lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)[C@@H](C)Cn1c(=O)[nH]/c(=N\c2ccc(Oc3cc(F)ccn3)cc2)n(Cc2ccc(Cl)cc2)c1=O
InChIKey: AIBRJIHJFNKFDK-INIZCTEOSA-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 PDL-coated microtiter plate at a concentration of 3000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, and 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, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 0.5% BSA, and 0.04% Pluronic P-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, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 20 μL of the washing buffer. The plate was placed in High-Throughput Screening System FLIPR 384 (Molecular Device Co.). Measurement of fluorescence intensity by FLIPR 884 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, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 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.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 539.95, ALogP 3.41, HBA 9, HBD 1, PSA 120.57, QED 0.34
Compound (SMILES): CN(c1ncnc2[nH]ccc12)[C@H]1C[C@@H](NS(=O)(=O)[C@H]2C[C@H](F)C2)C1
InChIKey: SLHUZYLEKZXHHM-NEBACSHRSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Caliper Enzyme Assay: Test article and assay controls were added to a 384-well plate. Reaction mixtures contained 20 mM HEPES, pH 7.4, 10 mM magnesium chloride, 0.01% bovine serum albumin (BSA), 0.0005% Tween 20, 1 mM ATP and 1 μM peptide substrate. The JAK1 and TYK2 assays contained 1 μM of the IRStide peptide (5FAM-KKSRGDYMTMQID) and the JAK2 and JAK3 assays contained 1 μM of the JAKtide peptide (FITC-KGGEEEEYFELVKK). The assays were initiated by the addition of 20 nM JAK1, 1 nM JAK2, 1 nM JAK3 or 1 nM TYK2 enzyme and were incubated at room temperature for three hours for JAK1, 60 minutes for JAK2, 75 minutes for JAK3 or 135 minutes for TYK2. Enzyme concentrations and incubation times were optimized for each new enzyme preps and were modified slightly over time to ensure 20%-30% phosphorylation. The assays were stopped with a final concentration of 10 mM EDTA, 0.1% Coating Reagent and 100 mM HEPES, pH=7.4. The assay plates were placed on a Caliper Life Science Lab Chip 3000 (LC3000) instrument, and each well was sampled using appropriate separation conditions to measure the unphosphorylated and phosphorylated peptide.
Measurement: IC50 = 18 nM (pChEMBL 7.75)
Descriptors: MW 353.42, ALogP 1.34, HBA 5, HBD 2, PSA 90.98, QED 0.85
Compound (SMILES): O=C1COCCN1c1ccc(N2CCN(CCCc3c[nH]c4ccc(F)cc34)CC2)cc1
InChIKey: QUFSKMSPAXLJKB-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 1A (SINGLE PROTEIN), Rattus norvegicus
UniProt: P19327
Assay: Rat Synaptosome Binding Assay: The synaptosomes (150 μg) prepared from a rat brain were incubated at 37° C. for 15 minutes with 0.1 μCi [3H]5-HT in the absence or presence of the test compound or the reference compound in a buffer solution containing 106.2 mM NaCl, 4.5 mM KCl, 2.25 mM MgSO4, 1.08 mM NaH2PO4, 22.5 mM NaHCO3, 9.9 mM glucose, 9 μM EGTA and 45 μM ascorbic acid (pH 7.4).The basal control activity was determined by incubating the same mixture at 4° C. for 15 minutes in the presence of 10 μM imipramine to block the uptake of 5-HT, which was taken as the standard reference compound and tested in each experiment at several concentrations to obtain an inhibition curve.Following the incubation, the samples were filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) and rinsed twice with an ice-cold incubation buffer using a 96-sample cell harvester (Unifilter, Packard) to eliminate free [3H]5-HT. The filters were dried and the retained radioactivity was measured in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The experimental results were expressed as a percent inhibition of the control uptake of [3H]5-HT.Data AnalysisInhibition of serotonin transporter in rat synaptosome was measured by the concentrations of [3H]5-HT. The test compounds were required to be tested at least twice in the case of the concentration thereof being greater than 6 log, and the obtained data were subjected to a nonlinear regression analysis via a curve of Hill equation, to obtain IC50 value.
