lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cccc(-c2nc3nc(C(=O)O)nc(N[C@H](C)C4CCC4)c3n2Cc2ccc(C(F)(F)F)cc2F)c1
InChIKey: CXLPSYBXRDCUKJ-OAHLLOKOSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 74.35 nM (pChEMBL 7.130000114440918)
Descriptors: MW 527.52, ALogP 6.31, HBA 6, HBD 2, PSA 92.93, QED 0.27
Compound (SMILES): O=C(NCCC1CCN(Cc2ccccc2)CC1)N1CCC(c2c[nH]c3ccccc23)CC1
InChIKey: XUTTUPJDRCAGCU-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Binding Assay: Binding affinity (Ki) of compounds was measured by inhibition of radioligand binding to membranes from CHO cells expressing human M1, M2, M3, M4 and M5 receptors. Membranes were prepared by nitrogen cavitation and differential centrifugation as previously described (Hoare et al., Mol. Pharmacol. 2003 March; 63(3): 751-65). The radioligand employed was tritiated N-methylscopolamine, used at a concentration of 1.5 nM. A dose-response of twelve concentrations of compound was used, ranging from 10 Î¼M to 32 Î¼M. The assay buffer was 50 mM HEPES, 100 mM NaCl, 5 mM MgCl2, 1 mM ethylenediaminetetraacetic acid, pH-adjusted to pH 7.4.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 444.62, ALogP 5.36, HBA 2, HBD 2, PSA 51.37, QED 0.54
Compound (SMILES): Cc1nc2cnc(N(C)C3CCN(C4CCC4(F)F)CC3)c(C#N)c2c(=O)n1CC#Cc1ccc(Cl)cc1-c1ccnc2c(C(=O)O)csc12
InChIKey: MYBQJKZELXSWEB-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 28.6 nM (pChEMBL 7.539999961853027)
Descriptors: MW 714.2, ALogP 6.35, HBA 10, HBD 1, PSA 128.24, QED 0.2
Compound (SMILES): CO[C@]1(c2ccc(Cl)cc2)c2c(F)cc([C@@](C)(O)C3CCOCC3)cc2C(=O)N1[C@@H](CC(=O)O)c1ccc(Cl)cc1
InChIKey: AVDHVIXZENQIRS-HOSFBAFGSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: MDM2-p53 Interaction Using a 96-Well Plate Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 μl per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20° C.) for 20 min with 190 μl aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4° C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20° C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20° C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminescence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sampleRLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]×100 or as the (RLU detected in the compound treated sample÷RLU of DMSO controls)×100. The IC50 was calculated using a plot of % MDM2 inhibition vs concentration and is the average of two or three independent experiments.
Measurement: IC50 = 0.79 nM (pChEMBL 9.100000381469727)
Descriptors: MW 602.49, ALogP 6.29, HBA 5, HBD 2, PSA 96.3, QED 0.32
Compound (SMILES): Cc1cccc(-c2nc3nc(C(=O)O)nc(N[C@H](C)C(C)C)c3n2Cc2ccc(C(F)(F)F)cc2)c1
InChIKey: LIYYWAXIZLMFQT-MRXNPFEDSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 145.2 nM (pChEMBL 6.840000152587891)
Descriptors: MW 497.52, ALogP 6.02, HBA 6, HBD 2, PSA 92.93, QED 0.32
Compound (SMILES): Cc1cccc2cc(Cn3cnc4c(N)ncnc43)c(N3CCC4CCCCC4C3)nc12
InChIKey: CIRNNZNMAHQKKF-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Luciferase-Coupled Chemiluminescence Assay: Kinase activity is measured as the percent of ATP consumed following the kinase reaction using luciferase-luciferin-coupled chemiluminescence. Reactions were conducted in 384 or 1536-well white medium binding microtiter plates (Greiner). Kinase reactions were initiated by combining test compounds, kinase, and ATP in a 20 μL volume (6 μL volume for 1536-well plate). The reaction mixture was incubated at ambient temperature for 2 h. Following the kinase reaction, a 20 μL (or 3 μL for 1536-well plate) aliquot of KinaseGlo (Promega) was added and the chemiluminescence signal measured using an EnVision plate reader (Perkin Elmer). Total ATP consumption was limited to 25-60% and the IC50 values correlate well with those determined by radiometric assays.
