lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C=CC(=O)N1CC(NC(=O)Cn2cc(C(=O)NCCOc3c(C)cccc3C)c3cc(Br)ccc32)C1
InChIKey: SGQSERVAWXGVQF-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (2 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for either 2 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 2580 nM (pChEMBL 5.590000152587891)
Descriptors: MW 553.46, ALogP 3.34, HBA 5, HBD 2, PSA 92.67, QED 0.31
Compound (SMILES): O=C(O)c1nn[nH]c1Oc1cccc(C#Cc2cccc3[nH]ncc23)c1
InChIKey: ZBNWGOBPVSADAT-UHFFFAOYSA-N
Target: 2-Hydroxyacid oxidase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UJM8
Assay: In-Vitro Human Glycolate Oxidase (hGOX) Assay: The in-vitro glycolate oxidase assay was performed using recombinant full-length human hydroxyacid oxidase 1 (HAO1), the equivalents of hGOX. The enzyme was obtained from AbCam (Catalog #ab113144) and was purified using conventional chromatography to >95% purity. Purified HAO1 was dissolved in assay buffer consisting of 10 mM NaCl, 110 mM KCl, 2 mM MgCl2, 50 mM HEPES (pH 7.4), and 0.01% Triton X-100. The assay used Corning 3575 384-well flat bottom, low flange, non-binding surface, black polystyrene plates.Test compounds in DMSO were preincubated at different concentrations with purified recombinant human GO (6 nM) for 10 min, followed by the addition of glycolate substrate (85 μM) to start the reaction. The plates were incubated for 10 min at room temperature, at which point Amplex red reagent (50 μM) was added.The fluorescence intensity signal was measured on a VariosKan LUX instrument using an excitation of 560 nm and an emission of 590 nm. The IC50 values were calculated using Graphpad Prism.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 345.32, ALogP 2.57, HBA 5, HBD 3, PSA 116.78, QED 0.49
Compound (SMILES): COc1ccccc1-n1c(NS(=O)(=O)CCc2ccc(Cl)cc2)nnc1-c1ccc(F)s1
InChIKey: YXFXNHRXHKLRLG-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.). All the results presented are means of several independent experiments and analyzed by non-linear regression methods using the commercially available program Prism (GraphPad, San Diego, Calif.) providing the EC50 values detailed in Table 40.
Measurement: EC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 492.99, ALogP 4.78, HBA 7, HBD 1, PSA 86.11, QED 0.38
Compound (SMILES): CS(=O)(=O)c1ccc(NC(=O)N2CCn3nc(-c4ccc(F)c(Cl)c4)c(C(N)=O)c3C2)cc1
InChIKey: PCRNLQJHJYBNFL-UHFFFAOYSA-N
Target: Casein kinase I isoform epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: P49674
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 μl prepared from 15 μl additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 μl of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product. Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 μM ATP, 1.5 μM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 491.93, ALogP 2.89, HBA 6, HBD 2, PSA 127.39, QED 0.58
Compound (SMILES): CCOCc1nc(O)c(-c2nnc(Cc3noc4cccnc34)o2)c(O)c1-c1c(OC)cccc1OC
InChIKey: UQQXSRJGLQXUQN-UHFFFAOYSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyr1)apelin-13) peptide, which was set to 100%.The examples disclosed below were tested in the APJ in vitro assays described above and were found having human APJ cyclic AMP (hcAMP) activity. The EC50 value of each compound is presented at the end of the example description.
Measurement: EC50 = 5.5 nM (pChEMBL 8.260000228881836)
Descriptors: MW 505.49, ALogP 3.89, HBA 12, HBD 2, PSA 158.88, QED 0.3
Compound (SMILES): COC[C@]12CC[C@@](C)(O)C[C@H]1CC[C@H]1[C@@H]3CC[C@H](C(=O)Cn4nnc5cc(Cl)cnc54)[C@@]3(C)CC[C@@H]12
InChIKey: NWESTZPKTBDNON-CFLHAHIFSA-N
Target: Gamma-aminobutyric acid type B receptor subunit 1 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9Z0U4
Assay: Steroid Inhibition of TBPS Binding: TBPS binding assays using rat brain cortical membranes in the presence of 5 μM GABA has been described (Gee et al, J. Pharmacol. Exp. Ther. 1987, 241, 346-353; Hawkinson et al, Mol. Pharmacol. 1994, 46, 977-985; Lewin, A. H et al., Mol. Pharmacol. 