lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCOC(=O)OC(C)OC(=O)C1CCC(C(C)(O)c2ccc(-c3cc(C)cc(Nc4cc(C(F)F)ccn4)n3)cn2)CC1
InChIKey: ZPTJKDMLTICQMZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: Homogeneous Time-Resolved Fluorescence (HTRF) Assay: A recombinant GST-hSyk fusion protein was used to measure potency of compounds to inhibit human Syk activity. The recombinant human GST-Syk (Carna Biosciences #08-176) (5 pM final concentration) was incubated with various concentrations of the inhibitor diluted in DMSO (0.1% final concentration) for 10 minutes at room temperature in 15 mM Tris-HCl (pH 7.5), 0.01% tween 20, 2 mM DTT in 384 well plate format. To initiate the reaction the biotinylated substrate peptide (250 nM final concentration) that contains the phosphorylation site for Syk was added with magnesium (5 mM final concentration) and ATP (25 uM final concentration). Final volume of the reaction was 10 uL. Phosphorylation of the peptide was allowed to proceed for 45x at room temperature. To quench the reaction and detect the phosphorylated product, 2 nM of a Europium-anti-phosphotyrosine antibody (Perkin Elmer #AD0161) and 70 nM SA-APC (Perkin-Elmer #CR130-100) were added together in 15 mM Tris pH 7.5.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 598.65, ALogP 6.6, HBA 10, HBD 2, PSA 132.76, QED 0.19
Compound (SMILES): Cc1ccc(-n2nc(C(C)(C)C)cc2NC(=O)Nc2ccc(Oc3ccnc(Nc4ccc5[nH]cnc5c4)n3)c3ccccc23)cc1
InChIKey: GACWECUHBHHMBL-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 > 1600 nM (pChEMBL 5.796000003814697)
Descriptors: MW 623.72, ALogP 8.48, HBA 8, HBD 4, PSA 134.67, QED 0.14
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1cccc(CO)n1)c1nc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: OQMNLCCRUUKRFC-KRWDZBQOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 523.98, ALogP 3.87, HBA 9, HBD 3, PSA 131.84, QED 0.3
Compound (SMILES): CC#CC(=O)Nc1cccc(-c2cnc3[nH]cc(Cl)c3c2)c1
InChIKey: GBUXKROHQAKMOJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: Biochemical Inhibition Assay: 1H-pyrrolo[2,3-b]pyridine, 1H-pyrazolo[3,4-b]pyridine and 5H-pyrrolo[2,3-b]pyrazine series of compounds exhibited over >100 fold selective against Janus and TEC family of kinases except for the BMX and TXK it had activity of 30 and 45 nM respectively.
Measurement: IC50 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 309.76, ALogP 3.85, HBA 2, HBD 2, PSA 57.78, QED 0.71
Compound (SMILES): O=C(O)COC[C@H]1CC[C@@H](COC(=O)N(c2ccccc2)c2ccc(Cl)cc2)CC1
InChIKey: NPDKXVKJRHPDQT-HDICACEKSA-N
Target: Prostacyclin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P43119
Assay: Agonist activity at recombinant human IP receptor expressed in CHO-K1 cells assessed as increase in intracellular cAMP level after 1 hr incubation by HTRF method
Measurement: EC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 431.92, ALogP 5.52, HBA 4, HBD 1, PSA 76.07, QED 0.59
Compound (SMILES): COc1cc(OC)cc(N(Cc2cccc(Cl)c2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: SFHBVSAUUQEUEZ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 485.98, ALogP 6.04, HBA 7, HBD 0, PSA 65.3, QED 0.28
Compound (SMILES): C=CC(=O)Nc1cccc(Nc2nc(Nc3cccc(OC)c3OC)ncc2Cl)c1
