lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cn1cc(-c2ccc3cnc(NC(=O)CN4CC[C@H](F)C4)cc3c2)cn1
InChIKey: AHVVQOPDAHKCJF-KRWDZBQOSA-N
Target: Proto-oncogene Wnt-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P04628
Assay: The screening assay for Wnt activity: The screening assay for Wnt activity is described as follows. Reporter cell lines can be generated by stably transducing cancer cell lines (e.g., colon cancer) or primary cells (e.g., IEC-6 intestinal cells) with a lentiviral construct that includes a Wnt-responsive promoter driving expression of the firefly luciferase gene.SW480 colon carcinoma cells were transduced with a lentiviral vector expressing luciferase with a human Sp5 promoter consisting of a sequence of eight TCF/LEF binding sites. SW480 cells stably expressing the Sp5-Luc reporter gene and a hygromycin resistance gene were selected by treatment with 150 μg/mL of hygromycin for 7 days. These stably transduced SW480 cells were expanded in cell culture and used for all further screening activities. Each compound was dissolved in DMSO as a 10 mM stock and used to prepare compound source plates. Serial dilution (1:3, 10-point dose-response curves starting from 10 μM) and compound transfer was performed using the ECHO 550 (Labcyte, Sunnyvale, Calif.) into 384-well white solid bottom assay plates (Greiner Bio-One) with appropriate DMSO backfill for a final DMSO concentration of 0.1%. For Sp5-Luc reporter gene assays, the cells were plated at 4,000 cells/well in 384-well plates with a DMEM medium containing 1% fetal bovine serum, and 1% Penicillin-Streptomycin and incubated for 36 to 48 hours at 37° C. and 5% CO2. Following incubation, 15 μl of BriteLite Plus luminescence reagent (Perkin Elmer) was added to each well of the 384-well assay plates. The plates were placed on an orbital shaker for 2 min and then luminescence was quantified using the Envision (Perkin Elmer) plate reader. Readings were normalized to DMSO only treated cells, and normalized activities were utilized for EC50 calculations using the dose-response log (inhibitor) vs. response variable slope (four parameters) nonlinear regression feature available in GraphPad Prism 5.0 (or Dotmatics).
Measurement: EC50 = 3700 nM (pChEMBL 5.429999828338623)
Descriptors: MW 353.4, ALogP 2.62, HBA 5, HBD 1, PSA 63.05, QED 0.78
Compound (SMILES): Cc1nc2nonc2c(N)c1Cc1cccc(C(C)(C)C#N)n1
InChIKey: AJOUAWCIWIHYAN-UHFFFAOYSA-N
Target: Membrane-bound ghrelin O-acyltransferase MBOAT4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96T53
Assay: hGOAT Activity in HEK293 Cells after Incubation: 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 ELISAThe medium sample is diluted 1:25 in Elisa buffer, A 25 ul 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 enzymeThe analysis of the data is performed by calculation of the percentage of acyl-ghrelin produced in the presence of test compound compared to the amount of acyl-ghrelin produced in the vehicle control samples. An inhibitor of the GOAT enzyme will give values between 100% CTL (no inhibition) and 0% CTL (complete inhibition). IC50 values are calculated with Assay Explorer or other suited software based on curve fitting of results of 8 different compound concentrations.
Measurement: IC50 = 0.48 nM (pChEMBL 9.319999694824219)
Descriptors: MW 308.35, ALogP 2.3, HBA 7, HBD 1, PSA 114.51, QED 0.79
Compound (SMILES): CC1(C)CCC(CN2CCN(c3ccc(C(=O)NS(=O)(=O)c4ccc(OCC5(F)CCN(C6COC6)CC5)c([N+](=O)[O-])c4)c(Oc4cnc5[nH]ccc5c4)c3)CC2)=C(c2ccc(Cl)cc2)C1
InChIKey: OBDIOCGTOVVKBJ-UHFFFAOYSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: TR-FRET (Time-Resolved Fluorescence-Resonance-Energy-Transfer) assay: TR-FRET (Time-Resolved Fluorescence-Resonance-Energy-Transfer) assay.
Measurement: Ki < 0.01 nM (pChEMBL 11)
Descriptors: MW 942.51, ALogP 8.4, HBA 12, HBD 2, PSA 172.47, QED 0.08
Compound (SMILES): CCc1nc2ccc(C3CCN(CC(=O)N4CC(CO)C4)CC3)cn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: QTJAMAFKHQUIGW-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: Human ENPP2 (hENPP2) Assay: Compound IC50 values are determined in a hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using LPC as substrate. 5 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 1 μg/mL or 3 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 8.5, 500 mM NaCl, 5 mM KCl, 10 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 10 μL. the reaction is started by the addition of 10 μL of 150 μM LPC (palmitoyl 16:0) diluted in the same buffer as described above and the mixture is incubated at 37° C. for 30 min. The reaction is terminated and choline quantified by the addition of a 25 μL of a mixture containing 0.6 U/mL of choline oxidase, 0.6 U/mL of peroxydase, 1.8 mM TOOS, 1.2 mM amino-antipyrine, 20 mM EGTA (stop-developer solution) diluted in the buffer described above. Luminescence is read on the Envision after an incubation of 30 min at room temperature (Excitation 555 nm, excitation light=70%).
