lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C#C[C@@]1(O)[C@@H](COC(Cc2ccc(N3CCCNC3=O)cc2)(C(=O)O)C(=O)O)O[C@@H](n2ccc(N)nc2=O)[C@@H]1O
InChIKey: QJHITONMZBYIIB-HOBQYKGVSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Inhibition of the CD73 Enzyme In Vitro: For measurements of soluble CD73 enzyme activity, recombinant CD73 was obtained from R&D Systems, Cat. No. 5795-EN-010. Serial dilutions of test compounds were incubated with recombinant CD73 and AMP in reaction buffer (25 mM Tris HCl pH7.5, 5 mM MgCl2, 50 mM NaCl, 0.25 mM DTT, 0.005% Triton X-100). The final reaction volume was 25 μL and the final concentrations of recombinant CD73 and AMP were 0.05 nM and 50 μM, respectively. Reactions were allowed to proceed for 1 hour at 37° C. before the addition of 100 μL Malachite Green (Cell Signaling Technology, Cat. No. 12776). After 5 minutes at room temperature, absorbance at 630 nm was determined on a microplate spectrophotometer.
Measurement: IC50 = 91 nM (pChEMBL 7.039999961853027)
Descriptors: MW 557.52, ALogP -1.46, HBA 11, HBD 6, PSA 226.77, QED 0.15
Compound (SMILES): O=C(NC/C=C/c1ccc2[nH]c(=O)[nH]c2c1)c1cccn(Cc2cc(F)c(F)c(F)c2)c1=O
InChIKey: PLFGUVKJZUTCEW-HNQUOIGGSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: Kinase Assay: A PDK1 kinase assay was performed as follows. PDK1 (amino acids 51-360) 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 greater than 90% homogeneity. PDK1 protein was divided into two fractions, one of which was subsequently dephosphorylated. To generate dephosphorylated PDK1, the PDK1 was reacted with GST-tagged lambda-phosphatase in vitro. GST was subsequently cleaved proteolytically for both phosphorylated and dephosphorylated PDK1. Protein preparations were run on glutathione Sepharose columns to remove GST and GST-tagged lambda-phosphatase, if present. Phosphorylated PDK1 and dephosphorylated PDK1 were verified by mass-spectrometry. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay using either phosphorylated or unphosphorylated PDK1 and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 3-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: 25 nM un-phosphorylated PDK1 or 0.5 nM phosphorylated PDK1, 30 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 24 μM for both forms 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.
Measurement: IC50 < 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 454.41, ALogP 2.93, HBA 4, HBD 3, PSA 99.75, QED 0.39
Compound (SMILES): COc1cccc(NC2CCN(c3ccc(-c4ccnc(F)c4)c(OC)n3)C2=O)c1
InChIKey: QJJXXBSEOZHTMX-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assays: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH (SEQ ID NO: 1)). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 408.43, ALogP 3.52, HBA 6, HBD 1, PSA 76.58, QED 0.63
Compound (SMILES): O=C1N(Cc2cccc(F)c2)CCN1c1ccc2c(n1)OCc1n[nH]cc1-2
InChIKey: YKEWGIDEEPMQGF-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL, assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound.
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 365.37, ALogP 2.95, HBA 4, HBD 1, PSA 74.35, QED 0.77
Compound (SMILES): Cc1nnc2ccc3c(cc(-c4ccn(C)n4)n3Cc3cc(Cl)cc(CN4CCC4)c3)n12
InChIKey: WXSUJAXZYZLBLM-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: BRD4-BD1 and BRD4-BD2 assays were conducted in white 384-well polystyrene plate in a final volume of 20 μL for BD1 and 40 μL for BD2. Inhibitors were first serially diluted in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 1.25% (BD1) and 0.83% (BD2). The assays were carried out at room temperature for 75 min. in the assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.05% CHAPS, 0.01% BSA), containing 50 nM Biotin-labeled tetra-acetylated histone H4 peptide (H4Ac4), 3.8 nM (BRD4-BD1, BPS Bioscience #31040) or 20 nM (BRD4-BD2, BPS Bioscience #31041). The reaction followed by the addition of 20 μL of assay buffer supplemented with Streptavidin donor beads (PerkinElmer 6760002) and GSH Acceptor beads (PerkinElmer-AL109C) at 4 μg/mL under reduced light. After plate sealing, the plate was incubated in the dark at room temperature for 75 min. before reading on a PHERAstar FS plate reader (BMG Labtech).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 445.96, ALogP 4.3, HBA 7, HBD 0, PSA 56.18, QED 0.4
Compound (SMILES): O=C(NCC1CC1)c1cccc([C@@H](CO)NC(=O)c2ccc(-c3ccncc3)cc2)c1
InChIKey: AODCBNYRCGABLY-HSZRJFAPSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: Kinase Inhibition Assay: The following assay protocol is for measuring the phosphorylation of a peptide substrate (FAM-KKLRRTLSVA-OH wherein FAM is carboxyfluorescein). The peptide is >98% purity by Capillary Electrophoresis. The peptide is phosphorylated by the protein kinase ROCK1 or ROCK2. The ROCK1 or ROCK2 enzyme, substrate, and cofactors (ATP and Mg2+) are combined in a well of a microtiter plate and incubated for 3 hours at 25° C. in the presence or absence of an inhibitor compound. At the end of the incubation, the reaction is quenched by the addition of an EDTA-containing buffer. The substrate and product are separated and quantified electrophoretically using the microfluidic-based LABCHIP® 3000 Drug Discovery System from Caliper Life Sciences (Hopkinton, Mass.).The components of the assay mixture are:100 mM HEPES, pH 7.50.1% BSA0.01% Triton X-1001 mM DTT10 mM