lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(NC1(c2c[nH]nn2)CC(F)(F)C1)C(=O)N1CC[C@H](C(=O)Nc2ccc(F)c(Cl)c2)C1
InChIKey: ATJCZTXQVVBHSE-JTQLQIEISA-N
Target: Core antigen (SINGLE PROTEIN), Hepatitis B virus
UniProt: Q03732
Assay: Anti-HBV Activity: The anti HBV activity was measured using the HepG2.117 cell line, a stable, inducible HBV producing cell line, which replicates HBV in the absence of doxicycline (Tet-off system). The HepG2 cell line is available from ATCC®) under number HB-8065. Transfection of the HepG2 cell line can be as described in Sun and Nassal 2006 Journal of Hepatology 45 (2006) 636-645 Stable HepG2- and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus.For the antiviral assay, HBV replication was induced, followed by a treatment with serially diluted compound in 96-well plates. After 3 days of treatment, the antiviral activity was determined by quantification of intracellular HBV DNA using real-time PCR and an HBV specific primer set and probe.Cytotoxicity of the compounds was tested using HepG2 or HepG2.117 cells, incubated for 3 days in the presence of compounds. The viability of the cells was assessed using the Perkin Elmer ATPlite Luminescence Assay System.
Measurement: EC50 = 40 nM (pChEMBL 7.400000095367432)
Descriptors: MW 470.84, ALogP 1.82, HBA 5, HBD 3, PSA 120.08, QED 0.59
Compound (SMILES): O=C(O)CCC1CCc2cc(-c3cccnc3OCC3CC3)ccc2O1
InChIKey: NBOZHWVCTZCXCV-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: Measurement of Activity of GPR120 Agonist (Cell-Based Assay): CHO-K1 cells expressing Ga16 and hGPR120 were dispensed into each well of a 96-well plate (3×104 cells/100 μl/well) and then incubated in 5% CO2, 37° C. incubator for 18 hours. Each well was treated with 100 μl of Calcium 5 dye (Molecular Devices) solution including 2% DMSO and then incubated in 5% CO2, 37° C. incubator for 1 hour. Serially diluted GPR120 agonists were prepared to a final concentration of 0.5% DMSO in a 96-well plate. Each well was treated with 50 μl of the agonist compounds using Plexstation II, and then fluorescence was measured at Ex 485 nm and Em 525 nm.Fluorescence increased by the serially diluted GPR120 agonists is calculated as a relative percent (%) value based on the fluorescence represented by the treatment of 1% DMSO only. EC50 refers to the concentration of agonist which shows 50% of maximum fluorescence increased by the treatment of agonist. The calculation of measurement was carried out by using statistical software (Prizm).
Measurement: EC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 353.42, ALogP 4.1, HBA 4, HBD 1, PSA 68.65, QED 0.81
Compound (SMILES): Cc1cc(N2C[C@@H](C)n3c(c(CCCOc4cc(C)c(Cl)c(C)c4)c4ccc(Cl)c(-c5c(C)ncnc5C)c43)C2=O)c2c(c1)cc(C(N)=O)n2C
InChIKey: ZDARWRXLAMAYOH-HSZRJFAPSA-N
Target: Induced myeloid leukemia cell differentiation protein Mcl-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07820
Assay: Fluorescence Polarization Anisotropy Competition Assay: Fluorescence Polarization Anisotropy Competition Assay. Anisotropy measurements were carried out in 384-well, black, flat-bottom plates (Greiner Bio-one, Monroe, N.C., USA). The assay was run using either a fluorescein isothiocyanate-labeled BH3 peptide derived from Bak (FITC-AHx-GQVGRQLAIIGDDINR-NH2) or a fluorescein isothiocyanate-labeled BH3 peptide derived from Bim (FITC-AHx-EARIAQELRRIGDEFNETYTR-NH2)that were purchased from GenScript (Piscataway, N.J.) at >95% purity and used without further purification. 10 nM FITC-Bak peptide and 15 nM recombinant Mcl-1 (residues 172-327) were added to assay buffer (3 mM dithiothreitol, 50 mM NaCl, 20 mM Tris, pH 7.5). The Bim based assay was run with 1 nM FITC-Bim peptide and 1.5 nM recombinant Mcl-1 (residues 172-327) added to assay buffer (20 mM TRIS pH 7.5, 50 mM NaCl, 3 mM DTT, 0.01% CHAPS). For selectivity assays, 40 nM Bcl-2 (residues 1-207 A96T,G110R, Δ35-91, replaced with Bcl-xL35-50) or 4 nM Bcl-xL (residues 1-209, loop 45-86 deleted) were incubated with 10 nM FITC-Bak in assay buffer. Compounds are diluted in DMSO in a 10-point, 3-fold serial dilution scheme. For the FITC-BAK assay 2.5 uL compound is added to 47.5 μL of assay buffer containing FITC-Bak and protein, for a final DMSO concentration of 5% and a top concentration of 20 μM. A FITC-Bak peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. For the FITC-Bim assay, compound is added to 40 uL of assay buffer containing protein, 15 minutes prior to addition of 10 μL of the FITC-Bim peptide, for a final DMSO concentration of 0.165% and a top concentration of 200 nM. A FITC-Bim peptide alone (100% inhibition) and peptide plus protein (0% inhibition) control is included on each assay plate. The plate was mixed and incubated for 90 minutes at room temperature.
