radiolabeled substrate and compound working solution.HER293-URAT1 and MOCK cells were seeded onto 24-well plates at the density of 1.5×106 cells per well. The cells were incubated at 37° C., 5% CO2 overnight. After cultured for approximately 2 to 3 days, cells were used for the experiments. The culture medium were removed from the wells, and cells were washed with HBSS (Cl− free) and incubated in 37° C. HBSS (Cl− free) for 10 min. HBSS was replaced with 500 μL of the mixture of radiolabeled substrate and compound working solution. The final concentration of 14C-Uric acid in the assay was 5.0 μmol/L. Plates were incubated at 37° C., 5% CO2 for 2 min, and the reaction was stopped by the addition of pre-chilled HBSS (Cl− free) by washing three times. 400 μL NaOH (0.1 mmol/L) was added to lyse the cells and the cell lysate was collected to scintillation vials, and 3 ml scintillant (Aquasol-2, PerkinElmer) was added and after mixing completely, the radioactivity was counted by Tri-Carb 2910TR liquid scintillation counter. Each concentration of compounds, positive control and negative control were repeated in two wells (n=2). Inhibition % data were calculated using the formula:Inhibition=[100×(U−U 0)/(U c −U 0)]%, and analyzed using Prism5 software.U0: Average of signals of MOCK cells;Uc: Average of signals of radiolabeled substrate. The half inhibition concentration of the tested compounds to URAT1 were analyzed using Prism 5 software.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 374.43, ALogP 3.84, HBA 6, HBD 1, PSA 102.18, QED 0.74
Compound (SMILES): O=C(CCC(=O)c1ccccc1F)c1ccc2[nH]ccc2c1
InChIKey: FGVWABHVZBXRRG-UHFFFAOYSA-N
Target: Catenin beta-1/Protein Wnt-3a (PROTEIN COMPLEX), Homo sapiens
UniProt: P56704
Assay: Evaluation of Biological Activity: Compounds that enhance the Wnt activity, or Activators, were assayed as follows. Reporter cell lines were generated by stably transducing cells of colon cancer cell lines (with a lentiviral construct that included a Wnt-responsive promoter driving expression of the firefly luciferase gene).Lentiviral constructs were made in which the SP5 promoter, a promoter having eight TCF/LEF binding sites derived from the SP5 promoter, was linked upstream of the firefly luciferase gene. The lentiviral constructs also included a hygromycin resistance gene as a selectable marker. The SP5 promoter construct was used to transduce SW480 cells, a colon cancer cell line having a mutated APC gene that generates a truncated APC protein, leading to de-regulated accumulation of β-catenin.Cultured SW480 cells bearing a reporter construct were distributed at approximately 10,000 cells per well into 384- or 96-well multiwell plates. Compounds from a small molecule compound library were then added to the wells in half-log dilutions using a maximum concentration of three or ten micromolar. A series of control wells for each cell type received only buffer and compound solvent DMSO. Twenty-four hours after the addition of compound, reporter activity for luciferase was assayed, for example, by addition of the BrightGlo luminescence reagent (Promega) and the Victor3 plate reader (Perkin Elmer). Readings were normalized to DMSO only treated cells, and any activities above DMSO were considered activation. Compounds were considered activators if reporter activities were 2× fold or greater than DMSO. EC50 is the concentration at half maximal activation.
Measurement: EC50 = 1 nM (pChEMBL 9)
Descriptors: MW 295.31, ALogP 4.15, HBA 2, HBD 1, PSA 49.93, QED 0.72
Compound (SMILES): N[C@H]1CC[C@H](C(=O)N2[C@@H]3CC[C@H]2C[C@@H](NC(=O)c2ccc4c(c2)CC(=O)N4)C3)CC1
InChIKey: QTTSPUCCQMVMSB-YMMLNBCLSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).
Measurement: IC50 = 962 nM (pChEMBL 6.019999980926514)
Descriptors: MW 410.52, ALogP 1.95, HBA 4, HBD 3, PSA 104.53, QED 0.71
Compound (SMILES): CC(C)c1c(-c2cc(C(F)(F)F)c3ncnn3c2)[nH]c2ccc(C3CCN(C(=O)CN4CCOCC4)CC3)cc12
InChIKey: BRPJTQAEZJNIAC-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: 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 = 1.4 nM (pChEMBL 8.850000381469727)
Descriptors: MW 554.62, ALogP 5.06, HBA 6, HBD 1, PSA 78.76, QED 0.37
Compound (SMILES): CC(F)(F)c1ccc(C2=C(c3ccc(O[C@H]4CCN(CCCF)C4)cc3)c3ccc(C(=O)O)cc3CCC2)cc1
InChIKey: QANHCDDKESZFOG-NDEPHWFRSA-N
Target: Type-2 angiotensin II receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P50052
Assay: AT2 Receptor Binding Assay: 120 μL membrane (5 mg protein/well) was incubated with 15 μL of [125I]-CGP42112A and 15 μL of compound at RT for 1.5 hrs. The binding reaction was stopped by rapid filtration through Unifilter GF/C plates (presoaked in 0.3% (v:v) BSA).Plate was washed three times with ice cold wash buffer.The filtration plates were dried at 37° C. overnight.50 μL of scintillation cocktail was added to each well.Radioactivity was determined using MicroBetaTriluxmicroplate scintillation counter.
