lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): N#Cc1cnc2cc(OC[C@@H](O)CO)c(NC(=O)C[C@@H]3CCSS3)cc2c1Nc1ccc(OCc2cccc(F)c2)c(Cl)c1
InChIKey: KVMBBUXCOHWOBJ-GOTSBHOMSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Kinase assay: KINOMEscan is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag.For most assays, kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32° C. until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 M non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 655.17, ALogP 6.44, HBA 10, HBD 4, PSA 136.73, QED 0.13
Compound (SMILES): CCCSCC[C@H]1CCc2cc([C@H]3CC[C@](N)(COP(=O)(O)O)C3)ccc2C1
InChIKey: VERHMUKBDSBJBI-TYCQWZJGSA-N
Target: Sphingosine-1-phosphate lyase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O95470
Assay: hS1P1 ERK Phosphorylation (S1P1 pERK): hS1P1/CHO cells were plated into BD Amine 384-well plates the day before the assay. On the day of the assay, growth medium was removed and replaced with serum-free medium (Ham's F-12 Invitrogen) and incubated for 2 hours. Test compounds pre-diluted in HBSS (Gibco)/20 mM HEPES (Gibco) were transferred to the cell plates and incubated for 7 minutes at 37° C. Cells were lysed in lysis buffer (Perkin Elmer) and phospho-ERK was measured using the SureFire pERK kit (Perkin Elmer) as described by the manufacturer. Data was plotted as percentage activation of the test compound relative to the efficacy of 10 μM S1P. The EC50 is defined as the concentration of test compound which produces 50% of the maximal response and was quantified using the 4 parameter logistic equation to fit the data.
Measurement: EC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 427.55, ALogP 4.4, HBA 4, HBD 3, PSA 92.78, QED 0.4
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Br)cc3F)ncnc2cc1OC[C@@H]1CN2CCOCC2CO1
InChIKey: NJQIBYWJBXKSRO-KZUDCZAMSA-N
Target: Ephrin type-B receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P54760
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 553.82, ALogP 4.42, HBA 8, HBD 1, PSA 77.97, QED 0.45
Compound (SMILES): COc1cccc(Sc2nnc(Cc3c[nH]c4cc(F)ccc34)n2CCCc2c[nH]cn2)c1
InChIKey: IBFJVEXBVBFLNC-UHFFFAOYSA-N
Target: Somatostatin receptor type 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P35346
Assay: Radioligand Binding Assay: Competitive radioligand binding experiments were performed using MEMBRANE TARGET Systems (Perkin-Elmer, Boston, Mass.) for human somatostatin receptors: SSTR1 (ES-520-M400UA), SSTR2A (ES-521-M400UA), SSTR3 (ES523-M400UA), SSTR4 (ES-524-M400UA) and SSTR5 (ES-522-M400UA) receptors. (See Table 1). Respective membrane receptor preparations were prepared in assay buffer (25 mM HEPES, 10 mM MgCl2, 1 mM CaCl2, 0.5% BSA, pH=7.4) at a 1:150 dilution. Binding assays were performed using 1251-Tyr-SRIF 14 (Perkin-Elmer, NEX 3890) dissolved in 1 mM HCl. Binding assays were performed in triplicate for each concentration of ligand in a total volume of 200 μL radioligand (25 μL ligand, 25 μL radioligand, 150 μL receptors) and incubated at room temperature for 90 minutes using a shaking table. Binding was terminated by filtration through GF/B glass fiber filters that were presoaked in 0.5% polyethyleneimine for a minimum of 4 hours. Filters were washed 9× with 1000 μL ice cold wash buffer (50 mM Tris-HCl, pH=7.4, 0.2% BSA). Filters were scored, transferred into plastic test tubes and counted in a gamma counter (Wizard2, Perkin-Elmer). Determination of the Ki for each compound was performed using non-linear regression with GraphPad PRISM-5 software (GraphPad Software, Inc., La Jolla, Calif.).