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 562.72, ALogP 4.56, HBA 8, HBD 1, PSA 77.21, QED 0.34
Compound (SMILES): COc1cccc(-c2nnc(N(CC[Si](C)(C)C)S(=O)(=O)C[C@H](O)c3ccc(F)cn3)n2-c2c(OC)cccc2OC)n1
InChIKey: BYLVMELSMPPODN-QFIPXVFZSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPγS 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 assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 M GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 M GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.) to obtain EC50.
Measurement: EC50 = 0.32 nM (pChEMBL 9.489999771118164)
Descriptors: MW 630.78
Compound (SMILES): CNCCC(Oc1cccc2ccncc12)c1ccc(C(F)(F)F)nc1
InChIKey: BJLVBEIYYZAPSE-UHFFFAOYSA-N
Target: Voltage-dependent calcium channel subunit alpha-2/delta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P54289
Assay: Binding Assay: Human α2δ-1 enriched membranes (2.5 μg) were incubated with 15 nM of radiolabeled [3H]-Gabapentin in assay buffer containing Hepes-KOH 10 mM, pH 7.4.NSB (non specific binding) was measured by adding 10 μM pregabalin. The binding of the test compound was measured at five different concentrations. After 60 min incubation at 27° C., binding reaction was terminated by filtering through Multiscreen GF/C (Millipore) presoaked in 0.5% polyethyleneimine in Vacuum Manifold Station, followed by 3 washes with ice-cold filtration buffer containing 50 mM Tris-HCl, pH 7.4. Filter plates were dried at 60° C. for 1 h and 30 μl of scintillation cocktail were added to each well before radioactivity reading.Readings were performed in a Trilux 1450 Microbeta radioactive counter (Perkin Elmer).
Measurement: Ki > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 361.37, ALogP 4.38, HBA 4, HBD 1, PSA 47.04, QED 0.71
Compound (SMILES): Nc1ncnc2c1c(-c1[nH]c3cc(C(=O)NC4CC4)ccc3c1Cl)nn2C1CCOC1
InChIKey: QDIPGXPRBSWHHQ-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: Enzyme Assay: Kinase activity was detected using CisBio HTRF kinEASE kit based on time-resolved fluorescence transfer (FRET). The assay was performed in 384-well white plates (Corning #3574) in a reaction volume of 10 μL containing 1× CisBio enzymatic buffer supplemented with a final concentration of 5 mM MgCl2, 1 mM DTT, 10 nM SEB and 0.01% Triton X100 for RET. The same buffer conditions were used for KDR with the addition of 2 mM MnCl2. RETV804M buffer used 1× CisBio enzymatic buffer supplemented with a final concentration of 2 mM MgCl2, 1 mM DTT, 20 nM SEB and 0.01% Triton X100.
Measurement: IC50 = 9.88 nM (pChEMBL 8.010000228881836)
Descriptors: MW 437.89, ALogP 3.06, HBA 7, HBD 3, PSA 123.74, QED 0.45
Compound (SMILES): Cc1ccc(-c2cc(N3CCOC[C@H]3C)nc3c(-c4ccn[nH]4)nccc23)c(F)n1
InChIKey: JPJAHPRMACPWEW-CQSZACIVSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Time-Resolved Fluorescence Energy Transfer (TR-FRET Tracer A): To determine of binding activity of the test compounds, full-length human ATR protein was expressed and purified together with ATRIP as described above. Furthermore, a fluorescently labelled compound (tracer A) was used as a tracer molecule. Detection of the binding event of the tracer was achieved by time-resolved fluorescence energy transfer (TR-FRET). We used an anti-GST-Terbium antibody (CisBio) that binds to the GST-tag at the N-terminus of ATR-kinase. Excitation of Terbium with 337 nm light results in emission of fluorescent light with 545 nm. In case a tetrameric complex has formed (antiGST-Tb+GST-ATR+Strp2-ATRIP+tracer), part of the energy will be transferred from the Terbium to the fluorophore that itself emits light of 570 nm. Displacement of the fluorescent tracer by a test compound leads to a reduction of the TR-FRET-signal.For the assay 50 nl of a 100-fold concentrated solution of the test compound in DMSO was pipetted into a black low volume 384well microtiter plate (MTP, Greiner Bio-One, Frickenhausen, Germany). To prepare the ATR-working solution, ATR/ATRIP stock solution was diluted in assay buffer [50 mM HEPES (pH 7.0), 10 mM MgCl2, 1 mM DTT, 0.01% (w/v) Igepal, 0.01% (w/v) BSA] to 4.2 nM protein concentration (concentration may vary from lot to lot of protein preparation). AntiGST-Tb antibody was diluted to 4.2 nM. The ATR-working solution was incubated for 30 min at 22° C. prior to dispensing to pre-form the complex of antiGST-Tb+GST-ATR+ATRIP. Then, 3 μl of the ATR-working solution were added to the test compound and the mixture was incubated for 10 min at 22° C. to allow pre-binding of the test compounds to ATR/ATRIP. Then, 2 μl of a 100 nM solution of either tracer A or B in assay buffer were added to the ATR-working solution. The resulting mixture was incubated for 30 min at 22° C. The measurement of the TR-FRET signal was performed in a standard HTRF-compatible MTP reader instrument (e.g. BMG Pherastar) by recording the fluorescence emissions at 545 nm and 570 nm after excitation at 337-350 nm. The ratio between emission at 570 nm divided by emission at 545 nm was calculated to give the well ratio. The experimental data (well ratios) were normalised by the following way: positive control contained ATR-working solution plus either tracer A or B solution (=0% inhibition), the negative control contained all components except GST-ATR/ATRIP (=100% inhibition).
