lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC1(O)CCC(CN)(c2ccc(OC(F)(F)F)cc2)CC1
InChIKey: AJLDCFDWOAROQU-UHFFFAOYSA-N
Target: Sodium-dependent dopamine transporter (SINGLE PROTEIN), Homo sapiens
UniProt: Q01959
Assay: Functional Uptake Assay (hDAT): Inhibition of human dopamine reuptake transporter was assayed using the recombinant human dopamine transporter expressed in either CHO-K1 or HEK293 cells using a published method (Pristupa, Z. B. et al., Mol. Pharmacol. 45: 125-135, 1994). Either CHO-K1 or HEK293 cells expressing human recombinant dopamine transporter were plated before the assay. Test compound and/or vehicle was preincubated with cells in modified HEPES buffer pH 7.1 or pH 7.4 for 20 minutes at 18 to 25° C. and 50 nM [3H]dopamine was then added for an additional timed incubation period (10 to 30 minutes). After washing the cells to remove [3H]dopamine not internalized, the cells were lysed, and the amount of tritium in the lysate was measured using a liquid scintillation counter to determine [3H]dopamine uptake. Non-specific binding of tritium was measured in a control reaction containing 10 uM nomifensine, and was subtracted from the counts for assays to correct for non-specific binding of tritium. Reduction of [3H]dopamine uptake by 50 percent or more (50%) relative to an uninhibited control reaction indicates significant inhibitory activity. Compounds were screened at 10, 1, 0.1, 0.01 and 0.001 uM. The reference compound for the assay was nomifensine, for which the IC50 value of 11 nM was obtained in a typical experiment.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 303.32, ALogP 3.11, HBA 3, HBD 2, PSA 55.48, QED 0.9
Compound (SMILES): C[C@@H]1CN(c2nnc(-c3cc(F)cc(Cl)c3Cl)c(N)n2)C[C@H](C)N1
InChIKey: ADQIYYHQDQQCFT-OCAPTIKFSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Ex-Vivo Potencies in Inactivated state: The potencies of the compounds provided herein to block specific Na+ channels was measured using automated patch clamp electrophysiologically of Nav1.7 sodium channels (human SCN9A gene) stably expressed in CHO cells using the IonWorks Barracuda system. Currents were measured from the whole cell patch configuration. From a holding potential of −90 mV, membranes were subjected to a 200 ms pre-pulse to −120 mV, then pulsed to 0 mV to measure blockade of the tonic (resting) state. Inactivated state block was elicited by repolarization to −100 mV (20 ms pulse duration), followed by depolarization to 0 mV (20 ms pulse duration). Peak inward Na+ currents were measured in the presence of vehicle or various concentrations of the test compound to determine the percent block of current.
Measurement: IC50 < 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 371.25, ALogP 2.75, HBA 6, HBD 2, PSA 79.96, QED 0.79
Compound (SMILES): C[C@@H]1CCC[C@@H](c2ccc(-c3cc(Cl)ccc3-n3cnnn3)c[n+]2[O-])c2cccc(c2)-c2cc(F)ccc2NC1=O
InChIKey: WOJYOHCXQCTJSI-KBMIEXCESA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: Kallikrein Assay: The effectiveness of a compound of the present invention as an inhibitor of Kallikrein can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at rt or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Kallikrein determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; Fisher Scientific). Determinations were made using purified Human plasma Kallikrein at a final concentration of 0.5 nM (Enzyme Research Laboratories) and the synthetic substrate, Acetyl-K-P-R-AFC (Sigma # C6608) at a concentration of 100 mM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration ≤0.2 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. The reactions were performed under linear progress curve conditions and fluorescence increase measured at 405 Ex/510 Em nm. Values were converted to percent inhibition of the control reaction (after subtracting 100% Inhibition value). IC50 was determined by inflection point from a four parameter logistic curve fit. Ki was calculated using the Cheng Prusoff equation, Ki=IC50/(1+([S]/Km)).
Measurement: Ki = 79.8 nM (pChEMBL 7.099999904632568)
Descriptors: MW 569.04, ALogP 6.31, HBA 6, HBD 1, PSA 99.64, QED 0.2
Compound (SMILES): CCNCc1csc(C(O)c2cc(-c3ncnc4cc(N5CCOCC5)ccc34)c(F)cc2Cl)n1
InChIKey: QYNNKWUEYHNEOA-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Patch Clamp Assay: The patch-clamp measurement was carried out at room temperature in whole-cell configuration on human embryonic kidney cells (HEK293) which have been transfected in a stable manner with the hERG gene.The whole-cell configurations were carried out using an automated patch clamp device (Patchliner, Nanion Technologies, Munich). This is a glass chip-based system with which automated whole-cell measurements on up to 8 cells simultaneously are possible. The glass chip has a hole of defined size to which the cell is transferred into the Gigaseal by application of a reduced pressure and brought into the whole-cell configuration. Buffer, cell suspension and test substances were added to microchannels of the chip using a Teflon-coated pipette. The cells were clamped to a holding potential of -80 mV. For measurement of substance-promoted inhibition of the Kv11.1 channel, the following voltage protocol was applied at 10-second intervals: 51 ms/-80 mV, 500 ms/+40 mV, 500 ms/-40 mV, 200 ms/-80 mV. The leakage current is subtracted by means of the P4 method. The cells were resuspended in extracellular buffer (EC) and applied to the chip. After the cell had been collected, the seal was improved by addition of a seal enhancer buffer. As soon as the whole-cell configuration had been reached, the seal enhancer buffer was washed out and replaced by extracellular buffer. The measurement started in EC for 1.5 min. DMSO (vehicle control, 0.1% of DMSO) was then applied, and the control current was recorded for 3 min. The test substance was subsequently added twice in the same concentration, and the potassium current was measured for 3.5 min in each case.