Measurement: Ki > 10000 nM (pChEMBL 5)
Descriptors: MW 462.55, ALogP 5.01, HBA 6, HBD 2, PSA 84.41, QED 0.32
Compound (SMILES): COc1ccc([C@H](Cc2c(Cl)c[n+]([O-])cc2Cl)OC(=O)c2ccc(CNC(CO)(C(=O)OCC3CCN(C)CC3)c3ccccc3)s2)cc1OC
InChIKey: ATTNXGSCKDJLQO-XRRNFBIGSA-N
Target: Muscarinic acetylcholine receptor M3 (SINGLE PROTEIN), Homo sapiens
UniProt: P20309
Assay: M3 Binding Assay: Human M3 receptor membranes (15 μg/well) from Perkin Elmer are incubated with 0.52 nM Scopolamine Methyl Chloride, [N-methyl-3H] with or without test compounds, or a saturating concentration of Atropine (5 μM) for the determination of non-specific binding. The assay is carried out in 96-well polypropylene plates in a volume of 250 μl. The assay buffer used is 50 mM Tris-HCl, 154 mM NaCl (pH 7.4). The final assay concentration of DMSO is 0.5% (v/v). The plates are sealed and incubated for 2 h at room temperature on an orbital shaker (slow speed). Membranes are harvested onto 96-well unifilter GF/C filter plates pre-treated with 0.5% polyethyleneimine (v/v), using a filter manifold, washed four times with 200 μl of assay buffer. The plates are dried before addition of 50 μl of microscint-0, sealed then read in a Trilux Microbeta scintillation counter. IC50 values are determined from competition curves using a non-linear curve fitting program. Ki values are calculated from IC50 values by the Cheng and Prusoff equation.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 758.72, ALogP 5.71, HBA 11, HBD 2, PSA 133.5, QED 0.09
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4nccn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: MATHZBUSFDRTGM-CQSZACIVSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 533.5, ALogP 3.62, HBA 9, HBD 4, PSA 147.05, QED 0.24
Compound (SMILES): CC(C(=O)N1CCN(C)CC1)n1cc(Nc2nc3c(N4CC5CCC(C4)N5C(=O)C4CC(C(F)(F)F)C4)cccn3n2)cn1
InChIKey: MATYHLHHWZXCAX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: JAK Enzyme Assays: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications. Ki values were then determined using the Morrison tight binding model (Morrison, J. F., Biochim. Biophys. Acta. 185:269-296 (1969); William, J. W. and Morrison, J. F., Meth. Enzymol., 63:437-467 (1979)) modified for ATP-competitive inhibition [Ki=Ki,app/(1+[ATP]/Km,app)].
Measurement: Ki = 2.5 nM (pChEMBL 8.600000381469727)
Descriptors: MW 614.68, ALogP 2.77, HBA 10, HBD 1, PSA 107.14, QED 0.45
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cncc(-n5ccnc5)c4)cc3C3(CC3)C2=O)c1F
InChIKey: IMIWAPPULTUBIS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: FGFR Enzymatic Assay 2: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 3 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 5 to 10 minutes.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 489.48, ALogP 4.2, HBA 7, HBD 0, PSA 82.37, QED 0.42
Compound (SMILES): NC(=O)c1ccc(Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1
InChIKey: YPSCKJWJIXZMSC-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 5 activator 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15078
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in 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. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 78 nM (pChEMBL 7.110000133514404)
Descriptors: MW 391.44, ALogP 2.39, HBA 7, HBD 4, PSA 126.96, QED 0.54
Compound (SMILES): CN1C(=O)Cc2cc(-c3cncc(-c4ccc(=O)n(C5CC5)c4)c3)ccc21
InChIKey: JNHOSPJQAAEXMO-UHFFFAOYSA-N
Target: Bone morphogenetic protein receptor type-1B (SINGLE PROTEIN), Homo sapiens
UniProt: O00238
Assay: In Vitro Enzyme Inhibition Using a Biochemical Peptide Phosphorylation Assay- Caliper Assay: Example 54:The assays were performed in 384-well, low volume microtiter assay plates in a final reaction volume of 9 ul. Dose-response curves were generated by incubating 10 nM of each kinase together with 2 uM of the fluorescently labeled substrate peptide 5-Fluo-Ahx-KKYQAEEN-T-YDEYENKK-amid (10 mM stock solution in DMSO) in 50 mM Hepes pH 7.5, 0.02% Tween 20, 0.02% BSA, 1 mM DTT, 10 uM Na3VO4, 10 mM ß-Glycerolphosphate, 1 mM MgCl2, 12 mM MnCl2 (ALK1 and ALK6 7 mM) and 15 uM ATP for 60 min at 30° C. in the presence or absence of compound diluted in DMSO.Kinase reactions were terminated by adding 15 ul STOP buffer (100 mM HEPES pH 7.5, 5% DMSO, 0.1% Caliper coating reagent, 10 mM EDTA, and 0.015% Brij35.