Measurement: IC50 = 7.3 nM (pChEMBL 8.140000343322754)
Descriptors: MW 436.53, ALogP 3.43, HBA 4, HBD 1, PSA 51.81, QED 0.64
Compound (SMILES): CCN1CCN(c2ccc(-c3nc(-c4cnn(C)c4)cn4ncc(C#N)c34)cn2)CC1
InChIKey: BFYDTSLYELGBTB-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF KinEASE-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TKOPEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1× TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TKOPEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 162 nM (pChEMBL 6.789999961853027)
Descriptors: MW 413.49, ALogP 2.21, HBA 9, HBD 0, PSA 91.17, QED 0.51
Compound (SMILES): CN(C)C(=O)c1csc(Nc2nc(OC3CCOCC3)c3c(ccn3-c3cnn(C)c3)n2)c1
InChIKey: HXEDEPQHZCJPBS-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Activity Inhibition Assay: IRAK4 kinase (purchased from Life Technologies, Cat. No.: PR5612U) was diluted to 2 folds of the final concentration (the final concentration is 0.76 ng/4) with reaction buffer (40 mM Tris-HC1, pH 7.5; 20 mM MgCl2; 0.1 mg/mL BSA; 1 mM DTT), and added to a 384-well plate at 5 μL/well. The test drugs were 10-fold serial diluted from 10 μM to set six concentration points and added to the test wells of the 384-well plate at 2.5 μL/well. After incubation for 10 min at 25° C., 10 μM of ATP and 0.1 μg/μL of an enzyme reaction substrate, myelin basic protein MBP (purchased from Signal Chem, Cat. No.: M42-51N) were added at 2.5 μL/well, and reacted at 25° C. for 60 minutes. After the reaction was completed, the kinase activity assay was performed using the ADP-Glo kinase assay kit (purchased from Promega, Cat. No.: V9102) according to the manufacture's instruction, i.e., 10 μL of ADP-Glo reaction reagent was first added and reacted at 25° C. for 40 minutes, 10 μL of the reaction mixture was taken and mixed with 10 μL of ADP-Glo detection reagent, and reacted at 25° C. for 30 minutes.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 467.56, ALogP 3.22, HBA 10, HBD 1, PSA 99.33, QED 0.46
Compound (SMILES): CCc1c(F)ccc2cc(O)cc(-c3ncc4c(N5CC6CCC(C5)N6)nc(OCC56CCCN5CCC6)nc4c3F)c12
InChIKey: BBOAOMYQBXIELJ-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: KRas G12D Binding Assay: Table 2: The ability of a compound to bind to KRAS G12D was measured using a TR-FRET displacement assay. Biotinylated GDP-loaded recombinant human KRAS G12D (corresponding to amino acids 1:169, produced at Array BioPharma) was incubated with a custom-made Cy5 labelled tracer, europium labelled streptavidin and compound (2% DMSO final) in buffer (50 mM HEPES [pH 7.5], 5mM MgCl2, 0.005% Tween-20 & 1 mM DTT). After a 60 minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) calculated using a ratiometric emission factor. 100 POC is determined using no test compound and 0 POC is determined using a concentration of control compound that completely inhibits binding of the tracer to KRAS. The POC values were fit to a 4-parameter logistic curve and the IC50 value was determined as the concentration where the curve crosses 50 POC.
Measurement: IC50 = 0.6 nM (pChEMBL 9.220000267028809)
Descriptors: MW 586.69, ALogP 5.34, HBA 8, HBD 2, PSA 86.64, QED 0.31
Compound (SMILES): COc1ccc(C2COc3cc(Cn4cnc5cc(-c6ccc(F)nc6)cnc54)cc(OC)c3O2)cn1
InChIKey: LUPFROSUMULLKF-UHFFFAOYSA-N
Target: Macrophage colony-stimulating factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P07333
Assay: In Vitro Studies: All assay reaction conditions for IC50 determinations were within the linear range with respect to time and enzyme concentration. In a 384 well polypropylene plate, c-FMS (0.14 nM, Carna 08-155) was pre-incubated in a 100 mM Hepes-NaOH pH 7.5 buffer containing 0.01% Triton X-100, 10 mM MgCl2, 0.1% BSA, 1 mM DTT, 10 μM sodium orthovanadate and 10 μM beta-glycerophosphate and compound with a concentration of 2.5% DMSO for 15 minutes at room temperature. The reaction was initiated with an equal volume of peptide substrate (Caliper Life Sciences catalog no. 760430) and ATP in the aforementioned buffer. The final concentrations in the reaction were 70 pM c-FMS, 1.5 μM peptide substrate and 500 μM ATP (ATP Km). The reaction was incubated at room temperature for 120 minutes and terminated with a buffer containing excess EDTA (100 mM Hepes-NaOH pH 7.5, 0.02% Brij, 0.1% CR-3, 0.36% DMSO and 100 mM EDTA). The plate was run for one cycle on a LabChip 3000 (Caliper Life Sciences, Hopkinton, Mass.) in an off-chip mobility shift type assay with an upstream voltage of −2250 volts, a downstream voltage of −500 volts and a vacuum pressure of −1.6 psi.