Measurement: IC50 = 431 nM (pChEMBL 6.369999885559082)
Descriptors: MW 549.63, ALogP 7.6, HBA 3, HBD 1, PSA 49.77, QED 0.3
Compound (SMILES): CC(=O)c1cn(CC(=O)N2C[C@H](F)C[C@H]2C(=O)Nc2cccc(-c3ccc(F)c(F)c3Cl)c2F)c2ccccc12
InChIKey: MYFVAOKXGLCURE-GYCJOSAFSA-N
Target: Complement factor D (SINGLE PROTEIN), Homo sapiens
UniProt: P00746
Assay: Inhibition Assay: Human factor D (purified from human serum, Complement Technology, Inc.) at 80 nM final concentration is incubated with test compound at various concentrations for 5 minutes at room temperature in 50 mM Tris, 1M NaCl, pH 7.5. A synthetic substrate Z-L-Lys-SBzl and DTNB (Ellman's reagent) are added to final concentrations of 10 μM each. The increase in color is recorded at OD405 nm in a microplate in kinetic mode over 30 minutes with 30 second time points in a spectrofluorimeter. IC50 values are calculated by non-linear regression from the percentage of inhibition of complement factor D activity as a function of test compound concentration.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 571.96, ALogP 6.16, HBA 4, HBD 1, PSA 71.41, QED 0.17
Compound (SMILES): C[C@@H]1COC[C@@H](C)N1C(=O)c1ccc(Nc2cc(-c3c(F)cccc3F)nc3c2C(=O)NC3)cc1
InChIKey: GRHAOYWBLSICFB-HUUCEWRRSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Tyk2 & JAK2 Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki < 10 nM (pChEMBL 8)
Descriptors: MW 478.5, ALogP 4.26, HBA 5, HBD 2, PSA 83.56, QED 0.59
Compound (SMILES): CN1CCc2c(C3CCc4c(cnn(-c5ncccn5)c4=O)C3)cccc21
InChIKey: CTYJJNJFRPXQIH-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14831
Assay: In Vitro Pharmacology: The HTRF-based assays from CisBio Bioassays allow the measure of different cell signaling pathways, notably those allowing the determination of the production of the second messenger inositol phosphate (IP). IP1 production was determined using the IP-One HTRF kit (CisBio Bioassays), a competitive immunoassay using cryptate-labeled anti-IP1 antibody and d2-labeled IP.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 359.43, ALogP 2.29, HBA 6, HBD 0, PSA 63.91, QED 0.7
Compound (SMILES): COc1cc([C@@H](O)[C@@H](CN2CCCC2)NC(=O)CC2Cc3ccccc3C2)ccc1F
InChIKey: NANWPOJHGMQXNP-RCZVLFRGSA-N
Target: Ceramide glucosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q16739
Assay: Broken Cell Assay: Inhibition of GlcCer synthesis in broken wild-type MDCK cell preparations
Measurement: IC50 > 300 nM (pChEMBL 6.5229997634887695)
Descriptors: MW 426.53, ALogP 3.25, HBA 4, HBD 2, PSA 61.8, QED 0.68
Compound (SMILES): COc1ccc(-c2[nH]c3ccc(-c4nnc(C5CCN(CC(=O)N(C)C)CC5)[nH]4)cc3c2C(C)C)cc1OC
InChIKey: RBVBMNOHMFXJNR-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κc Band AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Bue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 530.67, ALogP 5.03, HBA 6, HBD 2, PSA 99.37, QED 0.33
Compound (SMILES): O=c1nc(NCC2CC2)c2ccc(OC(F)(F)F)cc2n1-c1ccccc1Cl
InChIKey: NZRKXIHCKJTDFZ-UHFFFAOYSA-N
Target: S-adenosylmethionine synthase isoform type-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P31153
Assay: Phosphate Sensor Fluorescence Assay : MAT2A enzyme is incubated with a test compound in DMSO or DMSO and its substrates (L-methionine and ATP) in a microtiter plate. The enzymatic reaction is stopped by the addition of Working Phosphate Sensor Mixture. The plate is analyzed for fluorescence at 450 nm. The high control (DMSO with enzyme and its substrates) gives high fluorescence which represents no inhibition of enzymatic activity while the low control (DMSO with MAT2A substrates and no enzyme) gives low fluorescence which represents full inhibition of enzymatic activity.Materials:Human MAT2A: Cepter, amino acids 1-395Tris, pH 7.5: Invitrogen cat #15567-027KCl: Ambion cat #AM9640GMgCl2: Ambion cat #AM9530GBrij-35: Sigma cat B4184-10MLDTT: Goldbio cat #DTT100BGG: Sigma cat #G5009-25GPNP: Novus