Measurement: IC50 = 6400 nM (pChEMBL 5.190000057220459)
Descriptors: MW 357.41, ALogP 3.43, HBA 4, HBD 0, PSA 55.2, QED 0.72
Compound (SMILES): O=C1CC[C@@H](c2ccccc2)N1c1ccc(-c2noc(C(F)(F)F)n2)cn1
InChIKey: MJXZGOILTUBEPA-ZDUSSCGKSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Enzymatic Assay: HDAC1 and 6 reagents: FLAG-tagged HDACs 1 and 6 were prepared in-house by protein expression in HEK293F cells followed by anti-FLAG affinity purification. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. In addition to the above buffer ingredients, TCEP [CalBiochem, catalog #580561, 500 mM stock] was added at a final concentration of 0.5 mM to the buffer for the HDAC6 assays. HDAC1, 2, 3, and 6 enzymes were run at the final concentrations of 0.3 nM, 1.5 nM, 0.3 nM, and 1.333 nM, respectively. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-104], used to evaluate enzyme activity, was added at the final concentrations of 20 uM, 40 uM, 20 uM, and 2.5 uM for HDACs 1, 2, 3, and 6. To enable detection of the signal, Developer [BioMol Research Laboratories, catalog #KI-105] was added at a 1:250 dilution to the stop solution, which also included 10 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 7200 nM (pChEMBL 5.139999866485596)
Descriptors: MW 374.32, ALogP 4.02, HBA 5, HBD 0, PSA 72.12, QED 0.69
Compound (SMILES): CCC(CC)n1cc(-c2nc(-c3cnn(CC4(O)CCN(C(C)=O)CC4)c3)cn3nccc23)cn1
InChIKey: ODXJPWQJGRKWJV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK2 Inhibition Assay: Compounds of Formula I were screened for their ability to inhibit JAK2 using the general enzyme inhibition assay method, in which the assay mixture contained 1 mM ATP, 10 μM Omnia Y7 peptide (Catalog # IVGN KNZ3071C, Invitrogen Corporation, Carlsbad, Calif.) and 4 nM JAK2 in a total volume of 25 μL. JAK2 was purchased from Invitrogen Corporation, Carlsbad, Calif. (catalog # IVGN PV4288).
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 476.59, ALogP 3.19, HBA 9, HBD 1, PSA 106.37, QED 0.44
Compound (SMILES): COCC(C)(C)C(=O)NCCN1CC=C(c2cccc(C(F)(F)F)n2)CC1
InChIKey: QKTXOFXCKWUIEE-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: Binding Activity Assay: The binding affinity of the present compounds to human 5-HT1A receptor, human D4 receptor, and human D2 receptor was measured in a manner mentioned below.CHO cell membrane fraction in which human 5-HT1A receptor, human D4 receptor, and human D2 receptor were expressed was purchased from PerkinElmer Co., Ltd. In the evaluation test of binding, the test compound dissolved in DMSO, each receptor membrane preparation diluted with buffer solution, and [3H] 8-OH-DPAT (for 5-HT1A receptor), [3H] dopamine (for D4 receptor), or [3H] spiperone (for D2 receptor) (all were obtained from PerkinElmer Co., Ltd.) were mixed, and each mixture was incubated at room temperature for 30 or 60 minutes. The nonspecific binding to each receptor was evaluated by a competition binding experiment in the presence of 10 μmol/L 8-OH-DPAT, 10 μmol/L dopamine, or 10 μmol/L spiperone, respectively. The radioactivity of each receptor-binding sample was measured with a liquid scintillation counter (PerkinElmer Co., Ltd.), the 50% inhibitory concentration was calculated, and Ki value was evaluated based on the dissociation constant and the substrate concentration caluculated in the saturated bond test, which was used as binding affinity.
Measurement: Ki = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 385.43, ALogP 2.98, HBA 4, HBD 1, PSA 54.46, QED 0.78
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CC1(O)CCS(=O)(=O)CC1)C(=O)N2CC1(O)CCC1
InChIKey: KVPUDSWARNXTPX-FEGDYQJNSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co. St. Louis. Mo.). The final assay volume (250 ul/well) included 1 nM of [N-allyl-2.3-3H]naloxone as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 25 uM unlabelled naloxone for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 90 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ??-counter (PerkinElmer Life Sciences/Wallac. Turku. Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]naloxone-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation. (Cheng and Prusoff. 1973).