1989, 35, 189-194).Briefly, cortices are rapidly removed following decapitation of carbon dioxide-anesthetized Sprague-Dawley rats (200-250 g). The cortices are homogenized in 10 volumes of ice-cold 0.32 M sucrose using a glass/teflon homogenizer and centrifuged at 1500×g for 10 min at 4° C. The resultant supernatants are centrifuged at 10,000×g for 20 min at 4° C. to obtain the P2 pellets. The P2 pellets are resuspended in 200 mM NaCl/50 mM NaK phosphate pH 7.4 buffer and centrifuged at 10,000×g for 10 min at 4° C. This washing procedure is repeated twice and the pellets are resuspended in 10 volumes of buffer. Aliquots (100 μL) of the membrane suspensions are incubated with 3 nM [35S]-TBPS and 5 μL aliquots of test drug dissolved in dimethyl sulfoxide (DMSO) (final 0.5%) in the presence of 5 μM GABA. The incubation is brought to a final volume of 1.0 mL with buffer. Nonspecific binding is determined in the presence of 2 μM unlabeled TBPS and ranged from 15 to 25%. Following a 90 min incubation at room temp, the assays are terminated by filtration through glass fiber filters (Schleicher and Schuell No. 32) using a cell harvester (Brandel) and rinsed three times with ice-cold buffer. Filter bound radioactivity is measured by liquid scintillation spectrometry. Non-linear curve fitting of the overall data for each drug averaged for each concentration is done using Prism (GraphPad). The data are fit to a partial instead of a full inhibition model if the sum of squares is significantly lower by F-test. Similarly, the data are fit to a two component instead of a one component inhibition model if the sum of squares is significantly lower by F-test. The concentration of test compound producing 50% inhibition (IC50) of specific binding and the maximal extent of inhibition (Imax) are determined for the individual experiments with the same model used for the overall data and then the means±SEM.s of the individual experiments are calculated. Picrotoxin serves as the positive control for these studies as it has been demonstrated to robustly inhibit TBPS binding.Various compounds are or can be screened to determine their potential as modulators of [35S]-TBPS binding in vitro. These assays are or can be performed in accordance with the above discussed procedures.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 515.1, ALogP 5.09, HBA 7, HBD 1, PSA 90.13, QED 0.6
Compound (SMILES): CC1(C(=O)N2CCN[C@@H](c3nc(-c4ccc(C(=O)Nc5cc(C(F)(F)F)ccn5)cc4)c4c(N)nccn34)C2)CC1
InChIKey: MFKXMGVLUDJTRJ-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Enzyme Activity Assay B: BTK enzymatic activity was determined in an IMAP-FP assay (Immobilized Metal ion Affinity-based fluorescence Polarization; Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, final compound concentration range in assay from 10 μM to 0.508 nM) titration curve using the following outlined procedure. To each well of a black Corning 384-well microplate (Corning Catalog #3575), 200 nL of compound (100 fold dilution in final assay volume of 20 μL) was dispensed, followed by the addition of 10 μL of 1× kinase buffer (10 mM Tris pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM MnCl2, and 1 mM DTT) containing 0.16 ng/μL (2.1 nM) of BTK enzyme (recombinant protein from baculovirus-transfected Sf9 cells: full-length BTK, 6HIS-tag cleaved). Following a 60 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 μL kinase buffer containing 100 nM Blk/Lyntide IMAP substrate peptide (5-carboxyfluorescein-EFPIYDFLPAKKK-NH2; Molecular Devices Catalog #R7188), and 10 μM ATP. The final reaction in each well of 20 μL consists of 1.05 nM hBTK, 50 nM Blk/Lyntide IMAP substrate, and 5 μM ATP. Phosphorylation reactions were allowed to proceed for 120 minutes and were immediately quenched by the addition of 40 μL IMAP detection beads resuspended in IMAP progressive binding buffer (beads were diluted 1:600 in 75% and 25% progressive binding buffer A and B, respectively; Molecular Devices). Plates were read on Packard microplate reader after 60 minutes binding equilibration using Fluorescence Polarization protocol. Specifically, fluorescence at 535 nm is measured using emission filters oriented both parallel and perpendicular to the polarized excitation filter. This allows detection of changes in Fluorescence Polarization due to binding of the phosphorylated substrate peptide to the beads. Values are calculated as percentage of the difference in readout (ΔmP) of the controls with and without hBTK enzyme. IC50 values are determined by curve fitting of the experimental results using Activity Base.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 564.57, ALogP 3.92, HBA 8, HBD 3, PSA 130.54, QED 0.34
Compound (SMILES): O=C(O)COc1ccc2c(c1)N(C(=O)Cc1ccc(-c3n[nH]c(=O)c4ccccc34)cc1)CC2
InChIKey: GDCGZXVOEQJVGM-UHFFFAOYSA-N
Target: Rho-associated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13464
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 455.47, ALogP 3.19, HBA 5, HBD 2, PSA 112.59, QED 0.46
Compound (SMILES): Cc1cc(Nc2cc(C(F)F)cc(N3CC(F)(C(=O)NCc4ccc(-n5cc(F)cn5)nc4)C3)n2)n[nH]1