MgCl210 μM Sodium Orthovanadate10 LM Beta-Glycerophosphate5 μM ATP (for ROCK1) or 7 μM ATP (for ROCK2)1% DMSO (from compound)1.25 μM FAM-KKLRRTLSVA-OH3 nM ROCK1 or 2.5 nM ROCK2 enzymeSubstrate and product peptides present in each sample are separated electrophoretically using the LABCHIP® 3000 capillary electrophoresis instrument. As substrate and product peptides are separated two peaks of fluorescence are observed. Change in the relative fluorescence intensity of the substrate and product peaks is the parameter measured reflecting enzyme activity. Capillary electrophoregramms (RDA acquisition files) are analyzed using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). The kinase activity in each sample is determined as the product to sum ratio (PSR): P/(S+P), where P is the peak height of the product peptide and S is the peak height of the substrate peptide. For each compound, enzyme activity is measured at various concentrations (12 concentrations of compound spaced by 3× dilution intervals).
Measurement: IC50 = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 415.49, ALogP 3.35, HBA 4, HBD 3, PSA 91.32, QED 0.53
Compound (SMILES): O[C@@H]1[C@H](O)CS[C@H]1c1nnc2c(NCc3cccc(OC(F)(F)F)c3)nccn12
InChIKey: JXAJYMYABNFNSL-JHJVBQTASA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Assay for Binding Affinity for Adenosine Receptor A3: HEK-293 cell membrane homogenates (32 μg protein), in which A3 adenosine receptors were expressed, are incubated for 120 min at 22° C. with 0.15 nM [1251]AB-MECA in the absence or presence of the test compound in a buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM EDTA and 2 UI/ml ADA. Nonspecific binding is determined in the presence of 1 μM IB-MECA. Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). The results are expressed as a percent inhibition of the control radioligand specific binding. The standard reference compound is IB-MECA, which is tested in each experiment at several concentrations to obtain a competition curve from which its IC50 is calculated.
Measurement: Ki = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 427.41, ALogP 2.14, HBA 9, HBD 3, PSA 104.8, QED 0.57
Compound (SMILES): CNc1cc2c(cn1)c(-c1cnn(C)c1)nn2C1CCC(Oc2cccc(OCC(F)(F)F)n2)CC1
InChIKey: VZDLPJYBPUCWAE-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 = 350 nM (pChEMBL 6.460000038146973)
Descriptors: MW 501.51, ALogP 4.77, HBA 9, HBD 1, PSA 91.91, QED 0.39
Compound (SMILES): CC(C)Nc1cc(Nc2cc(F)c3ncsc3c2)ncc1C(=O)NC[C@@H](F)C(C)(C)O
InChIKey: KJXPZKJJTBVFOT-QGZVFWFLSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Inhibition Assay: The assays were performed in U-bottom 384-well plates. The final assay volume was 30 μL prepared from 15 μL additions of enzyme and substrates (fluoresceinated peptide and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.2, 10 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was initiated by the combination of IRAK4 with substrates and test compounds. The reaction was incubated at room temperature for 60 min. and terminated by adding 45 μL of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIP® 3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the fluorescent substrate and phosphorylated product. Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentrations of reagents in the assays are ATP, 500 μM; FL-IPTSPITTTYFFFKKK peptide 1.5 μM; IRAK4, 0.6 nM; and DMSO, 1.6%.
Measurement: IC50 = 5.9 nM (pChEMBL 8.229999542236328)
Descriptors: MW 449.53, ALogP 4.23, HBA 7, HBD 4, PSA 99.17, QED 0.41
Compound (SMILES): CONC(=O)Nc1c(F)cncc1-c1ccc2ncc(-c3cc(C)cc(F)c3)c(N3CCC(N)CC3)c2c1
InChIKey: XRLKSACYNSIOII-UHFFFAOYSA-N
Target: Somatostatin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P30874
Assay: cAMP Assay : Four days prior to the assay, 5,000 Chinese hamster ovary cells (CHO-K1, ATCC #CCL-61) stably expressing the human SSTR2 are plated in each well of a 96-well tissue culture-treated plate in Ham's F12 growth media (ThermoFisher #10-080-CM) supplemented with 10% donor bovine serum (Gemini Bio-Products #100-506), 100 U/mL penicillin; 100 ug/mL streptomycin; 2 mM L-glutamine (Gemini Bio-Products #400-110) and 0.2 mg/mL hygromycin B (GoldBio #31282-04-9). The cells are cultured at 37° C., 5% CO2 and 95% humidity. On the day of the assay, the media is aspirated and the cells are treated with 50 μL of 1.6 μM NKH477 (Sigma #N3290) plus various dilutions of compounds of the invention in assay buffer [1× Hank's Balanced Salt Solution (ThermoFisher #SH3058802), 0.5 mM HEPES pH 7.4, 0.1% bovine serum albumin, 0.2 mM 3-Isobutyl-1-methylxanthine (IBMX, VWR #200002-790)]. The cells are incubated for 20 minutes at 37° C. (the final concentration of the compounds of the invention are typically 0-10,000 nM). The cells are treated with 50 μL of lysis buffer (HRTF cAMP kit, Cisbio). The lysate is transferred to 384-well plates and cAMP detection and visualization antibodies are added and incubated for 1-24 hours at room temperature. The time-resolved fluorescent signal is read with a Tecan M1000Pro multiplate reader. The intracellular cAMP concentrations are calculated by regression to a standard curve and are plotted vs. the concentration of the compounds of the invention and the EC50 of the compounds are calculated using standard methods. All data manipulations are in GraphPad Prism v6.