Measurement: Ki = 265 nM (pChEMBL 6.579999923706055)
Descriptors: MW 519.71, ALogP 2.35, HBA 6, HBD 2, PSA 101.39, QED 0.6
Compound (SMILES): COc1cc2ncc3c(c2cc1-c1cnn(C)c1)n(-c1c(C)cncc1F)c(=O)n3C
InChIKey: BGHCQTQTZXMZFG-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: ATM Kinase Assay: PI3K p110α/p85α (human) was incubated in an assay buffer comprising 10 μM phosphatidylinositol 4,5-bisphosphate and MgATP (concentration as necessary). The reaction was initiated by addition of MgATP solution. After incubation at room temperature for 40 minutes, the reaction was stopped by addition of a solution consisting of EDTA and biotinylated phosphatidylinositol 3,4,5-trisphosphate. Finally, the detection buffer, consisting of a europium-labelled anti-GST monoclonal antibody, a GST-labelled GRP1 PH domain and streptavidin allophycocyanin, was added. The plate was read out via homogeneous time-resolved fluorescence (HTRF) and the corresponding signals were evaluated via the formula HTRF=10000×(Em665 nm/Em620 nm).
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 418.43, ALogP 3.13, HBA 8, HBD 0, PSA 79.76, QED 0.45
Compound (SMILES): N#Cc1cnc2cc(OC[C@@H](O)CO)c(NC(=O)C[C@@H]3CCSS3)cc2c1Nc1ccc(OCc2cccc(F)c2)c(Cl)c1
InChIKey: KVMBBUXCOHWOBJ-GOTSBHOMSA-N
Target: Receptor tyrosine-protein kinase erbB-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P04626
Assay: Kinase assay: KINOMEscan is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand. The assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PCR of the DNA tag.For most assays, kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32° C. until lysis. The lysates were centrifuged and filtered to remove cell debris. The remaining kinases were produced in HEK-293 cells and subsequently tagged with DNA for qPCR detection. Streptavidin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecific binding. Binding reactions were assembled by combining kinases, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). All reactions were performed in polystyrene 96-well plates in a final volume of 0.135 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (1×PBS, 0.05% Tween 20, 0.5 M non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes. The kinase concentration in the eluates was measured by qPCR.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 655.17, ALogP 6.44, HBA 10, HBD 4, PSA 136.73, QED 0.13
Compound (SMILES): COc1cc2c(Nc3cc(Cl)c(Br)cc3F)ncnc2cc1OC[C@@H]1CN2CCOCC2CO1
InChIKey: NJQIBYWJBXKSRO-KZUDCZAMSA-N
Target: Ephrin type-A receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29317
Assay: AlphaScreen Assay: In AlphaScreen, when the donor and acceptor beads are close in proximity, a series of photochemical events will give rise to a fluorescent signal upon light activation. Here we use biotinylated poly-(Glu, Tyr) 4:1 as the kinase substrate, streptavidin-coated donor beads and anti-phosphortyrosine antibody PY100-coated acceptor beads. Upon phosphorylation, the peptide substrate can bind to both donor and acceptor beads, thus gives rise to fluorescence. Compounds, ATP, biotinylated poly-(Glu, Tyr) and kinases are mixed in a volume of 20 μL for 1 hr at ambient temperature using Greiner 384-well white clear bottom medium binding plates. Then 10 uL solution containing 15-30 mg/mL AlphaScreen beads, 75 mM Hepes, pH 7.4, 300 mM NaCl, 120 mM EDTA, 0.3% BSA and 0.03% Tween-20 is added to each well. After 2-16 hr incubation of the beads, plates are read in a Perkin Elmer AlphaQuest reader (available from PerkinElmer Life and Analytical Sciences, Inc., of Boston Mass.).