Measurement: Ki = 6.5 nM (pChEMBL 8.1899995803833)
Descriptors: MW 723.7, ALogP 8.77, HBA 7, HBD 1, PSA 108.27, QED 0.16
Compound (SMILES): CC(C)(C)COc1nc(N2CC3(COC3)C2)c2ncn(Cc3ccccc3Cl)c2n1
InChIKey: IXHAARFCUFFFNK-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1x HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30° C. for 30 min. Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated as follows: FRET=T730-Alexa730-P(T645-B645) with P=Ru730-B730/Ru645-B645, where T730 is the test well measured at 730 nM, T645 is the test well measured at 645 nm, B730 and B645 are the buffer controls at 730 nm and 645 nm, respectively. cAMP content is determined from the function of a standard curve spanning from 10 uM to 0.13 nM cAMP. EC50 values were determined using Activity Base analysis (ID Business Solution, Limited). The EC50 values for a wide range of cannabinoid agonists generated from this assay were in agreement with the values published in the scientific literature. The compounds of the invention are CB2 receptor agonists with EC50 below 1 uM and selectivity versus CB1 in the corresponding assay of at least 10 fold. Particular compound of the invention are CB2 receptor agonists with EC50 below 0.05 uM and selectivity versus CB1 in the corresponding assay of at least 500 fold.
Measurement: EC50 = 268 nM (pChEMBL 6.570000171661377)
Descriptors: MW 427.94, ALogP 3.79, HBA 7, HBD 0, PSA 65.3, QED 0.62
Compound (SMILES): Cc1ccc(C2(O)CCCC2F)cc1-c1cnc2c(N)nc(C(F)(F)F)cn12
InChIKey: JJFCMBOEXQORLM-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Scintillation Proximity Assay: PI3K-γ: [γ-33P]ATP (10 mCi/mL) and Wheat Germ Agglutinin (WGA) YSi SPA Scintillation Beads was purchased from PerkinElmer (Waltham, Mass.). Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2), CAS 204858-53-7, was purchased from Echelon Biosciences (Salt Lake City, Utah). PI3Kγ (p110γ) Recombinant Human Protein was purchased from Life technology (Grand Island, N.Y.). ATP, MgCl2, DTT, EDTA, MOPS and CHAPS were purchased from SigmaAldrich (St. Louis, Mo.).The kinase reaction was conducted in polystyrene 384-well Greiner Bio-one white plate from Thermo Fisher Scientific in a final volume of 25 μL. Inhibitors were first diluted serially in DMSO and added to the plate wells before the addition of other reaction components. The final concentration of DMSO in the assay was 2%. The PI3Kγ assay was carried out at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl2, 5 mM DTT and CHAPS 0.03%. Reactions were initiated by the addition of ATP, the final reaction mixture consisted of 20 μM PIP2, 2 μM ATP, 0.5 μCi [γ-33P] ATP, 13 nM PI3Kγ. Reactions were incubated for 120 min and terminated by the addition of 40 μL SPA beads suspended in quench buffer: 163 mM potassium phosphate pH 7.8, 20% glycerol, 25 mM EDTA. The final concentration of SPA beads is 1.0 mg/mL. After the plate sealing, plates were shaken overnight at room temperature and centrifuged at 1500 rpm for 10 min, the radioactivity of the product was determined by scintillation counting on Topcount (Perkin-Elmer).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 394.37, ALogP 4.02, HBA 5, HBD 2, PSA 76.44, QED 0.64
Compound (SMILES): Cc1cc(F)cc(N2CCC3(CCC(CC(=O)O)CC3)CC2)c1Cl
InChIKey: VSRVIEOUQPXSDL-UHFFFAOYSA-N
Target: Free fatty acid receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5NUL3
Assay: FLIPR Assay: On the day before the measurement day, CHO cells expressing human GPR120 were plated at 10000 cells per well in a 384-well black plate (#3702, Corning) and incubated overnight in a CO2 incubator. On the day of the measurement, 4 μM Fluo-4 AM (fluorescence calcium indicator reagent) was incubated to be introduced into the human GPR120 expression CHO cells in the presence of 0.08% Pluronic F-127 in a CO2 incubator for 90 minutes. To the cells was added the test compound diluted with HBSS solution containing 20 mM HEPES and 2.5 mM probenecid. Variations in the intracellular calcium concentration were measured by Fluorescence Imaging Plate Reader (FLIPR; Molecular Devices) to examine the agonist action, and EC50 values were calculated.