InChIKey: BHJUAKDTALSDNG-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Omnia Assay: Briefly, 10x stocks of EGFR-WT (PV3872) from Invitrogen and EGFR-T790M/L858R (40350) from BPS Bioscience, San Diego, Calif., 1.13x ATP (AS001A) and appropriate Tyr-Sox conjugated peptide substrates (KCZ1001) were prepared in 1x kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl2, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10x stock, KB002A) and 0.2 mM DTT (DS001A). 5 uL of each enzyme were pre-incubated in a Corning (#3574) 384-well, white, non-binding surface microtiter plate (Corning, N.Y.) for 30 min. at 25° C. with a 0.5 uL volume of 50% DMSO and serially diluted compounds prepared in 50% DMSO. Kinase reactions were started with the addition of 45 uL of the ATP/Tyr-Sox peptide substrate mix and monitored every 71 seconds for 60 minutes at λex360/λem485 in a Synergy4 plate reader from BioTek (Winooski, Vt.). At the conclusion of each assay, progress curves from each well were examined for linear reaction kinetics and fit statistics.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 425.88, ALogP 4.76, HBA 7, HBD 3, PSA 97.4, QED 0.45
Compound (SMILES): COc1ccc(C[C@@H]2COCCN2C(=O)c2ccccc2-n2nccn2)cc1-c1ncccn1
InChIKey: OREIPZHDUFKPTP-LJQANCHMSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: CYP3A4 Inhibition Assay: The CYP3A4 inhibition assay was performed using human liver microsomes and testosterone 6'-hydroxylation as a P450 isoform-specific marker. In a total volume of 150 uL, 14C-testosterone at a final concentration of 40 uM in a 100 mM phosphate buffer (pH 7.4) was incubated in a 96-well plate with 0.3 mg/mL of human liver microsomes in an Eppendorf thermomixer at 37° C. and 400 rpm. A 1.0 uL-aliquot of the 150-fold concentrated compound stock solution, prepared in DMSO, was added to yield final inhibitor concentrations of 0, 0.1, 0.5, 1.0, 5.0, 10, 25, and 50 uM. The reaction was initiated by addition of 15 uL of the NADPH-regenerating system containing the glucose-6-phosphate dehydrogenase and terminated after 7 min with a 75 uL-aliquot of methanol. After centrifugation at 465 g and 4° C. for 20 min, a 50 uL-aliquot of the supernatant was submitted to HPLC according to the method described below.
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 456.51, ALogP 2.82, HBA 8, HBD 0, PSA 95.26, QED 0.44
Compound (SMILES): Cc1ccc(C2=C(C#N)C(=O)NC(c3ccc(OCCCC(F)(F)F)cc3)(C(F)(F)F)C2)cc1
InChIKey: DQXSGSIJXJWQFI-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in CORNING Falcon 96-well Polypropylene plates, in a total volume of 60 uL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 uM 2-oleoylglycerol, 15 uM oleoyl-Coenzyme A and 0.0013 ug/uL Human or Mouse MGAT-2 or 0.0026 ug/uL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 uL of ice cold methanol containing 1 ug/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50x2.1 mm column, was used for the chromatography of enzyme products.
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 482.42, ALogP 5.97, HBA 3, HBD 1, PSA 62.12, QED 0.41
Compound (SMILES): O=C1NCCCc2c1c1ccccc1n2CCc1ccc(F)cc1
InChIKey: LTIQFVDJDQJRPP-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P06276
Assay: Inhibition of human BChE pretreated for 20 mins followed by butyrylthiocholine iodide substrate addition measured for 5 mins by Ellman's method
Measurement: IC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 322.38, ALogP 3.7, HBA 2, HBD 1, PSA 34.03, QED 0.78
Compound (SMILES): O=C(c1cccc(C(F)(F)F)c1Cl)N1CCc2c(ncn2-c2ccc(F)cn2)C1
InChIKey: YWVQILGNCOWLGD-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Radioligand Binding Assay: human or rat P2X7-1321 N1 cells were collected and frozen @−80° C. On the day of the experiment, cell membrane preparations were made according to standard published methods. The total assay volume was 100 μl: 10 μl compound (10×)+(b) 40 μl tracer (2.5×)+50 μl membrane (2×). The tracer used for the assay was tritiated A-804598. The compound can be prepared as described in the literature. (Donnelly-Roberts, D. Neuropharmacology 2009, 56 (1), 223-229.) Compounds, tracer and membranes were incubated for 1 hour @ 4° C. The assay was terminated by filtration (GF/B filters pre-soaked with 0.3% PEI) and washed with washing buffer (Tris-HCl 50 mM).