Measurement: IC50 = 525 nM (pChEMBL 6.28000020980835)
Descriptors: MW 553.82, ALogP 4.42, HBA 8, HBD 1, PSA 77.97, QED 0.45
Compound (SMILES): COc1cccc(Sc2nnc(Cc3c[nH]c4cc(F)ccc34)n2CCCc2c[nH]cn2)c1
InChIKey: IBFJVEXBVBFLNC-UHFFFAOYSA-N
Target: Somatostatin receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P32745
Assay: Activity Assays: Measurement of forskolin stimulated inhibition of cAMP was performed via time-resolved fluorescence resonance energy transfer (TR-FRET) LANCE assay (AD0262, PerkinElmer Life Science, Inc., Boston Mass.). Recombinant Chinese hamster ovary (CHO-K1) cells expressing human somatostatin SSTR4 cells (ES-524-CF, PerkinElmer Life Science, Inc., Boston Mass.) were thawed (37° C.), resuspended in 10 mL Hanks' balanced salt solution no phenol red (HBSS, Invitrogen, Carlsbad Calif.), and then centrifuged (150×g, 5 minutes). Cellular pellet resuspended in stimulation buffer containing HBSS 1×, HEPES 5 mM, Protease free BSA 0.1% (PerkinElmer), and 3-Isobutyl-1-methylxanthine 0.5 mM (pH 7.4) and seeded in 96-well plates at 4000 cells/well. LANCE cAMP assay was performed per manufacture instruction with assessment of respective compound, somatostatin-28 (Sigma-Aldrich Co., St. Louis, Mo.) as control agonist, against 5 μM forskolin, performed in triplicate across concentration curve. Fluorescence signal was measured at 20 hours (excitation 340 nm and emission 665 nm, 400-μs delay) via TR-FRET (FLUOSTAR Omega-F, BMG Labtech, Inc., Cary, N.C.). Data was calculated via GraphPad PRISM-5 software.
Measurement: EC50 = 0.992 nM (pChEMBL 9)
Descriptors: MW 462.55, ALogP 5.01, HBA 6, HBD 2, PSA 84.41, QED 0.32
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4nccn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: MATHZBUSFDRTGM-CQSZACIVSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 533.5, ALogP 3.62, HBA 9, HBD 4, PSA 147.05, QED 0.24
Compound (SMILES): CC(C(=O)N1CCN(C)CC1)n1cc(Nc2nc3c(N4CC5CCC(C4)N5C(=O)C4CC(C(F)(F)F)C4)cccn3n2)cn1
InChIKey: MATYHLHHWZXCAX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assays: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications. Ki values were then determined using the Morrison tight binding model (Morrison, J. F., Biochim. Biophys. Acta. 185:269-296 (1969); William, J. W. and Morrison, J. F., Meth. Enzymol., 63:437-467 (1979)) modified for ATP-competitive inhibition [Ki=Ki,app/(1+[ATP]/Km,app)].
Measurement: Ki = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 614.68, ALogP 2.77, HBA 10, HBD 1, PSA 107.14, QED 0.45
Compound (SMILES): COc1cc(OC)c(F)c(N2Cc3cnc(-c4cncc(-n5ccnc5)c4)cc3C3(CC3)C2=O)c1F
InChIKey: IMIWAPPULTUBIS-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: FGFR Enzymatic Assay 2: FGFR1, FGFR2, and FGFR4 are measured under equivalent conditions with the following changes in enzyme and ATP concentrations: FGFR1, 0.02 nM and 210 uM respectively, FGFR2, 0.01 nM and 100 uM, respectively, and FGFR4, 0.04 nM and 600 uM respectively. The enzymes were purchased from Millipore or Invitrogen. GraphPad prism3 was used to analyze the data. The IC50 values were derived by fitting the data to the equation for a sigmoidal dose-response with a variable slope. Y=Bottom+(Top−Bottom)/(1+10^((Log IC50−X)*HillSlope)) where X is the logarithm of concentration and Y is the response. Compounds having an IC50 of 1 μM or less are considered active. The compounds of the invention were found to be selective inhibitors of FGFR3 and/or FGFR4 according to the FGFR Enzymatic Assays. Compounds of Formula (I′) and (I) and all the compounds as described herein have been tested and exhibit an IC50 of less than 1 μM. Table 3 provides IC50 data for compounds of the invention assayed in the FGFR Enzymatic Assay after dilution in assay buffer, added to the plate and pre-incubated for 5 to 10 minutes.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 489.48, ALogP 4.2, HBA 7, HBD 0, PSA 82.37, QED 0.42
Compound (SMILES): NC(=O)c1ccc(Nc2ncc3cc4n(c3n2)C2(CCCCC2)C(=O)NN4)cc1
InChIKey: YPSCKJWJIXZMSC-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: P50613
Assay: CDK Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in 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. Specifics of each assay are as described below:CDK1/Cyclin B1: Enzyme concentration: 0.08 nM; ATP concentration: 40 μM; Incubation time: 3 hr. CDK2/Cyclin A: Enzyme concentration: 0.1 nM; ATP concentration: 50 μM; Incubation time: 3 hr. CDK2/Cyclin E: Enzyme concentration: 0.15 nM; ATP concentration: 100 μM; Incubation time: 3 hr. CDK4/Cyclin D1: Enzyme concentration: 1 nM; ATP concentration: 200 μM; Incubation time: 3 hr. CDK6/Cyclin D3: Enzyme concentration: 2 nM; ATP concentration: 300 μM; Incubation time: 3 hr. CDK9/Cyclin T1: Enzyme concentration: 5 nM; ATP concentration: 10 μM; Incubation time: 17 hr.