Measurement: EC50 < 10 nM (pChEMBL 8)
Descriptors: MW 518.57, ALogP 5.16, HBA 6, HBD 3, PSA 105.4, QED 0.32
Compound (SMILES): Cc1ccc(-c2c(C)c3c(c(C)c2[C@H](OC(C)(C)C)C(=O)O)CN(C(=O)c2cccc4ccoc24)C3)cc1
InChIKey: XRIBFOQPSVAYFA-LJAQVGFWSA-N
Target: Human immunodeficiency virus type 1 integrase (SINGLE PROTEIN), Human immunodeficiency virus 1
UniProt: Q7ZJM1
Assay: Anti-HIV Activity MT4 Assay: Antiviral HIV activity and cytotoxicity values for compounds of the invention from Table 1 were measured in parallel in the HTLV-1 transformed cell line MT-4 based on the method previously described (Hazen et al., 2007, In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV (Hazen et al., In vitro antiviral activity of the novel, tyrosyl-based human immunodeficiency virus (HIV) type 1 protease inhibitor brecanavir (GW640385) in combination with other antiretrovirals and against a panel of protease inhibitor-resistant HIV, Antimicrob. Agents Chemother. 2007, 51: 3147-3154; and Pauwels et al., Sensitive and rapid assay on MT-4 cells for the detection of antiviral compounds against the AIDS virus, J. of Virological Methods 1987, 16: 171-185).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 511.62, ALogP 7.12, HBA 4, HBD 1, PSA 79.98, QED 0.31
Compound (SMILES): CCS(=O)(=O)c1ccc(OC)cc1Cn1cnc2c(Br)cc(C(F)(F)F)cc2c1=O
InChIKey: YZTLABCPIIFPID-UHFFFAOYSA-N
Target: Epithelial discoidin domain-containing receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q08345
Assay: LanthaScreen Assay: DDR1 binding activity was measured using the LanthaScreen (Registered trademark) Eu Kinase Binding Assay (manufactured by Life Technologies Corporation). The test compound and the Alexa Fluor® 647-labeled Kinase Tracer 178 (manufactured by Life Technologies Corporation) were added to a mixture of DDR1 and the LanthaScreen (Registered trademark) Eu-anti-GST antibody. After one-hour reaction at room temperature, the fluorescence resonance energy transfer was measured. The 50% inhibitory concentration (IC50) was calculated from the inhibition rate relative to the test compound-free control.
Measurement: IC50 = 1420 nM (pChEMBL 5.849999904632568)
Descriptors: MW 505.31, ALogP 4.03, HBA 6, HBD 0, PSA 78.26, QED 0.52
Compound (SMILES): O=C(c1cccc(F)c1-c1ncccn1)N1CC2CC(Nc3ccc(Cl)cn3)C1C2
InChIKey: BVFVSYKYGCTVQL-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Human Orexin 1 Receptor Ca2+ Mobilization Assay: CHO cells stably transfected with the human orexin 1 receptor (Genebank accession number NM_001526) were grown to confluency in DMEM/F12, 10% FBS, 1×pen-strep, 400 μg/ml G418. Cells were seeded on to 384-well Packard viewplates at a density of 10,000 cells/well and incubated overnight at 37° C., 5% CO2. The cells were dye-loaded with BD Calcium Assay kit (BD, cat #640178) in HBSS (Gibco, cat #14025-092) with 2.5 mM probenecid and incubated at 37° C., 5% CO2 for 45 min. Cells were pre-incubated with compounds (diluted in DMEM/F-12) for 15-30 minutes before agonist (orexin A, 10 nM) stimulation. Ligand-induced Ca2+ release was measured using a Fluorometric Imaging Plate Reader (FLIPR, Molecular Devices, Sunnyvale, Calif.). Functional responses were measured as peak fluorescence intensity minus basal. The concentration of agonist that produced a half-maximal response is represented by the EC50 value. Antagonistic potency values were converted to apparent pKB values using a modified Cheng-Prusoff correction. Apparent pKB=−log IC50/1+[conc agonist/EC50].