Measurement: Ki = 20000 nM (pChEMBL 4.699999809265137)
Descriptors: MW 514.03, ALogP 4.57, HBA 8, HBD 2, PSA 83.4, QED 0.37
Compound (SMILES): CC(=O)N(C)c1cnn(C(=O)N2C[C@H]3C[C@H](N(C)Cc4ccccc4Cl)C[C@H]3C2)c1
InChIKey: DHSCQPKQHALXCZ-BWTSREIZSA-N
Target: Monoglyceride lipase (SINGLE PROTEIN), Mus musculus
UniProt: O35678
Assay: In Vitro Competitive Activity-Based Protein Profiling: Proteomes (mouse brain membrane fraction or cell lysates for mouse assays; human prefrontal cortex or cell membrane fractions for human assays) (50 μL, 1.0 mg/mL total protein concentration) were preincubated with varying concentrations of inhibitors at 37° C. After 30 min, FPRh or HT-01 (1.0 μL, 50 μM in DMSO) was added and the mixture was incubated for another 30 min at 37° C. Reactions were quenched with SDS loading buffer (15 μL-4×) and run on SDS-PAGE. Following gel imaging, serine hydrolase activity was determined by measuring fluorescent intensity of gel bands corresponding to MAGL and FAAH using ImageJ 1.43u software.Preparation of Mouse Brain Proteomes from Inhibitor Treated Mice.Inhibitors were administered to wild-type C57Bl/6J by oral gavage in a vehicle of polyethylene glycol. Each animal was sacrificed 4 h following administration and brain proteomes were prepared and analyzed according to previously established methods (See Niphakis, M. J., et al. (2011) ACS Chem. Neurosci. and Long, J. Z., et al. Nat. Chem. Biol. 5:37-44).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 429.95, ALogP 3.33, HBA 5, HBD 0, PSA 61.68, QED 0.75
Compound (SMILES): C=CC(=O)N1CC[C@@H](F)[C@@H](Nc2nc(Nc3cnn(CC)c3)nc3[nH]cc(C#N)c23)C1
InChIKey: UCVZUKQNQSYEDV-CABCVRRESA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibitory Activity Assay: JAK3 and BTK kinases inhibitory activities were measured for the compounds prepared in the Examples through in vitro analysis on the ADP Glow (Glo) platform.Specifically, the inhibitory activities against JAK3 and BTK kinase were measured using a JAK3 kinase assay kit (Promega, V9441) and a BTK kinase assay kit (Promega, V9071) which were purchased from Promega. Recombinant purified human JAK3 and BTK were diluted with 1× kinase reaction buffer (JAK3: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA and 50 uM DTT/BTK: 40 mM Tris-Cl, pH 7.5, 20 mM MgCl2, 0.1 mg/mL BSA, 2 mM MnCl2 and 50 uM DTT) and added to 96 well plates (JAK3: final concentration of 4 ng per reaction/BTK: final concentration of 8 ng per reaction). The compounds prepared in the previous Examples were treated so as to be finally a 1% DMSO aqueous solution, and a substrate cocktail containing ATP (JAK3: final concentration of 5 uM/BTK: final concentration of 10 uM) and 0.2 ug/uL of Poly(Glu4, Tyr1)peptide (JAK3 and BTK final concentration) in the total 25 uL reactants was added to 96-well plates to initiate enzymatic reaction. After incubation (30° C.) for 1 hour, equivalent volume (25 uL per reaction) of ADP Glo was added and incubated (30° C.) for 40 minutes at room temperature. Then, a kinase detection reagent (50 uL per reaction) was added and incubated (30° C.) for 30 minutes at room temperature. The kinase activity was measured by chemiluminescence according to the instructions of ADP Glo kinase assay kit, and the inhibitory activity of the compounds according to the present invention was calculated.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 423.46, ALogP 2.33, HBA 8, HBD 3, PSA 127.55, QED 0.52
Compound (SMILES): CC[C@H](C)NC(=O)O[C@@H]1CC[C@H](c2cc(NC(=O)c3ccnn3CCO)n[nH]2)C1
InChIKey: VICCUAJAMMEXNX-MELADBBJSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: Biochemical Assay: The purpose of GSK3β assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. GSK3β catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-15 (5-FAM-KRREILSRRPpSYR-COOH, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:2). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Active GSK3β (H350L) was purchased from Upstate/Millipore. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 40 μM ATP (ATP Km=9.43 μM), 0.005% Tween-20, 2 μM FL-Peptide-15, and 0.6 nM GSK3β in 25 mM HEPES buffer at pH 7.5. The assay was initiated with the addition of ATP, following 15 minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 30 minutes at room temperature by the addition of 50 μL of 80 mM EDTA. The Ki value was determined from the fit of the data to the Morrison tight-binding competitive inhibition equation with the enzyme concentration as a variable.