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 499.5, ALogP 4.61, HBA 9, HBD 0, PSA 93.41, QED 0.31
Compound (SMILES): N#CN1CC2CN(C(=O)Oc3ccc(C(F)(F)F)cc3)C[C@H]2C1
InChIKey: ZNHXRGOGYANLFL-NFJWQWPMSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 30 (SINGLE PROTEIN), Homo sapiens
UniProt: Q70CQ3
Assay: Biochemical Fluorescence Intensity 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 0.001 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 Ubiquitin-Rhodamine 110 (U-555; Boston Biochem) at a final concentration of 100 nM. 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 487 nm; λ Emission 535 nm.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 325.29, ALogP 2.55, HBA 4, HBD 0, PSA 56.57, QED 0.74
Compound (SMILES): COc1ncnc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](OC)c2ncc(Cl)cn2)nnc1[C@@H]1CC[C@@H]1F
InChIKey: SQCSKRGVXRXOIS-QJZXMWHDSA-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.
Measurement: EC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 542.98, ALogP 2.25, HBA 12, HBD 1, PSA 156.13, QED 0.4
Compound (SMILES): COc1ccc(Cn2c(-c3cccc(C)c3)nc3nc(C(=O)O)nc(N[C@H](C)C4CCC4)c32)cc1
InChIKey: ZBSNSHYAYWFGHV-QGZVFWFLSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: FRET Assay: An HDM2 FRET assay was developed to assess the compounds' inhibitory activity towards binding of p53 protein. A truncated version of HDM2 with residues 17 to 125 (containing p53 binding surface, Science (1994) 265, 346-355), with N-terminal His and Thioredoxin tag was generated in pET32a expression vector and expressed in E. coli strain BL21(DE3)Rosetta. Protein was purified using Ni-affinity chromatography, followed by size exclusion chromatography using Superdex 75 26/60 column. To assess inhibition of p53 binding to HDM2, a FITC labeled 8-mer peptide (sequence: Ac-Phe-Arg-Dpr-Ac6c-(6-Br)Trp-Glu-Glu-Leu-NH2; Anal Biochem. 2004 Aug. 1; 331(1):138-46) with strong affinity towards p53 binding pocket of HDM2 was used. The HDM2 assay buffer contained 1× Phosphate Buffered Saline (Invitrogen, Cat#14190), 0.01% BSA (Jackson ImmunoResearch, Cat#001-000-162), 0.01% Tween-20. In the 1× assay buffer recombinant HDM2 protein, peptide and Lumi4-Tb Cryptate-conjugate mouse anti-6×His antibody (cisbio, Cat# Tb61HISTLB) were added and transferred to ProxiPlate PLUS (PerkinElmer, Cat#6008269), containing compounds so that final DMSO concentration is 0.1%. Final concentrations of reagents in the assay wells are 0.5 nM HDM2, 0.25 nM anti HIS (Tb label) antibody and 3 nM peptide. After two hour incubation at room temperature in a humidified chamber plates were read on EnVision plate reader with the following settings: excitation: UV, 340 nM, two emission filters: 520 nm and 495 nm respectively. Ratio of em520/em495 was used to calculate % inhibition and to obtain IC50 with 4-parameter logistic equation.