Measurement: Ki = 12 nM (pChEMBL 7.920000076293945)
Descriptors: MW 423.88, ALogP 4.05, HBA 5, HBD 1, PSA 71.01, QED 0.69
Compound (SMILES): COc1ccc(CN2CCN(c3ccc(-c4cc(OCC5(CO)COC5)cn5ncc(C#N)c45)cn3)CC2)cn1
InChIKey: ZMTHMRJXIRHQNZ-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. 62TK0PEC) 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. 62TK0PEC). 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 = 50.3 nM (pChEMBL 7.300000190734863)
Descriptors: MW 541.61, ALogP 2.38, HBA 11, HBD 1, PSA 121.27, QED 0.34
Compound (SMILES): C/C=C\C(=O)N1CCC[C@@H](NC(=O)c2sc3nccc4c3c2NC(=O)N4c2ccc(Oc3ccccc3)cc2C)C1
InChIKey: XXHFALQMUFKABT-ZUSXEKFQSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: TBD: TBD
Measurement: IC50 = 63.1 nM (pChEMBL 7.199999809265137)
Descriptors: MW 567.67, ALogP 6.38, HBA 6, HBD 2, PSA 103.87, QED 0.26
Compound (SMILES): CC(C)C(C)(O)c1cc([C@@H]2CC[C@@H](C)N(C(=O)c3ccccc3-n3nccn3)C2)no1
InChIKey: SJIHDYMYGWGYTB-QVSFKUMNSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: FLIPR Ca2+ Flux Assay: n a typical experiment the OX1 and OX2 receptor antagonistic activity of the compounds of the present invention was determined in accordance with the following experimental method. For intracellular calcium measurements, Chinese hamster ovary (CHO) cells expressing the rat orexin-1 receptor or the human orexin-2 receptor, are grown in Iscove's modified DMEM containing 2 mM L-glutamine, 0.5 g/ml G418, 1% hypoxanthine-thymidine supplement, 100 U/ml penicillin, 100 μg/ml streptomycin and 10% heat-inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into Becton-Dickinson black 384-well clear bottom sterile plates coated with poly-D-lysine. All reagents are from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70 pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist.
Measurement: IC50 = 24.3 nM (pChEMBL 7.610000133514404)
Descriptors: MW 423.52, ALogP 3.53, HBA 7, HBD 1, PSA 97.28, QED 0.67
Compound (SMILES): NC(=O)Cc1ccccc1-c1cnc(N)c(-c2nnc(-c3ccccc3)o2)n1
InChIKey: UYBIYWSFLCNCBG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase ATR (SINGLE PROTEIN), Homo sapiens
UniProt: Q13535
Assay: Inhibition Assay: Compounds were screened for their ability to inhibit ATR kinase using a radioactive-phosphate incorporation assay. Assays were carried out in a mixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT. Final substrate concentrations were 10 M [γ-33P]ATP (3mCi 33P ATP/mmol ATP, Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).Assays were carried out at 25° C. in the presence of 5 nM full-length ATR. An assay stock buffer solution was prepared containing all of the reagents listed above, with the exception of ATP and the test compound of interest. 13.5 μL of the stock solution was placed in a 96 well plate followed by addition of 2 μL of DMSO stock containing serial dilutions of the test compound (typically starting from a final concentration of 15 μM with 3-fold serial dilutions) in duplicate (final DMSO concentration 7%). The plate was pre-incubated for 10 minutes at 25° C. and the reaction initiated by addition of 15 μL [γ-33P]ATP (final concentration 10 μM).The reaction was stopped after 24 hours by the addition of 30 μL 0.1M phosphoric acid containing 2 mM ATP. A multiscreen phosphocellulose filter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with 100 μL 0.2M phosphoric acid prior to the addition of 45 μL of the stopped assay mixture. The plate was washed with 5×200 μL 0.2M phosphoric acid. After drying, 100 μL Optiphase SuperMix liquid scintillation cocktail (Perkin Elmer) was added to the well prior to scintillation counting (1450 Microbeta Liquid Scintillation Counter, Wallac).