InChIKey: CXSOZKDRXHKNEG-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Wild Type Assay at KM: In each well of a 384-well plate, 7.5 nM-10 nM of wild type RET (ProQinase 1090-0000-1) was incubated in a total of 12.5 μL of buffer (100 mM HEPES pH 7.5, 0.015% BriJ 35, 10 mM MgCl2, 1 mM DTT) with 1 μM CSKtide (FITC-AHA-KKKKD DIYFFFG-NH2) (SEQ ID NO: 5) and 25 μM ATP at 25° C. for 120 minutes in the presence or absence of a dosed concentration series of compound (1% DMSO final concentration). The reaction was stopped by the addition of 70 μL of Stop buffer (100 mM HEPES pH 7.5, 0.015% BriJ 35, 35 mM EDTA and 0.2% of Coating Reagent 3 (Caliper Lifesciences)). The plate was then read on a Caliper EZReader 2 (protocol settings: −1.7 psi, upstream voltage −500, downstream voltage −3000, post sample sip 35 s). Data was normalized to 0% and 100% inhibition controls and the IC50 calculated using a 4-parameter fit in the CORE LIMS.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 515.48, ALogP 3.36, HBA 8, HBD 3, PSA 116.65, QED 0.31
Compound (SMILES): Cc1cccc2c(=O)[nH]c(CSC3CCN(C(=O)CCN)CC3)nc12
InChIKey: MXONQWINCZSMBQ-UHFFFAOYSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 360.48, ALogP 1.8, HBA 5, HBD 2, PSA 92.08, QED 0.85
Compound (SMILES): CCn1c(-c2cccnc2[C@H](C)OC)c2c3cc(ccc31)-c1cc(O)cc(c1)C[C@H](NC(=O)[C@H](C(C)C)N(C)C(=O)[C@H]1CCN(C(=O)C#CCN3[C@@H](C)COC[C@@H]3C)C1)C(=O)N1CCC[C@H](N1)C(=O)OCC(C)(C)C2
InChIKey: UCAHTEITWCOOLI-CSZAFGDPSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Biochemical Assay: A variety of Ras proteins may be inhibited by compounds of the present invention (e.g., K-Ras, N-Ras, H-Ras, and mutants thereof at positions 12, 13 and 61, such as G12C, G12D, G12V, G12S, G13C, G13D, and Q61L, and others described herein). The purpose of this biochemical assay is to measure the ability of test compounds to covalently label nucleotide-loaded K-Ras isoforms. In assay buffer containing 12.5 mM HEPES pH 7.4, 75 mM NaCl, 1 mM MgCl2, 1 mM BME, 5 uM Cyclophilin A and 2 uM test compound, a 5 uM stock of GMP-PNP-loaded K-Ras (1-169) G12C is diluted 10-fold to yield a final concentration of 0.5 uM; with final sample volume being 100 uL. The sample is incubated at 25�� C. for a time period of up to 24 hours prior to quenching by the addition of 10 uL of 5% Formic Acid. Quenched samples are centrifuged at 15000 rpm for 15 minutes in a benchtop centrifuge before injecting a 10 uL aliquot onto a reverse phase C4 column and eluting into the mass spectrometer with an increasing acetonitrile gradient in the mobile phase. Analysis of raw data may be carried out using Waters MassLynx MS software, with % bound calculated from the deconvoluted protein peaks for labeled and unlabeled K-Ras.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 1029.29
Compound (SMILES): CCCOc1cc(-n2ccc(C3COB(O)C3)n2)ccc1OC
InChIKey: GHPKSQCKGHRBGJ-UHFFFAOYSA-N
Target: N-formyl peptide receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25090
Assay: Cytokine Assays: Cytokine inhibitory activity is determined by measuring the effect of test agent on the release of the cytokines IL-4, IL-13 and IFNγ from human peripheral blood mononuclear cells (PBMCs) stimulated with the T-cell mitogen phytohemagglutinin-L (PHA-L). At the time of assay, human PBMCs (Astarte Biologics) are removed from cryopreservation, thawed quickly at 37° C., diluted in assay medium (RPMI-Gibco 1640 medium, 10% heat inactivated fetal bovine serum (HIFBS), 2 mM glutamine and 10 mM HEPES pH 7.4) and then centrifuged at 250×g for 5 minutes. The resulting cell pellet is re-suspended in assay medium to a concentration of approximately 5×106 cells/mL and 50 μL of this cell suspension (approximately 250,000 cells) is added to each well of a 384-well cell culture microtiter plate (Perkin Elmer). Varying concentrations of test compound are then diluted in assay medium and added to the assay plate wells in a volume of 25 μL. After −10 minutes at room temperature (21° C.) the cells are stimulated by the addition of 25 μL of PHA-L (14 μg/mL; Millipore) and the assay plate is placed in an incubator at 37° C. in a humidified environment in 5% carbon dioxide. The final assay conditions are approximately 250,000 human PBMCs per well in assay medium containing 3.5 μg/mL PHA-L and the indicated final concentration of test compound (approximately 10 μM to 9.5 μM by 4-fold dilution). The final concentration of DMSO in the assay is approximately 0.25%. After 48 hours, the assay plate is removed from the incubator and centrifuged at 250×g for 5 minutes. A portion of the resulting cell supernatant is then used to determine the amount of IL-4, IL-13 and IFNγ in each well. Cytokine measurements are made using human IL-4, IL-13 or IFNγ HTRF assay kits (CisBio) following the manufacturer's assay protocol. The concentrations and resulting effect values for tested compounds are plotted and the concentration of compound required for 50% effect (IC50) is determined with a four-parameter logistic dose response equation (E-WorkBook, ID Business Solutions Ltd.).