Measurement: IC50 = 10.9 nM (pChEMBL 7.960000038146973)
Descriptors: MW 404.45, ALogP 3.75, HBA 6, HBD 1, PSA 79.82, QED 0.52
Compound (SMILES): Cc1ncc(NC(=O)c2cccc(C3CC3NCC3CCOCC3)c2)s1
InChIKey: OPDRXEFPTJAKHS-UHFFFAOYSA-N
Target: Lysine-specific histone demethylase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: O60341
Assay: LSD1 Inhibition Assay: A test compound dissolved in 2.5% DMSO was added by 4 μL to 3 μL reaction solution (50 mM Tris-HCl (pH 8.0), 0.1% BSA, 1 mM DTT) containing 7.5 ng of LSD1, and the mixture was reacted at room temperature for 60 min. Biotin-histone H3 mono methylated K4 peptide solution (NH2-ART(me-K)QTARKSTGGKAPRKQLAGGK(Biotin)-CONH2) (3.3 μM) was added by 3 μL to start the reaction. After reaction at room temperature for 5 min, 1 mM 2-PCPA solution was added 5 μL to terminate the reaction. A detection solution (BOO mM potassium fluoride, 0.1% BSA) containing europium-labeled anti-histone H3 antibody (Wako Pure Chemical Industries, Ltd.) and Streptavidin-XL665 (Cisbio) was further added by 5 μL, and the mixture was left standing for 60 min. A time-resolved fluorescence (excitation 320 nm, emission 615 nm, 665 nm) was measured by Envision (PerkinElmer). The LSD1 inhibitory rate (%) of the test compound was calculated by the following formula.inhibitory rate(%)=(1−(test compound count−blank)÷(control−blank))×100
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 371.51, ALogP 3.58, HBA 5, HBD 2, PSA 63.25, QED 0.81
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3ccc(-c4ccc(C(=O)N(C)C)cc4)cc3)nn2C)C1
InChIKey: HWPRLXIVOJPGBP-UHFFFAOYSA-N
Target: CREB-binding protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q92793
Assay: TR-FRET Binding Assay: His/Flag epitope tagged CBP was cloned, expressed, and purified to homogeneity. CBP binding and inhibition was assessed by monitoring the engagement of a biotinylated small molecule compound with the target using the TR-FRET assay technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate CBP (4 nM final) was combined with biotin-ligand (60 nM final) in 50 mM HEPES (pH 7.5), 50 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.2% DMSO) or compound dilution series in DMSO. After 10 minutes incubation at room temperature, a mixture Eu-W1024 Anti-6×His antibody (6×His disclosed as SEO ID NO: 3) (Perkin Elmer AD0110) and SureLight Allophycocyanin-Streptavidin (APC-SA, Perkin Elmer CR130-100) were added to a final concentrations of 0.2 nMolar antibody and 50 nMolar APC-SA, respectively. After twenty minutes of equilibration, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 417.51, ALogP 3.44, HBA 5, HBD 1, PSA 70.47, QED 0.71
Compound (SMILES): Nc1nc2cc(-c3ccc4ncc(=O)[nH]c4c3)ccc2o1
InChIKey: HMEZXLJRWWPYKX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: LanthaScreen Kinase Assay: The kinase reaction is performed in a white 384 well microtitre plate. The total reaction volume is 20 ul per well and the reaction buffer composition is 50 mM HEPES pH7.5, 0.01% Polysorbate 20, 1 mM EGTA, 10 mM MnCl2, and 2 mM DTT. In the first step, each well receives 2 ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration. Next, 8 ul of mTOR diluted in reaction buffer is added per well for a 60 ng/ml final concentration. To start the reaction, 10 ul of an ATP/GFP-4EBP1 mixture (diluted in reaction buffer) is added per well for a final concentration of 10 uM ATP and 0.5 uM GFP-4EBP1. The plate is sealed and incubated for 1 hour at room temperature. The reaction is stopped by adding 10 ul per well of a Tb-anti-pT46 4EBP1 antibody/EDTA mixture (diluted in TR-FRET buffer) for a final concentration of 1.3 nM antibody and 6.7 mM EDTA. The plate is sealed, incubated for 1 hour at room temperature, and then read on a plate reader set up for LanthaScreen TR-FRET.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 278.27, ALogP 2.31, HBA 5, HBD 2, PSA 97.8, QED 0.56
Compound (SMILES): CC[C@@H](NC(=O)c1cc(=O)[nH]c(Cc2ccc(OC)c(F)c2)n1)c1ccc(OC(F)(F)F)cc1
InChIKey: OSMXUCDTWIMEOP-QGZVFWFLSA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 479.43, ALogP 4.29, HBA 5, HBD 2, PSA 93.31, QED 0.47
Compound (SMILES): Cc1ccc(NC(=O)c2ccc(CN3CCNCC3)cc2)cc1Nc1nccc(-c2cncc(-c3ccccn3)c2)n1
InChIKey: JSAIPLUIDULEAD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Kinase Assay: Kinase base buffer (50 mM HEPES, pH 7.5 0.0015% Brij-35; 10 mM MgCl2 2 mM DTT) and Stop buffer, (100 mM HEPES, pH 7.5 0.015% Brij-35; 0.2% Coating Reagent (50 mM EDTA) are prepared. Test compound is diluted in 100% DMSO to 50-times the desired final inhibitor concentration (the Stock Solution) and serially diluted in half-log increments resulting in final concentrations 250 μM to 75 μM, 25 μM, 7.5 μM, 2.5 μM, 0.75 μM, 0.25 μM, 75 nM, 25 nM, 7.5 nM in DMSO. 10 μl of each compound is placed in a 96-well plate as the intermediate plate. 90 μl of Kinase Buffer is added to to each well to prepare the intermediate plate.Figure US10906896-20210202-P00001Mix the compounds in intermediate plate for 10 min on shaker. For the assay of enzyme inhibitions, 5 μl of each well from the intermediate plate is transferred to a 384-well plate in duplicates, 10. Then 10 μl of 2.5× enzyme solution is added to each well of the 384-well assay plate and incubated for 10 min. Then enzyme substrate is added as 10 μl of 2.5×FAM-labeled peptide+ATP solution to each well of the 384-well assay plate. The reaction is allowed to proceed at 28° C. and quenched with the addition of 25 μl of stop buffer.