Measurement: Ki = 15.8 nM (pChEMBL 7.800000190734863)
Descriptors: MW 424.79, ALogP 4.28, HBA 4, HBD 0, PSA 51.02, QED 0.58
Compound (SMILES): COc1ccc2ccc(CC(O)/C=C/[C@H]3CCC(=O)[C@@H]3CCc3ccc(C(=O)O)cc3)cc2c1
InChIKey: ZBKLWSJFQROXNG-ZSYIDUMUSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Radioligand Binding Assay: The cell homogenate was centrifuged at 19000 r.p.m. for 20 min at 4° C. using a Beckman Ti-60 rotor. The resultant pellet was resuspended in TME buffer to give a final 1 mg/ml protein concentration, as determined by Biorad assay. Radioligand binding competition assays vs. [3H-]17-phenyl PGF2a , (5 nM) were performed in a 100 μl volume for 60 min. Binding reactions were started by adding plasma membrane fraction. The reaction was terminated by the addition of 4 ml ice-cold TRIS-HCl buffer and rapid filtration through glass fiber GF/B filters using a Brandel cell harvester. The filters were washed 3 times with ice-cold buffer and oven dried for one hour. [3H-] PGE2 (specific activity 180 Ci mmol) was used as the radioligand for EP receptors. [3H] 17-phenyl PGF2a, was employed for FP receptor binding studies. Binding studies employing EP1, EP2, EP4 and FP receptors were performed in duplicate in at least three separate experiments. A 200 μl assay volume was used. Incubations w
Measurement: Ki = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 458.55, ALogP 5.23, HBA 4, HBD 2, PSA 83.83, QED 0.43
Compound (SMILES): O=C(Nc1ccc(C#Cc2ccc3nc(NC(=O)Nc4cc(C(F)(F)F)ccc4F)cn3n2)cc1)Nc1cc(C(F)(F)F)ccc1F
InChIKey: IICMOVMVDDUJJO-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: PDGFRbeta: Automated FLIPR (Fluorometric Imaging Plate Reader) technology was used to screen for inhibitors of VEGF induced increases in intracellular calcium levels in fluorescent dye loaded endothelial cells. HUVEC (human umbilical vein endothelial cells) (Clonetics) were seeded in 384-well fibronectin coated black-walled plates overnight at 37° C./5% CO2. Cells were loaded with calcium indicator Fluo-4 for 45 minutes at 37° C. Cells were washed 2 times (Elx405, Biotek Instruments) to remove extracellular dye. For screening, cells were pre-incubated with test agents for 30 minutes, at a single concentration (10 uM) or at concentrations ranging from 0.0001 to 10.0 uM followed by VEGF165 stimulation (10 ng/mL). Changes in fluorescence at 516 nm were measured simultaneously in all 384 wells using a cooled CCD camera. Data were generated by determining max-min fluorescence levels for unstimulated, stimulated, and drug treated samples.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 659.5, ALogP 7.73, HBA 5, HBD 4, PSA 112.45, QED 0.12
Compound (SMILES): Cc1nn(C)c(C)c1S(=O)(=O)N1CCC(Oc2ccc(Br)cc2F)CC1
InChIKey: MKOGIKITDSUWCY-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Biological Assay: The ability of a compound to block the intracellular release of calcium mediated by PK1 in RBL2H3 cells expressing human PKR1 receptors is determined as a measure of the compound's antagonist activity in vitro.Approximately 10,000 cells per assay well are seeded in normal culture medium in a 384 well plate (Corning). Twenty-four hours after seeding, the cells are loaded with a calcium sensitive fluorescent dye by replacing the culture medium with assay buffer (1× Hanks buffered saline, 25 mM HEPES, 0.1% w/v fatty acid free BSA (bovine serum albumin), pH 7.4) containing 1 mM probenecid and 1× Calcium 5 Reagent (Molecular Devices). Cells are incubated at 37° C. for 1 hour to allow for dye uptake.To test for antagonist activity, test compounds at a final concentration range between 0.32 nM-10 μM (diluted in assay buffer) are added to the assay wells and allowed to incubate for 10 minutes prior to stimulation with PK1. After incubation with test compounds the assay plate is placed in a FLIPR Tetra (Molecular Devices) and PK1 (diluted in assay buffer) is added at the determined EC80 concentration (final). Ligand-dependent changes in intracellular calcium levels are determined by measuring changes in fluorescence of the dye at 525 nM following to excitation at 485 nM.