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 427.56, ALogP 4.33, HBA 7, HBD 1, PSA 85.75, QED 0.52
Compound (SMILES): COc1cnc2c(C(=O)Nc3cnccc3N3CCC[C@H](N)C3)c(N)sc2c1
InChIKey: AJMSCDFNBUJULL-NSHDSACASA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μm Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was pre-incubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.Pim-2 kinase assay-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM MgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μm Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 398.49, ALogP 2.46, HBA 8, HBD 3, PSA 119.39, QED 0.62
Compound (SMILES): COc1ncc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N1CCCN(Cc2ccccc2)C1
InChIKey: FYNMOMVGAHLKPW-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 82000 nM (pChEMBL 4.090000152587891)
Descriptors: MW 511.51, ALogP 3.71, HBA 8, HBD 1, PSA 101.88, QED 0.44
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3ccc(-c4cnn(C)c4)cc3F)nn2C2COC2)C1
InChIKey: UFZWZKKZDXMKTE-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 410.45, ALogP 2.64, HBA 7, HBD 1, PSA 77.21, QED 0.72
Compound (SMILES): COc1cc(Nc2cc(CO)[nH]n2)nc(N(C)[C@@H]2C[C@H]3CCC[C@@H](C2)N3C(=O)C(F)F)n1
InChIKey: IJJCLTDAJAAXMS-BTTYYORXSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Biochemical Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for 1h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for 1h before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate
Measurement: Ki = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 451.48, ALogP 2.06, HBA 8, HBD 3, PSA 119.5, QED 0.59
Compound (SMILES): C[C@@H]1C[C@H](N)C[C@H](c2ccncc2NC(=O)c2ccc(F)c3cc(N4CCC[C@H](C)C4)cnc23)C1
InChIKey: BHVZVDKVUSRKQL-HAGHYFMRSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 475.61, ALogP 5.49, HBA 5, HBD 2, PSA 84.14, QED 0.52
Compound (SMILES): CC(=O)N[C@@H](C)CO[C@H]1C[C@H](COc2ccc3nc(O[C@@H]4CO[C@@H]5CCO[C@@H]54)sc3n2)C1
InChIKey: LQEHSZUHWJNBJK-FAVYMQKBSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Inhibitory Activity on Human ACC1 and the ACC2: Recombinant human ACC1 and recombinant human ACC2, which were prepared by the method mentioned above, were preincubated with assay buffer solution (50 mM HEPES-KOH (pH 7.4), 10 mM magnesium chloride, 6-10 mM potassium citrate, 4 mM reduced form of glutathione, 1.5 mg/ml bovine serum albumin) for one hour. Then, 0.2 μL of each this invention compound solution (in DMSO) were dispensed to 384-well microplate, 5 μL of the preincubated enzyme solution and 5 μL of substrate solution (50 mM HEPES-KOH (pH 7.4), 1 mM ATP, 0.8 mM acetyl CoA and 25-50 mM potassium bicarbonate) were added to microplate. After centrifugation and shaking, the reaction mixtures were incubated in a humidified box at room temperature for 1 to 3 hours. After the incubation, the enzyme reactions were stopped by the addition of EDTA. Then, after the samples were cocrystallized with CHCA (α-cyano-4-hydroxy cinnamic acid) matrices on MALDI target plate, by using the matrix assist laser deionization time-of-flight mass spectrometer (MALDI-TOF MS), samples were measured in reflector negative mode. Deprotonated ions of acetyl CoA (AcCoA) of substrate and malonyl CoA (MalCoA) of the reaction product were detected, then, the conversion rates of acetyl CoA to malonyl CoA was calculated by the intensity of [MalCoA-H]−/(Intensity of [MalCoA-H]−+Intensity of [AcCoA-H]−) using each signal strength. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate of the enzymatic reaction at each concentration of the compounds. In addition, potassium citrate concentrations in assay buffer solution, potassium hydrogen carbonate concentrations in substrate solution and incubation time were adjusted by each lot of enzyme.