Measurement: IC50 = 203 nM (pChEMBL 6.690000057220459)
Descriptors: MW 556.67, ALogP 5.31, HBA 8, HBD 3, PSA 107.96, QED 0.24
Compound (SMILES): CC(C)(C)c1noc(C23CCC(CN(C(=O)C45CC(F)(C4)C5)c4cccc(-c5ccc(NS(C)(=O)=O)cc5)c4)(CC2)CC3)n1
InChIKey: HSPSTKUBMPXVRL-UHFFFAOYSA-N
Target: Bile acid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RI1
Assay: transient human FXR/Gal4-luciferase reporter assay: A Gal4-hFXR fusion construct reporter system was used as the primary assay to characterize compound activity. A construct including 5 copies of the Gal4 promoter response element upstream of a firefly luciferase reporter cDNA was stably expressed in HEK293 cells. This reporter cell line was maintained in Dulbecco's Modified Eagle's medium (DMEM; Gibco) supplemented with 1% penicillin-streptomycin (P/S) solution, 500 μg/mL Zeocin and 10% charcoal/dextran-treated fetal bovine serum (cs-FBS) at 37° C. in a humidified 5% CO2 atmosphere. Another plasmid was constructed in which the human cytomegalovirus promoter in the pcDNA3.1 vector directs the expression of the cDNA encoding a fusion protein comprised of the DNA binding domain from the Gal4 transcription factor fused to the ligand binding domain from human FXR.The day prior to transfection, the reporter cells in culture are detached from the plate with trypsin and plated into a T75 flask at a sufficient density to achieve approximately 90% confluence the next morning. The transfection reagents are prepared by separately diluting 25 μg of the pcDNA3.1-Gal4-FXR plasmid into 1.87 mL of Opti-MEM (Thermo-Fisher), and 40 μL of Lipofectamine 2000 (Thermo-Fisher) into 1.87 mL of Opti-MEM, and then adding the diluted DNA solution into the diluted Lipofectamine 2000 solution and incubating at room temperature for 15-20 minutes. The mixture is further diluted with 10 mL of a solution comprised of DMEM, 10% cs-FBS, and 1% P/S immediately prior to transferring to the cells. The maintenance culture media is aspirated from the cells and the final transfection mixture is added before the cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. This protocol can be scaled up, and the transiently transfected cells can be cryopreserved in an assay-ready format.For compound testing, 100 nL of the compounds (serial dilutions in DMSO) are dispensed with an Echo acoustic dispenser (Labcyte) into the wells of a Corning/Costar clear bottom 384-well white plate. The transfected cells are harvested, counted, and diluted such that 10-25,000 cells in 25 μL are plated into each well of the 384-well compound assay plate. The compound-treated cells are incubated overnight at 37° C. in a humidified 5% CO2 atmosphere. The next morning 25 μL of Steady-Glo (Promega) are added to each well of the plate, the mixture is incubated for 15 min. with shaking, and luminescence is measured on an Envision (Perkin Elmer) plate reader. Background counts from cells treated with DMSO alone are subtracted from all raw counts, and the corrected values are converted to a percentage of the control response attained with 8 μM GW-4064. These data are fit to a 4-parameter log agonist-response equation to calculate an EC50 value.
Measurement: EC50 = 843 nM (pChEMBL 6.070000171661377)
Descriptors: MW 620.79, ALogP 6.92, HBA 6, HBD 1, PSA 105.4, QED 0.29
Compound (SMILES): COc1ncnc(-c2cnn(C)c2C(=O)Nc2cc3nc(-c4ccccc4)nn3cc2F)c1C
InChIKey: HNKMWNJVVZKLRX-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Phosphodiersterase Evaluation Assay: IMAP TR-FRET Phosphodiersterase Evaluation Assay Kit (Molecular Device) was used for the measurement. Ten μL of a diluted test compound having each concentration and 5 μL of the enzyme PDE10 (BPA Bioscience) that had been diluted to 2 ng/mL with 1×IMAP Reaction Buffer (prepared with 5×included with the kit, 10 mM Tris-HCl, pH=7.2, 10 mM MgCl2, 0.05% NaN3, and 0.1% BSA) containing 0.1% BSA were added to a 384-well plate (Corning). The obtained mixture was then pre-incubated at room temperature for 5 minutes. Thereafter, 5 μL of cAMP substrate solution which was included with the kit and diluted to 400 nM with 1×IMAP Reaction Buffer containing 0.1% BSA, was added to the reaction solution, and the obtained mixture was reacted at room temperature for 60 minutes. Thereafter, 60 μL of an IMAP TR-FRET Binding solution included with the kit was further added to the reaction solution, and the obtained mixture was then left for 3 hours or more. Subsequently, using ARVO SX (PerkinElmer), the fluorescent intensity of Terbium (Emission=490 nm) and TR-FRET (Emission=520 nm) in the reaction solution were measured at an excitation wavelength of 340 nm, so as to calculate the amount of the generated 5′-AMP. The count of a well to which a solvent had been added instead of the test compound was set at 0%, and the count of a well to which the enzyme PDE10 had not been added was set at 100%. Thus, the inhibitory activity of each test compound was calculated.