Measurement: Ki = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 372.39, ALogP 2.47, HBA 7, HBD 2, PSA 133.81, QED 0.55
Compound (SMILES): CCN(CC)S(=O)(=O)N1CC[C@H](NC(=O)c2cc(C3CC3)on2)C[C@@H]1C
InChIKey: KQVIYOKJTPCSGH-JSGCOSHPSA-N
Target: Histone-lysine N-methyltransferase SMYD3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7B4
Assay: SMYD3 Biochemical Assay: The assays were all performed in a buffer consisting of 25 mM Tris-Cl pH 8.0, 1 mM TCEP, 0.005% BSG, and 0.005% Tween 20, prepared on the day of use. Compounds in 100% DMSO (1 ul) were spotted into a 384-well white opaque OptiPlate using a Bravo automated liquid handling platform outfitted with a 384-channel head (Agilent Technologies). DMSO (1 ul) was added to Columns 11, 12, 23, 24, rows A-H for the maximum signal control and 1 ul of SAH, a known product and inhibitor of SMYD3, was added to columns 11, 12, 23, 24, rows I-P for the minimum signal control. A cocktail (40 ul) containing the SMYD3 enzyme was added by Multidrop Combi (Thermo-Fisher). The compounds were allowed to incubate with SMYD3 for 30 min at room temperature, then a cocktail (10 ul) containing SAM and MEKK2 was added to initiate the reaction (final volume=51 ul). The final concentrations of the components were as follows: SMYD3 was 0.4 nM, 3H-SAM was 8 nM, MEKK2 was 12 nM, SAH in the minimum signal control wells was 1 mM, and the DMSO concentration was 2%. The assays were stopped by the addition of non-radiolabeled SAM (10 ul) to a final concentration of 100 uM, which dilutes the 3H-SAM to a level where its incorporation into MEKK2 is no longer detectable. Radiolabeled MEKK2 was detected using a scintillation proximity assay (SPA). 10 uL of a 10 mg/mL solution of SPA beads in 0.5 M citric acid was added and the plates centrifuged at 600 rpm for 1 min to precipitate the radiolabeled MEKK2 onto the SPA beads. The plates were then read in a PerkinElmer TopCount plate reader to measure the quantity of 3H-labeled MEKK2 as disintegrations per minute (dpm) or alternatively, referred to as counts per minute (cpm).% Inhibition Calculation% ⁢ ⁢ inh = 100 - ( dpm cmpd - dpm min dpm max - dpm min ) × 100Where dpm=disintegrations per minute, cmpd=signal in assay well, and min and max are the respective minimum and maximum signal controls.Four-Parameter IC50 FitY = Bottom + ( Top - Bottom ) ( 1 + ( X IC 50 ) Hill ⁢ ⁢ CoefficientWhere top and bottom are the normally allowed to float, but may be fixed at 100 or 0 respectively in a 3-parameter fit. The Hill Coefficient normally allowed to float but may also be fixed at 1 in a 3-parameter fit. Y is the % inhibition and X is the compound concentration.
Measurement: IC50 = 3718 nM (pChEMBL 5.429999828338623)
Descriptors: MW 384.5, ALogP 1.72, HBA 5, HBD 1, PSA 95.75, QED 0.77
Compound (SMILES): NCc1nccc2cc(-c3cccc(S(N)(=O)=O)c3-c3nn[nH]n3)ccc12
InChIKey: NOSBXEYDFQQFLO-UHFFFAOYSA-N
Target: Metallo-beta-lactamase type 2 (SINGLE PROTEIN), Bacteria
UniProt: C7C422
Assay: Class B Enzyme Assay: The Class B enzyme activities were measured in the presence of the test inhibitor in a fluorescence assay against a commercially available substrate consisting of a cephalosporin core linking 7-hydroxycoumarin to fluorescein (CCF2-FA). The enzyme (NDM-1, IMP-1 or VIM-1) and the substrate were diluted in 100 mM KH2PO4 buffer (pH 7) containing 0.005% Tween-20 and 10 μM ZnSO4. In the assay, the final concentration of enzyme was 1 pM, 2 pM and 30 pM for NDM-1, IMP-1 and VIM-1, respectively, and the final concentration of CCF2-FA was 1.25 μM. The test inhibitor was dissolved in dimethylsulfoxide and diluted 1:50 in the assay, resulting in a final concentration range of 20 uM to 0.00063 uM. In a 384-well microplate, the test inhibitor was incubated with the metallo-β-lactamase enzyme and the substrate for 2 hours at 25° C. Fluorescence at 460 nm following excitation at 405 nm was measured. The IC50 value was determined from semi-logarithmic plots of enzyme inhibition versus inhibitor concentration, with a curve generated using a 4-parameter fit.
Measurement: IC50 = 1.44 nM (pChEMBL 8.84000015258789)
Descriptors: MW 381.42, ALogP 1.19, HBA 7, HBD 3, PSA 153.53, QED 0.48
Compound (SMILES): COc1cc(Cn2cnc3cc(CO)cnc32)cc2c1OC(c1cnn(C3CCC3)c1)CO2
InChIKey: WLKVUNXUWHCHJE-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 = 35 nM (pChEMBL 7.460000038146973)
Descriptors: MW 447.5, ALogP 3.41, HBA 9, HBD 1, PSA 96.45, QED 0.48
Compound (SMILES): COc1cc(C(F)(F)F)ccc1COC(c1ccc(C2(S(=O)(=O)c3ccc(F)cc3)CCCC2)cc1)(C(F)(F)F)C(F)(F)F
InChIKey: RNRKPPOLPVNAEW-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: SPA Binding Assay: The binding of potential ligands to RORγ is measured by competition with [3H] 25-hydroxycholesterol (Perkin Elmer NET674250UC) using a scintillation proximity assay (SPA) binding assay. The ligand binding domain of human RORγ (A262-S507) with an N-terminal His tag is expressed in E. coli and purified using nickel affinity chromotography. 15 ug/well RORγ (A262-S507) is incubated with test compound at varying concentrations in 3-fold serial dilution, with final concentrations ranging from 16.6 μM to 0.28 nM for 10 min at room temperature in PBS buffer (Invitrogen #14190-144) containing 0.5% fatty acid free BSA (Gemini Bio-Products, Cat. #700-107P) and 0.1% Glycerol (Sigma Cat# G5516). 10 nM of [3H] 25-hydroxycholesterol is then added, and the reaction is incubated for 10 min. 10 mg/mL of Copper-His Tag-PVT beads (Perkin Elmer cat # RPNQ0095) are added, and the mixture is incubated for 60 min. The reaction is read on a TopCount Microplate scintillation plate reader (Perkin Elmer).