Measurement: EC50 = 2864 nM (pChEMBL 5.539999961853027)
Descriptors: MW 353.87, ALogP 5.04, HBA 2, HBD 1, PSA 40.54, QED 0.83
Compound (SMILES): Cn1cc(NC(=O)c2cnn3ccc(N4CCOCC4)nc23)c(C(N)=O)n1
InChIKey: YOSCKXRZGYSONH-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IRAK4 Kinase Assay: The kinase activity of IRAK4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP).20 μL reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DDT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μL of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 370.37, ALogP -0.35, HBA 9, HBD 2, PSA 132.67, QED 0.64
Compound (SMILES): CSc1ccc2nc([C@@H]3CCCN3C(=O)c3cc(C(F)(F)F)ccc3-n3nccn3)[nH]c2c1
InChIKey: APGFFSBHRCPRCL-IBGZPJMESA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 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 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 763 nM (pChEMBL 6.119999885559082)
Descriptors: MW 472.5, ALogP 4.86, HBA 6, HBD 1, PSA 79.7, QED 0.43
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@@H](OC(C)C)c2cnc(C)cn2)nnc1[C@H]1CCCO1
InChIKey: NHAAXZRCHPDFEG-AMHTUMDSSA-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 assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, and 0.1% (w/v) BSA with 200 mM NaCl, 3 μM GDP] and membranes expressing human APJ receptor/well along with WGA PS beads. The reaction was initiated by addition of 0.2 nM [35S]GTPγS (Perkin Elmer Life and Analytical Sciences, Waltham USA) in the absence or presence of various ligands and incubated at RT for 90 min. Nonspecific binding was determined in the presence of 100 μM GTPγS and was always less than 0.2% of total binding. All the results presented are means of several independent experiments and analyzed by non-linear regression methods using commercially available program Prism (GraphPad, San Diego, Calif.).
Measurement: EC50 = 6.9 nM (pChEMBL 8.15999984741211)
Descriptors: MW 546.65, ALogP 3.53, HBA 11, HBD 1, PSA 139.58, QED 0.38
Compound (SMILES): CCc1c(F)cc(-c2cc(C(F)(F)F)c3c(N)ncnn23)cc1C(=O)N[C@@H]1CN(C(=O)C2CC(F)C2)C[C@@H]1F
InChIKey: GZHLOVIVQKBUDX-KBUGRDCFSA-N
Target: Receptor-interacting serine/threonine-protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13546
Assay: RIPK1 HTRF Binding Assay: A solution was prepared containing 0.2 nM Anti GST-Tb (Cisbio, 61GSTTLB), 90.6 nM probe and 1 nM His-GST-TVMV-hRIPK1(1-324) in FRET Buffer (20 mM HEPES, 10 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 0.05 mg/mL BSA). Using Formulatrix Tempest, the detection antibody/enzyme/probe solution (2 mL) was dispensed into wells of a 1536 plate (Black Low Binding Polystyrene 1536 Plate (Corning, 3724)) containing 10 nL of compounds of interest at appropriate concentration in DMSO. The plate was incubated at rt for 1 h. FRET was measured using the EnVision plate reader (Excitation: 340 nM, Emission: 520 nM/495 nM). Total signal (0% inhibition) was calculated from wells containing 10 nL DMSO only. Blank signal (100% inhibition) calculated from wells containing 10 nL of 15 nM staurosporine and internal controls.