Biologicals cat #NBP1-508727-MEG: Cayman Chemical cat #15988L-Methionine: Alfa Aesar cat #J61904ATP: Alfa Aesar cat #J60336Phosphate Sensor: Thermo Fisher cat #PV4407EDTA: Life Tech cat #15575-038Assay plate: 384-well black polypropylene plate: Thomas Scientific cat #1149Q35Final Assay Conditions:Assay Buffer: 50 mM Tris, pH 7.5/50 mM KCl/10 mM MgCl2/0.01% Brij-35/1 mM DTT/0.1% BGG/40 nM PNP/6 uM 7-MEGMAT2A: 10 nM for Cepter clone ID 329, lot 00023-123 before the addition of Working Phosphate Sensor Mixture    5 nM for Cepter clone ID 334, lot 00023-148 before the addition of Working Phosphate Sensor MixtureL-methionine: 500 uM before the addition of Working Phosphate Sensor MixtureATP: 500 uM before the addition of Working Phosphate Sensor MixtureProcedure:For the assay, a mixture of 1 mM L-methionine/1 mM ATP (2× final pre-stopped concentration) in assay buffer; MAT2A (2× final pre-stopped concentration) in Assay Buffer and Working Phosphate Sensor Mixture (1.5 uM Phosphate Sensor/30 mM EDTA in Assay Buffer, which is 3× final concentrations) were prepared. Test compounds or DMSO were added to the appropriate well suing D300e digital dispenser. 5 μl/well of Assay Buffer was added to the wells corresponding to the negative control and 5 μl/well of MAT2A was added to all the wells except for those corresponding to the negative control. After incubating the plate at room temperature for 15 minutes, 5 μl/well of the 1 mM L-methionine/1 mM ATP mixture was added to all wells. The plate was centrifuged at 1000 rpm for 1 minute and then incubated at room temperature for 1 hour. 5 μl of the Working Phosphate Sensor Mixture was added to all wells and the plate was centrifuged at 1000 rpm for 1 minute. The plate was read for fluorescence at 450 nm after exciting at 430 nm.
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 409.8, ALogP 4.76, HBA 5, HBD 1, PSA 56.15, QED 0.66
Compound (SMILES): c1cc(Nc2ncc3ccc4ncc(C5CCCC5)n4c3n2)ncc1N1CCNCC1
InChIKey: TUVPQAXSMZXTMF-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 4/cyclin D1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24385
Assay: Biochemical Kinase Inhibition Assay: Potency determination of biochemical kinase inhibition test. Kinase activity test and IC50 determination. Firstly, 10 ng of recombinant CDK4/Cyclin D1 (Life Technologies PV4204) was diluted in kinase buffer (20 mM Tris pH 7.5, 10 mM MgCl2, 0.01% NP-40, 2 mM DTT), and was incubated with inhibitors at the indicated concentration at room temperature for 30 minutes. The kinase reaction was initiated by adding 1 μg (1.5 μM) of recombinant retinoblastoma protein, 5 μM ATP and 10μ Ci γ-32P-ATP. The reaction was incubated at 30° C. for 20 minutes, and was terminated by adding 2×Laemmli sample buffer, heated at 95° C. for three minutes, and the resulting solution was dissolved with 12% acrylamide SDS-PAGE and autoradiographed. A densitometer (Bio-Rad) was used to quantify the corresponding phosphorylated substrate protein bands. The resulting density values were plotted as a function of the log drug concentration using Prism 4 Graphpad software, IC50 values were determined by plotting a non-linear regression curve with a variable slope.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 414.52, ALogP 3.48, HBA 8, HBD 2, PSA 83.27, QED 0.53
Compound (SMILES): CC1(C)c2cnc(NC3CCOCC3)nc2CN1C(=O)Nc1cnn(Cc2ccccc2F)c1
InChIKey: OWRPUJHUSMEXQA-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Activated ERK2 Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R- [NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 μM ATP (all concentrations are final in the reaction) in a total volume of 10.25 μL. A 16-point, half-log dilution series of compounds at 41× final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 2-3 hours at ambient temperature. Reaction was terminated by addition of 10 μL of a 2× stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 μg/mL of AlphaScreen Protein A Acceptor Beads, 10 μg/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 μg/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 465.53, ALogP 3.73, HBA 7, HBD 2, PSA 97.2, QED 0.6