Measurement: IC50 = 12.9 nM (pChEMBL 7.889999866485596)
Descriptors: MW 316.17
Compound (SMILES): O=c1[nH]c(NCc2ccccc2)nc2c1cnn2C1CCCC1
InChIKey: AEYQRMFSCXRCPC-UHFFFAOYSA-N
Target: High affinity cGMP-specific 3',5'-cyclic phosphodiesterase 9A (SINGLE PROTEIN), Homo sapiens
UniProt: O76083
Assay: Inhibition Assay: Inhibition activities of all the N-substituted pyrazolo [3,4-d] pyrimidine ketone compounds according to the present invention to the phosphodiesterase IX are tested.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 309.37, ALogP 2.85, HBA 5, HBD 2, PSA 75.6, QED 0.78
Compound (SMILES): CC(C)=CC(CC(C)(C)c1ccc(C(F)(F)F)cc1)N1CCC(O)CC1
InChIKey: LAFQLJGWEFOSBF-UHFFFAOYSA-N
Target: Sigma non-opioid intracellular receptor 1 (SINGLE PROTEIN), Cavia porcellus
UniProt: Q60492
Assay: Competitive Radioligand Binding Assay 2: The affinity of test compounds at sigma-1 and sigma-2 receptors was also determined by displacement of different known labeled sigma-2 or sigma-1 ligands. Filtration assays were conducted according the previously published procedure (Xu, et al., 2005). Test compounds were dissolved in N,N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or ethanol and then diluted in 50 mM Tris-HCl pH 7.4 buffer containing 150 mM NaCl and 100 mM EDTA. Membrane homogenates were made from guinea pig brain for sigma-1 binding assay and rat liver for sigma-2 binding assay. Membrane homogenates were diluted with 50 mM Tris-HCl buffer, pH 8.0 and incubated at 25° C. in a total volume of 150 uL in 96 well plates with the radioligand and test compounds with concentrations ranging from 0.1 nM to 10 uM. After incubation was completed, the reactions were terminated by the addition of 150 uL of ice-cold wash buffer (10 mM Tris HCl, 150 mM NaCl, pH 7.4) using a 96 channel transfer pipette (Fisher Scientific, Pittsburgh, Pa.) and the samples harvested and filtered rapidly through 96 well fiber glass filter plate (Millipore, Billerica, Mass.) that had been presoaked with 100 uL of 50 mM Tris-HCl buffer. Each filter was washed four times with 200 uL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4). A Wallac 1450 MicroBeta liquid scintillation counter (Perkin Elmer, Boston, Mass.) was used to quantitate the bound radioactivity.
Measurement: Ki = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 369.47, ALogP 5.16, HBA 2, HBD 1, PSA 23.47, QED 0.72
Compound (SMILES): Cc1nc(-c2ccccc2C(C)C)nc2c1cnn2Cc1ccc(-c2nc(C(F)(F)F)cn2C)cc1
InChIKey: KTCDCWKVKTYLED-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O94782
Assay: Inhibitor Assay: Deubiquitinase activity was measured using ubiquitin-rhodamine 110 as a substrate. Cleavage of the amide bond between rhodamine and the c-terminal glycine of ubiquitin yields an increase in fluorescence signal. The assay was conducted in 20 ul total volume of assay buffer (50 mM Tris-HCl, pH 7.8, 0.5 mM EDTA, 0.01% Bovine Serum Albumin, 1 mM DTT, 0.01% Tween-20), and 0.05 nM USP1/UAF1 enzyme. Reaction was initiated by addition of 150 nM Ubiquitin-rhodamine (Boston Biochem) substrate.Compounds, dissolved in DMSO were tested in dose response format, beginning at 10 uM.Compounds depicted below were added to enzyme/assay buffer mix and incubated 10 min. Substrate mix was added, and reaction mix was read in kinetic mode for 30 min at Ex480/Em540 and IC50 response curves were plotted.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 490.53, ALogP 6.39, HBA 6, HBD 0, PSA 61.42, QED 0.29
Compound (SMILES): CC(C)Oc1ccc(C(OC(=O)N(Cc2ccc(F)cc2)C2CCN(C)CC2)C(F)(F)F)cc1
InChIKey: HVCKPENRUUCWPI-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 2A (SINGLE PROTEIN), Homo sapiens
UniProt: P28223
Assay: Receptor Selection and Amplification (R-SAT) Assays: R-SAT's were generally performed with 50 ng/well of receptor and 20 ng/well of β-galactosidase plasmid DNA. All receptor constructs used were in the pSI mammalian expression vector (Promega Inc) as described previously. The 5-HT2A receptor gene was amplified by nested PCR (polymerase chain reaction) from brain cDNA using the oligodcoxynuclcotides based on the published sequence (Saltzman et al., Biochem. Biophys. Res. Comm. 1991, 181, 1469). For large-scale transfections, cells were transfected for 12-16 h, then trypsinized and frozen in DMSO. Frozen cells were later thawed, plated at 10,000-40,000 cells per well of a 96 well plate that