Measurement: IC50 = 11000 nM (pChEMBL 4.960000038146973)
Descriptors: MW 554.5, ALogP 3.73, HBA 6, HBD 2, PSA 105.62, QED 0.47
Compound (SMILES): Cc1cc(Nc2cc3ncccc3c(N[C@@H]3C[C@H]4CCC[C@@H](C3)N4CC(=O)OC(C)C)n2)n[nH]1
InChIKey: TVMGOXSFWNIENE-LDLYASANSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK Assay: A panel of four LanthaScreen JAK biochemical assays (JAK1, 2, 3 and Tyk2) were carried in a common kinase reaction buffer (50 mM HEPES, pH 7.5, 0.01% Brij-35, 10 mM MgCl2, and 1 mM EGTA). Recombinant GST-tagged JAK enzymes and a GFP-tagged STAT1 peptide substrate were obtained from Life Technologies.Serially diluted compounds were pre-incubated with each of the four JAK enzymes and the substrate in white 384-well microplates (Corning) at ambient temperature for lh. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAK1, 2, 3 and Tyk2 are 4.2 nM, 0.1 nM, 1 nM, and 0.25 nM respectively; the corresponding Km ATP concentrations used are 25 μM, 3 μM, 1.6 μM, and 10 μM; while the substrate concentration is 200 nM for all four assays. Kinase reactions were allowed to proceed for 1 hour at ambient temperature before a 10 μL preparation of EDTA (10 mM final concentration) and Tb-anti-pSTAT1 (pTyr701) antibody (Life Technologies, 2 nM final concentration) in TR-FRET dilution buffer (Life Technologies) was added. The plates were allowed to incubate at ambient temperature for lh before being read on the EnVision reader (Perkin Elmer). Emission ratio signals (520 nm/495 nm) were recorded and utilized to calculate the percent inhibition values based on DMSO and background controls.For dose-response analysis, percent inhibition data were plotted vs. compound concentrations, and IC50 values were determined from a 4-parameter robust fit model with the Prism software (GraphPad Software). Results were expressed as pIC50 (negative logarithm of IC50) and subsequently converted to pKi (negative logarithm of dissociation constant, Ki) using the Cheng-Prusoff equation.Test compounds having a higher pKi value in each of the four JAK assays show greater inhibition of JAK activity. Compounds of the invention tested in this assay typically exhibited pKi values between about 7 and about 10.3
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 463.59, ALogP 4.15, HBA 8, HBD 3, PSA 108.06, QED 0.45
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1CCc2ccccc2N(Cc2c(OC)ccc3ccc(C(=O)OC)cc23)C1=O
InChIKey: VIISZVVKHOSIGK-SBUREZEXSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA (Peptide A) was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK (SEQ ID NO: 3) was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA as the substrate for the TR-FRET assay.The XIAP protein sequence was obtained from the SWISS-PROT protein sequence database and the BIR2 and BIR3 domains were derived from that. The sequence of the BIR2 domain used for the TR-FRET assay is MRHHHHHHRDHFALDRPSETHADYLLRTGQVVDISDTIYPRNPAMYSEEARLKSFQNW PDYAHLTPRELASAGLYYTGIGDQVQCFACGGKLKNWEPGDRAWSEHRRHFPNCFFVL GRNLNIRSE (SEQ ID NO: 4).The sequence of the BIR3 domain used for the TR-FRET assay is MRHHHHHHRSDAVSSDRNFPNSTNLPRNPSMADYEARIFTFGTWIYSVNKEQLARAGF YALGEGDKVKCFHCGGGLTDWKPSEDPWEQHAKWYPGCKYLLEQKGQEYINNIHLTH SLEECLVRTT (SEQ ID NO: 5).Ten nanomolar of 6× Histidine-tagged (SEQ ID NO: 2) BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH (SEQ ID NO: 1) 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/niL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 10110 nM (pChEMBL 5)
Descriptors: MW 489.57, ALogP 3.21, HBA 6, HBD 2, PSA 96.97, QED 0.5
Compound (SMILES): COC[C@H](NC(=O)[C@H](CCc1ccccc1)NC(=O)c1cc(CN2CCOCC2)on1)C(=O)N[C@@H](Cc1ccccc1)C(=O)[C@@]1(C)CO1
InChIKey: FKXXDZMVRQMUDF-CWBQFHJISA-N
Target: Chymotrypsin-like protease CTRL-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P40313
Assay: Proteasome-Activity Inhibitory Assay: Inhibition of the chymotrypsin-like (CT-L), peptidylglutamyl peptide hydrolyzing activity (PGPH), and trypsin-like (T-L) activities of the 20S proteasome in enzymatic assays were determined using succinyl-Leu-Leu-Val-Tyr-AMC (10 μmol/L), Z-Leu-Leu-Glu-AMC (10 μmol/L) and Boc-Leu-Arg-Arg-AMC (50 μmol/L), respectively, as the substrates with purified human 20S proteasome at 2, 4 and 8 nmol/L, respectively, in the assay buffer containing 25 mM HEPES (pH7.5), 0.5 mM EDTA, 0.002% sodium dodecyl sulfate (SDS) and 0.05% NP-40. Stock solutions of the 20S proteasome inhibitors disclosed herein were prepared in dimethyl sulfoxide (DMSO) and the final DMSO concentration in the assay mixture was 1%. Reaction was conducted at room temperature for one hour. Proteasome activity was measured based on detection of the fluorophore 7-Amino-4-methylcoumarin (AMC) after cleavage from the substrates with a plate-based spectrofluorometer. IC50 is a quantitative measure indicating the concentration of an inhibitor at which the catalytic activity of the 20S proteasome is inhibited by 50%.