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 458.46, ALogP 3.3, HBA 9, HBD 1, PSA 112.12, QED 0.43
Compound (SMILES): CC(C)n1nc(Nc2nccc(-c3cc(C#N)c4c(c3)[C@@](C)(CO)CN4)n2)cc1C(=O)NC1CC1
InChIKey: DCNIYQPESFNOKH-RUZDIDTESA-N
Target: Mitogen-activated protein kinase kinase kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99558
Assay: Inhibition of auto-phosphorylation of recombinant human NF-kappaB-inducing kinase (NIK/MAP3K14) activity: NIK/MAP3K14 auto-phosphorylation activity was measured using the AlphaScreen® (αscreen) format (Perkin Elmer). All compounds tested were dissolved in dimethyl sulfoxide (DMSO) and further dilutions were made in assay buffer. Final DMSO concentration was 1% (v/v) in assays. Assay buffer was 50 mM Tris pH 7.5 containing 1 mM EGTA (ethylene glycol tetraacetic acid), 1 mM DTT (dithiothreitol), 0.1 mM Na3VO4, 5 mM MgCl2, 0.01% Tween® 20. Assays were carried out in 384 well Alphaplates (Perkin Elmer). Incubations consisted of compound, 25 microM Adenosine-5′-triphosphate (ATP), and 0.2 nM NIK/MAP3K14. Incubations were initiated by addition of GST-tagged NIK/MAP3K14 enzyme, carried out for 1 h at 25° C. and terminated by addition of stop buffer containing anti-phospho-IKK Ser176/180 antibody. Protein A Acceptor and Glutathione-Donor beads were added before reading using an EnVision® Multilabel Plate Reader (Perkin Elmer). Signal obtained in the wells containing blank samples was subtracted from all other wells and IC50's were determined by fitting a sigmoidal curve to % inhibition of control versus Log10 compound concentration.
Measurement: IC50 = 2.6 nM (pChEMBL 8.59000015258789)
Descriptors: MW 472.55, ALogP 3.1, HBA 9, HBD 4, PSA 140.78, QED 0.41
Compound (SMILES): C[C@@H](C1CC1)N1Cc2cc(-c3cnc4c(N)nc(-c5cn[nH]c5)cn34)cc(S(C)(=O)=O)c2C1=O
InChIKey: UGUBPXSVDONJPV-LBPRGKRZSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 ��L. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3K�� assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 ��M PIP2, 2 ��M ATP, 0.5 ��Ci [��-33P]ATP, 3.4 nM PI3K��. Reactions were incubated for 120 min and terminated by the addition of 40 ��L SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (PerkinElmer).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 477.55, ALogP 2.53, HBA 8, HBD 2, PSA 139.34, QED 0.45
Compound (SMILES): CN1CCC(NC(=O)C(C)(C)COc2ccc(F)cc2)CC1
InChIKey: GRZOBDCYGKQKSV-UHFFFAOYSA-N
Target: Somatostatin receptor type 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P31391
Assay: Inhibition of Forskolin Stimulated cAMP in Cells Overexpressing SSTR4: This cell-based assay measures the ability of compounds to inhibit forskolin stimulated cAMP in CHO-K1 cells overexpressing SSTR4. CHO-K1 cells overexpressing SSTR4 (CHO-SSTR4) are purchased from DiscoveRx (product code 95-0059C2). The CHO-SSTR4 cells are maintained in F12K media with 10% Fetal Bovine Serum (Hyclone), 1% Pen/Strep (Life Technologies), and 800 μg/mL G418 (Life Technologies). To perform the assay, 3000 cells are plated per well in white 384-well plate (Corning 3570) in 50 μL complete media and the cells are allowed to attach for 16 hours in a 37° C., 5% CO2 incubator. The next day, the culture media is removed from the cells and the cells are washed (added then removed) with Kreb's Ringer Buffer (ZenBio, KRB-1000 mL). Test compounds are suspended in DMSO and diluted in stimulation buffer: Kreb's Ringer Buffer plus 0.5% BSA (Roche), 300 μM IBMX (Sigma), and 350 nM forskolin (Sigma). The cells are incubated in L compound/stimulation buffer for 30 minutes at room temperature. Cellular cAMP levels are detected with a HTRF LANCE Ultra cAMP kit (Perkin Elmer, catalog number TRF0264).The assay is performed in accordance with the manufacturer's instructions. Five μL of diluted Eu-W8044 labeled streptavidin (dilution: 1:50 in cAMP Detection Buffer) is added to each well. Then 5 μL of diluted biotin cAMP (dilution: 1:150 in cAMP Detection Buffer) is added to each well. The plates are covered and allowed to incubate for 60 minutes at room temperature on a shaker. HTRF (665 nm/615 nm) is read on a Perkin Elmer ENVISION plate reader. The pEC50 values are generated using Activity Base for Screening Data Management.