contained a compound according to Formula (I). To run functional antagonist assays, cells and compounds were additionally combined with a fixed concentration (approximately 3× the previously determined EC50) of an agonist (usually 5-CT) at 5-HT2A or other appropriate agonists for other receptors. With both methods, cells were then grown in a humidified atmosphere with 5% ambient CO2 for five days. Media was then removed from the plates and marker gene activity was measured by the addition of the b-galactosidase substrate o-nitrophenyl b-D-galactopyranoside (ONPG, in PBS with 5% NP-40). The resulting colorimetric reaction was measured in a spectrophotometric plate reader (Titertek Inc.) at 420 nM. All data were analyzed using the computer program XLFit (IDBSm). Efficacy is the percent maximal repression compared to repression by a control compound (ritanserin in the case of 5-HT2A). pIC50 is the negative of the log(IC50), where IC50 is the calculated concentration in molar that produces 50% maximal repression. The compounds as provided herein were assayed as described herein. Compounds of Formulas (I) and (II), demonstrated high inhibition of the 5-HT2A receptor activity as shown in the table below. This data below indicates that compounds as provide herein may be useful as pharmaceutical agents.
Measurement: Ki = 31.6 nM (pChEMBL 7.5)
Descriptors: MW 482.52, ALogP 5.95, HBA 4, HBD 0, PSA 42.01, QED 0.46
Compound (SMILES): COc1ccc([C@H](C)Nc2nc3nc(C)cc(C4CCOCC4)n3n2)cc1
InChIKey: FNMVOXDUJFBNEB-AWEZNQCLSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: IMAP TR-FRET-based phosphodiesterase assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 367.45, ALogP 3.51, HBA 7, HBD 1, PSA 73.57, QED 0.74
Compound (SMILES): Cc1nc2ncnn2c(N)c1Cc1ccc(Br)c(OC(F)F)c1
InChIKey: PEJFNXJQZLEXON-UHFFFAOYSA-N
Target: Membrane-bound ghrelin O-acyltransferase MBOAT4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96T53
Assay: Pharmacological Activity Assay: Methods: Cells are plated with a density of 5000 cells/well in 384-well poly-D-lysin plates and incubated for 1 day at 37° C., 5% CO2 in DMEM medium, 10% FCS, 1×NEAA, Puromycin (0.5 μg/ml) and G418 (1 mg/ml). Then the medium is changed to a identical medium without FCS and containing Octanoate-BSA (final concentration 100 μM each) and compound in DMSO (final DMSO concentration 0.3%). After incubation for 5 hours acylghrelin in the medium is measured by ELISA. The medium sample is diluted 1:25 in Elisa buffer, a 25 μl aliquot is transferred to a 384-well ELISA plate previously washed 4 times with 100 μL wash buffer, and 25 μl tracer-solution is added. After incubation overnight (20 h) at 4° C. temperature the plate is washed 4 times with 100 μl wash-buffer per well. Finally 50 μl Ellman's reagent is added to each well and the plate is incubated in the dark for 20 minutes. The absorbance is measured at 405 nm in an Envision multilabel reader and the amount of acylated ghrelin is calculated according to a acylated ghrelin standard curve provided in the same plate. Each assay plate contains wells with vehicle controls (1% DMSO) for the measurement of non-inhibited transfer reaction (=100% Ctl) and wells with 10 μM ([Dap3]-Ghrelin) as controls for fully inhibited GOAT enzyme.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 384.18, ALogP 2.97, HBA 6, HBD 1, PSA 78.33, QED 0.75
Compound (SMILES): N#Cc1c(N)nc(N)nc1N[C@H](c1nc2c(F)ccc(CCCN3CCCC3)c2c(=O)n1-c1ccccc1)C1CC1
InChIKey: SXBAVVZKRLYUCT-DEOSSOPVSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: TR-FRET assay : TR-FRET monitored the formation of 3,4,5-inositol triphosphate molecule that competed with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin homology domain protein. An increase in phosphatidylinositide 3-phosphate product resulted in a decrease in TR-FRET signal as the labeled fluorophore was displaced from the GRP-1 protein binding site. Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 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, Compound X ((S)-2-(1-((9H-purin-6-yl)amino)ethyl)-5-chloro-3-phenylquinazolin-4(3H)-one) and Compound Y ((S)-4-amino-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. Additionally, compounds 19 to 116 were added to the assay solution and incubated for 30 minutes at 25° C. The reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 μs delay and 500 μs read window).