Compound (SMILES): COc1ccc(-n2cnc3cc(C(=O)N4CCCCC4)ccc32)cc1Cl
InChIKey: QEPFIXNMGGOYSX-UHFFFAOYSA-N
Target: 15-hydroxyprostaglandin dehydrogenase [NAD(+)] (SINGLE PROTEIN), Homo sapiens
UniProt: P15428
Assay: Biochemical Assay: A hydroxyprostaglandin dehydrogenase inhibition screening biochemical assay can be performed to assess the synthesized inhibitors provided herein. Provided herein is an exemplary biochemical assay for hPGDH inhibitor screening.The in vitro biochemical assay can be performed in white, 384 plates in total 20 μl reaction volume consisting of 10 nM of 15-PGDH/HPGD (R&D System #5660-DH), 15 μM Prostaglandin E2 (Sigma, Cat #P5640-10MG) and 0.25 mM (3-Nicotinamide adenine dinucleotide sodium salt (Sigma, Cat #N0632-5G) made in reaction buffer (50 mM Tris-HCl, pH 7.5, 0.01% Tween 20) at 10-point dose response curve for test/tool compounds. Briefly, 5 μl (4×) of compounds solution and 5 μl (final concentration, 10 nM) of enzyme solution is added to white 384 well plates and incubated for 10 mins at 37° C. 5 μl (4×) of Prostaglandin E2 and 5 μl (4×) of β-Nicotinamide adenine dinucleotide sodium salt is added to the wells and incubated for 10 mins at room temperature. Fluorescence is recorded at ex/em=340 nm/485 nm. The percentage (%) inhibition of enzyme activity was determined relative to positive control (1% DMSO) and IC50 was calculated using GraphPad prism software (four parameter-variable slope equation).
Measurement: IC50 = 47.1 nM (pChEMBL 7.329999923706055)
Descriptors: MW 369.85, ALogP 4.31, HBA 4, HBD 0, PSA 47.36, QED 0.69
Compound (SMILES): Cc1ccc(NC(=O)c2cccn(Cc3ccc(F)c(F)c3)c2=O)cc1-c1ccc2[nH]c(=O)[nH]c2c1
InChIKey: WHMXQKNUUYORCD-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: Kinase Assay: A PDK1 kinase assay was performed as follows. PDK1 (amino acids 51-360) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to greater than 90% homogeneity. PDK1 protein was divided into two fractions, one of which was subsequently dephosphorylated. To generate dephosphorylated PDK1, the PDK1 was reacted with GST-tagged lambda-phosphatase in vitro. GST was subsequently cleaved proteolytically for both phosphorylated and dephosphorylated PDK1. Protein preparations were run on glutathione Sepharose columns to remove GST and GST-tagged lambda-phosphatase, if present. Phosphorylated PDK1 and dephosphorylated PDK1 were verified by mass-spectrometry. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay using either phosphorylated or unphosphorylated PDK1 and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 3-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide. (final concentrations: 25 nM un-phosphorylated PDK1 or 0.5 nM phosphorylated PDK1, 30 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 24 μM for both forms of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm.
Measurement: IC50 = 5250 nM (pChEMBL 5.28000020980835)
Descriptors: MW 486.48, ALogP 4.57, HBA 4, HBD 3, PSA 99.75, QED 0.34
Compound (SMILES): C[C@]1(O)CC[C@H](CNc2ccc(S(=O)(=O)NC(=O)c3ccc(C4=CCC(N5CCCC5c5cccc(Cl)c5C5CC5)CC4)cc3Oc3cnc4[nH]ccc4c3)cc2[N+](=O)[O-])CC1