Measurement: IC50 = 470 nM (pChEMBL 6.329999923706055)
Descriptors: MW 446.34, ALogP 3.17, HBA 5, HBD 0, PSA 64.43, QED 0.72
Compound (SMILES): CCn1nc(-c2cccc(-c3cc(C(=O)NCCO)cc(C(=O)NCCN4CCCCC4)c3)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: HIIBWSPDPMOXIQ-UHFFFAOYSA-N
Target: Adenosine receptor A1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30542
Assay: Antagonist activity at adenosine A1 receptor (unknown origin)
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 721.65, ALogP 4.88, HBA 9, HBD 5, PSA 170.58, QED 0.14
Compound (SMILES): Cc1cc(OC2CCN(C)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: DKRYPHFBCYWHAB-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 645.71, ALogP 7.51, HBA 8, HBD 2, PSA 96.68, QED 0.18
Compound (SMILES): O=S(=O)(Cc1ccccc1)Nc1ccc(-c2nc3cc(NS(=O)(=O)Cc4ccccc4)ccc3o2)cc1
InChIKey: PJYIRVITEBAYJG-UHFFFAOYSA-N
Target: Phosphotyrosine protein phosphatase (SINGLE PROTEIN), Mycobacterium tuberculosis
UniProt: A0A045ISB3
Assay: Inhibition of Mycobacterium tuberculosis His-tagged PtpB expressed in Escherichia coli Rosetta 2 (DE3) using pNPP as substrate after 30 mins by spectrophotometric method
Measurement: IC50 = 27000 nM (pChEMBL 4.570000171661377)
Descriptors: MW 533.63, ALogP 5.38, HBA 6, HBD 2, PSA 118.37, QED 0.26
Compound (SMILES): CC(C)Nc1nnc(-c2ccc3[nH]cc(-c4nccc(C5CC5)n4)c3c2)o1
InChIKey: YXZSPNLYIYHRNS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 1.394 nM (pChEMBL 8.859999656677246)
Descriptors: MW 360.42, ALogP 4.37, HBA 6, HBD 2, PSA 92.52, QED 0.55
Compound (SMILES): COC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(NS(=O)(=O)O)cc1)c1nc(C2CCCCC2)cs1
InChIKey: OMJLGUFRLBKFON-ZEQRLZLVSA-N
Target: Receptor-type tyrosine-protein phosphatase beta (SINGLE PROTEIN), Homo sapiens
UniProt: P23467
Assay: Inhibitory Activity to HPTP-β: Non-limiting examples of the HPTP-β IC50(μM) activity for illustrative compounds are listed in TABLE 2.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 586.74, ALogP 4.77, HBA 7, HBD 4, PSA 146.72, QED 0.24
Compound (SMILES): COC(=O)c1cnc(N2CCN(c3nnc(-c4ccccc4-c4ccccc4)c(C)c3C)C[C@H]2C)cn1
InChIKey: NSEMGRHHSHKGNG-LJQANCHMSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 494.6, ALogP 4.72, HBA 8, HBD 0, PSA 84.34, QED 0.37
Compound (SMILES): O=C1NCC2(CCCCC2)n2c1cc1cnc(Nc3ccc(N4CCC(N5CCOCC5)CC4)cn3)nc12
InChIKey: KPEABKWUJCTBRM-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P24941
Assay: Caliper Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK6/CycD3 CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara, Calif.) to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology (Caliper Assay Platform). The compounds were tested in 12-point dose-response format in singlicate at Km for ATP. Phosphoacceptor substrate peptide concentration used was 1 μM for all assays and Staurosporine was used as the reference compound for all assays. Specifics of each assay are as described below:CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM; Incubation time: 3 hr.CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM; Incubation time: 1 hr.CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 10 hr.CDK6/CyclinD3: Enzyme concentration: 1 nM; ATP concentration: 300 μM; Incubation time: 3 hr.