Measurement: IC50 = 984 nM (pChEMBL 6.010000228881836)
Descriptors: MW 463.56, ALogP 2.33, HBA 9, HBD 1, PSA 101.03, QED 0.6
Compound (SMILES): COC(=O)N1CC=C(c2cc3c(-c4cc(F)ccc4OC)ccnc3[nH]2)CC1
InChIKey: QBSIMYIEZLKPRJ-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 381.41, ALogP 4.23, HBA 4, HBD 1, PSA 67.45, QED 0.74
Compound (SMILES): C[C@]1(c2cc(/C=C(\F)c3ccc(C#N)cn3)ccc2F)N=C(N)S[C@@]2(C(=O)N3CC[C@@H]4CCCC[C@H]4C3)C[C@H]21
InChIKey: OKCROLBQEYZDEH-GVYKSHRCSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE1 and BACE2 FRET (Fluorescence Resonance Energy Transfer) Assays: The cDNAs for both human recombinant BACE1 and 2 with C-terminal 6-His Tags were cloned into transient protein expression vectors, which were subsequently transfected into mammalian cell lines. These recombinant proteins were further purified using Ni-NTA affinity chromatography (Qiagen). The assay buffer used in these screens was 0.05M acetate, pH 4.5, 8% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.02 nM for BACE1 and 0.64 nM for BACE2) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 590 nm).
Measurement: IC50 = 0.527 nM (pChEMBL 9.279999732971191)
Descriptors: MW 547.68, ALogP 5.63, HBA 6, HBD 1, PSA 95.37, QED 0.54
Compound (SMILES): Cn1cc(NC(=O)c2csc3ncc(N4CCNCC5(CC5)C4)nc23)c(C(F)(F)F)n1.O=C(O)C(F)(F)F
InChIKey: XBXMFGCFCIAXKT-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 451.48, ALogP 2.89, HBA 8, HBD 2, PSA 87.97, QED 0.64
Compound (SMILES): Nc1nccc2cc(CNC(=O)c3cn(Cc4ccc(N5CC[C@@H](CO)C5)nc4)nc3C(F)(F)F)ccc12
InChIKey: LQCMCNVSZTUNJE-GOSISDBHSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Sturzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 66.9 nM (pChEMBL 7.170000076293945)
Descriptors: MW 525.54, ALogP 3.22, HBA 8, HBD 3, PSA 122.19, QED 0.34
Compound (SMILES): O=C(O)C[C@@H](NC(=O)c1cccn(Cc2ccc(F)c(F)c2)c1=O)c1nnc(-c2c[nH]c3ncccc23)o1
InChIKey: KUFBVOOJVBLKJY-LJQANCHMSA-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 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 520.45, ALogP 3.05, HBA 8, HBD 3, PSA 156, QED 0.28
Compound (SMILES): CC(C)Cn1nccc1-c1ccnc(N[C@@H](C)c2cc3cc(Cl)ccc3[nH]c2=O)c1
InChIKey: WWWFRQPTFRLINL-HNNXBMFYSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: IDH1-R132H and IDH1-R132C Enzymatic Assay: Assays were performed in a 384-well black plate. An aliquot of 250 nL of compound was incubated with 10 μL of 30 nM IDH1-R132H or 10 nM IDH1-R132C recombinant protein in assay buffer (50 mM Tris pH=7.5, 150 mM NaCl, 5 mM MgCl2, 0.1% (w/v) Bovine Serum Albumin, and 0.01% Triton X-100) in each well at 25° C. for 15 minutes. After the plate was centrifuged briefly, an aliquot of 10 μL of 2 mM α-ketoglutarate and 20 μM NADPH solution prepared in assay buffer was then added to each well and the reaction was maintained at 25° C. for 45 minutes. An aliquot of 10 μL of diaphorase solution (0.15 U/mL diaphorase and 30 μM Resazurin in assay buffer) was added to each well. The plate was maintained at 25° C. for 15 minutes and then read on a plate reader with excitation and emission wavelengths at 535 nm and 590 nm, respectively. The IC50 of a given compound was calculated by fitting the dose response curve of inhibition of NADPH consumption at a given concentration with the four parameter logistic equation.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 421.93, ALogP 5.27, HBA 5, HBD 2, PSA 75.6, QED 0.44