InChIKey: KHORRHPUZNJPAF-BRZVJBEHSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Bcl-2/Bcl-X Fluorescence Polarization Assay: Compounds disclosed herein were tested for blocking of Bcl-2/Bcl-xl protein with its ligand in an assay based on fluorescence polarization methodology. Recombinant human 2.7 nM Bcl-2/1.3 nM Bcl-xl protein was pre-incubated with a serial dilution of compounds disclosed herein (maximum concentration is 1 μM for Bcl-2 assay, and 10 μM or 1 μM for Bcl-xl assay, 3-fold serially diluted, 10 points) at room temperature for 0.5 hour in an assay buffer containing 20 ml potassium phosphate buffer, pH 7.5, 50 mM NaCl, 1 mM EDTA, 0.05% Tween-20, 0.01% BSA. Then the FITC labeled Bak peptide Ac-GQVGRQLAIIGDK (FITC)INR-amide (1 nM for Bcl-2, 0.82 nM for Bcl-xl) was added to plate and further incubated at room temperature for 0.5 h. The FP signals (485 nm-520 nm-520 nm) were read on BMG PHERAstar FS or BMG PHERAstar FSX instalment. The inhibition percentage of Bcl-2/Bcl-xl interaction with its ligand in presence of increasing concentrations of compounds was calculated based on the FP signals. The IC50 for each compound was derived from fitting the data to the four-parameter logistic equation by Graphpad Prism software.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 879.48, ALogP 10.04, HBA 10, HBD 4, PSA 179.79, QED 0.07
Compound (SMILES): Cc1nn2c(-c3ccnc4cc(-c5ccncc5)ccc34)cnc2cc1-c1ccc(C(=O)N2CCN(C)CC2)cc1
InChIKey: OWFXMEAGTPPEFW-UHFFFAOYSA-N
Target: Activin receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q04771
Assay: ALK2 HTRF Assay: ALK2 (aa 147-end) was obtained from BPS biosciences. The enzymatic assays were conducted in white 384-well polystyrene plates in a final volume of 8 μL. The inhibitors were serially diluted in DMSO and added to the plate wells prior to addition of the other reaction components. The assays were carried out at 25° C. in the assay buffer (50 mM HEPES, pH 7.0, 10% Glycerol, 0.01% Brij50, 10 mM MgCl2, 1 mM EGTA, 5 mM DTT, and 0.01% BSA), containing 50 nM LANCE Ultra ULight-DNA Topoisomerase 2-alpha peptide (Perkin Elmer TRF0130), and 3 μM ATP. The final concentration of DMSO in the assay was 1% and the enzyme concentration was 0.5 nM for ALK2. The reactions were allowed to proceed for 2 hr for ALK2 after which, the reaction was quenched by addition of EDTA at a final concentration of 20 mM along with 1.5 nM LANCE Ultra Europium-anti-phospho-DNA Topoisomerase 2-alpha (Thr1342) antibody (Perkin Elmer TRF0218). The reaction was incubated at 25° C. for 1 hr and read on a PHERAstar FS plate reader (BMG Labtech). IC50 determination was performed by fitting percent control activity versus the log of the inhibitor concentration using the IDBS XLFit and GraphPad Prism 5.0 software.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 539.64, ALogP 5.37, HBA 7, HBD 0, PSA 79.52, QED 0.3
Compound (SMILES): O=c1[nH]c2cc(CN3CCN(c4ccc5[nH]ncc5c4)CC3)ccc2c2c1CCCN2
InChIKey: LRRAZXKYVVVMLY-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Inhibition Assay: The PARP-1 enzyme inhibitory activities of the compounds of the present invention were assayed in the following manner by use of a kit (cat. 80551) purchased from BPS Bioscience.The 96-well plate provided in BPS Bioscience kit was coated with histone and incubated at 4° C. for 16 hours. Then, the plate was washed four times with PBST (7.5 mM Na2HPO4, 2.5 mM NaH2PO4, 145 mM NaCl, 0.05% Tween 20, pH 7.4), and blocking buffer (provided in BPS Bioscience kit) was added thereto in order to block nonspecific reaction, and was then incubated at 25° C. for 1 hour. After incubation for 1 hour, the plate was washed four times with PBST, and varying concentrations of each of the compounds of the Examples were added to a reaction solution containing PARP-1 enzyme (50 ng/well), an assay mixture and activated DNA, and allowed to react at 25° C. for 1 hour. After 1 hour, each well was washed four times with PBST, and in order to measure the level of ribosylation by PARP enzyme, streptavidin-linked peroxidase (Strep-HRP, 1:50 dilution) was added and allowed to react at 25° C. for 30 minutes. The plate was washed four times with PBST, and then finally an HRP chemiluminescent substrate was added and allowed to react. The level of histone ribosylation formed by each enzyme was quantified using Synergy H4 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Inc., USA).