Measurement: IC50 = 1.46 nM (pChEMBL 8.84000015258789)
Descriptors: MW 516.65, ALogP 3.27, HBA 9, HBD 2, PSA 100.44, QED 0.55
Compound (SMILES): CCOc1ccc(-c2nc3ccccn3c2-c2ccnc(Nc3ccc(CCN4CCN(C)CC4)cc3OC)n2)cc1C(=O)Nc1c(F)cccc1F
InChIKey: DREHWHRYPXJTBI-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human full length EGFR expressed in baculovirus expression system assessed as decrease in transfer of gamma-phosphate from ATP onto tyrosine residue of biotinylated peptide substrate preincubated for 1 hr followed by substrate addition measured after 90 mins by HTRF method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 718.81, ALogP 6.93, HBA 10, HBD 2, PSA 109.15, QED 0.14
Compound (SMILES): Cn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CC(N)C3)n2)cn1
InChIKey: NRDBSOIRIVKMSF-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.507 nM (pChEMBL 9.289999961853027)
Descriptors: MW 346.4, ALogP 1.36, HBA 8, HBD 1, PSA 90.68, QED 0.6
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#N)c1nc2c(F)ccc(Cl)c2c(=O)n1-c1cnc(N)cn1
InChIKey: DDDXGRJVFAVYKV-QMMMGPOBSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The activities were measured using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay. TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site. Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (75-500 uM), 2 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, and 1% (v/v) DMSO at the following concentrations for each isoform: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 uM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) and Compound Y ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-(pyridin-3-yl)-3,4-dihydroquinazolin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile) were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window). The results were normalized based on positive (1 uM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3K α, β, δ and γ were calculated from the fit of the dose-response curves to a four-parameter equation.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 452.84, ALogP 1.97, HBA 11, HBD 3, PSA 174.31, QED 0.41
Compound (SMILES): CCn1c(-c2ccc(OC)c(F)c2)cs/c1=N/c1ccccc1
InChIKey: GOYJPZQMECJLBY-CZIZESTLSA-N
Target: Aldehyde dehydrogenase, dimeric NADP-preferring (SINGLE PROTEIN), Homo sapiens
UniProt: P30838
Assay: Inhibiton Assay: IC50 values were determined for CB29 and its analogs using propionaldehyde as the substrate for ALDH1A1 and ALDH2 or benzaldehyde as the substrate for ALDH3A1. The assays were performed on a Beckman DU-640 spectrophotometer at various concentrations of inhibitors ranging from 50 nM to 250 μM following a 1 minute pre-incubation. There was no pre-incubation time-dependence to the inhibition. All reactions were initiated by the addition of the aldehyde substrate. The inhibition curves were fit to the Logistic four parameter IC50 equation using the SigmaPlot (v11, StatSys). We characterized the mode of inhibition using steady-state kinetics through co-variation of inhibitor and substrate concentrations. The steady state kinetic measurements were performed in 100 mM Na2HPO4 buffer, pH 7.5. The reaction mixture contained 10 nM ALDH3A1, varied benzaldehyde (50 μM-800 μM; fixed NADP+, 1.5 mM) and varied inhibitor concentrations. In all cases−including the control reactions lacking inhibitors, the final reaction mixture contained 2% (v/v) DMSO. The reaction was initiated by addition of substrate and the initial rate of product formation was determined on a Beckman DU-640.
Measurement: IC50 = 60000 nM (pChEMBL 4.21999979019165)
Descriptors: MW 328.41, ALogP 4.62, HBA 4, HBD 0, PSA 26.52, QED 0.69
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccc(Cl)c(Cl)c4)[C@@H](C2)N(C)CC[C@]315
InChIKey: IVHIGBBUFYHGNB-NWCUSBOBSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: Binding assay of certain compounds of this invention to the opioid receptor was determine using radioligand binding assay (screening and dose-displacement).
Measurement: Ki = 0.04 nM (pChEMBL 10.399999618530273)
Descriptors: MW 530.49, ALogP 5.66, HBA 5, HBD 0, PSA 40.16, QED 0.48
Compound (SMILES): N#CCc1ccc(Nc2ncnc3ccc(-c4cccc(O)c4)cc23)cc1
InChIKey: GWZSCITZLNTBSJ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Inhibition of PI3Kalpha (unknown origin) using diC8 PIP2 as substrate after 80 mins by ADP-glo assay
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 352.4, ALogP 4.81, HBA 5, HBD 2, PSA 81.83, QED 0.55
Compound (SMILES): O=C(Nc1cccc(C(F)(F)F)c1)c1ccc(CF)c(-c2ccnc(N3CCOCC3)c2)c1
InChIKey: HADQWOUFZYOCND-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 0.332 nM (pChEMBL 9.479999542236328)
Descriptors: MW 459.44, ALogP 5.33, HBA 4, HBD 1, PSA 54.46, QED 0.52
Compound (SMILES): COc1cccc(S(=O)(=O)Nc2ccc(-c3nc4cc(NS(=O)(=O)c5cccc(OC)c5)ccc4o3)cc2)c1
InChIKey: QGKPLHUADRKTKJ-UHFFFAOYSA-N
Target: Phosphotyrosine protein phosphatase (SINGLE PROTEIN), Mycobacterium tuberculosis
UniProt: A0A045ISB3
Assay: Inhibition of Mycobacterium tuberculosis His-tagged PtpB expressed in Escherichia coli Rosetta 2 (DE3) using pNPP as substrate after 30 mins by spectrophotometric method
Measurement: IC50 = 9400 nM (pChEMBL 5.03000020980835)
Descriptors: MW 565.63, ALogP 5.11, HBA 8, HBD 2, PSA 136.83, QED 0.25
Compound (SMILES): CCC(C)Sc1nc2c(c(=O)n1-c1ccccc1)SCC2
InChIKey: UWXVMIBCJRNBBP-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: In Vitro Enzyme Assay: In vitro enzyme assays were conducted via the Ba(OH)2 precipitation method of Wang et al. (32) using recombinant human PDE11A4, PDE10A1, PDE5A1, PDE6C, PDE8A (BPS Bioscience Inc.), PDE7A (BIOMOL International), and PDE4A10 enzymes (gift from HengmingKe), in the presence of 100 nM cGMP, 30 nM cAMP, 500 nM cGMP, 1.7 μM cGMP, 10 nM cAMP, 15 nM cAMP, and 625 nM cAMP, respectively. Inhibitor concentrations that reduce enzyme activity by 50% (IC50) are presented. The values are means of at least three independent experiments.