Measurement: Ki = 45.7 nM (pChEMBL 7.340000152587891)
Descriptors: MW 404.47, ALogP 2.01, HBA 7, HBD 4, PSA 134.16, QED 0.53
Compound (SMILES): COCc1nn(Cc2ccc(Cn3ccccc3=O)cc2)cc1C(=O)NCc1cccc(OC)c1C#N
InChIKey: YBWZNXCRUYMFTB-UHFFFAOYSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: Determination of the IC50 for KLK1: KLK1 inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Sturzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human KLK1 (Callbiochem) was incubated at 25° C. with the fluorogenic substrate H-DVal-Leu-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 > 4000 nM (pChEMBL 5.3979997634887695)
Descriptors: MW 497.56, ALogP 3.1, HBA 8, HBD 1, PSA 111.17, QED 0.36
Compound (SMILES): CN1CCC(N(C)C(=O)c2ccc(Nc3nc4c(C5=CCN(C(=O)CCO)CC5)cccn4n3)cc2)CC1
InChIKey: OLUOKFCYAQMVSI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 1.48 nM (pChEMBL 8.829999923706055)
Descriptors: MW 517.63, ALogP 2.64, HBA 8, HBD 2, PSA 106.31, QED 0.5
Compound (SMILES): CC[C@H](Nc1nc(N)nc(C)c1C#N)c1nc2cccc(Cl)c2c(=O)n1N1CCN(Cc2ccc(F)cc2)CC1
InChIKey: SIBRPOVZYPMOBN-QFIPXVFZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: TR-FRET PIK3 Assay: TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIPS 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 2× Km ATP (75-500 μM), 2 μM 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 pM, and PI3Kγ at 2 nM. The compounds of Table 1 and Compound X ((S)-2,4-diamino-6-((1-(5-chloro-4-oxo-3-phenyl-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-PIPS, 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 is delay and 500 is read window).The results were normalized based on positive (1 μM wortmanin) and negative (DMSO) controls, and the IC50 values for PI3Kα, β, δ, and γ were calculated from the fit of the dose-response curves to a four-parameter equation. These assays generally produced results within 3-fold of the reported mean.
Measurement: IC50 = 23000 nM (pChEMBL 4.639999866485596)
Descriptors: MW 562.05, ALogP 3.76, HBA 10, HBD 2, PSA 128.99, QED 0.35
Compound (SMILES): Cc1nc(N2CC3CC3C2)ncc1Cn1cc(C(=O)N[C@@H]2CCc3c2nn(COCC[Si](C)(C)C)c3C)nn1
InChIKey: LXWFSNVUCGMOGD-YABDQXBESA-N
Target: Thymidine kinase, cytosolic (SINGLE PROTEIN), Homo sapiens
UniProt: P04183
Assay: Inhibition Assay: Prior to the assay, human TK1 (R&D Systems) was activated by incubation with human trypsin (Calbiochem) in a 1:10,000 ratio for 15 min at 37° C. For assaying TK1 inhibitory activity, activated TK1 (31.25 nM or 1 U/mL) was incubated at 24° C. with 0.1 mM fluorogenic substrate H-Pro-Phe-Arg-AMC (11295 from Bachem) and various concentrations of the test compound in assay buffer. Measurements were performed in a kinetic interval every 2nd minute for 16 min using a Spectramax M5 (Molecular Devices) with the following settings of the wavelength excitation of 350 nm and wavelength emission of 450 nm.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 549.76
Compound (SMILES): Cc1ncc(-c2nc(C3CCOCC3)c(NC(=O)N[C@@H]3c4cc(F)c(F)cc4C(C)(C)C[C@H]3O)cc2C)cn1
InChIKey: YFBAJNRWLYSVDN-SHQCIBLASA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Pharmacological Assay: Measurement was carried out using TrkA LanthaScreen (registered trademark) Eu Kinase Binding Assay (ThermoFisher SCIENTIFIC). To a 384 well plate (Corning), 2.5 μL of the test compound having various concentrations and diluted with Kinase buffer (ThermoFisher SCIENTIFIC) and 2.5 μL of 15 nM TrkA enzyme (ThermoFisher SCIENTIFIC) were added. Moreover, 5 μL of 3 nM Eu-anti-His Tag antibody (ThermoFisher SCIENTIFIC) and 5 μL of 30 nM Kinase (registered trademark) Tracer 236 (ThermoFisher SCIENTIFIC) were added, followed by reaction at room temperature for 60 minutes. After the reaction, the fluorescence intensity of Europium (Emission wavelength 615 nm) and TR-FRET (Emission wavelength 665 nm) with an excitation wavelength of 340 nm were measured with EnVision 2100 (PerkinElmer) to calculate the fluorescence ratio as the amount of the test compound and the TrkA enzyme bonded. The inhibitory activity (IC50 value) of each test compound was calculated with the fluorescence ratio of the well to which a solvent was added instead of the test compound being 0% and the fluorescence ratio of the well without addition of the TrkA protein being 100%.