Measurement: IC50 = 11.1 nM (pChEMBL 7.960000038146973)
Descriptors: MW 661.76, ALogP 1.45, HBA 10, HBD 3, PSA 164.63, QED 0.18
Compound (SMILES): COCCN1C[C@@H](NC(=O)Nc2c(Cl)c(-c3c(C)noc3C)nn2-c2ccccc2)[C@H](c2ccc(F)c(F)c2)C1
InChIKey: SSGVKUGOZGUERZ-NZQKXSOJSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: TrkA Omnia Assay: Trk enzymatic selectivity was assessed using Omnia Kinase Assay reagents from Invitrogen Corp. Enzyme (TrkA from Invitrogen Corp.) and test compound (various concentrations) were incubated for 10 minutes at ambient temperature in a 384-well white polypropylene plate (Nunc catalog#267462). Omnia Tyr Peptide #4, as well as ATP, were then added to the plate. Final concentrations were as follows: 20 nM enzyme, 500 μM of ATP, 10 μM peptide substrate. The assay buffer consisted of 25 mM MOPS pH 7.5, 0.005% (v/v) Triton X-100 and 5 mM MgCl2. The production of phosphorylated peptide was monitored continuously for 70 minutes using a Molecular Devices FlexStation II384 microplate reader (excitation=360 nm; emission=485 nm). Initial rates were calculated from the progress curves. IC50 values were calculated from these rates using either a 4 or 5-parameter logistic curve fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 571.03, ALogP 5.31, HBA 7, HBD 2, PSA 97.45, QED 0.3
Compound (SMILES): Cc1nc(N)n2ncn(Cc3nc([C@@H]4CO[C@@H](c5ccc(F)cc5)C4)no3)c(=O)c12
InChIKey: ZBIRHILYHCONPB-GXTWGEPZSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (90 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 90 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC80 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 75 nM (pChEMBL 7.119999885559082)
Descriptors: MW 411.4, ALogP 1.6, HBA 10, HBD 1, PSA 126.36, QED 0.54
Compound (SMILES): CC(C)n1cnc(C(=O)N2CC3C(C2)C3C2=NOC(C)(C)N2C)c1
InChIKey: KPTGVIIQAARMAH-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5A (SINGLE PROTEIN), Homo sapiens
UniProt: P29375
Assay: Biochemical KDM5A Inhibition Assay: Using 384-well white Greiner784075 (Greiner), representative compounds were characterized for their inhibition of KDM5A using HTRF® technology (Cisbio Bioassays). Briefly, the test compounds, reference compounds, and DMSO control (typical compound concentration range 1 μM-10 M, final assay concentration (FAC*) of DMSO 0.5%) were added to 384-well plate by the Echo® acoustic dispensing platform (Labcyte). Five (5) 1l of KDM5A protein (produced by the method described in Nat Chem Biol 12, 531-538 (2016)). (5-20 nM FAC*) in assay buffer (50 mM MES, 50 mM NaCl, 1 mM TCEP, 0.01% v/v Tween 20, 0.03% BSA, pH 6.5) was added to wells and plates were incubated for 10-20 minutes at 25 degrees celsius. Then, 5 μl of alpha-ketoglutaric acid (100 μM FAC*), biotin-labelled H3K4-Me3 substrate (Anaspec Cat #AS-64357-1; 200 nM FAC*), Fe(II)SO4 (100 μM FAC*) and ascorbic acid (2 mM FAC*) in assay buffer was added to wells and plates were incubated for 20 minutes at room temperature. The reaction was stopped with the addition of 10 μl of anti-H3K4-Me2-Eu(K) (Cisbio Bioassays Cat #61KA2KAH; 0.75 nM FAC**)+Streptavidin XL665 (Cisbio Bioassays Cat #610SAXLB; 25 nM FAC**) in HTRF detection buffer (Cisbio Bioassays Cat #62SDBRDF). The mixture was incubated for 30 minutes at room temperature before 340 nm excitation and measurement of dual emission at 620 nm and 665 run. *FAC: final assay concentration calculated based on 10 μl of the assay buffer**FAC: final assay concentration calculated based on 20 μl of the detection buffer.