Measurement: IC50 = 5.65 nM (pChEMBL 8.25)
Descriptors: MW 414.51, ALogP 3.08, HBA 5, HBD 3, PSA 80.05, QED 0.48
Compound (SMILES): NC/C(=C/F)COc1ccc(S(=O)(=O)CC23CCC(C(=O)NC4CCC4)(CC2)CC3)cc1
InChIKey: HGBOKPIXTCYJSE-JXAWBTAJSA-N
Target: Amine oxidase [copper-containing] 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75106
Assay: SSAO Activity Assay: Amine oxidase activity of recombinant hSSAO, hMAO-A, and hMAO-B isoforms are measured using the MAO-Glo assay kit (Promega, V1402). Ten nL test compounds (with DMSO as vehicle, 0.1% v/v for hSSAO, hMAO-A and hMAO-B) were added into assay plates by Echo, and pre-incubated with 5 μL of enzyme for 30 mins at room temperature before the addition of 5 μL of luminogenic substrate. The substrate concentration is 40 μM for human recombinant SSAO, 40 μM for human recombinant MAO-A, and 4 μM for human recombinant MAO-B. The hSSAO and hMAO-A assays were conducted in the MAO-A reaction buffer in the kit, and the MAO-B assay was conducted in the MAO-B reaction buffer. Oxidation of the substrate was conducted for 1 hr, before the addition of detecting reagent according the manufacture's protocol. The IC50 value of the tested compounds was calculated by fitting the dose response curve using a 4-parameter non-linear regression routine.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 464.6, ALogP 3.66, HBA 5, HBD 2, PSA 98.49, QED 0.58
Compound (SMILES): NC(=O)[C@@H](Nc1cc2c3c(c1)nc(-c1ccc4ncncc4c1)n3CCCO2)C1CC1
InChIKey: VLPLIYWQHOSKQI-FQEVSTJZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: p110alpha (alpha) PI3K Binding Assay: PI3K Binding assays are intended for determining the biochemical potency of small molecule PI3K inhibitors. The PI3K lipid kinase reaction is performed in the presence of PIP2:3PS lipid substrate (Promega #V1792) and ATP. Following the termination if the kinase reaction, turnover of ATP to ADP by the phosphorylation of the lipid substrate is detected using the Promega ADP-Glo¿ (Promega #V1792) assay.
Measurement: Ki = 215 nM (pChEMBL 6.670000076293945)
Descriptors: MW 414.47, ALogP 3.1, HBA 7, HBD 2, PSA 107.95, QED 0.52
Compound (SMILES): CNC(=O)c1c(F)c(Cl)c(-c2ccc3nc(NC(=O)[C@@H]4C[C@@H]4F)sc3c2)c2cn[nH]c12
InChIKey: MCICKIDMRHCGQS-SCZZXKLOSA-N
Target: Mitogen-activated protein kinase kinase kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92918
Assay: ADP-Glo kinase assay: HPK1 kinase activity was measured by Promega's ADP-Glo kinase assay. In this assay, 5 ng of recombinant human HPK1 (signalchem) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of MBP (0.5 μg/μl) and 5 μL of ATP (25 μM) in buffer (40 mM Tris, 7.5; 20 mM MgCl2; 0.1 mg/ml BSA; 5 μM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30′ C for 40 minutes. After the incubation, 25 uL ADP-Glo reagent was added and the reaction was incubated at room temperature for 40-min to stop the reaction and degrade residual ATP. The ADP product was then converted to ATP by adding 50 uL per well of detection reagent. Luminescence was detected after 30-min room temperature incubation with the Molecular device I3X plate reader. The IC50 values were calculated from a series of percent inhibition values determined at a range of inhibitor concentration using software routines as implemented in the GraphPad Prism 7 software and SigmaPlot13.0.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 461.88, ALogP 4.29, HBA 5, HBD 3, PSA 99.77, QED 0.42
Compound (SMILES): FC(F)(F)c1[nH]nnc1-c1ccnc(-c2cn(CCc3ccc(I)cc3)cn2)c1
InChIKey: MCRWUHRVWUZEBE-UHFFFAOYSA-N
Target: Lysine-specific demethylase 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NHM5
Assay: FBXL10 Assay: The ability of test compounds to inhibit the activity of FBXL10 was determined in 384-well plate format under the following reaction conditions: 0.3 nM FBXL10, 30 nM H3K36me2-biotin labeled peptide (Anaspec cat #64442), 0.2 μM alpha-ketoglutaric acid in assay buffer of 50 mM HEPES, pH7.3, 0.005% Brij35, 0.5 mM TCEP, 0.2 mg/ml BSA, 50 μM sodium L-ascorbate, 5 μM ammonium iron(II) sulfate. Reaction product is determined quantitatively by AlphaScreen detection after the addition of detection reagents anti-H3K36me1 antibody, ALPHASCREEN Streptavidin-coated Donor beads, and ALPHASCREEN Protein A Acceptor beads in 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 5 mM EDTA, 2 mg/ml BSA to final 10 μg/ml beads.The assay reaction was initiated by the following: 3 μl of the mixture of 90 nM H3K36me2-biotin labeled peptide and 0.6 μM alpha-ketoglutaric acid with 3 μl of 