Measurement: IC50 = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 537.61, ALogP 5.23, HBA 6, HBD 3, PSA 109.26, QED 0.42
Compound (SMILES): CCn1c(-c2cc(Cl)c(F)c(C(=O)NNS(=O)(=O)C3CCCCC3)c2)cnc1C
InChIKey: PKSUHOMXTJVQGE-UHFFFAOYSA-N
Target: Histone acetyltransferase KAT6A (SINGLE PROTEIN), Homo sapiens
UniProt: Q92794
Assay: KAT-6A Enzyme Assay: KAT-6A. Enzyme assay buffer was 50 mM Tris pH 8.0, 0.002% Tween20, 0.005% bovine skin gelatin, and 1 mM dithiothreitol (DTT). For determination of IC50values, compounds were serially diluted with 2% (v/v) DMSO in the final reaction, pre-incubating each dilution of each compound with 40 μL of assay buffer containing KAT-6A enzyme (12.5 nM final concentration). 10 μL of assay buffer containing 1 μM peptide substrate and 1 μM acetyl coenzyme A (final concentrations) was added. Reactions (50 μL total) were then carried out at 25° C. for 90 minutes. Reactions were terminated by the addition of 0.5% formic acid (final concentration), and a sample of each reaction was analyzed by SAMDI Tech, Inc. (Chicago, Ill.) using self-assembled monolayer desorption/ionization time-of-flight mass spectrometry (Mrksich, M. (2008) Mass spectrometry of self-assembled monolayers: a new tool for molecular surface science. ACS Nano 2, 7-18).
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 442.94, ALogP 3.57, HBA 5, HBD 2, PSA 93.09, QED 0.67
Compound (SMILES): COCC(COC)Nc1n[nH]c2nccc(Oc3ccc(NC(=O)c4ccnn(-c5ccc(F)cc5)c4=O)cc3F)c12
InChIKey: WBZMYAQDVHLJKG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
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 = 4900 nM (pChEMBL 5.309999942779541)
Descriptors: MW 577.55, ALogP 3.9, HBA 10, HBD 3, PSA 145.28, QED 0.21
Compound (SMILES): CNC(=O)CSc1nonc1/C(=N/O)NC1CCc2ccc(F)cc21
InChIKey: LEOBEOFKYFJQLM-UHFFFAOYSA-N
Target: Indoleamine 2,3-dioxygenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P14902
Assay: Enzyme Assay: HIS-tagged IDO1 protein was recombinantly expressed in Escherichia coli using ZYP5052 autoinduction media supplemented with 500 μM delta aminolevulinic acid for 48 hours at 16 degrees Celsius. IDO1 protein was purified using Ni2+-affinity resin and size exclusion chromatography. Purified protein was then diluted in assay buffer (50 mM Tris pH 7.0, 1% glycerol, 20 μM methylene blue, 0.05% Tween-20, 20 mM sodium ascorbate, 100 units/mL catalase to obtain a final IDO1 concentration of 40 nM. IDO1 solution (30 μM) or buffer alone (30 μM) were dispensed to wells of the assay plate using a BioRAPTR liquid dispenser (Beckman Coulter). Assay plates containing compound and IDO1 enzyme were incubated at room temperature for 30 minutes. Afterwards, 10 μL of 400 μM tryptophan in assay buffer were added to each well of the assay plate using a BioRAPTR liquid dispenser. Plates were incubated at room temperature for 60 minutes and reactions were quenched by addition of 10 μL of 0.5 M methyl isonipecotate in dimethyl sulfoxide. Plates were sealed and incubated at 37° C. for 4 hours or 50° C. for 2 h. The plates are allowed to cool and then centrifuged for 1 minute at 1000×g. The resulting fluoresence was measured in an Envision plate reader (Perkin Elmer) with a 400/25 nm excitation filter and an 510/20 nm emission filter.
Measurement: IC50 = 10410 nM (pChEMBL 4.980000019073486)
Descriptors: MW 365.39, ALogP 1.46, HBA 7, HBD 3, PSA 112.64, QED 0.24
Compound (SMILES): c1cc(N2CCN(CCCCc3nnnn3C3CCCCC3)CC2)c2ccsc2c1
InChIKey: LGNQDGZEDYGQHO-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: D2 Receptor Antagonism Activity Test: The D2 receptor antagonism activity test (The antagonism activity of test compounds on D2 receptor expressing human recombinant D2 receptor in HEK293 cells) was performed using LANCE cAMP 384 Kit (Product of USA PerkinElmer Inc.). The D2 antagonism activity of test compounds was evaluated through their inhibition on dopamine-induced decrease of cAMP production in HEK293 cells. cAMP concentration test was performed according to the method documented in the kit instructions. The concentration of the test compounds was 0.1 nM-10000 nM, risperidone was used as a positive control, IC50 value was calculated by the Excelfit software
Measurement: IC50 = 9.4 nM (pChEMBL 8.029999732971191)
Descriptors: MW 424.62, ALogP 4.54, HBA 7, HBD 0, PSA 50.08, QED 0.52
Compound (SMILES): C[C@@H](N)C(=O)N1CC[C@H]1C(=O)NCc1ccc(C(=N)N)cc1
InChIKey: QEMGPWICPGOIAI-SKDRFNHKSA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: Enzymatic Assay for Thrombin: The thrombin assay utilizes a fluorogenic peptide substrate (Boc-VPR-AMC (R&D Systems) and was run at room temperature in an assay buffer containing 20 mM Hepes, pH 7.4, 140 mM NaCl and 0.1% Tween 20. Assay parameters were adjusted such that the assay was linear with respect to time, enzyme and substrate concentrations. Under these optimized assays conditions, IC50values were equivalent to Ki values, except in a few cases of tight binding inhibitors. Cases of tight binding or possible slow binding inhibitors were handled by the methods described in Copeland R. A. (2013) Evaluation of Enzyme Inhibitors in Drug Discovery. 2nd Ed. John Wiley and Sons, Inc., Chapters 5-7.