Measurement: EC50 = 398 nM (pChEMBL 6.400000095367432)
Descriptors: MW 308.4, ALogP 2.44, HBA 3, HBD 1, PSA 41.57, QED 0.91
Compound (SMILES): Cc1nn(-c2ccccc2F)c(N)c1C(=O)Nc1ccc(C(F)(F)F)cn1
InChIKey: DLCCFCROKVWBFD-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: Nav1.7 and Nav1.5 Screening Assay: Cell LinesStable cell lines expressing the full-length protein of the voltage-gated sodium channels Nav1.7 or Nav1.5, with or without one beta-1, beta-2, beta-3, or beta-4 subunit, are created by transfected CHO cells or HEK293 cells, or any other suitable cell line, with a vector construct containing the complete open reading frame under a suitable promoter, as well known in the art.In Vitro ElectrophysiologyElectrophysiological studies are performed with an IonWorks Quattro (Molecular Devices Corp.) automated patch-clamp electrophysiology platform as described by (Schroeder K et al. Journal of Biomolecular Screening 2003, 8 (1), 50-64). Buffers for the experiments have the following composition (mM): Internal solution; K-gluconate 100, KCl 40, MgCl2 3.2, HEPES 5, EGTA 3, pH 7.3. To this amphotericin B is added to final concentration of 0.1 mg/ml to generate access solution. External solution; Dulbecco's Phosphate buffered saline (D-PBS) NaCl 137.93, KCl 2.67, KH2PO4 1.47, Na2HPO4 8.06, CaCl2 0.90, MgCl2 0.49. Prior to the experiment the cells expressing the voltage-gated ion channel of interest are detached from the tissue culture flasks, centrifuged and resuspended in D-PBS. Compounds are prepared and serially diluted in DMSO and finally diluted 1:100 in D-PBS. Cells are exposed to compounds through the pipetting system integrated into the platform and the voltage-gated ion channel of interest is activated with specific voltage stimulation protocols. The following voltage stimulation protocol is used for testing compounds against the voltage-gated sodium channel Nav1.7; from a holding potential of −100 mV a train of eight 60 ms depolarising steps to −20 mV at a frequency of 14 Hz are employed followed by a further step to −20 mV for 2000 ms. After which the voltage is returned to −100 mV for 10 ms before another voltage step to −20 mV for 60 ms is applied. The following voltage stimulation protocol is used for testing compounds against the voltage-gated sodium channel Nav1.5; from a holding potential of −120 mV a train of twenty-six 120 ms depolarising steps to −20 mV at a frequency of 5 Hz are employed. Recordings are made before and after compound addition with the compound incubation time being 5 minutes.Percent block was calculated for each concentration in duplicate for peak 1 and peak 10 and peak 1 and 25 for Nav 1.7 and Nav 1.5 respectively in order to assess compound activity at close and inactivated states and IC50 curves were fitted to percent block as a function of concentration.
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 379.32, ALogP 3.57, HBA 5, HBD 2, PSA 85.83, QED 0.68
Compound (SMILES): CC(C)(O)c1cnnn1[C@H]1C[C@@H](C(=O)NC(CCCCNC(=O)OCc2ccccc2)C(=O)C(N)=O)N(C(=O)[C@@H](CC2CCCCC2)NC(=O)c2ccc(C(N)=O)cc2)C1
InChIKey: SMINWORMBXUMEV-KZRJWHRGSA-N
Target: Serine protease HTRA1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92743
Assay: Full Length HTRA1 Assay: Serial dilutions (1/3) from 1000 μM down to 0.051 μM of test compounds were prepared in dimethyl sulfoxide (DMSO). Then 2 μL of solution from each dilution were added to 100 μL of 4 nM full-length human His-HTRA1 in assay buffer (50 mM Tris, pH 7.5, 200 mM NaCl and 0.25% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate or CHAPS) in white non-binding 96-well plates. The assay solutions were mixed for 5 seconds on a shaker plate and incubated for 10 minutes at room temperature. Mca-H2OPT (Mca-Ile-Arg-Arg-Val-Ser-Tyr-Ser-Phe-Lys(Dnp)-Lys-OH trifluoroacetate salt) (Mca=7-methoxycoumarin-4-acetic acid; Dnp=dinitrophenyl) (5 μM) in 100 μL of assay buffer was added to the assay solutions. The reaction mixture was shaken for 5 seconds on a shaker plate and cleavage of Mca-H2OPT was monitored by spectrofluorometry (SpectraMax M3 by Molecular Devices, CA) for 10 minutes (Exλ=330 nm; Emλ=420 nm). Percent inhibition was calculated by fitting values to a standard mathematical model for determining the dose response curve.