Measurement: IC50 = 581.7 nM (pChEMBL 6.239999771118164)
Descriptors: MW 471.56, ALogP 5.16, HBA 7, HBD 2, PSA 102.16, QED 0.37
Compound (SMILES): COc1cc2c(Oc3ccc(NC(=O)C4(C(=O)Nc5ccc(F)cc5)CC4)cc3F)ccnc2cc1OCCN1CC(N2CCOCC2)C1
InChIKey: DUUFUKIIRDHEAQ-UHFFFAOYSA-N
Target: Hepatocyte growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08581
Assay: Inhibitory Activity Assay: 1. Methods(1) 4 μL of prepared kinase buffer solution or 4 μL of kinase solution (100% inhibition control) were added into a 384-hole plate; 2 μL of the compound or 2 μL of buffer without the compound (0% inhibition control) was added into the holes; 2 repetitive holes were set for all the samples or control above.(2) Incubated at 25° C. for 5 min;(3) 2 μL of ATP/substrate/MgCl2/MnCl2/SEB/DTT mixed solution were added into the holes;(4) Centrifugated at 1000 rpm for 1 min, and incubated by vibration at 30° C. for 30 min;(5) 8 μL of XL-665/antibody mixed solution were added into the holes;(6) Incubated at 25° C. for 1 h;(7) The signals at 665 nm and 620 nm were read on PHERAstar FS;(8) The data was analyzed according to the instructions of the kit, and the fitting calculation of IC50 was performed using GraphPad Prism5.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 673.72, ALogP 5.07, HBA 9, HBD 2, PSA 114.49, QED 0.2
Compound (SMILES): CC1CN(C(=O)CN2C(=O)C3(CCN(C(=O)c4ccc5[nH]ncc5c4)CC3)c3c(Br)cccc32)c2ccccc21
InChIKey: YRPDHWXVRBKAQB-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: In Vitro Binding Assay for DDR1: An in vitro binding competition assay was performed to evaluate the effect of the compounds of present invention on DDR1 protein.Binding Competition Assay:This assay is based on the intracellular domain of the DDR1 protein which contains the kinase active site. The recombinant protein additionally carries a GST-tag that can be recognized by an Eu-labeled anti-GST antibody. A tracer compound binding to the active site is labeled with a dye so that a FRET donor acceptor pair can be formed. Excitation energy absorbed by the Europium complex (350 nm flash light or pulsed laser) is transferred to a suitable fluorescent dye, if it is in close proximity. Compounds binding competitively with the tracer molecule will displace the bound tracer molecules and reduce the FRET signal in a dose dependent manner. Due to the long lifetime of the Eu excited state, the emission of the donor and the acceptor can be measured in time-gated mode such that most of the intrinsic fluorescence contributions have already decayed. This results in high sensitivity, excellent reproducibility and high data quality. This sensitive detection method enables protein concentrations below 20 nM.Protein, tracer and labeled antibody were obtained from commercial sources. The assay was performed in 384 low volume microtiter-plates with a final volume of 15 al. Dose response curves were generated from 16 compound dilutions in DMSO as solvent compound dilutions, a solution containing protein and labeled antibody, and a solution containing the tracer which is added in the last step. The fluorescence of donor and acceptor were then measured after one hour incubation at room temperature. Every assay run was quality-controlled with dose response curves for two reference compounds.
Measurement: IC50 = 26.8 nM (pChEMBL 7.570000171661377)
Descriptors: MW 598.5, ALogP 5, HBA 4, HBD 1, PSA 89.61, QED 0.36
Compound (SMILES): C[C@@H]1OCC2(CCN(c3cnc4c(N5CCN(C)c6nc(-c7cnn(C)c7)ccc65)n[nH]c4n3)CC2)[C@@H]1N
InChIKey: DBJQSUAFLDYBJA-KSFYIVLOSA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phorphorylated peptides to its Src Homology 2 (SH2) domains. The latter activation step leads to the release of the auto-inhibitory interface of SHP2, which in turn renders the SHP2 protein tyrosine phosphatase (PTP) active and available for substrate recognition and reaction catalysis. The catalytic activity of SHP2 was monitored using the surrogate substrate DiFMUP in a prompt fluorescence assay format.The inhibition of SHP2 by compounds of the disclosure (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.25 nM of SHP2 was incubated with of 0.5 μM of peptide IRS1_pY1172(dPEG8)pY1222 (sequence: H2N-LN(pY)IDLDLV(dPEG8)LST(pY)ASINFQK-amide) After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat #D6567, 100 μM final) was added to the reaction and the conversion of DiFMUP to 6,8-difluoro-7-hydroxyl-4-methyl coumarin (DiFMU) was monitored continuously for 10 minutes with excitation at 355 nm and emission at 460 nm using a microplate reader (PolarStar, BMG).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 515.63, ALogP 2.07, HBA 11, HBD 2, PSA 130.14, QED 0.42
Compound (SMILES): O=C(N(CC12CCC(c3nc(C4CC4)no3)(CC1)CC2)c1cccc(-c2cc(Br)cn3ncnc23)c1)C12CC(F)(C1)C2
InChIKey: CMSSNHBWEADERZ-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 = 782 nM (pChEMBL 6.110000133514404)
Descriptors: MW 631.55, ALogP 6.94, HBA 7, HBD 0, PSA 89.42, QED 0.22
Compound (SMILES): O=C(O)c1c(O)c(=O)n(Cc2cccc(F)c2)c2ccsc12
InChIKey: NKMOEVRSSRBOAJ-UHFFFAOYSA-N
Target: Flap endonuclease 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P39748
Assay: Enzymatic IC50 Assays: FEN1, EXO1 or XPG enzyme was incubated with compound or vehicle (DMSO) and the FAM-labeled DNA oligomer substrate in a microtiter plate. The stop buffer contains EDTA to stop the enzymatic reaction. The plate is read for fluorescence intensity. The high control (DMSO) with high fluorescence intensity represents no inhibition of enzymatic reaction while the low control (10 μM) with low fluorescence intensity represents full inhibition of enzymatic reaction.