Measurement: IC50 = 8400 nM (pChEMBL 5.079999923706055)
Descriptors: MW 318.47, ALogP 3.77, HBA 5, HBD 0, PSA 34.89, QED 0.63
Compound (SMILES): O=C(O)c1ccc(-c2c(/C=C/c3ccc4ccccc4n3)nc3c(N4CCOCC4)ccnn23)cc1
InChIKey: MJTJOIPBOVHNCN-VAWYXSNFSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: Inhibition of human PDE2A
Measurement: IC50 = 5900 nM (pChEMBL 5.230000019073486)
Descriptors: MW 477.52, ALogP 4.65, HBA 7, HBD 1, PSA 92.85, QED 0.39
Compound (SMILES): COc1cncc(-n2c([C@H](C)Nc3nc(N)nc(N)c3C#N)nc3cccc(Cl)c3c2=O)c1
InChIKey: VDVLPUFQZDYPCJ-JTQLQIEISA-N
Target: PI3-kinase p110-alpha/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P42336
Assay: Inhibition of human full length PI3K p110alpha/p85alpha using phosphatidylinositol 3,4,5-trisphosphate as substrate measured after 30 mins by TR-FRET assay
Measurement: IC50 = 10000 nM (pChEMBL 5)
Descriptors: MW 463.89, ALogP 2.44, HBA 11, HBD 3, PSA 170.65, QED 0.4
Compound (SMILES): COc1cc2c(cc1OC)C(=O)C(CC1CCN(Cc3cccc(C)n3)CC1)C2
InChIKey: ZBPKNQIMVQSMLB-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition of human erythrocytes AChE using acetylthiocholine iodide as substrate preincubated for 5 mins followed by substrate addition measured up to 3 mins by Ellman's method
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 394.51, ALogP 4.06, HBA 5, HBD 0, PSA 51.66, QED 0.74
Compound (SMILES): CCOc1ccc(-c2nc(-c3cccc4c3CCC4O)no2)cc1OCC
InChIKey: RFNAOFFZMHTEFM-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99500
Assay: Agonist activity at human S1P3 Galpha5 expressed in NFAT-bla HEK293T cells assessed as inhibition of cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 5274 nM (pChEMBL 5.28000020980835)
Descriptors: MW 366.42, ALogP 4.18, HBA 6, HBD 1, PSA 77.61, QED 0.71
Compound (SMILES): O=C(NO)c1ccc(CNC(=O)c2nc(-c3nc4c(s3)CCCC4)sc2C2CC2)cc1
InChIKey: BRGHNGRVZSFLGW-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzyme Assay: Enzyme activity of HDAC1,3 is determined using the substrate Ac-Lys-Tyr-Lys(Ac)-AMC while the enzyme activity of HDAC6 is assayed using the substrate Boc-lys(Ac)-AMC. The reaction is carried out in flat-bottom, 96-well, or 384-well microplates. After the substrate was deacetylated by HDACs, the product AMC which was obtained from hydrolysis by trypsin can generate fluorescence signal. Measurement is taken in using an multilabe plate reader a 355 nm excitation filter and a 460 nm emission filter. The initial rate of fluorescence should accurately reflect the rate of product formation and enzyme activity. Sample processing: The sample was dissolved in DMSO and kept at a low temperature. DMSO in the final system is limited under a low concentration which won't affect the enzyme activity.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 454.58, ALogP 4.07, HBA 7, HBD 3, PSA 104.21, QED 0.38