Measurement: IC50 = 579 nM (pChEMBL 6.239999771118164)
Descriptors: MW 391.44, ALogP 2.39, HBA 7, HBD 4, PSA 126.96, QED 0.54
Compound (SMILES): O=C1CC[C@@H](c2ccccc2)N1c1ccc(-c2noc(C(F)(F)F)n2)cn1
InChIKey: MJXZGOILTUBEPA-ZDUSSCGKSA-N
Target: Histone deacetylase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UQL6
Assay: Enzymatic Assay: HDAC5 reagents: N-terminal GST tagged HDAC5 was purchased from BPS Bioscience [catalog #50045]. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 137 mM NaCl [Sigma, catalog #S5150, 5M stock], 2.7 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], and 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock]. The HDAC5 enzyme was run at the final concentration of 0.447 nM. Boc-Lys(TFA)-AMC substrate [Bachem, catalog #1-1985.0050], used to evaluate enzyme activity, was added at the final concentration of 60 uM. To enable detection of the signal, Developer II [BioMol Research Laboratories, catalog #KI-176] was added at a 1:200 dilution to the stop solution, which also included 20 uM trichostatin A (TSA) [Sigma, catalog #T8552] to ensure complete termination of the reaction.
Measurement: IC50 = 200 nM (pChEMBL 6.699999809265137)
Descriptors: MW 374.32, ALogP 4.02, HBA 5, HBD 0, PSA 72.12, QED 0.69
Compound (SMILES): COCC(C)(C)C(=O)NCCN1CC=C(c2cccc(C(F)(F)F)n2)CC1
InChIKey: QKTXOFXCKWUIEE-UHFFFAOYSA-N
Target: D(4) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P21917
Assay: Binding Activity Assay: The binding affinity of the present compounds to human 5-HT1A receptor, human D4 receptor, and human D2 receptor was measured in a manner mentioned below.CHO cell membrane fraction in which human 5-HT1A receptor, human D4 receptor, and human D2 receptor were expressed was purchased from PerkinElmer Co., Ltd. In the evaluation test of binding, the test compound dissolved in DMSO, each receptor membrane preparation diluted with buffer solution, and [3H] 8-OH-DPAT (for 5-HT1A receptor), [3H] dopamine (for D4 receptor), or [3H] spiperone (for D2 receptor) (all were obtained from PerkinElmer Co., Ltd.) were mixed, and each mixture was incubated at room temperature for 30 or 60 minutes. The nonspecific binding to each receptor was evaluated by a competition binding experiment in the presence of 10 μmol/L 8-OH-DPAT, 10 μmol/L dopamine, or 10 μmol/L spiperone, respectively. The radioactivity of each receptor-binding sample was measured with a liquid scintillation counter (PerkinElmer Co., Ltd.), the 50% inhibitory concentration was calculated, and Ki value was evaluated based on the dissociation constant and the substrate concentration caluculated in the saturated bond test, which was used as binding affinity.
Measurement: Ki = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 385.43, ALogP 2.98, HBA 4, HBD 1, PSA 54.46, QED 0.78
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CC1(O)CCS(=O)(=O)CC1)C(=O)N2CC1(O)CCC1
InChIKey: KVPUDSWARNXTPX-FEGDYQJNSA-N
Target: Nociceptin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41146
Assay: Receptor Binding Assay: The hNOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl. 10 mM MgCl2. 1 mM EDTA (pH 7.4). The final assay volume (250 μl/well) included 0.5 nM of [leucyl-3H]nociceptin as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 1 μM unlabelled nociceptin for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA).