Measurement: IC50 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 331.42, ALogP 1.79, HBA 6, HBD 0, PSA 62.96, QED 0.85
Compound (SMILES): COCCNc1cc(Nc2ccc(C(=O)N3CCC(N4CCN(C)CC4)CC3)cc2OC)nc2[nH]cc(C(F)(F)F)c12
InChIKey: ITNBUYOLAQWTQX-UHFFFAOYSA-N
Target: Dual specificity tyrosine-phosphorylation-regulated kinase 1A (SINGLE PROTEIN), Homo sapiens
UniProt: Q13627
Assay: Kinase Binding Assay: Table 6: LanthaScreen Eu-anti-GST Antibody (Invitrogen, PV5594) and DYRK1A (Invitrogen, PV3785) were diluted to make the final concentrations of 6 nM and 15 nM respectively in 1× kinase buffer A, resulting in the preparation of antibody/kinase mixed solution. This antibody/kinase mixed solution was added to the assay plate where the diluted compound was loaded at the concentration of 5 μl/well. At this time, the final concentrations of the antibody and the DYRK1A were 2 nM and 5 nM respectively.Next, kinase tracer 236 solution (Invitrogen, PV5592) was diluted in 1× kinase buffer A to make the concentration of 45 nM. This diluted solution was added to the assay plate at the concentration of 5 μl/well. At this time, the final concentration of kinase tracer 236 was 15 nM and Kd value of Kinase tracer 236 was determined through tracer titration assay.Finally, after reacting at room temperature for 1 hour, fluorescence was measured (Excitation 340 nm, Kinase Tracer Emission 665 nm, LanthaScreen Eu-anti-GST Antibody Emission 620 nm) using Synergy neo (BioTek). Emission ratio (Kinase Tracer Emission Antibody Emission) was calculated based on the measured values, which was presented as a dose-response curve.
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 589.66, ALogP 4.24, HBA 8, HBD 3, PSA 97.99, QED 0.32
Compound (SMILES): COc1cc(C(F)(F)F)ccc1[C@H](C)NC(=O)c1cc(=O)[nH]c(C2CC2)n1
InChIKey: OYHNNFPESHJCAJ-VIFPVBQESA-N
Target: GMP-stimulated 3',5'-cyclic nucleotide phosphodiesterase (SINGLE PROTEIN), Macaca mulatta
UniProt: B3VTJ2
Assay: Fluorescence Polarization (FP) Assay: In a typical experiment the PDE2 inhibitory activity of the compounds of the present invention was determined in accordance with the following experimental method. Rhesus PDE2A3 was amplified from rhesus macaque brain cDNA (Biochain Institute, Hayward, Calif.) using primers based on human PDE2A sequence (accession NM_002599.3) where the forward primer containing a Kozak consensus was 5′-gccaccatggggcaggcatgtggc-3′ and the reverse primer was 5′-tcactcagcatcaaggctgca-3′. Amplification with Easy-A High-Fidelity PCR cloning enzyme (Stratagene, La Jolla, Calif.) was 95° C. for 2 minutes followed by thirty three cycles of 95° C. for 40 seconds, 52° C. for 30 seconds, and 72° C. for 2 minutes 48 seconds. Final extension was 72° C. for 7 minutes. The PCR product was TA cloned into pcDNA3.3-TOPO (Invitrogen, Carlsbad, Calif.) according to standard protocol. A consensus sequence was developed from multiple clones and then deposited into GenBank (EU812167). AD293 cells (Stratagene, La Jolla, Calif.) with 70-80% confluency were transiently transfected with rhesus PDE2A3/pcDNA3.3-TOPO using Lipofectamine 2000 according to manufacturer specifications (Invitrogen, Carlsbad, Calif.).