Compound (SMILES): C[C@]1(NCC(F)(F)F)CC[C@H](NC(=O)c2nc(-n3ccnc3)nc3cn[nH]c23)CC1
InChIKey: YRURLNLWMCYYHP-QQVUYCAZSA-N
Target: ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28907
Assay: Enzyme Assay: The CD38 enzyme assay was performed as described previously (Becherer, J D, et al. J. Med. Chem. 2015, 58, 7021-7056). Briefly, 200 nL of a dose response titration of each test compound dissolved in 100% DMSO was spotted in clear polystyrene 384-well plate (Thermo #264704) using a Mosquito (TTP Labtech). A 10 μL solution of 2 nM CD38 (BPS Biosciences #71227) suspended in 100 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH=7.5), 4 mM EDTA (2,2′2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid) and 1 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) was incubated with test compound at 25° C. for 30 min. The enzyme reaction was initiated by adding 10 μL of 400 μM nicotinamide adenine dinucleotide (NAD+), 1000 μM (E)-2-(2-(pyridin-4-ylmethylene)hydrazinyl)pyridine in buffer containing 5 mM sodium acetate (pH=5.2) and 1 mM CHAPS. The reactions were incubated at 25° C. and the absorbance at 405 nm was measured after 15 minutes and 60 min on an Envision plate reader (Perkin Elmer) The absorbance values from the 15 min timepoint were subtracted from the absorbance value from the 60 min timepoint.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 422.42, ALogP 2.12, HBA 7, HBD 3, PSA 113.41, QED 0.58
Compound (SMILES): C[C@H](CO)NC(=O)c1nc(OC[C@H]2CCCO2)nc(N2CCC(c3n[nH]c4ncccc34)CC2)n1
InChIKey: PKCYGGOPLFKBBU-GDBMZVCRSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL enzyme inhibition (% inhibition, Kiapp and Ki values) by small molecule inhibitors was evaluated using a fluorescence-based microfluidic mobility shift assay. AXL catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-30 (5-FAM-KKKKEEIYFFF-CONH2, CPC Scientific, Sunnyvale, Calif.). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Human wild-type receptor tyrosine kinase protein Axl comprising residues 505-811 was produced in-house using the baculoviral expression vector system that incorporated a hexahistidine affinity tag into the protein (LJIC-1916B1.1). The enzyme was preactivated by auto-phosphorylation of 34 uM non-activated enzyme in the presence of 2 mM ATP, 4 mM MgCl2, 50 mM NaCl and 1 mM TCEP in 20 mM HEPES, pH 7.3 at 4° C. for 30 minutes. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 10 mM MgCl2, 1 mM DTT, 120 μM ATP (ATP Km=70.4 μM), 0.01% Tween-20, 3 μM FL-Peptide-30, and 0.5 nM phosphorylated AXL enzyme in 100 mM HEPES buffer at pH 7.3. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 30 minutes at 25° C. by the addition of 50 μL of 200 mM EDTA, pH 7.5. The Ki values were determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable. (See, Morrison, J. F. (1969) Kinetics of the reversible inhibition of enzyme-catalysed reactions by tight-binding inhibitors, Biochimica et biophysica acta 185, 269-286; and Murphy, D. J. (2004) Determination of accurate KI values for tight-binding enzyme inhibitors: an in silico study of experimental error and assay design, Analytical biochemistry 327, 61-67.)