Measurement: IC50 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 553.65, ALogP 3.94, HBA 10, HBD 3, PSA 151.77, QED 0.28
Compound (SMILES): OCC1O[C@H](Sc2cnc(C(F)(F)F)c(Br)c2)C(O)[C@@H](n2cc(-c3cc(F)c(F)c(F)c3)nn2)[C@H]1O
InChIKey: QTJVBNJMWATIRF-VFAFTYBQSA-N
Target: Galectin-3 (SINGLE PROTEIN), Homo sapiens
UniProt: P17931
Assay: fluorescence anisotropy assay : The affinity of Example 1-106 for galectins were determined by a fluorescence anisotropy assay where the compound was used as an inhibitor of the interaction between galectin and a fluorescein tagged saccharide probe as described Sörme, P., Kahl-Knutsson, B., Huflejt, M., Nilsson, U. J., and Leffler H. (2004) Fluorescence polarization as an analytical tool to evaluate galectin-ligand interactions. Anal. Biochem. 334: 36-47, (Sörme et al., 2004) and Monovalent interactions of Galectin-1 By Salomonsson, Emma; Larumbe, Amaia; Tejler, Johan; Tullberg, Erik; Rydberg, Hanna; Sundin, Anders; Khabut, Areej; Frejd, Torbjorn; Lobsanov, Yuri D.; Rini, James M.; et al, From Biochemistry (2010), 49(44), 9518-9532, (Salomonsson et al., 2010). The assay was also adapted to be able to measure the high affinity of compounds for galectin-3 by using the below probe constructed to have high affinity for galectin-3 which made it possible to use a low concentration of galectin-3 (50 nM). 100 nM albumin was included as a carrier to prevent protein loss at such low concentration of galectin.
Measurement: Kd = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 601.32, ALogP 3.31, HBA 9, HBD 3, PSA 113.52, QED 0.3
Compound (SMILES): COc1cccc(Oc2ccc(/N=c3\[nH]cc(C(N)=O)c(=O)n3Cc3ccc(Cl)cc3)cc2C)n1
InChIKey: IXDJVXWVLFEKRN-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 384-well microtiter plate at a concentration of 3000 cells/well and cultured in the medium (7.0% fetal bovine serum, 7.0% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37° C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 μM Fluo-3-AM solution (20 mM HEPES, 1.37 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic F-127, pH 7.5) and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH7.5), and each well was added with 20 μL of this buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 384 was started, and 20 μL of DMSO solutions containing different concentrations of the test compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 5.27 mM KCl, 0.9 mM MgCl2, 1.26 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.1% Pluronic F-127, pH 7.5) were dispensed to each well through the built-in automatic dispenser. Five minutes after, 150 nM ATP solution (25 μL) prepared by dilution with the dilution buffer was dispensed through the built-in automatic dispenser, and the measurement of fluorescence intensity was continued for 4 min. For each well, the specific maximum fluorescence intensity was calculated as the ratio of the maximum fluorescence intensity after addition of the ATP solution to the fluorescence intensity at the starting of the measurement. The 50% inhibitory concentration (IC50) was calculated under the assumption that the specific maximum fluorescence intensity without test compound is 0% inhibition and that the specific maximum fluorescence intensity when the dilution buffer was added in place of ATP solution is 100% inhibition, to evaluate the inhibitory activity of the test compound.
Measurement: IC50 = 99 nM (pChEMBL 7)
Descriptors: MW 491.94, ALogP 3.71, HBA 7, HBD 2, PSA 124.59, QED 0.41
Compound (SMILES): CCOC(=O)C1CCCC(n2nc(-c3ccc(CNC(=O)c4ccccc4OC)cc3)c3c(N)ncnc32)C1
InChIKey: FPLDSFVCJJWFOY-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: BTKWT Binding Affinity: BTKWT binding affinity of each compound tested was determined using a time-resolved fluorescence resonance energy transfer (TR-FRET) methodology. 2.5 nM Recombinant BTKWT kinase, varying concentrations of inhibitor, 2 nM LanthaScreen Eu anti-His Antibody and 15 nM Kinase Tracer 236 was incubated in 1× LanthaScreen Kinase Buffer A for 5 h. Recombinant BTK kinase and all LanthaScreen components were purchased from Invitrogen. Measurements were performed in a reaction volume of 30 μL using half-area 96-well assay plates. The TR-FRET signal was read on a plate reader with an excitation wavelength of 340 nm and detection wavelengths of 615 and 665 nm. Binding affinity was determined for each compound by measuring TR-FRET signal at various concentrations of compound and plotting the relative fluorescence units against the inhibitor concentration to estimate the IC50 from log [Inhibitor] vs response using the Variable Slope model in Graphpad prism from Graphpad software (SanDiego, Calif.).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 528.61, ALogP 4.31, HBA 9, HBD 2, PSA 134.25, QED 0.32