Measurement: IC50 = 85 nM (pChEMBL 7.070000171661377)
Descriptors: MW 658.55, ALogP 8.63, HBA 4, HBD 0, PSA 52.6, QED 0.18
Compound (SMILES): Cc1cc(F)ccc1-c1cnc2[nH]c(=O)n(CCN3CCC3)c2c1
InChIKey: VPTXKSSRQKRRBF-UHFFFAOYSA-N
Target: Glutamate receptor ionotropic, NMDA 2B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13224
Assay: Standard cell-based calcium flux assay: NMDA receptors are ion channels that are highly permeable to Ca2+ ions, rendering it possible to monitor NMDA receptor function using cell-based calcium flux assay. In this assay, co-agonists glutamate and glycine are added to cells heterologously expressing human GluN1/GluN2B NMDA receptors to initiate cellular Ca2+ influx. The time course of the changes in intracellular calcium is measured using a fluorescent dye and a FLIPR (Fluorometric Imaging Plate Reader) device.Twenty four hours before measurements, the expression of the NMDA receptors in the stable cell line is induced with Tet-On inducible system in the presence of a non-selective NMDA receptor blocker. On the day of the experiment, cell culture media is carefully washed and the cells are loaded with Calcium 5 Dye Kit (Molecular Devices) in dye loading buffer containing 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2 (standard assay) or 1.5 mM MgCl2 (HTS assay), 10 mM HEPES and 5 mM D-glucose; pH 7.4. After 1 h incubation at the room temperature, the dye is washed away with the assay buffer (137 mM NaCl (standard assay) or 150 mM (HTS assay), 4 mM KCl (standard assay) or 3 mM (HTS assay), 2 mM CaCl2, 0.01 mM EDTA, 10 mM HEPES and 5 mM D-glucose; pH 7.4) In the FLIPR TETRA reader, various concentrations of the test compounds are added to the cells for 5 min while fluorescence is monitored to detect potential agonist activity. Next, co-agonists, glutamate and glycine are added for another 5 minutes. The concentration of glutamate corresponding to EC40 (standard assay) is used to maximize the assay's signal window and ability to detect NMDA receptor antagonists and negative allosteric modulators. A saturating concentration (10 μM) of glycine is also present in the assay. A non-selective NMDA receptor antagonist, (+)MK-801 is used as a positive control for antagonist activity. The fluorescent signal in the presence of test compounds is quantified and normalized to the signal defined by the appropriate control wells.
Measurement: IC50 = 115 nM (pChEMBL 6.940000057220459)
Descriptors: MW 326.38, ALogP 2.54, HBA 4, HBD 1, PSA 53.92, QED 0.8
Compound (SMILES): NCC(=O)N1CCN(c2ccc(Nc3ncc4c(n3)OCN(c3c(Cl)cccc3Cl)C4=O)cc2)CC1
InChIKey: VYXLDWNGKUECTP-UHFFFAOYSA-N
Target: Wee1-like protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P30291
Assay: WEE1 IC50 Determination: IC50 values of compounds against WEE1 kinase enzyme were determined by LanthaScreen Terbium Labeled TR-FRET assay. Kinase assays were performed in 1× kinase buffer (# PV6135, Invitrogen, Life Technologies Grand Island, N.Y.) where total reaction volume was 10 μl in low-volume 384-well plates (#4511, Corning). Serially diluted compounds (3-fold) were incubated with WEE1 Enzyme (1 nM) (# PR7373A, Invitrogen, Life Technologies Grand Island, N.Y.) for 10 min, following which a mixture of ATP (10 M) (# A1852, Sigma, St-Louis, Mo.) and fluorescent-PolyGT substrate (200 nM) (# PV3610, Invitrogen, Life Technologies Grand Island, N.Y.) was added and incubated in dark at room temperature for 1 h. After 1 h, 10 μl stop solution containing Terbium labeled antibody (4 nM) (# PV3529, Invitrogen, Life Technologies Grand Island, N.Y.) and EDTA (# E5134, Sigma, St-Louis, Mo.) (20 mM) in TR-FRET dilution buffer (# PV3574, Invitrogen, Life Technologies Grand Island, N.Y.) was added. Readings were taken in a Synergy Neo Plate reader (BioTek, Winooski) at single excitation of 340 nm and Dual emission at 495 nm and 520 nm respectively.