Compound (SMILES): CN1CCN(c2ccc(Nc3cc(-n4cnc5ccccc54)ncn3)nc2)CC1
InChIKey: AWYBHCVXZFMJHV-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: Inhibition of recombinant full-length human C-terminal His6-tagged CDK9/full-length human cyclin T1 expressed in baculovirus infected insect Sf21 cells using KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC as substrate in presence of [gamma-33P]-ATP after 40 mins by scintillation counting analysis
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 386.46, ALogP 2.71, HBA 8, HBD 1, PSA 75, QED 0.58
Compound (SMILES): N#Cc1cnc2[nH]cc(C(=O)c3cccc(NS(=O)(=O)c4ccccc4F)c3F)c2c1
InChIKey: KUBNUYYKOBXLOM-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of C-Raf1 (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 438.42, ALogP 3.74, HBA 5, HBD 2, PSA 115.71, QED 0.46
Compound (SMILES): CN1CN([C@H]2CC[C@H](CNS(C)(=O)=O)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: ASXUMHICBABNOJ-JOCQHMNTSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 8.9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 391.5, ALogP 1.52, HBA 5, HBD 2, PSA 98.4, QED 0.82
Compound (SMILES): C[C@@]1(c2cc(Nc3cccc4cc(OC(F)F)cnc34)ccc2F)COCC(N)=N1
InChIKey: CHPYWNCYTLASBD-NRFANRHFSA-N
Target: Cathepsin E (SINGLE PROTEIN), Homo sapiens
UniProt: P14091
Assay: Fluorescent Substrate Kinetic Assay: A substrate peptide is labeled at the N-terminus with tryptophan and at the C-terminus with the fluorophore MR121 (for cathepsin D the 10 amino acid peptide WTSVLMAAPC-MR121 was used; for cathepsin E, MR121-CKLVFFAEDW was used). The fluorescent substrate cathepsin D and cathepsin E kinetic assays were performed at room temperature in 384-well microtiter plates (black with clear flat bottom, non binding surface plates from Corning) in a final volume of 51 ul. The test compounds were serially diluted in DMSO (15 concentrations, 1/3 dilution steps) and 1 ul of diluted compounds were mixed for 10 min with 40 ul of cathepsin D (from human liver, Calbiochem) diluted in assay buffer (100 mM sodium acetate, 0.05% BSA, pH 5.5; final concentration: 200 nM) or with 40 ul of recombinant human cathepsin E (R&D Systems) diluted in assay buffer (100 mM sodium acetate, 0.05% BSA, pH 4.5; final concentration: 0.01 nM). After addition of 10 ul of the cathepsin D substrate WTSVLMAAPC-MR121 diluted in cathepsin D assay buffer (final concentration: 300 nM) or 10 ul of the cathepsin E substrate MR121-CKLVFFAEDW diluted in cathepsin E assay buffer (final concentration: 300 nM), the plates were strongly shaken for 2 minutes. The enzymatic reaction was followed in a plate: vision reader (Perkin Elmer) (excitation wavelength: 630 nm; emission: 695 nm) for at least 30 minutes in a kinetic measurement detecting an increase of MR121 fluorescence during the reaction time.