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 319.31, ALogP 2.65, HBA 5, HBD 2, PSA 79.53, QED 0.78
Compound (SMILES): COc1nc(Nc2ccc(CN3CCN(C)CC3)cc2)nc2[nH]cc(-c3cccc(Cl)c3)c12
InChIKey: APAZLBBJADULHZ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Axl Kinase Assay: Certain compounds of formula (I) were tested for their potency in inhibiting Axl kinase. Inhibition of Axl kinase activity by a test compound was calculated by AlphaScreen (PerkinElmer 6760620M). Standard assay conditions included 1 ng of recombinant Axl kinase (SignalChem) with 3-10 ng Poly-(GT)-Biotin (Cisbio) in the assay buffer, i.e., 40 μM ATP (Sigma), 10 mM MOPs, pH7.0 (Sigma M-1254), 0.21 mM EDTA (Sigma E-6758), 0.5% glycerol (J. T Baker 2136-1), 1 mg/ml BSA (Bio-Rad 500-0007), 0.01% 2-mercaptoethanol (BDH 441433A), and 0.001% Brij35 (Sigma P1254) in a final volume of 25 μL. Reactions were incubated at 30° C. for 30 minutes and stopped by adding 5 μL of 50 mM EDTA. Product analysis was performed using the AlphaScreen kit and counted with Enspire Alpha (PerkinElmer). Screen values of inhibitors were determined after carrying out assays with eight serially diluted concentrations in duplication. Results were analyzed using linear regression software (GraphPad Prism 4; GraphPad Software Inc.).
Measurement: IC50 = 65 nM (pChEMBL 7.190000057220459)
Descriptors: MW 462.99, ALogP 4.78, HBA 6, HBD 2, PSA 69.31, QED 0.43
Compound (SMILES): Cc1c(COc2ccc(CN3CCC(C(=O)O)CC3)cc2Cl)cccc1-c1ccccc1
InChIKey: KQDCXYBUWMBUSN-UHFFFAOYSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Homogenous Time-Resolved Fluorescence (HTRF) Binding Assay: All binding studies were performed in an HTRF assay buffer consisting of dPBS supplemented with 0.1% (withv) bovine serum albumin and 0.05% (v/v) Tween-20. For the PD-1-Ig/PD-L1-His binding assay, inhibitors were pre-incubated with PD-L1-His (10 nM final) for 15 m in 4 μl of assay buffer, followed by addition of PD-1-Ig (20 nM final) in 1 μl of assay buffer and further incubation for 15 m. PD-L1 from either human, cyno, or mouse were used. HTRF detection was achieved using europium crypate-labeled anti-Ig (1 nM final) and allophycocyanin (APC) labeled anti-His (20 nM final). Antibodies were diluted in HTRF detection buffer and 5 μl was dispensed on top of binding reaction. The reaction mixture was allowed to equilibrate for 30 minutes and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer. Additional binding assays were established between PD-1-Ig/PD-L2-His (20 & 5 nM, respectively), CD80-His/PD-L1-Ig (100 & 10 nM, respectively) and CD80-His/CTLA4-Ig (10 & 5 nM, respectively). Competition studies between biotinylated SEQ ID NO:71 and human PD-L1-His were performed as follows. Inhibitors were pre-incubated with PD-L1-His (10 nM final) for 60 m in 4 μl of assay buffer followed by addition of biotinylated SEQ ID NO:71 (0.5 nM final) in 1 μl of assay buffer. Binding was allowed to equilibrate for 30 m followed by addition of europium crypated labeled Strepatavidin (2.5 pM final) and APC-labeled anti-His (20 nM final) in 5 μl of HTRF buffer. The reaction was allowed to equilibrate for 30 m and signal (665 nm/620 nm ratio) was obtained using an EnVision fluorometer.