Measurement: Ki = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 519.71, ALogP 2.35, HBA 6, HBD 2, PSA 101.39, QED 0.6
Compound (SMILES): COc1cc2ncc3c(c2cc1-c1cnn(C)c1)n(-c1c(C)cncc1F)c(=O)n3C
InChIKey: BGHCQTQTZXMZFG-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: ATM Kinase Assay: PI3K p110β/p85α (human) was incubated in an assay buffer comprising 10 μM phosphatidylinositol 4,5-bisphosphate and MgATP (concentration as necessary). The reaction was initiated by addition of MgATP solution. After an incubation time of 30 min at room temperature, the reaction was stopped by addition of a solution consisting of EDTA and biotinylated phosphatidylinositol 3,4,5-trisphosphate. Finally, the detection buffer, consisting of a europium-labelled anti-GST monoclonal antibody, a GST-labelled GRP1 PH domain and streptavidin allophycocyanin, was added. The plate was read out via homogeneous time-resolved fluorescence (HTRF) and the corresponding signals were evaluated via the formula HTRF=10000×(Em665 nm/Em620 nm).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 418.43, ALogP 3.13, HBA 8, HBD 0, PSA 79.76, QED 0.45
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4nccn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: MATHZBUSFDRTGM-CQSZACIVSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 36.2 nM (pChEMBL 7.440000057220459)
Descriptors: MW 533.5, ALogP 3.62, HBA 9, HBD 4, PSA 147.05, QED 0.24
Compound (SMILES): O=C1CC[C@@H](c2ccccc2)N1c1ccc(-c2noc(C(F)(F)F)n2)cn1
InChIKey: MJXZGOILTUBEPA-ZDUSSCGKSA-N
Target: Histone deacetylase 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9BY41
Assay: Enzymatic Assay: HDAC8 reagents: HDAC8 was purchased from Enzo Life Sciences [catalog #BML-SE145]. Assays were performed with buffer containing 20 mM HEPES, pH 8.0 [Boston BioProducts, catalog #BB-104, 1M stock], 100 mM NaCl [Sigma, catalog #S5150, 5M stock], 20 mM KCl [BioChemika, catalog #87526, 4M stock], 1 mM MgCl2 [Fluka, catalog #63020, 1M stock], 0.05% BSA (Fraction V) [Invitrogen, catalog #15260, 7.5% stock], and 0.1% n-Octyl-β-D-glucopyranoside (N-OG) [Anatrace, catalog #O311, 10% stock]. The HDAC8 enzyme was run at the final concentration of 1.333 nM. Fluor-de-Lys substrate [BioMol Research Laboratories, catalog #KI-178], used to evaluate enzyme activity, was added at the final concentration of 200 uM. To enable detection of the signal, Developer II [BioMol Research Laboratories, catalog #KI-176] was added at a 1:200 dilution to the stop solution, which also included 20 uM SAHA [Sigma, catalog #SML0061] to ensure complete termination of the reaction.
Measurement: IC50 = 2900 nM (pChEMBL 5.539999961853027)
Descriptors: MW 374.32, ALogP 4.02, HBA 5, HBD 0, PSA 72.12, QED 0.69
Compound (SMILES): COCC(C)(C)C(=O)NCCN1CC=C(c2cccc(C(F)(F)F)n2)CC1
InChIKey: QKTXOFXCKWUIEE-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P08908
Assay: Binding Activity Assay: The binding affinity of the present compounds to human 5-HT1A receptor, human D4 receptor, and human D2 receptor was measured in a manner mentioned below.CHO cell membrane fraction in which human 5-HT1A receptor, human D4 receptor, and human D2 receptor were expressed was purchased from PerkinElmer Co., Ltd. In the evaluation test of binding, the test compound dissolved in DMSO, each receptor membrane preparation diluted with buffer solution, and [3H] 8-OH-DPAT (for 5-HT1A receptor), [3H] dopamine (for D4 receptor), or [3H] spiperone (for D2 receptor) (all were obtained from PerkinElmer Co., Ltd.) were mixed, and each mixture was incubated at room temperature for 30 or 60 minutes. The nonspecific binding to each receptor was evaluated by a competition binding experiment in the presence of 10 μmol/L 8-OH-DPAT, 10 μmol/L dopamine, or 10 μmol/L spiperone, respectively. The radioactivity of each receptor-binding sample was measured with a liquid scintillation counter (PerkinElmer Co., Ltd.), the 50% inhibitory concentration was calculated, and Ki value was evaluated based on the dissociation constant and the substrate concentration caluculated in the saturated bond test, which was used as binding affinity.