Measurement: Ki = 4.13 nM (pChEMBL 8.380000114440918)
Descriptors: MW 482.55, ALogP 1.2, HBA 10, HBD 3, PSA 151.27, QED 0.43
Compound (SMILES): Nc1c(F)cccc1-c1ccc2nc(NC(=O)[C@@H]3C[C@@H]3F)cn2c1
InChIKey: LSCRWKPOMPLSSS-YPMHNXCESA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Kinase Assay: c-abl: ADP-Glo assay kit was purchased from Promega. Magnesium chloride (MgCl2), bovine serum albumin (BSA), ethylene glycol-bis(p-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), triton X-100, 1,4-dithiothreitol (DTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. HEPES buffer was purchased from Gibco. ABL1 kinase and Abltide were purchased from Signalchem.c-Abl kinase activity was measured by Promega's ADP-Glo Assay. In this assay, His-tagged recombinant human ABL1 (0.25 ng/μl) is incubated with 5 μL of compounds (0.5% DMSO), 5 μL of Abltide (0.01 μg/μl) and 5 μL of ATP (25 μM) in buffer (50 mM HEPES, 7.5; 10 mM MgCl2; 1 mM EGTA; 0.05% BSA; 0.01% Triton X-100; 2 mM DTT). The assay was started by incubating the reaction mixture in a 96-well plate at 30° C. for 30-min. After the incubation, 25 μL 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 μL 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 Sigma Plot 13.0.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 328.32, ALogP 3.02, HBA 4, HBD 2, PSA 72.42, QED 0.73
Compound (SMILES): CCOc1ccc2cc(-c3nn(C(C)C(C)(C)O)c(N)c3C(N)=O)ccc2n1
InChIKey: AMGMAKSGXBFINM-UHFFFAOYSA-N
Target: Calmodulin-domain protein kinase 1 (SINGLE PROTEIN), Toxoplasma gondii
UniProt: Q9BJF5
Assay: Luminescent Kinase Assay: Inhibition of TgCDPK1 and CpCDPK1 was determined using a luminescent kinase assay which measures ATP depletion in the presence of the Syntide 2 peptide substrate (KinaseGlo). For description of the methods and assays, see WO 2011/094628, incorporated by reference herein. Similar to TgCDPK1, exogenous calcium was necessary for CpCDPK1 to possess maximum catalytic activity (data not shown). Notably, both kinases were tested at the same ATP concentration which allows direct comparison of inhibitor potencies due to these enzymes possessing similar Kms for this cofactor.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 383.45, ALogP 2.51, HBA 7, HBD 3, PSA 129.28, QED 0.6
Compound (SMILES): CC(C)COC(=O)N1CC=C(c2cc(NC(=O)c3c[nH]c(=O)cc3C(F)(F)F)c(N3C[C@@H](C)N(C)[C@@H](C)C3)cc2F)C1
InChIKey: MNAVPWLEHUTGHS-HDICACEKSA-N
Target: WD repeat-containing protein 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P61964
Assay: Surface Plasmon Resonance (SPR) Assay: Exemplary compounds of the application were dissolved in 100% DMSO at 10 mM, assayed fresh, and then stored at −20° C. for repeat studies and other experiments. Full length WDR5 with an N-terminal His tag and C-terminal AviTag (Avidity Inc.) was expressed in E. coli with coexpression of BirA to biotin labelled protein in vivo. Purification of the protein was performed using Ni-NTA. The purified WDR5 protein has a molecular weight of 41976 Da.SPR studies were performed using a Biacore T200 instrument (GE Health Sciences Inc.). Biotinylated WDR5 protein (approximately 3000 RU) was stably captured to streptavidin coupled SA chips according to the manufacture's protocol (GE Health Sciences Inc.). The running buffer used was HBS-EP (20 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P-20) plus 5% DMSO with a flow rate of 40 μl/min. For SPR analysis, 5 different concentrations of each exemplary compound of the application were sprayed into 96 or 384 well plates using an HP D300 digital dispenser. The concentration ranged from about 195 nM to about 12 nM in a two-fold series. Concentration ranges were adjusted higher or lower for weaker or more potent compounds respectively when necessary. For the KD determinations, single cycle kinetic analysis was performed with an on time of 60 seconds, and an off time of 300 or 600 seconds. Curve fitting and KD calculations were performed with the Biacore T200 Evaluation software (GE Health Sciences Inc).