Measurement: Ki = 1018 nM (pChEMBL 5.989999771118164)
Descriptors: MW 381.35, ALogP 3.17, HBA 4, HBD 2, PSA 84.08, QED 0.83
Compound (SMILES): CC(C)(C)C#Cc1cncc([C@H]2NC(=O)O[C@@H]2c2cccc(F)c2)c1
InChIKey: UMXNPJZPWDBHPR-QZTJIDSGSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: mGluR5 FLIPR Assay: HEK293 (ZF) cells stably transfected with human mGluR5A (pIRES neo) and the rat glutamate-aspartate transporter (GLAST; pIRES puro) are grown in a monolayer culture at 37° C. in 5% CO2 and fed with Minimum Essential Medium (MEM) supplemented with 10% dialysed fetal bovine serum. 24 hours prior to assay, cells are enzymatically dissociated from the culture flask (Trypsin, 0.25%), spun down (1000 rpm, 3 min), resuspended, and plated on Greiner black clear bottomed PDL-coated 384-well plates at a density of 30 thousand cells/well. On the day of the experiment, media is removed from the cell plates and replaced with Molecular Devices Calcium 4 microfluorometric Ca++ sensitive dye in assay buffer (HBSS; Gibco #14025+20 mM HEPES and 250 uM probenacid). Plates are incubated in dye at 37° C. in 5% CO2 for 60 minutes prior to delivery of test compounds in assay buffer. Test compounds are incubated with cells in the presence of dye for 10 minutes prior to being read on the FLIPR platform (Molecular Devices). A Ca++ signal is induced in the assay plates via the delivery of an ¿EC10 concentration of the endogenous agonist 1-glutamate; images are acquired at 1 Hz for 100 seconds post-delivery of agonist stimulus. Positive modulator activity (i.e. the ability of test compounds to increase the Ca++ response to a sub-maximal concentration of agonist) is normalized to a saturating concentration of a known mGluR5 PAM run in each assay plate.
Measurement: EC50 > 3226 nM (pChEMBL 5.491000175476074)
Descriptors: MW 338.38, ALogP 4.14, HBA 3, HBD 1, PSA 51.22, QED 0.84
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3=CCC(OCC(=O)N4CCOCC4)CC3)cc12
InChIKey: GANWMSGKVWITOL-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay: CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 160 nM (pChEMBL 6.800000190734863)
Descriptors: MW 465.53, ALogP 4.19, HBA 5, HBD 1, PSA 76.68, QED 0.59
Compound (SMILES): CN(C[C@H](Nc1ncc(-c2nnc(C(F)F)o2)cn1)c1ccc(F)cc1)C(=O)C1CC1
InChIKey: ORYAGXJXLUZPHD-HNNXBMFYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Enzymatic Activity Assay: All enzymatic reactions were conducted in duplicate at room temperature for 17 hours in a 50 μl mixture containing HDAC assay buffer (50 mM Tris-HCl, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.05% Tween 20, 5 μg BSA), an HDAC substrate, an HDAC enzyme, and a test compound. Compound dilution was prepared ten-fold higher than the final concentration of the compounds with 10% DMSO in HDAC assay buffer and 5 μl of the dilution was added to a 50 μl reaction so that the final concentration of DMSO is 1% in all of reactions. After enzymatic reactions, 50 μl of HDAC Developer was added to each well and the plate was incubated at room temperature for an additional 20 minutes. Fluorescence intensity was measured at an excitation of 360 nm and an emission of 460 nm using a Tecan Infinite M1000 or Biotek Synergy microplate reader.The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (Ft) in each data set was defined as 100% activity. In the absence of HDAC, the fluorescent intensity (Fb) in each data set was defined as 0% activity. The percent activity in the presence of each compound was calculated according to the following equation: % activity=(F−Fb)/(Ft−Fb), where F=the fluorescent intensity in the presence of the compound.