Compound (SMILES): CCS(=O)(=O)c1ccc(CNC(=O)c2ccc3c(c2)nc([C@H]2C[C@H]2C#N)n3Cc2cccc3c2OC(F)(F)O3)cc1
InChIKey: GJLSTTJUPCFLFP-UNMCSNQZSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: Binding Assay : Compounds described herein were tested for ability to bind to RORγ in a cell-free competition assay with commercially available radio-ligand (RL), 25-hydroxy [26,27-3H]-cholesterol (PerkinElmer, Cat. #NET674250UC), for a ligand binding site on a recombinant RORγ Ligand Binding Domain (LBD) protein expressed as a 6×His-Glutathione-S-Transferase (GST) fusion. The assay was performed in 96-well SPA plates (PerkinElmer, Cat. #1450-401) in 50 mM HEPES buffer, pH 7.4, containing 150 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 2 mM CHAPS, 0.5 mM β-octylglucopyranoside and 5 mM DTT. Tested compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions of the compounds were prepared in the same solvent. Two μL of the DMSO solutions were mixed with 28 μL of 8.6 nM 25-hydroxy [26,27-3H]-cholesterol and 50 μL of 24 nM RORγ LBD. The plate was shaken at 700 rpm for 20 min and incubated for 10 min at rt, after which 40 μL of poly-Lys YSi SPA beads (PerkinElmer, Cat. #RPNQ0010) were added to achieve 50 ag of the beads per well. The plate was incubated on an orbital shaker for 20 min and then for 10 min without agitation at rt. SPA signal for tritium beta radiation was registered on PerkinElmer Microbeta plate reader. Percent inhibition values were calculated based on the high signal obtained with DMSO control and the low signal observed with 10 μM standard RORγ inverse agonist T0901317 (SigmaAldrich, Cat. #T2320). The percent inhibition vs. concentration data were fit into a four-parameter model, and IC50 values were calculated from the fit as the concentrations corresponding to the inflection points on the dose-response curves.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 578.6, ALogP 4.76, HBA 8, HBD 1, PSA 123.31, QED 0.32
Compound (SMILES): C[C@H](c1ccc(C(F)(F)F)cc1)N1CC[C@H](C2CC(Br)=NO2)C1
InChIKey: CTBBFFROWIEUEU-RBOJZIDYSA-N
Target: Transcriptional enhancer factor TEF-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28347
Assay: TEAD Inhibition Assay: TEAD inhibition can be assayed using Hippo Pathway TEAD ReporterMCF7 Cell Line (BPS Bioscience, Catalog #: 60618).
Measurement: EC50 > 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 391.23, ALogP 4.59, HBA 3, HBD 0, PSA 24.83, QED 0.75
Compound (SMILES): Cc1nc(F)ccc1[C@H](Nc1cc(C#N)c2ncc(C#N)c(NCC(C)(C)C)c2c1)C1=CN(C2CC2)NN1
InChIKey: GYQHEZZYJFLULD-MHZLTWQESA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: High Throughput Biochemical Assay: EGFR activity was measured using KinEASE (Cisbio), a time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay. In this assay, EGFR-catalyzes the phosphorylation of a universal Tyrosine kinase peptide substrate labeled with XL665. Europium conjugated phosphor-tyrosine specific antibody binds the resulting phosphorylated peptide. Formation of phosphorylated peptide is quantified by TR-FRET with Europium as the donor and XL665 the acceptor. The assay was performed in two main steps. The first step is the kinase reaction step and the second step is the detection step with TR-FRET reagents. In brief, test compounds 1:3 serially diluted in DMSO were delivered into Corning white, low volume, non-binding 384 well plates using the Echo 550 acoustic liquid dispenser (Labcyte®). EGFR enzyme (Human EGFR, cytoplasmic domain [669-1210] from Carna Biosciences Cat. No. 08-115) and substrates TK substrate-biotin (included in Cisbio HTRF KinEASE-TK kit Cat. No. 62TK0PEJ) were dispensed into assay plates using a Multi-Flo (Bio-Tek Instruments). The standard 10 μL reaction mixture contained 6 μM ATP (1×Km) or 12 μM ATP (2×Km), 1 μM biotinylated peptide, 0.3 nM EGFR (for 1×Km ATP) or 0.1 nM EGFR (for 2×Km ATP) in reaction buffer (10 mM MOPs, pH 7.0, 1.5% Glycerol, 0.5 mg/ml BSA, 10 mM Mg-Acetate, 1 mM DTT, 0.025% NP-40). After 60 min of incubation at room temperature, 10 μL of Stop and Detect Solution (1:400 Europium Cryptate labeled anti-phosphorylated peptide antibody solution and 125 nM strepavidin-XL665 Tracer in a 50 mM Hepes pH 7.0 detection buffer containing sufficient EDTA) was added. The plate was then further incubated for over 60 minutes at room temperature and read using an Envision 2103 Multilabeled reader (PerkinElmer) with excitation/emission/FRET emission at 340 nm/615 nm/665 nm, respectively.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 511.61, ALogP 4.76, HBA 9, HBD 4, PSA 124.72, QED 0.33