Measurement: Kd = 2.37 nM (pChEMBL 8.619999885559082)
Descriptors: MW 593.62, ALogP 4.81, HBA 6, HBD 2, PSA 97.98, QED 0.47
Compound (SMILES): C[C@@H]1CCCN(C(=O)c2c(F)cccc2-n2nccn2)[C@@H]1CNc1ncc(C(F)(F)F)cn1
InChIKey: PSTXYXVIQCDKBC-CXAGYDPISA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Cell-based Functional Assay: Measurement of [Ca2+]i using a FLIPR: CHO-OX1 or CHO-OX2 cells are seeded into black-walled clear-base 384-well plates (Costar) at a density of 20,000 cells per well in F12-K medium supplemented with 10% FBS and selection antibiotic (500 ug/ml G418 or 15 ug/ml Blasticidin) and cultured overnight. The cells are incubated with equal volume of calcium4 loading buffer (Molecular Devices, Inc.) containing 2.5 mM probenecid at 37° C. for 30 min, followed by putative OX1 or OX2 receptor antagonists (dose-range 0.1 nM-10 μM) for another 30 min.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 463.44, ALogP 3.57, HBA 7, HBD 1, PSA 88.83, QED 0.58
Compound (SMILES): Cc1cc2c(cc1-n1cnc(C3CC3)c1)C(=O)N(c1cccc(-c3nnc4n3C3(CC4)CC3)n1)C2
InChIKey: SGOXAEOBENHBPG-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: ASK1 Enzymatic Test: In this test, the fluorescence-resonance energy transfer (TR-FRET) method was used to determine the inhibition effect of the test compounds on ASK1 kinase activity, and the half maximal inhibitory concentration IC50 of the compounds on ASK1 kinase activity was obtained.
Measurement: IC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 463.55, ALogP 4.31, HBA 7, HBD 0, PSA 81.73, QED 0.45
Compound (SMILES): Cn1cnc(S(=O)(=O)N2CC(c3ccccc3)C(C)(NCc3ccc(C(F)(F)F)cc3Cl)C2)c1
InChIKey: LCBJXGQKIFIGMM-UHFFFAOYSA-N
Target: Alpha-1,3-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P26572
Assay: Radioligand Binding Assay: Radioligand binding assays using recombinant CHO cell membranes expressing human GlyT1: Radioligand binding to human GlyT1c transporter-expressing membranes was determined as described in Mezler et al., Molecular Pharmacology 74:1705-1715, 2008.
Measurement: Ki < 100 nM (pChEMBL 7)
Descriptors: MW 512.99, ALogP 4.43, HBA 5, HBD 1, PSA 67.23, QED 0.53
Compound (SMILES): O=C(c1ccc2c(ccn2-c2cccc(Cl)c2)c1)N1CCCCC1
InChIKey: JYDGFFBYTRTDAN-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 = 0.5 nM (pChEMBL 9.300000190734863)
Descriptors: MW 338.84, ALogP 4.91, HBA 2, HBD 0, PSA 25.24, QED 0.65
Compound (SMILES): Cc1cc(F)c(-c2nc3sc(N4CCC[C@@H]4C(=O)NCc4ccccc4)nc3c(=O)[nH]2)s1
InChIKey: JHYFZIGWZKKMFU-OAHLLOKOSA-N
Target: Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q8N5Z0
Assay: KAT-II inhibitory assay: The inhibitory action of the test compound on human recombinant KAT-II was determined by the following method.To a reaction mixture (45 μL) containing 3.0 μmol/L kynurenine, 10 μmol/L pyridoxal phosphate, 2.0 ng/μL human recombinant KAT-II, and 150 mmol/L tris(hydroxymethyl)aminomethane-acetate buffer (pH 8.0) was added a 10% dimethyl sulfoxide solution (5 μL) of each test compound prepared, and the mixture was reacted at 37° C. for 1 hr. After the reaction, 50% trichloroacetic acid (5 μL) was added to terminate the reaction.The resultant kynurenic acid was quantified as follows by high performance liquid chromatography. An enzyme reaction mixture was separated by an octadecylsilane reversed-phase column (SC-50DS, Eicom Corporation; mobile phase: 250 mmol/L zinc acetate, 50 mmol/L sodium acetate, and 5.0% acetonitrile (pH 6.2)) incubated at 30° C., and kynurenic acid was quantified using a fluorescence detector (RF-20Axs, Shimadzu Corporation) at excitation wavelength 354 nm, detection wavelength 460 nm. The analytical curve was drawn every time by an external standard method. Each test compound was tested by dual measurement at each concentration. The kynurenic acid level in the presence of a test compound at each concentration was converted into % relative to kynurenic acid resulting from a reaction with an enzyme alone as 100%, and the obtained values were fitted to S-curve to determine IC50.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 469.57, ALogP 3.84, HBA 7, HBD 2, PSA 90.98, QED 0.46