Measurement: IC50 = 555 nM (pChEMBL 6.260000228881836)
Descriptors: MW 449.98, ALogP 6.19, HBA 3, HBD 1, PSA 49.77, QED 0.47
Compound (SMILES): COc1ccc(-c2cnc3ccc(C(=O)Nc4cnccc4N4C[C@@H](N)[C@H](O)[C@@H](C)C4)nc3c2)cn1
InChIKey: JIYXIPLMAZJNTL-AKQVGLANSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Enzyme Assay: Pim-1 and Pim-3 kinase assays-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Biotin-labeled BAD peptide substrate (AnaSpec 62269), 1 mM ATP, and 2.5 pM (Pim-1, Invitrogen PV3503) or 1.25 pM (Pim-3, Millipore 14-738) enzyme for 1 h at 25° C. Reactions were stopped by addition of 10 μL STOP Buffer (150 mM Tris, pH=7.5, 150 mM NaCl, 75 mM EDTA, 0.01% Tween-20, 0.3% BSA,) supplemented with Phospho-Bad (Ser112) Antibody (Cell Signaling 9291) diluted 666-fold, and Streptavidin donor beads (PerkinElmer 6760002) along with Protein-A acceptor beads (PerkinElmer 6760137) at 15 μg/mL each. Supplementation of the STOP buffer with beads and stopping the reactions were done under reduced light. Prior to the stopping reactions STOP buffer with beads was preincubated for 1 h in the dark at room temperature. After stopping the reactions, plates were incubated for 1 h in the dark at room temperature before reading on a PHERAstar FS plate reader (BMG Labtech) under reduced light.Pim-2 kinase assay-20 μL reactions were run in white 384 well polystyrene plates dotted with 0.8 μL compound/DMSO in the assay buffer (50 mM Tris, pH 7.5, 0.01% Tween-20, 5 mM mgCl2, 0.01% BSA, 5 mM DTT), containing 0.05 μM Fluorescein-labeled CREBtide peptide substrate (Invitrogen PV3508), 1 mM ATP, and 1 nM enzyme (Invitrogen PV3649) for 2 h at 25° C. Reactions were stopped by addition of 10 μL TR-FRET Dilution Buffer (Invitrogen PV3574) with 30 mM EDTA and 1.5 nM LanthaScreen Tb-CREB pSer133 antibody (Invitrogen PV3566). After 30 min. incubation at room temperature, plates were read on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 485.55, ALogP 2.49, HBA 9, HBD 3, PSA 139.38, QED 0.39
Compound (SMILES): CC(C)(c1cccc(-c2cc(-c3ccnn3C3CCOCC3)c3c(N)ncnn23)c1)N1CCOCC1
InChIKey: LRBOTOPIFAXOTF-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 487.61, ALogP 3.76, HBA 9, HBD 1, PSA 95.73, QED 0.46
Compound (SMILES): O=C(Nc1cncc(C(F)(F)F)c1)c1cnn(-c2cccc3c(=O)[nH]ccc23)c1C(F)(F)F
InChIKey: KJRHMAZWQMZJLA-UHFFFAOYSA-N
Target: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UDY8
Assay: In Vitro Assay: MALT1 protease activity was assessed in an in vitro assay using a tetrapeptide as substrate and full-length MALT1 protein (Strep-MALT1(1-824)-His) purified from baculovirus-infected insect cells. The tetrapeptide LRSR is coupled to AMC (7-amino-4-methylcoumarin) and provides a quenched, fluorescent substrate for the MALT1 protease (SM Biochemicals). Cleavage of AMC from the Arginine residue results in an increase in coumarin fluorescence measured at 460 nm (excitation 355 nm). The final assay buffer consisted of 10 nM FL MALT1 protein, 200 μM Ac-LRSR-AMC, 50 mM Tris pH 7.5, 0.6 M Citrate, 1 mM DTT, 1 mM EDTA, 0.05% BSA and 1.5% DMSO. Test compounds were spotted at 50 nL in 100% DMSO per well of a black 384-Proxiplate (Perkin Elmer). Test compound concentrations ranged from 30 μM to 0.5 nM using 11 dilution steps (1:3). Background signal was measured from control wells containing assay buffer without enzyme which functions as low control (LC). High control (HC) values were generated using the reaction with enzyme but no compound treatment. Compounds were pre-incubated with MALT1 enzyme for 50 minutes at RT. Substrate was added subsequently and fluorescence was measured in Labsystems fluoroskan at excitation 355 nm and emission 460 nm to determine time 0. The reaction was subsequently incubated for 4 h at RT and fluorescence was measured.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 467.33, ALogP 4.4, HBA 5, HBD 2, PSA 92.67, QED 0.44
Compound (SMILES): Cc1cc(-c2ccc(CC(=O)Nc3ccc(C4CCN(C(=O)OC(C)(C)C)CC4)cn3)cc2)ccn1
InChIKey: ONTQFHLDAHOYRQ-UHFFFAOYSA-N
Target: Protein Wnt-3a (SINGLE PROTEIN), Homo sapiens
UniProt: P56704
Assay: Wnt-Luc Reporter Assay: Human embryonic kidney 293 cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are cultured in DMEM medium (Gibco/Invitrogen, Carlsbad, Calif.) supplemented with 10% FBS (Gibco/Invitrogen, Carlsbad, Calif.), 50 unit/mL penicillin and 50 μg/mL of streptomycin (Gibco/Invitrogen, Carlsbad, Calif.) at 37° C. with 5% CO2 in air atmosphere. 293 cells in a 10 cm dish are co-transfected with 8 μg of STF-reporter plasmid containing a luciferase gene driven by Wnt-responsive elements and 2 μg of pcDNA3.1-Neo (Gibco/Invitrogen, Carlsbad, Calif.) with 30 μL of FuGENE6 (Roche Diagnostics, Indianapolis, Ind.) following the manufacturer's protocol. Stable cell lines (293 Wnt-Luc) were selected with 400 μg/mL of G418 (Gibco/Invitrogen, Carlsbad, Calif.). The 293 Wnt-Luc cells and L-cell Wnt3a cells (obtained from American Type Culture Collection, ATCC, Manassas, Va.) are trypsinized and co-cultured into a 384-well plate with DMEM medium supplemented with 2% FBS, and treated with different concentrations of a compound of the invention. After 24 hours, the firefly luciferase activities are assayed with the Bright-Glo Luciferase Assay System (Promega, Madison, Wis.). The IC50 is measured when the effect of the compound reduces the luminescence signal by 50%.