Measurement: Ki = 1500 nM (pChEMBL 5.820000171661377)
Descriptors: MW 303.37, ALogP -0.47, HBA 4, HBD 4, PSA 125.3, QED 0.43
Compound (SMILES): O=C(c1cc(F)ccc1-n1nccn1)N1CC2CC(Nc3ncc(Cl)cn3)C1C2
InChIKey: QIVUNYIDXNJLLQ-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P56718
Assay: Radioligand Binding Assay: Human Embryonic Kidney 293 cells (HEK293) stably expressing rat orexin 1 receptor (Genebank accession number NM_001525) or Chinese ovary cells (CHO) stably expressing human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM (Hyclone, cat # SH30022), 10% FBS, 1× Pen/Strep, 1× sodium pyruvate, 10 mM HEPES, 600 μg/mL G418 and DMEM/F12 (Gibco, Cat #11039), 10% FBS, 1× Pen/Strep, 600 μg/mL G418 media, respectively on 150 cm2 tissue culture plates, washed with 5 mM EDTA in PBS (HyClone Dulbecco's Phosphate Buffered Saline 1× with Calcium and Magnesium, Cat # SH30264.01, hereafter referred to simply as PBS) and scraped into 50 ml tubes. After centrifugation (2K×G, 5 min at 4° C.), the supernatant was aspirated and the pellets frozen and stored at −80° C. Cells were resuspended in PBS in the presence of 1 tablet of protease inhibitor cocktail (Roche, Cat. #11836145001) per 50 mL. Each cell pellet from a 15 cm plate was resuspended in 10 mL, stored on ice, and homogenized for 45 sec prior to addition to the reactions. Competition binding experiments in 96 well polypropylene plates were performed using [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Moraveck Corporation, specific activity=35.3 Ci/mmol), diluted to a 10 nM concentration in PBS (4 nM final). Compounds were solubilized in 100% DMSO (Acros Organics, Cat. #61042-1000) and tested over a range of 7 concentrations (from 0.1 nM to 10 μM). The final concentration of DMSO in the reactions is equal to or less than 0.1%. Total and nonspecific binding was determined in the absence and presence of 10 μM almorexant. The total volume of each reaction is 200 μL (20 μL of diluted compounds, 80 μL of [3H]-(1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) diluted in PBS and 100 μL of the cell suspension). Reactions were run for 60 min at room temperature and terminated by filtration through GF/C filter plates (PerkinElmer, Cat. #6005174) presoaked in 0.3% polyethylenimine using the cell harvester (PerkinElmer Filtermate). The plates were washed 3 times by aspirating 30 ml PBS through the plates. Plates were dried in 55° C. oven for 60 min, scintillation fluid was added, and the radioactivity was counted on a Topcount (Packard).
Measurement: Ki = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 413.84, ALogP 2.56, HBA 7, HBD 1, PSA 88.83, QED 0.71
Compound (SMILES): Cc1ncsc1C(CNC(=O)c1c(F)cccc1Cl)N1CCC(Oc2ccccc2F)CC1
InChIKey: GSYVZCSOIPDPJP-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q64663
Assay: Screen Quest Fluo-8 No Wash Calcium Assay Kit: Ca2+ influx was measured in HEK-293 cells stably transfected with the receptor using Screen Quest Fluo-8 No Wash Calcium Assay Kit (AAt Bioquest®). Briefly, once inside the cells, the lipophilic blocking groups of Fluo-8 are cleaved by non-specific cell esterases, resulting in a negatively-charged fluorescent-dye that stays inside cells. Its fluorescence increases upon binding to calcium. When HEK-293/P2X7 cells were stimulated with BzATP, Ca2+ entered the cells and the fluorescence of Fluo-8 NW increaseed. The dye absorption spectrum was compatible with excitation at 488 nm by argon laser sources and its emission wavelength was in the range of 515-575 nm.To routinely test the compounds, HEK-293 cells stably transfected with rat P2X7R were seeded overnight in growth medium at 10000, 15000 or 20000 cells/well in 384-well plate, according to the level of response after thawing. 24 hours later, the medium was removed and the cells were pre-loaded at RT for 1 hour with 20 μL/w of Fluo-8 NW prepared in Tyrode 0.3 mM Ca2+/Mg2+-free. Compounds of the invention were tested at 8 concentrations (4 replicates for each concentration): 10-3.16-1-0.316-0.1-0.0316-0.01 and 0.00316 μM, in the same plate.Compounds were tested at FLIPRTETRA according to the following method:    first injection at FLIPRTETRA of 10 μL of 3× test compound (in Tyrode's buffer 0.3 mM Ca2+/Mg2+-free+DMSO 0.5% final concentration)    5′ incubation    second injection at FLIPRTETRA of 15 □L of 3× BzATP at ECK) (in Tyrode's buffer 0.3 mM Ca2+/Mg2+-free+BSA 0.0003% final concentration)    Fluorescence recording for 3′Between one plate and the following, tips were extensively washed with water, then with 100% DMSO and finally with water to avoid carry-over inside the tips.The effect of the test compounds was measured as percent inhibition vs a reference antagonist and IC50 values were calculated accordingly.