11-point serial diluted inhibitor in 3% DMSO are added to each well of 384 well Proxiplate (Perkin Elmer), followed by the addition of 3 μl of 0.9 nM FBXL10 to initiate the reaction. The reaction mixture is incubated at room temperature for 1 hour, and terminated by the addition of 3 μl of appropriate dilution of anti H3K36me1 antibody in 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 5 mM EDTA, 2 mg/ml BSA. Plates will then incubated at rt for 40 min, followed by addition of 3 μl of 50 μg/ml ALPHASCREEN Streptavidin-coated Donor beads and ALPHASCREEN Protein A Acceptor beads in 50 mM HEPES, pH7.3, 10 mM NaCl, 0.005% Brij35, 5 mM EDTA, 2 mg/ml BSA. Plates will then be read by EnVision Multilabel Reader in AlphaScreen mode after minimum 2 hour incubation at room temperature. The AlphaScreen signal for each well is used to determine inhibition constant (IC50).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 510.26, ALogP 4.6, HBA 5, HBD 1, PSA 72.28, QED 0.4
Compound (SMILES): Cn1cncc1-c1ccc2cnc(NC(=O)c3cccc(CN4CCCC4)c3)cc2c1
InChIKey: UQRILHRGULWRPX-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: The DYRK1A kinase assa: Briefly, recombinant DYRK1A kinase, ATP and Ser/Thr peptide 18 were prepared in 1× Kinase buffer to final concentrations of 0.19 μg/mL, 30 μM, and 4 μM respectively. The mixture was allowed to incubate with the representative compounds for one hour at room temperature. All reactions were performed in duplicate. Unphosphorylated (0% Control) and phosphorylated (100% control) forms of Ser/Thr 18 served as control reactions. Additionally, an 11-point dose-response curve of Staurosporine (1 uM top) was run to serve as a positive compound control.After incubation, Development Reagent A was diluted in Development Buffer then added to the reaction and allowed to further incubate for one hour at room temperature. The plate was read at Ex 400 Em 455 to detect the coumarin signal and Ex 400 Em 520 to measure the signal (EnVision Multilabel Plate Reader, PerkinElmer).
Measurement: EC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 411.51, ALogP 4.48, HBA 5, HBD 1, PSA 63.05, QED 0.53
Compound (SMILES): CC(C)n1cnc2cc(C(=O)Nc3ccc(OC(F)(F)Cl)cc3)cc(Br)c21
InChIKey: JAKWLUZBLWZBPN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Biochemical Kinase Assay: ABL1 WT protein (64-515aa) containing an N-terminal His tag was produced by co-expression with YopH in Sf9 insect cells. Cells were harvested by centrifugation and resuspended in 50 mM Tris, 500 mM NaCl, 5 mM β-ME, pH 8.2. Cells were lysed by sonication and clarified by centrifugation. ABL1 was purified by affinity chromatography using a HisTrap column with a wash step in 4% wash buffer (50 mM Tris, 500 mM NaCl, 500 mM imidazole, 5 mM β-ME, pH 8.2) and eluted in a linear gradient of the same buffer. Fractions containing ABL1 were pooled, concentrated and further purified using an ion exchange column washed with 50 mM Tris, pH 8.3 and eluted with a linear gradient of elution buffer (50 mM Tris, 1M NaCl, pH 8.3). Purified protein was stored at -80° C. in 50 mM Tris (pH 8.2), 300 mM NaCl, 1 mM DTT and 20% glycerol.
Measurement: IC50 = 0.16 nM (pChEMBL 9.800000190734863)
Descriptors: MW 458.69, ALogP 5.8, HBA 4, HBD 1, PSA 56.15, QED 0.49
Compound (SMILES): COCC1(CN[C@@H]2CC2c2ccccc2)CCN(CC2(C(=O)O)CCCC2)CC1
InChIKey: OUHFLPHJVJYMBF-BPGUCPLFSA-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 M Biotin-labeled Histone H3 peptide substrate: ART-K(Mel)-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. IC50 data for the example compounds is provided in Table 1 (+ refers to IC50≦100 nM; ++ refers to IC50>100 nM and ≦500 nM).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 400.56, ALogP 3.51, HBA 4, HBD 2, PSA 61.8, QED 0.66
Compound (SMILES): O=C(NC1CCOCC1)c1c[nH]/c(=N\c2ccc(Oc3cccc(F)n3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: VBCKSKUBZAYROJ-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 3000 cells/well and cultured in the medium (7.0% fetal bovine serum, 7.0% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 1.37 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH7.5), and each well was added with 20 μL of this buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 384 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 549.99, ALogP 4.35, HBA 7, HBD 2, PSA 110.6, QED 0.33
Compound (SMILES): C[C@@H]1CCc2ncc(F)cc2[C@H]2CCCN2c2ccn3nc(N)c(c3n2)C(=O)N1