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 829.96, ALogP 2.15, HBA 12, HBD 6, PSA 271.03, QED 0.08
Compound (SMILES): CN(C)C/C=C/C(=O)Nc1ccc(Oc2ncnc(N)c2-c2ccc(Oc3ccccc3)cc2)cc1
InChIKey: LMGOUZIGCIJXPG-RMKNXTFCSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzyme Assay: The following describes a microfluidic, off-chip mobility shift kinase assay used to measure inherent potency of compounds against BTK enzyme. Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent BTK Enzyme Assay IC50. These IC50 values are reported in ranges wherein: A<100 nM, B<1 uM, and C>1 uM.2.5× stocks of full-length human BTK (08-080) from CarnaBio USA, Inc., Natick, Mass., 1.6×ATP and appropriate kinKDR peptide substrate (FITC-AHA-EEPLYWSFPAKKK-NH2) were prepared in kinase reaction buffer consisting of 25 mM MgCl2, 0.015% Brij-35 (30%), 100 mM Hepes, pH 7.5, and 10 mM DTT.5 uL of enzyme buffer and 7.5 uL of ATP/kinKDR peptide substrate mix were added to Matrix (#115304) 384-well, sterile, polypropylene plates (Thermo Fisher Scientific, Hudson, N.H.) with 125 nL of serially diluted compounds prepared in 100% DMSO, and incubated for 90 min. at 27 C. Following the incubation period, reactions were stopped by adding 60 uL stop buffer consisting of 100 mM Hepes, pH 7.5, 0.015% Brij-35 (30%), 0.277% Coating Reagent #3 (Caliper Life Sciences, Mountain View, Calif.), 5% DMSO. Stopped reactions were monitored at −2 PSI, −3000 V/−700 V in a LabChip 3000 plate reader from Caliper Life Sciences, a PerkinElmer Company (Hopkinton, Mass.), and the activity was measured by off-chip mobility shift assay measuring the charge/mass difference between substrate and product resulting from peptide phosphorylation. IC50 and efficacy were determined by plotting log [Inhibitor] vs. % Activity in GeneData Screener (Basel, Switzerland). Compounds described by embodiments of the present invention were assayed using this protocol and the data from the same is recorded in Table 2 within the column labeled: Time Dependent PBMC BTK Enzyme Assay IC50.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 481.56, ALogP 5.37, HBA 7, HBD 2, PSA 102.6, QED 0.31
Compound (SMILES): Cc1cc(=O)cc(C)n1/C=C/c1ccc(OC(F)F)c(O[C@H]2CCSC2)c1
InChIKey: OSYBYWBWSUYNBA-UABRLCRWSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Determination of the inhibition of the compounds on PDE4B1 activity by using cAMP HTRF assay: The inhibitory effect of the compounds on human PDE4B1 enzyme activity was determined by quantifying the 5′-adenosine monophosphate (5′-AMP) formed from 3′,5′-cyclic adenosine monophosphate (cAMP).The test compound or water (control) and human recombinant PDE4B1 enzyme (4.8 U) were mixed in a buffer solution (pH 7.4) consisting of 1× Hanks' balanced salt solution (HBSS), 5 mM HEPES, 3 mM MgCl2, and 0.1% BSA and incubated for 10 min. A cAMP enzyme substrate (final concentration 40 nM) was added, and the mixture was incubated at room temperature for 60 min. Then a fluorescent acceptor (Dye2 labeled with cAMP), a fluorescent donor (anti-cAMP antibody labeled with europium cryptate) and a non-specific phosphodiesterase inhibitor IBMX (3-isobutyl-1-methyl xanthine; final concentration 1 mM) were added. After 60 min, the fluorescence transfer related to the amount of remaining cAMP was measured on a microplate reader (Rubystar, BMG) at λex=337 nm, λem=620 nm and λem=665 nm. The enzyme activity is calculated from the ratio of the signals measured at 665 nm and 620 nm. The results are expressed as percent inhibition of the enzyme activity of the control (without PDE4 inhibitor). The enzyme was omitted for measurement of the basic control. The IC50 value (IC50=the concentration that caused the half-maximum inhibition of the specific activity of the control) is derived from dose-response measurements with eight different concentrations (n=2; repeated 2 times).
Measurement: IC50 = 3.7 nM (pChEMBL 8.430000305175781)
Descriptors: MW 393.46, ALogP 4.58, HBA 5, HBD 0, PSA 40.46, QED 0.72
Compound (SMILES): C[C@H](NC(=O)c1cccnc1NCc1ccc(-c2ccc3ncnc(NC4CNC4)c3c2)s1)c1ccc(F)c(F)c1
InChIKey: LVXAFUUJZGYVKO-KRWDZBQOSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 571.66, ALogP 5.52, HBA 8, HBD 4, PSA 103.86, QED 0.18
Compound (SMILES): Nc1ccc2c(n1)CCC2NC(=O)c1cn(Cc2ccc(N3CCN4CCCCC4C3=O)cc2)nn1
InChIKey: YONRNOGERAUEOK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Inhibition TR-FRET (Time Resolved FRET) Assay: This BTK competition assay measures compound potency (IC50) for the inactivated state of Bruton's Tyrosine Kinase using FRET (Förster/Flouresence Resonance Energy Transfer) technology. The BTK-Eu complex was incubated on ice one hour prior to use at a starting concentration of 50 nM BTK-Bioease: 10 nM Eu-streptavidin (Perkin-Elmer Catalog AD0062). The assay buffer consisted of 20 mM HEPES (pH 7.15), 0.1 mM DTT, 10 mM MgCl2, 0.5 mg/ml BSA with 3% Kinase Stabilizer (Fremont Biosolutions, Catalog # STB-K02). After 1 h, the reaction mixture from above was dilated 10 fold in assay buffer to make 5 nM BTK: 1 nM Eu-Streptavidin complex (donor fluorophore), 18 μl of a mixture of 0.11 nM BTK-Eu and 0.11 nM Kinase Tracer 1.78 (Invitrogen, Catalog # PV5593,) with BTK-Eu alone as no negative control, was then dispensed into 384-well flat bottom plates (Greiner, 784076). Compounds to be tested in assay were prepared as 10× concentrations and serial dilution in half-log increments was performed in DMSO so as to generate 10 point curves. To initiate the FRET reaction, compounds prepared as 10× stock in DMSO was added to the plates and the plates were incubated 18-24 h at 14° C.