Measurement: IC50 = 583 nM (pChEMBL 6.230000019073486)
Descriptors: MW 491.99, ALogP 5.4, HBA 5, HBD 1, PSA 54.46, QED 0.48
Compound (SMILES): CC(C)n1cnnc1-c1cccc(N2Cc3ccc(-c4ccccc4C(=O)O)cc3C2=O)n1
InChIKey: IGMSWNVJCWUYPR-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: Kinase Activity Inhibition Assay: Inhibition of ASK1 kinase activity was determined radiometrically using 33P substrate incorporation (Reaction Biology Corp., Malvern, Pa.). Briefly, recombinant human ASK1 protein, 20 μM substrate (myelin basic protein (MBP)), and compound (diluted from DMSO stock to give 1% final DMSO) were incubated in Base Reaction buffer (20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO) at room temperature for 20 minutes. The reaction was initiated by addition of 10 μM 33P-ATP (specific activity 10 μCi/4l). The reaction was then allowed to proceed for 2 hours at room temperature. Upon reaction termination, the mixture was spotted on P81 exchange paper and kinase activity by incorporation of 33P into MBP substrate was detected by filter-binding method.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 439.48, ALogP 4.45, HBA 6, HBD 1, PSA 101.21, QED 0.49
Compound (SMILES): Nc1ccc(C2=CCCC2)cc1NC(=O)C1CCOCC1
InChIKey: SLQPKBLCLXMMNK-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: Inhibition of Histone Deacetylase Enzymatic Activity Assay: HDAC2: The following trypsin-coupled protocol and Caliper protocol described Herein was used to assay the compounds of the invention. HDAC2-Kinetic IC50 3Hr PreIncubation. Working Stocks: 1xHDAC Assay Buffer: 50 mM HEPES (GIBCO 15630-114), 100mM KCl, 0.001% Tween-20 (Zymed 00-3005), 0.005% BSA (Invitrogen, P2489), pH 7.4, 1.5xHDAC+1.5xTrypsin: 225nM Trypsin(Worthinton)+0.2 ug/ml HDAC2 (BPS Inc) in assay buffer 300x Trypsin stock is made up in HDAC Assay Buffer, aliquoted and stored at -80C. 3xSubstrate: 13.8uM Substrate in assay buffer BPS Enzyme and Substrate amounts (per well).
Measurement: IC50 = 555 nM (pChEMBL 6.260000228881836)
Descriptors: MW 286.38, ALogP 3.2, HBA 3, HBD 2, PSA 64.35, QED 0.84
Compound (SMILES): CC(=O)NCCc1ccc2c(c1)nc1n2Cc2cccc(c2)-c2ccc(=O)n(n2)Cc2cccc(c2)C(=O)N1
InChIKey: CVOQTMLCZVEEHH-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Enzymatic Assay: IRAK4 is a human purified recombinant enzyme (His-TEV-IRAK1 (194-712) IRAK4 hydrolyses ATP, autophosphorylates and phosphorylates a Serine/Threonine generic peptidic substrate (STK: 61ST1BLC from CisBio International based in Bagnols/Cèze FR).Measurement of IRAK-4 inhibition is performed in streptavidin coated 384 well FlashPlate (PerkinElmer #SMP410A). His-TEV-IRAK4 (20 ng/well), ATP (2 μM, [33P]ATP 0.25 μCi/well), STK1-biotin peptide (300 nM) and compounds in DMSO (range of concentrations from 20 μM to 1 nM) or controls (2% DMSO) are incubated for 3 hours at 30° C. in assay buffer: Hepes pH 7.0 50 mM, Fatty acid-free BSA 0.1%, Dithiothreitol DTT 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Tween-20 0.01%, MnCl2 5 mM.Kinase reaction is stopped by addition of EDTA. Supernatant is discarded, plates are washed three times with 150 mM NaCl and radioactivity is then measured in a Microbeta Trilux reader.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 518.58, ALogP 3.6, HBA 7, HBD 2, PSA 110.91, QED 0.38
Compound (SMILES): Cc1c(-c2cc3ccccc3n2Cc2ccccn2)nc2cc(C(=O)N3CCC[C@@H](N)C3)ccn12
InChIKey: YWTVIWJXOMYZPA-JOCHJYFZSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: FP Binding Assay: Binding of compounds with PAD4 was detected by FP assay. PAD4 was diluted to 1 uM in assay buffer (100 mM HEPES, 50 mM NaCl, 1 mM DTT, 5% Glycerol and 1 mM CHAPS) and added to wells containing various concentration of compounds or DMSO vehicle (1%) in a 384 well black plate. 10 nM of fluorescein labelled probe (JPAD-00085) was added to the plate. Assay plate was incubated for 60 minutes at RT before measuring FP reading at FP module (λex 485/λem 535 nm) on Pherastar. IC50 was calculated using XL-fit software model 205.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 464.57, ALogP 4.27, HBA 6, HBD 1, PSA 81.45, QED 0.43
Compound (SMILES): Cc1nc(NCc2c(F)cccc2CN2CCCC2)ccc1S(=O)(=O)Nc1cscn1
InChIKey: MAKANBDJFJSFTP-UHFFFAOYSA-N
Target: Sodium channel protein type 1 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P35498