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 486.62, ALogP 5.75, HBA 5, HBD 1, PSA 84.42, QED 0.5
Compound (SMILES): COc1ccc(S[C@H]2C[C@@H]3CN(CC(=O)c4ccc(O)cc4)C[C@@H]3C2)cc1
InChIKey: MVPIZAITHDYTNP-LFUKPOHLSA-N
Target: Glutamate NMDA receptor; GRIN1/GRIN2B (PROTEIN COMPLEX), Homo sapiens
UniProt: Q05586
Assay: NR2B NAM Assay: Cell Culture and plating: HEK293 cells expressing NR1/NR2B (Chantest, Cleveland, Ohio) were grown to 70-80% confluency as an adherent monolayer in standard tissue culture flasks at 37° C., 5% CO2 per supplier's instructions. NR2B expression was induced by incubation with 0.3-0.4 μg/ml tetracycline in the presence of 4 mM ARL-15896 for 18-24 hours under the same growth conditions, then transferred to 30° C. for another 3-5 hours.After induction, cell culture medium was removed and cells were rinsed once with Ca2+ and Mg2+-free Dulbecco's phosphate buffered saline. Cells were then removed from the flask using TrypLE Express (Life Technologies) according to the manufacturer's instructions and collected to 50 ml centrifuge tubes. Following two washes in Ca2+/Mg2+-free HBSS with 20 mM HEPES (HHnoCa), cells were counted and viability assessed using trypan blue. To load cells with Ca2+-sensitive dye, they were resuspended in fluo-8 plus Component B (AAT Bioquest Products) diluted in HHnoCa and incubated 15 minutes at 37° C., followed by 30 minutes at room temp (in dark). Cells were then washed and resuspended in HHnoCa to remove extracellular dye and plated in 384-well plates (Falcon, uncoated) at 20,000-30,000 cells/well in a final volume of 25 μl/well.
Measurement: IC50 = 230 nM (pChEMBL 6.639999866485596)
Descriptors: MW 383.51, ALogP 4.09, HBA 5, HBD 1, PSA 49.77, QED 0.76
Compound (SMILES): Cc1ccc(Cn2c(=O)n(CCCC(=O)O)c(=O)[nH]/c2=N\c2ccc(Oc3ccncc3)cc2)cc1
InChIKey: ZGADMCMZPYLKDE-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Human P2X3 Receptor Inhibition Assay: Stably expressing cell line (C6BU-1 transfected with human P2X3 receptor gene (GenBank accession number Y07683) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% 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, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% 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, 2.7 KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), each well was added with 40 μL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.). Measurement of fluorescence intensity by FDSS 3000 was started, and 40 μl, of DMSO solutions containing different concentrations of the test, compound as prepared by dilution with dilution buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 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, 50 nM ATP solution (50 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 3 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. FDSS software (Hamamatsu Photonics K.K.) was used for calculation of the specific maximum fluorescence intensity. IC50 was calculated using Microsoft Excel (Microsoft Corporation) and Ltd.).