Measurement: IC50 = 3200 nM (pChEMBL 5.5)
Descriptors: MW 486.58, ALogP 1.92, HBA 8, HBD 2, PSA 122.27, QED 0.56
Compound (SMILES): O=C(NC1(c2ccccc2)CC1)c1nc(-n2ccnc2)nc2cc[nH]c12
InChIKey: OELMOIWYWQCCHN-UHFFFAOYSA-N
Target: ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28907
Assay: Enzyme Assay: The CD38 enzyme assay was performed as described previously (Becherer, J D, et al. J. Med. Chem. 2015, 58, 7021-7056). Briefly, 200 nL of a dose response titration of each test compound dissolved in 100% DMSO was spotted in clear polystyrene 384-well plate (Thermo #264704) using a Mosquito (TTP Labtech). A 10 μL solution of 2 nM CD38 (BPS Biosciences #71227) suspended in 100 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH=7.5), 4 mM EDTA (2,2′2″,2′″-(ethane-1,2-diyldinitrilo)tetraacetic acid) and 1 mM CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) was incubated with test compound at 25° C. for 30 min. The enzyme reaction was initiated by adding 10 μL of 400 μM nicotinamide adenine dinucleotide (NAD+), 1000 μM (E)-2-(2-(pyridin-4-ylmethylene)hydrazinyl)pyridine in buffer containing 5 mM sodium acetate (pH=5.2) and 1 mM CHAPS. The reactions were incubated at 25° C. and the absorbance at 405 nm was measured after 15 minutes and 60 min on an Envision plate reader (Perkin Elmer) The absorbance values from the 15 min timepoint were subtracted from the absorbance value from the 60 min timepoint.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 344.38, ALogP 2.56, HBA 5, HBD 2, PSA 88.49, QED 0.6
Compound (SMILES): CCOc1ncccc1-c1cc(NCc2ccc(OC)cn2)c2c(n1)c(C)nn2C(C)C
InChIKey: HWSJSACIWPNYOB-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: P54750
Assay: PDE1 Inhibition Assay: PDE1A, PDE1B and PDE1C assays were performed as follows: the assays was performed in 60 μL samples containing a fixed amount of the PDE1 enzyme (sufficient to convert 20-25% of the cyclic nucleotide substrate), a buffer (50 mM HEPES pH 7.6; 10 mM MgCl2; 0.02% Tween20), 0.1 mg/ml BSA, 15 nM tritium labelled cAMP and varying amounts of inhibitors. Reactions were initiated by addition of the cyclic nucleotide substrate, and reactions were allowed to proceed for 1 hr at room temperature before being terminated through mixing with 20 μL (0.2 mg) yttrium silicate SPA beads (PerkinElmer). The beads were allowed to settle for 1 hr in the dark before the plates were counted in a Wallac 1450 Microbeta counter. The measured signals were converted to activity relative to an uninhibited control (100%) and IC50 values were calculated using XIFit (model 205, IDBS).
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 432.53, ALogP 4.8, HBA 8, HBD 1, PSA 86.98, QED 0.43
Compound (SMILES): NCC#Cc1cn(-c2c(Cl)cccc2Cl)c(=O)c2cnc(Nc3ccc(N4CCOCC4)cc3)nc12
InChIKey: JDJCNZJDYZFLQE-UHFFFAOYSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: Wee-1 kinase Assay: In the measurement of Wee-1 activity, a commercial peptide Poly(Lys Tyr(4:1)) hydrobromide was purchased from Sigma Aldrich and used as the substrate. Activated Wee-1 kinase was purchased from Invitrogen (PV3817) and an ADP-Glo luminescent kit was purchased from Promega.All reactions took place in 60 μL volumes in reaction buffer containing 40 mM Tris-HCl and 20 mM magnesium chloride, supplemented with 0.1 mg/mL bovine serum albumin and 2 mM DTT. Compounds were serially diluted in buffer and 5 μL of each concentration pipetted into a white 384 well plate (Sigma Aldrich M6186). A 5 μL aliquot of the Wee-1 enzyme was added to each well and the plate centrifuged for 1 min to ensure mixing of the enzyme and inhibitor.The plate was incubated at room temperature for 30 minutes before the addition of 2.0 μg/mL of substrate and 30 μM ATP in a 5 μL aliquot. The plate was centrifuged for one minute and incubated for 1 h at RT.15 μL of ADP-Glo stop reagent was added to each well to quench the reaction and deplete unconverted ATP. The plate was incubated for a further 40 min in the dark at RT.30 μL of ADP-Glo kinase detection reagent was added to each well, converting ADP to ATP, catalysing the generation of luciferin by luciferase. The plate was shaken for 1 min, and incubated in the dark for an additional hour.Luminescence from each well was detected using the Biotek Synergy4 HD plate reader and the percentage inhibition of kinase activity calculated for each inhibitor tested. Positive (kinase only) and negative (no kinase) controls were added to each plate to ensure specific interaction of kinase and inhibitor. The IC50 concentration for each inhibitor was calculated by plotting the percentage kinase inhibition against concentration of inhibitor and the curve generated by non-linear regression fitting.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 521.41, ALogP 3.98, HBA 8, HBD 2, PSA 98.3, QED 0.39