Assay: Sodium Influx Assay (In Vitro Assay): This sodium influx assay employs the use of the cell permeable, sodium sensitive dye ANG2 to quantify sodium ion influx through sodium channels which are maintained in an open state by use of sodium channel modulators. This high throughput sodium influx assay allows for rapid profiling and characterization of sodium channel blockers.In general, Trex HEK293 cells were stably transfected with an inducible expression vector containing the full-length cDNA coding for the desired human sodium channel α-subunit and with an expression vector containing full length cDNA coding for the β1-subunit. Sodium channel expressing cell lines were induced with tetracycline (1 μg/mL) and plated on 384-well PDL-coated plates at a density of 25K-30K cells/well in culture media (DMEM, containing 10% FBS and 1% L-glutamine). After overnight incubation (37° C., 5% CO2), culture media was removed and cells were loaded with 5 uM ANG2 dye for 1-1.5h in Buffer 1 (155 mM NMDG, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted with Tris to pH 7.4). Access dye was removed and cells were incubated with test compounds for 1 hr in buffer 1 containing sodium channel modulator(s) at room temperature. Hamamatsu FDSS μCell was used to perform a 1:1 addition of Na/K challenge buffer (140 mM NaCl, 20 mM HEPES, 1 mM CaC2, 15 mM KCl, 1 mM MgCl2, 10 mM glucose, adjusted with Tris to pH 7.4) and simultaneously read plates at excitation wavelength of 530 nm and emission wavelength set at 558 nm. Percent inhibition of sodium ion influx was calculated for each test compound at each test concentration to determine the IC50 values.
Measurement: IC50 = 8162 nM (pChEMBL 5.090000152587891)
Descriptors: MW 461.59, ALogP 3.99, HBA 7, HBD 2, PSA 87.22, QED 0.53
Compound (SMILES): CCc1cc2sc(C(=O)C[C@H](C)C(=O)O)cc2cc1OC
InChIKey: GOIQQKURADBKEJ-VIFPVBQESA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: STING Biochemical [3H]cGAMP Competition Assay: The individual compounds described in the Examples herein are defined as STING agonists by (i) binding to the STING protein as evidenced by a reduction in binding of tritiated cGAMP ligand to the STING protein by at least 20% at 20 uM (concentration of compound being tested) in a STING Biochemical 13H1-cGAMP Competition Assay and (ii) demonstrating interferon production with a 6% or greater induction of IFN-β secretion at 30 uM in the THP1 cell assay (where induction caused by cGAMP at 30 uM was set at 100%).The ability of compounds to bind STING is quantified by the ability to compete with tritiated cGAMP ligand for human STING receptor membrane using a radioactive filter-binding assay. The binding assay employs STING receptor obtained from Hi-Five cell membranes overexpressing full-length HAQ STING prepared in-house and tritiated cGAMP ligand also purified in-house.
Measurement: EC50 = 14290 nM (pChEMBL 4.840000152587891)
Descriptors: MW 306.38, ALogP 3.77, HBA 4, HBD 1, PSA 63.6, QED 0.83
Compound (SMILES): Cn1cnc2ncn(Cc3nc([C@H]4[C@@H]5C=C(c6cc(Cl)ccn6)C[C@@H]54)no3)c(=O)c21
InChIKey: JNKBSIPJIAHQTP-WWGRRREGSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (90 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 90 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC50 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 31 nM (pChEMBL 7.510000228881836)
Descriptors: MW 421.85, ALogP 2.43, HBA 9, HBD 0, PSA 104.52, QED 0.5
Compound (SMILES): CCc1nc2ccc(C3CCN(C(=O)CN4CC(F)C4)CC3)cn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: PTMWHHNXRBDPIL-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Biochemical Assay: TABLE VI: (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using the fluorogenic autotaxin substrate FS-3 as substrate. FS-3 is a doubly labeled analog of LPC wherein the fluorophore is quenched through intramolecular energy transfer. Without hENPP2, the emission of the probe is quenched. If the substrate is hydrolyzed by hENPP2, the emission of the probe is not quenched anymore resulting in a fluorescence increase. Inhibition of hENPP2 by compounds will result in a decrease of the signal.10 μL of a dilution series of compound, starting from 20 μM highest concentration, 1/5 dilution, is added to the wells. hENPP2 is used at a final concentration of 0.4 μg/mL or 0.64 μg/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.0, 250 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 20 μL. Enzyme mixture is added to compounds and the resulting mixture is incubated for 30 min at room temperature under shaking. The reaction is started by the addition of 20 μL of 0.75 μM FS-3 diluted in the same buffer as described above and the mixture is incubated at 30° C. for 30 min. Fluorescence is read on the Envision (Excitation 485 nm, emission 520 nM).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 550.68, ALogP 5.29, HBA 7, HBD 0, PSA 56.98, QED 0.31
Compound (SMILES): Cc1ncc(F)cc1[C@H]1CCCN1c1ccn2ncc(C(=O)NC3(C)CC3)c2n1