Compound (SMILES): N#Cc1cc(F)cc(-c2ccc3oc([C@@H]4CCCN4C#N)nc3c2)c1
InChIKey: NJEIYSNAEOYJRQ-KRWDZBQOSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: Biochemical IC50 Assay: Dilution plates were prepared at 21 times the final concentration (2100 μM for a final concentration of 100 μM) in 50% DMSO in a 96-well polypropylene V-bottom plate (Greiner #651201). A typical 8-point dilution series would be 100, 30, 10, 3, 1, 0.3, 0.1, 0.03 μM final. Reactions were performed in duplicate in black 384 well plates (small volume, Greiner 784076) in a final reaction volume of 21 μl. Either 1 μl of 50% DMSO or diluted compound was added to the plate. USP30 was diluted in reaction buffer (40 mM Tris, pH 7.5, 0.005% Tween 20, 0.5 mg/ml BSA, 5 mM beta-mercaptoethanol) to the equivalent of 0.05 μl/well and 10 μl of diluted USP30 was added to the compound. Enzyme and compound were incubated for 30 min at room temp. Reactions were initiated by the addition of 50 nM of TAMRA labelled peptide linked to ubiquitin via an iso-peptide bond as fluorescence polarisation substrate. Reactions were read immediately after addition of substrate and following a 2 h incubation at room temperature. Readings were performed on a Pherastar Plus (BMG Labtech). λ Excitation 540 nm; λ Emission 590 nm.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 332.34, ALogP 4.12, HBA 5, HBD 0, PSA 76.85, QED 0.66
Compound (SMILES): CN1CC[C@@](O)(C#Cc2cccc(-n3nc(C(N)=O)c4scnc43)c2)C1=O
InChIKey: FBGIDECZYBLUTL-SFHVURJKSA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: NIK Enzyme Inhibition Assay: The ability of the nuclear factor-kappa B (NF-kB)-inducing kinase (NIK) to catalyze the hydrolysis of adenosine-5′-triphosphate (ATP) was monitored using the Transcreener ADP (adenosine-5′-diphosphate) assay (BellBrook Labs). Purified NIK (0.5 nM) derived from a baculovirus-infected insect cell expression system was incubated with test compounds for 1-3.5 hours in 50 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid buffer (pH 7.2) containing 10 mM MgCl2, 2 mM dithiothreitol, 10 μM ATP, 0.01% Triton X-100, 0.1% gamma-globulins from bovine blood, 1% dimethylsulfoxide (DMSO), 7 μg/mLADP antibody and 5 nM ADP-MR121 633 tracer. Reactions were quenched by the addition of 20 mM 2,2′,2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid and 0.01% Brij 35. The tracer bound to the antibody was displaced by the ADP generated during the NIK reaction, which causes a decrease in fluorescence polarization that was measured by laser excitation at 633 nm with a Fluorescence Correlation Spectroscopy Plus reader (Evotec AG). Equilibrium dissociation constant (Ki) values for NIK inhibitors are calculated from plots of activity vs. inhibitor concentration using Morrison's quadratic equation that accounts for the potential of tight binding, and by also applying the conversion factor that accounted for competitive inhibition and the concentration of substrate used in the assay relative to its Michaelis constant (Km).
Measurement: Ki = 0.31 nM (pChEMBL 9.510000228881836)
Descriptors: MW 381.42, ALogP 0.53, HBA 7, HBD 2, PSA 114.34, QED 0.63
Compound (SMILES): COc1cc2nc3c(c(N[C@H]4CCCN(C)C4)c2cc1OC)CCC3
InChIKey: KHCLGFBLXGULLQ-ZDUSSCGKSA-N
Target: Histone-lysine N-methyltransferase EHMT2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KQ7
Assay: Enzymatic Activity Assay: For the assay, the following buffer was used to set up reactions: 50 mM Tris-HCl pH9, 50 mM NaCl, 0.01% Tween-20 and 1 mM DTT (added fresh prior to starting the reactions). The assay is set up by adding a final concentration of 0.15 nM G9a, 15 uM S-adenosyl-methionine and 100 nM biotinylated histone 3 peptide (1-21). The reaction is incubated at rt for 1 hour and subsequently quenched with the acceptor beads (anti-H3k9me2 AlphaLISA acceptor beads, Perkin Elmer #AL117) at a final concentration of 20 ug/mL. The acceptor beads are incubated for 1 hour. After 1 hour, the donor beads are added at a final concentration of 20 ug/mL (Alpha Streptavidin donor beads, PerkinElmer #6760002). Donor beads are incubated for 0.5 hours. Both donor and acceptor beads are resuspended in AlphaLISA 5X Epigenetics Buffer 1 Kit (PerkinElmer #AL008) prior to addition to the reaction. All manipulations and incubations with the donor and acceptor beads are done in subdued light. Signal is detected in an EnVision plate reader in Alpha mode.
Measurement: IC50 = 14.8 nM (pChEMBL 7.829999923706055)
Descriptors: MW 341.46, ALogP 3.25, HBA 5, HBD 1, PSA 46.62, QED 0.92