Measurement: IC50 = 53 nM (pChEMBL 7.28000020980835)
Descriptors: MW 528.4, ALogP 3.13, HBA 8, HBD 2, PSA 116.92, QED 0.52
Compound (SMILES): CNc1ncc2cc(-c3cc(NC(=O)N4CC[C@H](OC(F)(F)F)C4)ccc3C)cnc2n1
InChIKey: TYGYQWGSJQOWJD-INIZCTEOSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Kinase Assay: Protein kinase assay: Assay platform was used to measure kinase/inhibitor interactions as described previously (Anastassiadis et al., 2011). In brief, for each reaction, kinase and substrate were mixed in a buffer containing 20 mM HEPES (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO. All compounds were solubilized in DMSO. Compounds were then added to each reaction mixture via acoustic dispense using an ECHO 550 nanoliter dispenser. For human RAF1 testing, human MEK1 (K97R) was used as a substrate at a concentration of 3 micromolar, with a final ATP concentration of 10 micromolar. For human BRAF testing, human MEK1 (K97R) was used as a substrate at 1 micromolar concentration, with a final ATP concentration of 25 micromolar. Compounds were tested in 10-dose IC50 mode with a 3-fold serial dilution starting at 10 micromolar. After a 20-min incubation, ATP (Sigma-Aldrich, St. Louis, Mo. 63178) and [g33P] ATP (specific activity 10 microCi/microliter) purchased at PerkinElmer (Boston, Mass., 02118 Cat #BLU 003H250UC) were added at a final total concentration of 10 mM. Reactions were carried out at room temperature for 2 hr and spotted onto P81 ionexchange cellulose chromatography paper (Reaction Biology). Filter paper was washed in 0.75% phosphoric acid to remove unincorporated ATP. The percent remaining kinase activity relative to a vehicle-containing (DMSO) kinase reaction was calculated for each kinase/inhibitor pair.
Measurement: IC50 < 0.1 nM (pChEMBL 10)
Descriptors: MW 446.43, ALogP 4.18, HBA 6, HBD 2, PSA 92.27, QED 0.62
Compound (SMILES): Cc1cccc(-c2nc(C(C)(C)C)sc2CNC(=O)Nc2ccc(CNS(C)(=O)=O)c(F)c2)c1
InChIKey: HFRYZWTZVINCKM-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: FLIPR Assay: The FLIPR protocol consists of 2 substance additions during a kinetic measurement. First the compounds to be tested (5 μM) are pipetted onto the cells and the Ca2+ influx is determined in comparison to the control (capsaicin 10 μM), providing the result as % activation and representing the compound-alone effect (calculation at peak signal related to baseline). A Ca2+ influx of 10%-60% reveals a partial agonist (pAG), a Ca2+ influx of >60% relates to a pure agonist (AG). After 5 min incubation, the Ca2+ influx is related to an injection of 100 nM of capsaicin and thereby the antagonistic effect of the test compounds detected.
Measurement: Ki = 2.3 nM (pChEMBL 8.640000343322754)
Descriptors: MW 504.65, ALogP 4.93, HBA 5, HBD 3, PSA 100.19, QED 0.42
Compound (SMILES): Cc1cccc(-c2ncnc3c(S(=O)(=O)c4ccccc4)c(N)[nH]c23)c1
InChIKey: ZCHHGAHPZZJJFA-UHFFFAOYSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: Ca-Flux Functional Assay: GPR43 agonist/PAM activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by FLIPR (manufactured by Molecular Devices). GPR43 mediated increases in intracellular Ca2+ concentration were readily detected upon activation with sodium acetate. Prior to the assay (24 hours), CHO-K1 Gα16 cells stably expressing human GPR43 were-seeded in cell culture medium in black, clear-bottom 384-well plates (Corning Inc) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (Molecular Devices) diluted in HBSS containing 25 mM HEPES, 2.5 mM Probenecid, 0.1% BSA for 1 hour at 37° C., 5% CO2. 10 point half log concentration response curves of sodium acetate from 10 mM were conducted prior to the testing of compounds to calculate the sodium acetate concentration that produces 20% of the maximal response (EC20). Test compounds (at 10 point half log concentration response curves from 10 μM) were added in the presence of sodium acetate to achieve a final concentration that produces approximately 20% maximal response as calculated from the previous experiment. The changes in fluorescent signal were monitored by FLIPR upon addition of the compound/EC20 sodium acetate mix. The EC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: EC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 364.43, ALogP 3.35, HBA 5, HBD 2, PSA 101.73, QED 0.58
Compound (SMILES): C=CC(=O)Nc1cc(Nc2ncc(Cl)c(-c3cnc4c(ccn4C4CC4)c3)n2)c(OC)cc1N(C)CCN(C)C