InChIKey: OXVCBPXLNVQEGM-QGZVFWFLSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA ELISA Assay: An enzyme-linked immunosorbant assay (ELISA) was used to assess TrkA kinase activity in the presence of inhibitors. Immulon 4HBX 384-well microtiter plates (Thermo part #8755) were coated with a 0.025 mg/mL solution of poly (Glu, Ala, Tyr; 6:3:1; Sigma P3899). Various concentrations of test compound, 2.5 nM TrkA (Invitrogen Corp., histidine-tagged recombinant human TrkA, cytoplasmic domain), and 500 μM ATP were incubated for 25 minutes at ambient temperature in the coated plates while shaking. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The reaction mixture was removed from the plate by washing with PBS containing 0.1% (v/v) Tween 20. The phosphorylated reaction product was detected using 0.2 μg/mL of a phosphotyrosine specific monoclonal antibody (clone PY20) conjugated to horseradish peroxidase in conjunction with the TMB Peroxidase Substrate System (KPL). After the addition of 1M phosphoric acid, the chromogenic substrate color intensity was quantitated via absorbance at 450 nm. IC50 values were calculated using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 394.45, ALogP 3.2, HBA 6, HBD 1, PSA 75.42, QED 0.74
Compound (SMILES): NC(=O)c1cccc2c1OCC(c1cncc(CN3CCOC3=O)c1)O2
InChIKey: JQODTJVNGPAMJY-UHFFFAOYSA-N
Target: Cytochrome P450 11B1, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P15538
Assay: Inhibition Assay: Assays are performed in 96-well format in a final volume of 60 μL/well, containing 100 mM potassium phosphate, pH 7.4, 1% (v/v) DMSO, and additionally, 2 μM of corticosterone and 50 units of CYP11B2 activity. Reactions are started by the addition of NADPH to 1 mM and allowed to proceed for 90 minutes at 37° C. Reactions are terminated by the addition of 60 μL of MeCN containing an internal standard for mass spectrometry. One hundred microliters are then transferred to a glass filter plate and centrifuged at 570×g for 5 minutes and the filtrate is collected. Reaction product aldosterone is quantified by mass spectrometry. To determine the assay blank value (0% activity), NADPH is omitted from some reactions.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 355.35, ALogP 1.65, HBA 6, HBD 1, PSA 103.98, QED 0.89
Compound (SMILES): Cc1c(C#N)cccc1[C@@H](C)Nc1nnc(C)c2cnc(N3CC(N(C)C)C3)cc12
InChIKey: ZLLPYCJKNRWEAS-OAHLLOKOSA-N
Target: Son of sevenless homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07889
Assay: Inhibiting SOS1-Mediated GTP Nucleotide Exchange by Blocking Mant-GDP Exchange: Table 24: This Example illustrates that exemplary compounds of the present invention prevent KRas-mediated GTP nucleotide exchange mediated by SOS1 to inhibit KRas activity thereby inhibiting the generation of the downstream effector pERK. MKN1 cells (15,000/w) or H358 (30,000/w) were seeded in a black clear flat bottom 96-well cell culture plate (Corning, #3904) and incubated at 37° C. overnight. Assay day 1, cells were dosed with compounds of Formula (I) with a 10 μm starting concentration and serially diluted 3× for a total of 9 concentrations. The cells were incubated for approximately 0.5-1 hour with the compounds solubilized in DMSO at 37° C. Cells were immediately fixed by adding 50 μL of 4% formaldehyde to all wells in a fume hood and the plates were incubated for 20 minutes at room temperature. The formaldehyde was discarded from the plates and 150 μL of ice-cold methanol was added to permeabilize the cells for 10 minutes at −20° C. The methanol was discarded from each of the plates and any liquid remaining in the plate by tapping the plate against paper towels. Cells were then blocked with 150 μL of Odyssey blocking buffer (LI-COR Biosciences #927-50010) using 0.05% Tween for 1 hour at room temperature on a shaker. The blocking buffer was discarded and 50 μL of primary antibodies pERK (cell signaling Technology #9101L; Rabbit, 1:500) and GapDH (Millipore #MAB34; Mouse, 1:5000) diluted in Odyssey blocking buffer was added. The plates were incubated overnight at 4° C. on a shaker. On Assay Day 2, the primary antibody solution was removed. Each plate was washed 3× times with 150 μL of 1×PBST (PBS+0.1% Tween 20) and incubated with 50 μL of secondary antibodies: Anti-Rabbit (LI-COR Biosciences #926-32211) and Anti-Mouse (LI-COR Biosciences #68070) at 1:800 dilution in Odyssey blocking buffer with Tween at room temperature on a shaker for 2 hours (protected from light). The secondary antibody solution as removed and each plate was washed with PBST 3× times. Any liquid remaining was discarded and the plate was imaged using the Licor Odyssey machine according to the manufacturer's instruction, using a set focus length at 3 mm and both 800 nm and 700 nm filters. The GAPDH normalized scan values for each well were divided by the average of vehicle wells to get the % of pERK inhibition. The IC50 values were then calculated with the Graph pad Prism software.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 401.52, ALogP 3.44, HBA 7, HBD 1, PSA 80.97, QED 0.7