Measurement: Ki = 2209 nM (pChEMBL 5.659999847412109)
Descriptors: MW 385.43, ALogP 2.98, HBA 4, HBD 1, PSA 54.46, QED 0.78
Compound (SMILES): CN(C)[C@]1(c2ccccc2)CC[C@]2(CC1)CN(CC1(O)CCS(=O)(=O)CC1)C(=O)N2CC1(O)CCC1
InChIKey: KVPUDSWARNXTPX-FEGDYQJNSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Receptor Binding Assay: The hMOP receptor binding assay was performed as homogeneous SPA-assay (scintillation proximity assay) using the assay buffer 50 mM TRIS-HCl (pH 7.4) supplemented with 0.052 mg/ml bovine serum albumin (Sigma-Aldrich Co. St. Louis. Mo.). The final assay volume (250 ul/well) included 1 nM of [N-allyl-2.3-3H]naloxone as ligand (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). and either test compound in dilution series or 25 uM unlabelled naloxone for determination of unspecific binding. The test compound was diluted with 25% DMSO in H2O to yield a final 0.5% DMSO concentration. which also served as a respective vehicle control. The assay was started by adding wheat germ agglutinin coated SPA beads (GE Healthcare UK Ltd. Buckinghamshire. UK) which had been preloaded with hMOP receptor membranes (PerkinElmer Life Sciences. Inc. Boston. Mass. USA). After incubation for 90 minutes at RT and centrifugation for 20 minutes at 500 rpm the signal rate was measured by means of a 1450 Microbeta Trilux ??-counter (PerkinElmer Life Sciences/Wallac. Turku. Finland). Half-maximal inhibitory concentration (IC50) values reflecting 50% displacement of [3H]naloxone-specific receptor binding were calculated by nonlinear regression analysis and Ki values were calculated by using the Cheng-Prusoff equation. (Cheng and Prusoff. 1973).
Measurement: Ki = 805 nM (pChEMBL 6.090000152587891)
Descriptors: MW 519.71, ALogP 2.35, HBA 6, HBD 2, PSA 101.39, QED 0.6
Compound (SMILES): COc1cc2ncc3c(c2cc1-c1cnn(C)c1)n(-c1c(C)cncc1F)c(=O)n3C
InChIKey: BGHCQTQTZXMZFG-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ATM Kinase Assay: PI3K p110δ/p85α (human) was incubated in an assay buffer comprising 10 μM phosphatidylinositol 4,5-bisphosphate and MgATP (concentration as necessary). The reaction was initiated by addition of MgATP solution. After an incubation time of 30 min at room temperature, the reaction was stopped by addition of a solution consisting of EDTA and biotinylated phosphatidylinositol 3,4,5-trisphosphate. Finally, the detection buffer, consisting of a europium-labelled anti-GST monoclonal antibody, a GST-labelled GRP1 PH domain and streptavidin allophycocyanin, was added. The plate was read out via homogeneous time-resolved fluorescence (HTRF) and the corresponding signals were evaluated via the formula HTRF=10000×(Em665 nm/Em620 nm).
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 418.43, ALogP 3.13, HBA 8, HBD 0, PSA 79.76, QED 0.45
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4nccn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: MATHZBUSFDRTGM-CQSZACIVSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 533.5, ALogP 3.62, HBA 9, HBD 4, PSA 147.05, QED 0.24
Compound (SMILES): C[C@H](CO)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4nccn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: MATHZBUSFDRTGM-CQSZACIVSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Enzyme Assay: The affinity of compound binding to wild type and mutant human MET kinases is measured using Invitrogen's LanthaScreen Eu Kinase Binding technology. Briefly, GST-tagged recombinant human MET kinase domain from Signal Chem (see Table 8 below for concentration in assay) is incubated with 50 nM Alexa-Fluor® Tracer 236 (Invitrogen Cat No. PR9078A) and 2 nM Europium-anti-GST (Invitrogen Cat. No. A151 6) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, 1 mM DTT, and 2% DMSO. Compounds are typically prepared in a three-fold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction is measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compounds and 0 POC is determined using a concentration of control compound that completely inhibits the enzyme
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 533.5, ALogP 3.62, HBA 9, HBD 4, PSA 147.05, QED 0.24
Compound (SMILES): CN1CCN(c2ccc(Nc3nccc(N4CCNC(=O)C4)n3)nc2)CC1
InChIKey: NETXKRRZNKEDQO-UHFFFAOYSA-N
Target: CDK6/cyclin D3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q00534
Assay: Inhibition In Vitro Assay: Selected compounds disclosed herein were tested in CDK4/cyclinD1, CDK2/CycA and CDK2/cyclinE kinase assays to determine their inhibitory effect on these CDKs. The assays were performed using microfluidic kinase detection technology. 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.
Measurement: IC50 = 51000 nM (pChEMBL 4.289999961853027)
Descriptors: MW 368.45, ALogP 0.3, HBA 8, HBD 2, PSA 89.52, QED 0.8