Compound (SMILES): C[C@@H](COCCC(=O)N1CCN(c2ncc(C(F)(F)F)cn2)C[C@@H]1C)Nc1cn[nH]c(=O)c1C(F)(F)F
InChIKey: SETLFNSCEYLBOF-STQMWFEESA-N
Target: Protein mono-ADP-ribosyltransferase TIPARP (SINGLE PROTEIN), Homo sapiens
UniProt: Q7Z3E1
Assay: Enzymatic Assay for Inhibition of PARP7: 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 μL 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 6xHis 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 platereader 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. Control wells containing a negative control of 0.25% DMSO vehicle or a positive control of 100 μM Example 190 were used to calculate the % inhibition as described below:% ⁢ ⁢ inhibition = 100 × TRF cmpd - TRF min TRF max - TRF minwhere TRFcmpd is the TR-FRET ratio from the compound treated well, TRFmin is the TR-FRET ratio from the Example 190-treated positive control well and TRFmax is the TR-FRET ratio from the DMSO-treated negative control well.The % inhibition values were plotted as a function of compound concentration and the following 4-parameter fit was applied to derive the IC50 the values:Y = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ Coefficientwhere top and bottom are normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient is normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and x is the compound concentration.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 537.47, ALogP 2.54, HBA 8, HBD 2, PSA 116.34, QED 0.39
Compound (SMILES): O=C1Nc2ccccc2[C@@]12C[C@@H]2c1ccc2cn[nH]c2c1
InChIKey: RJUIETDWZAGQNR-CXAGYDPISA-N
Target: Serine/threonine-protein kinase PLK4 (SINGLE PROTEIN), Homo sapiens
UniProt: O00444
Assay: Inhibition Assay: PLK4 activity was measured using an indirect ELISA detection system. Dephosphorylated GST-PLK4 (4 nM) was incubated in the presence of 15 μM ATP (Sigma cat#A7699), 50 mM HEPES-Na2+ pH 7.4, 10 mM MgCl2, 0.01% Brij 35 (Sigma cat#03-3170), in a 96 well microtitre plate pre-coated with MBP (Millipore cat#30-011). The reaction was allowed to proceed for 30 minutes, followed by 5 washes of the plate with Wash Buffer (50 mM TRIS-C1 pH 7.4 and 0.2% Tween 20), and incubation for 30 minutes with a 1:3000 dilution of primary antibody (Cell Signaling cat#9381). The plate was washed 5 times with Wash Buffer, incubated for 30 minutes in the presence of secondary antibody coupled to horse radish peroxidase (BioRad cat#1721019, 1:3000 concentration), washed an additional 5 times with Wash Buffer, and incubated in the presence of TMB substrate (Sigma cat#T0440). The colourimetric reaction was allowed to continue for 5 minutes, followed by addition of stop solution (0.5 N sulphuric acid), and quantified by detection at 450 nm with either a monochromatic or filter based plate reader (Molecular Devices M5 or Beckman DTX880, respectively).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 275.31, ALogP 2.94, HBA 2, HBD 2, PSA 57.78, QED 0.72
Compound (SMILES): CCN(CC(NC)c1nccc(C(C)(C)O)n1)C(=O)c1ccccc1-n1nccn1
InChIKey: CPJXORATLMHDPZ-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: In a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 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 Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 409.49, ALogP 1.71, HBA 8, HBD 2, PSA 109.06, QED 0.58