InChIKey: KMCJVQYGVDKIHH-UHFFFAOYSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Wild Type EGFR and Mutant EGFR Kinase Inhibition Test: The inhibitory effects of compounds to be tested on double mutant EGFR kinase (EGFR T790M/L858R Kinase) (Invitrogen, PV4879) and wild-type EGFR kinase (EGFR WT) (Invitrogen, PV3872) were measured by z-lyte methods.The working concentration of each component in 10 μl T790M/L858R kinase reaction system was: 25 μM ATP, 0.08 (or 0.1) ng/μl EGFR T790M/L858R kinase, 2 μM Tyr04 substrate (Invitrogen, PV3193, similarly hereinafter). After the compounds (i.e., test compounds) prepared by the above-mentioned examples described herein were added, the concentration of DMSO was 2%.The working concentration of each component in 10 μl EGFR WT kinase reaction system was: 10 μM ATP, 0.8 ng/μl EGFR WT kinase, 2 μM Tyr04 substrate. After the test compounds were added, the concentration of DMSO was 2%.Test Methods:10 mM stock solutions of the test compounds dissolved at room temperature were gradiently diluted by 4% DMSO in water to final concentrations of 10-0.005 μM. To each well were added 2.5 μl of solution of the test compounds and 5 μl mixture of the EGFR T790M/L858R kinase (or EGFR WT kinase) and Tyr04 substrate diluted by reaction buffer, and then 2.5 μl of ATP was added to initiate the reaction. Reaction buffer instead of ATP were added to C1 wells, no drugs were added to C2 wells, and the phosphorylated substrates were added to C3 wells according to the instruction.The reaction was performed on a shaking table at room temperature for 60 min. Afterwards, 5 μl of Development Reagent B (Invitrogen) was added, and reacted on a shaking table at room temperature for 60 min. The plates were read in a VictorX5 Microplate Reader (PerkinElmer), for measuring the absorbance at excitation wavelength of 405 nm, and emission wavelength of 450 nm and 520 nm. (For example, C3520nm represents the reading at 520 nm of C3 well).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 561.09, ALogP 5.36, HBA 9, HBD 2, PSA 100.44, QED 0.24
Compound (SMILES): COc1cc(-c2[nH]c3ccc(C4CCN(C(=O)CN(C)C)CC4)cc3c2C(C)C)cn2ncnc12
InChIKey: DUGHAHUHPFRPOB-UHFFFAOYSA-N
Target: Toll-like receptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYK1
Assay: TLR7/8/9 Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 474.61, ALogP 4.28, HBA 6, HBD 1, PSA 78.76, QED 0.45
Compound (SMILES): COc1cc(-c2cccc(-c3cccc(NC(=O)c4c[nH]c(=O)n(C)c4=O)c3C)c2C)cc(F)c1CNC[C@H]1CCC(=O)N1
InChIKey: HRXMHQFFNYWWKA-OAQYLSRUSA-N
Target: Programmed cell death 1 ligand 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ7
Assay: Enzyme-Linked Immunosorbent AssayELISA: 96 Well plates were coated with 111 g/mL of human PD-L1 (obtained from R&D) in PBS overnight at 4° C. The wells were then blocked with 2% BSA in PBS (W/V) with 0.05% TWEEN-20 for 1 hour at 37° C. The plates were washed 3 times with PBS/0.05% TWEEN-20 and the compounds were serial diluted (1:5) in dilution medium and added to the ELISA plates. Human PD-1 and biotin 0.31 μg/mL (ACRO Biosystems) were added and incubated for 1 hour at 37° C. then washed 3 times with PBS/0.05% TWEEN-20. A second block was performed with 2% BSA in PBS (W/V)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HRP was added for 1 hour at 37° C. then the plates were washed 3 times with PBS/0.05% TWEEN-20. TMB substrate was added and reacted for 20 min at 37° C. A stop solution (2 N aqueous H2SO4) was added. The absorbance was read at 450 nm using a micro-plate spectrophotometer.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 599.66, ALogP 3.79, HBA 7, HBD 4, PSA 134.32, QED 0.23
Compound (SMILES): Cc1cc(OCCCc2c3n(c4c(-c5c(C)nn(C)c5C)c(Cl)ccc24)[C@H](C)CN(c2cn(CCN4CCN(C)C(=O)C4)c4ccc(-c5ncn(C)n5)cc24)C3=O)cc(C)c1Cl
InChIKey: UAURSKSSHTVAGR-GDLZYMKVSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: TR-FRET Binding Assay: Measurements were carried out in OptiPlate-384 White Opaque 384-well plates (Perkin Elmer. Shelton, Conn.). The assay was run using a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) that was purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification, 300 nM FITC-Bak peptide, 1 nM recombinant Maltose Binding Protein (MBP) tagged Mcl-1 (residues 172-327) and 1 nM terbium conjugated-MBP antibody were added to assay buffer (4.5 mM Monobasic potassium phosphate, 15.5 mM Dibasic potassium phosphate, 1 mM Sodium EDTA, 50 mM NaCl, 1 mM DTT, 0.05% Pluronic F-68, pH 7.5). Compounds are diluted in DMSO in a 16-point, semilog serial dilution scheme. For the BAK FITC TR-FRET assay 50 nL compound is added to 20 μL of assay buffer containing Bak-FITC. MBP tagged Mcl-1 protein and antibody, for a final DMSO concentration of 0.25% and a top concentration of 10 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 3 hours at room temperature. TR-FRET signal (Delta F) is measured on a Biotek Cytation 3 multimode plate reader equipped with a filter cube containing an Ex 340/30 Em 620/10 nm filter and an Ex 340/30 Em 520 filter.
Measurement: Ki = 0.89 nM (pChEMBL 9.050000190734863)
Descriptors: MW 875.91, ALogP 8.34, HBA 11, HBD 0, PSA 111.48, QED 0.12