Measurement: IC50 = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 487.52, ALogP 2.98, HBA 8, HBD 2, PSA 131.57, QED 0.37
Compound (SMILES): COc1cc(N2CCC(N3C[C@@H](C)N[C@@H](C)C3)CC2)ccc1Nc1ncc(Cl)c(Nc2ccccc2NS(C)(=O)=O)n1
InChIKey: CTUYJNXFAUWSJB-BGYRXZFFSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibitory Ability Assay: The composition of the assay buffer used in the activity measurement was 50 mM Tris-HCl pH 7.5, 100 mM NaCl, 7.5 mM MgCl2, 3 mM KCl, 0.01% Tween 20, 0.1% BSA, 1 mM DTT. The enzymatic reaction was carried out hereto by using the peptide substrate labeled with ATP of 50 mM concentration and biotin of 0.5 mM concentration. The analysis of an EGFR activity inhibitory effect of the compound was carried out in accordance with the analysis reaction recipe below.Component 1: 4 ml of EGFR wild-type or mutation enzymeComponent 2: 2 ml of compound solutionComponent 3: 4 ml of peptide labeled with ATP and biotinThe enzymatic reaction was initiated by mixing Component 1 and Component 2 first, and then, adding component 3. After the reaction for 2 hours at 37° C., 10 ml of a measurement solution consisting of streptavidin-XL665 provided by Cisbio and anti-phosphotyrosine antibody labeled with europium was added to the enzymatic reaction solution and reacted for 1 hour at ambient temperature. Finally, the ratio of fluorescence values at 615 nm and 665 nm was obtained by using the Envision equipment of Perkin-Elmer, and the enzymatic activity was quantitatively measured, and the inhibitory ability of the compound was confirmed. The measurement values measured at the seven concentrations of the compound were analyzed by using the Prism program (version 5.01, Graphpad Software, Inc.), and IC50 value of the compound was calculated, which is an inhibitory ability indicator.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 615.2, ALogP 4.65, HBA 10, HBD 4, PSA 123.75, QED 0.27
Compound (SMILES): CNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2ccc(C(F)(F)F)nc2)CC1
InChIKey: CPKPERZPHDRHKD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase receptor UFO (SINGLE PROTEIN), Homo sapiens
UniProt: P30530
Assay: Enzyme Assay: AXL: Compounds of Formula I were screened for their ability to inhibit AXL kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human AXL cytoplasmic domain was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 471.49, ALogP 4.85, HBA 8, HBD 1, PSA 82.68, QED 0.45
Compound (SMILES): COc1cccc(-c2nnc(NS(=O)(=O)[C@@H](C)[C@H](C)c3ncc(Cl)cn3)n2Cc2ccon2)n1
InChIKey: CFJHBMJPITUWHC-STQMWFEESA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: 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 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.
Measurement: EC50 = 5.1 nM (pChEMBL 8.289999961853027)
Descriptors: MW 504.96, ALogP 2.76, HBA 11, HBD 1, PSA 150.81, QED 0.36
Compound (SMILES): Cc1cccc(-n2nc(C(=O)NC3CCc4ccccc4NC3=O)c(=O)cc2C)c1
InChIKey: UOIRNHIPPDQVTA-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: ADP-Glo Kinase Assay: In order to measure RIPK1 activity the ADP-Glo kinase assay (Promega, Catalog #V7002) was used to measure the conversion of ATP to ADP. This enzymatic assay was performed in a 384-well white, Optiplate (Perkin Elmer, Catalog #6007299) with assay buffer consisting of 50 mM HEPES pH 7.5 (Gibco, Catalog #15630-080), 50 mM NaCl (Teknova, Catalog #S0252), 30 mM MgCl2 (Ambion, Catalog #AM9530G), 1 mM DTT (Santa Cruz Biotechnology, Catalog #sc-29089), 0.05% BSA (Sigma, Catalog #A3059-50G) and 0.02% CHAPS (Sigma, Catalog #C5070-5G). Stock solutions of the test compounds were prepared in 100% DMSO (Sigma, Catalog #D2650) and serially diluted 1:3 using 100% DMSO. Compounds were additionally diluted 1:40 in assay buffer, and 2 μL/well were transferred to the assay plate. 4 μL/well (final concentration of 5 nM) of RIPK1 protein (SignalChem, Catalog #R07-11G-05) diluted in assay buffer and added to the assay plate followed by a 10 minute preincutation at room temperature. 4 μL/well of ATP (Promega, Catalog #V7002) (final concentration of 50 μM) diluted in assay buffer were then added to the assay plate followed by a 6 hr reaction time. Final concentrations of RIPK1 and ATP refer to a 10 μL volume. Luminescence was measured using a BioTek Synergy NEO plate reader. IC50 values were calculated using a four-parameter logistic curve fit using Genedata Screener software.
Measurement: IC50 = 43.1 nM (pChEMBL 7.369999885559082)
Descriptors: MW 402.45, ALogP 2.53, HBA 5, HBD 2, PSA 93.09, QED 0.7