Measurement: IC50 = 17600 nM (pChEMBL 4.75)
Descriptors: MW 416.4, ALogP 4.32, HBA 6, HBD 2, PSA 81.76, QED 0.65
Compound (SMILES): COc1cc(OC)cc(N(CCCN2CC=CCC2)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: ZWYLWYKAEYANQR-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Inhibition of VEGFR2 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in absence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 = 309.03 nM (pChEMBL 6.510000228881836)
Descriptors: MW 484.6, ALogP 4.84, HBA 8, HBD 0, PSA 68.54, QED 0.32
Compound (SMILES): CCOC(=O)CCCNC(=O)c1cc2nccc(Oc3ccc(NC(=O)Nc4cc(C)ccc4F)c(F)c3)c2s1
InChIKey: IKVZCSGUEDRZOZ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17948
Assay: Kinase Assay: Kinase assays were performed in 96 well microtiter plates that were coated overnight with 30 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.2-7.4. The plates were incubated with 1% BSA and then washed four times with PBS prior to starting the reaction. Reactions were carried out in 120 μL reaction volumes containing 3.6 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 0.5 ng of purified protein. Following a ten minute incubation at 25 °C., the reactions were washed four times with PBS containing 0.05% Tween-20. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate was diluted 1:10000 in PBS-Tween-20 and added to the wells for 30 minutes. Following four washes with PBS-Tween-20, 100 μl of 0-phenylenediamine Dihydrochloride in Phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2 SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 568.6, ALogP 6.39, HBA 7, HBD 3, PSA 118.65, QED 0.15
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2ccc(Oc3cc(Nc4ccc5[nH]ncc5c4)ncn3)c3ccccc23)n(-c2ccc(OCCN3CCOCC3)cc2)n1
InChIKey: IRXWZAWSUMZCBD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 738.85, ALogP 7.88, HBA 11, HBD 4, PSA 156.37, QED 0.1
Compound (SMILES): CCC#CCN1CCOC(COc2cc3ncnc(Nc4ccc(Cl)c(Cl)c4)c3cc2OC)C1
InChIKey: UHDUHDKGRPVSSW-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 501.41, ALogP 5.18, HBA 7, HBD 1, PSA 68.74, QED 0.45
Compound (SMILES): COc1cc2c(Nc3ccc(Cl)c(Cl)c3)ncnc2cc1OCC1CCC1
InChIKey: GOTKNVOMZNDPKT-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of recombinant human N-terminal GST His6-tagged thrombin cleavage site-fused EGFR (H672 to A1210 residues) expressed in baculovirus using poly-EY as substrate after 210 mins by luciferase coupled chemiluminescence assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 404.3, ALogP 5.87, HBA 5, HBD 1, PSA 56.27, QED 0.56
Compound (SMILES): COCCN(CCOC)C(=O)CCCC#Cc1cccc2nc(Cn3nc(-c4cccc(O)c4)c4c(N)ncnc43)n(Cc3ccccc3Cl)c(=O)c12
InChIKey: UBLOHCIYTDRGJH-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Enzyme Inhibition Assay: The PI3K enzymes catalyse the phosphorylation of phosphatidylinositol 4,5-biphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) in the presence of ATP and Mg2+ ions. The PIP3 product can be detected by displacement of biotin-PIP3 from energy transfer complexes consisting of europium labelled anti-GST monoclonal antibody, a GST-tagged Pleckstrin homology (PH) domain, biotinylated PIP3 and streptavidin-allophycocyanin (APC) by the time-resolved fluorescence resonance energy transfer (TR-FRET) (HTRF PI3K enzyme assay, Millipore). Excitation, at 330 nm, of europium in the complex results in an energy transfer to the APC and a fluorescent emission at 665 nm although europium itself emits at its characteristic wavelength of 620 nm. The PIP3 product formed by PI3K activity displaces biotin-PIP3 from the complex and results in a loss of energy transfer (decreasing signal).The compound to be tested was added, at the desired final concentrations, to a mixture of PIP2 substrate.
Measurement: IC50 = 25 nM (pChEMBL 7.599999904632568)
Descriptors: MW 735.24, ALogP 4.88, HBA 12, HBD 2, PSA 163.51, QED 0.12
Compound (SMILES): Cn1nccc1-c1cc(C(F)(F)F)ccc1N1CCOc2cc(S(=O)(=O)Nc3ncns3)ccc21
InChIKey: ZRWPQWBCQJFRQE-UHFFFAOYSA-N
Target: Sodium channel protein type 5 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q14524
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 1870 nM (pChEMBL 5.730000019073486)
Descriptors: MW 522.53, ALogP 4.29, HBA 9, HBD 1, PSA 102.24, QED 0.42
Compound (SMILES): OCc1cncc(-c2cc3c(cn2)cnn3-c2cccc(N3CCNCC3)n2)n1
InChIKey: PBVKOULOPAMXJJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q86V86
Assay: Kinase Binding Assay: PIM-1, -2, and -3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 0.036 nM (pChEMBL 10.4399995803833)
Descriptors: MW 388.44, ALogP 1.17, HBA 9, HBD 2, PSA 104.88, QED 0.54
Compound (SMILES): CC(=O)c1cnc(N2CCN(c3nnc(Cc4ccc(F)cc4)c4c3CCC4)C[C@H]2C)cc1C(F)(F)F
InChIKey: BPBSSGDAWAXKLS-MRXNPFEDSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Displacement of radio-labeled smoothened agonist from mouse Smo by radio-ligand competition binding assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 513.54, ALogP 5.03, HBA 6, HBD 0, PSA 62.22, QED 0.35