InChIKey: BSZPUUAEOAZJPL-IAQYHMDHSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: A testing platform for TrkAWT kinase activity was established based on Homogeneous Time-Resolved Fluorescence (HTRF) assay, and the activities of the compounds were tested using the platform. The compounds were subjected to five-fold gradient dilution for eight times with 100% DMSO with a starting concentration of 200 μM (9 concentrations in total). 4 μL of diluted sample for each concentration was added to 96 μL of a reaction buffer (50 mM HEPES, pH7.4, 5 mM MgCl2, 0.1 mM NaVO3, 0.001% Tween-20, 0.01% BSA and 1 mM DTT) and mixed homogeneously to be used as a 4* compound. The reaction buffer was used to formulate 2* TrkA kinase (the final concentration thereof was 1 nM) and 4* substrate (ATP+TK peptide) (TK peptide, HTRF® KinEASE-TK, was purchased from Cisbio, and the final concentration of TK peptide was 1 μM, and the final concentration of ATP was 40 μM). 2.5 μL of the 4* compound was added to a 384-well plate (OptiPlate-384, purchased from PerkinElmer), and then 5 μL of the 2* TrkA kinase were added, and mixed homogeneously by centrifugation. Then 2.5 μL of the 4* substrate mixture was added to initiate the reaction (the total reaction volume was 10 L). The 384-well plate was placed in an incubator to react for 60 min at 23° C. Then the reaction was terminated by adding 5 μL of Eu3+ cryptate-labeled anti-phosphotyrosine antibody (HTRF® KinEASE-TK, purchased from Cisbio), and L of Streptavidin-XL-665 (HTRF® KinEASE-TK, purchased from Cisbio). After incubated for 1 hr in the incubator, the fluorescence values were read out on Envision (purchased from PerkinElmer). The excitation wavelength was 320 nm, and the emission wavelengths for detection were 665 nm and 620 nm. The enzymatic activity was represented by a ratio of the two readout at the two emission wavelengths.
Measurement: IC50 = 0.31 nM (pChEMBL 9.510000228881836)
Descriptors: MW 395.44, ALogP 2.25, HBA 7, HBD 2, PSA 101.44, QED 0.61
Compound (SMILES): CCN(CC)C(=O)Oc1nc(-c2ccc(F)c(Cl)c2)nc2ccccc12
InChIKey: QAWDYBCPLZRSAH-UHFFFAOYSA-N
Target: Translocator protein (SINGLE PROTEIN), Homo sapiens
UniProt: P30536
Assay: [3H]PK 11195 radio-assay: For the assays described herein systemic injections of kainic in rats were applied and also contusion (Veenman et al. 2002; Soustiel et al. Neuropathol Appl Neurobiol. 2008; Soustiel et al. Exp Neurol. 2008). These models present very well defined cell death in the brain. Cell death in the brain is the major pathological characteristic of progressing neurodegeneration common to brain diseases. As the compounds of the present invention are designed to prevent cell death in the brain as a consequence of disease and progressing brain damage after injury, including behavioral impairments, animal models displaying cell death in the brain are the models of choice for displaying the protective capabilities of the compounds of the invention. For these reasons the R6-2 model for Huntington Disease was chosen to test the effectiveness of Compound 1 and to compare the effectiveness of Compound 1 with those of the compound of U.S. Pat. No. 8,541,428 (Cpd A) and with the classical TSPO ligand PK 11195, as well as other compounds disclosed herein. Compounds 5 and 6 were also tested in the same R6-2 model.
Measurement: Ki = 380 nM (pChEMBL 6.420000076293945)
Descriptors: MW 373.82, ALogP 4.93, HBA 4, HBD 0, PSA 55.32, QED 0.65
Compound (SMILES): COc1ccc(-c2ccc3c(N4CC5CCC(C4)O5)nc(N4CC5CCC(C4)O5)nc3n2)cc1CO
InChIKey: YNKVZRVOZBAZDZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Kinase Assay: The test on activity against mTOR Kinase was performed using a 1.7 nM mTOR (Millipore, 14-770M), a 50 nM ULight-4EBP1 (Perkin-Elmer, TRF0128M) and a 100 μM ATP on a detection system LANCE® Ultra (PerkinElmer). The compound was first prepared into a 20 mM stock solution, and then gradientle diluted so as to be added into the mTOR enzyme reaction system on a 384 well plates. The test concentrations were 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, 0.000256 μM (n=3). After the mTOR enzyme reaction was performed for 1.5 hours, it was terminated, and LANCE® Ultra systeme (PerkinElmer) was used to perform the test for 1 hour. The test results were recorded on a multimode microplate reader Synergy II (BioTek). Relative activity against mTOR %=(Lance light value of the well with drug−light value of the blank group (without mTOR)/(light value of the DMSO group−light value of the blank group)×100%.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 489.58, ALogP 2.93, HBA 9, HBD 1, PSA 93.07, QED 0.58