Measurement: IC50 = 77 nM (pChEMBL 7.110000133514404)
Descriptors: MW 432.41, ALogP 3.62, HBA 7, HBD 1, PSA 97.04, QED 0.58
Compound (SMILES): Nc1nn(Cc2ccc(Cn3c(F)cccc3=O)cc2)cc1C(=O)NCCOc1cccc(Cl)c1
InChIKey: HYYVIMQLEHYSJS-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: Inhibitory Activity Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods. Human plasma kallikrein (Protogen) was incubated at 25° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 2070 nM (pChEMBL 5.679999828338623)
Descriptors: MW 495.94, ALogP 3.32, HBA 7, HBD 2, PSA 104.17, QED 0.27
Compound (SMILES): Cn1nc(C2CC2)cc1C12CCC(CN(C(=O)C34CC(F)(C3)C4)c3cccc(-c4noc(C(F)F)n4)c3)(CC1)CC2
InChIKey: PHSXOOOOAIGUTD-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 = 104 nM (pChEMBL 6.980000019073486)
Descriptors: MW 565.64, ALogP 6.8, HBA 6, HBD 0, PSA 77.05, QED 0.3
Compound (SMILES): CN(C)S(=O)(=O)c1cccc(-c2c(F)cc(Nc3nc(N)n[nH]3)cc2C(F)(F)F)c1
InChIKey: AOSJFFSAFYQPPZ-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: Pseudotyped HCV particle (HCVpp) reporter assay: Plasmids expressing HCV E1 and E2 envelope proteins of GT1a H77 strain (Proc Natl Acad Sci USA 1997 94:8738-43) or GT1b Con1 strain (Science 1999 285:110-3) were constructed by cloning the nucleic acids encoding the last 60 amino acids of HCV core protein and all of the HCV E1 and E2 proteins into pcDNA3.1(+) vector. Plasmid pVSV-G expressing the glycoprotein G of the vesicular stomatitis virus (VSV G) is from Clontech (cat #631530). The HIV packaging construct expressing the firefly luciferase reporter gene was modified based on the envelope defective pNL.4.3.Luc-Râ¿¿.Eâ¿¿ vector (Virology 1995 206:935-44) by further deleting part of the HIV envelope protein.
Measurement: EC50 = 430 nM (pChEMBL 6.369999885559082)
Descriptors: MW 444.41, ALogP 3.21, HBA 6, HBD 3, PSA 117, QED 0.52
Compound (SMILES): CC(c1ccc([C@H]2COc3ccccc3O2)cc1)N1CCCC1
InChIKey: NVJSSBQYOUBUKR-YQYDADCPSA-N
Target: Leukotriene A-4 hydrolase (SINGLE PROTEIN), Homo sapiens
UniProt: P09960
Assay: Enzyme Assay: The compounds of the invention are assessed for the ability to interact with human LTA4 hydrolase in an enzymatic assay that measures the ability of the enzyme to cleave the peptide bond of arginyl-aminomethylcoumarin (Arg-AMC). LTA4H Enzyme (1 nM final), Arg-AMC substrate (50 μM final), and compound are combined in a reaction buffer (50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.5% bovine serum albumin) at room temperature for 1 h. The formation of product is assessed by measuring the fluorescence of aminomethylcoumarin product (excitation wavelength 380 nm/emission wavelength 460 nm).
Measurement: IC50 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 309.41, ALogP 4.36, HBA 3, HBD 0, PSA 21.7, QED 0.84
Compound (SMILES): O=C1c2cc(-c3cncnc3)ccc2CN1c1cccc(-c2nncn2C2CC2)n1
InChIKey: BDWRMIDGXDFVOE-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase kinase kinase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99683
Assay: ASK1 Kinase Assay: The ASK1 enzymatic assay was run following Promega ASK1 Kinase Enzyme System (Cat #V3881). The kit provides the protocol, enzymes and all reagents necessary to run an assay. Firstly, the compounds, enzyme, substrate and ATP were diluted in provided assay buffer. The final concentration of the enzyme was 50 nM, substrate (Myelin basic protein) 1 μg/ml and ATP 10 μM. The compound and the enzyme were pre-incubated in a 384 well white solid bottom plate (Greiner, Cat#784075) for 10 minutes. After incubation, the substrate and ATP were added and incubated for further 60 minutes. After 60 minutes, ADP-Glo™ was added and plate was incubated for another 40 minutes. After 40 minutes, Kinase Detection Reagent was added and the plate was incubated for 45 minutes. After 45 minutes, plate was read on Perkin Elmer EnVision using luminescence read (0.5 seconds/well).
Measurement: IC50 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 395.43, ALogP 3.29, HBA 7, HBD 0, PSA 89.69, QED 0.53
Compound (SMILES): O=C(c1ccc(F)cc1-c1ncccn1)N1CC2CCC1C(Nc1cnc(C(F)(F)F)cn1)C2
InChIKey: IDNGUAZJDBXCKQ-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 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 472.45, ALogP 4.2, HBA 6, HBD 1, PSA 83.9, QED 0.57
Compound (SMILES): OCC1CCCCN1c1cnc2c(N3CCCc4ncccc43)n[nH]c2n1
InChIKey: BJNFWHSLAJNDAS-UHFFFAOYSA-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 365.44, ALogP 2.18, HBA 7, HBD 2, PSA 94.06, QED 0.73
Compound (SMILES): NC(=O)c1cc(COc2ccc(-c3[nH]ncc3C(F)F)nc2)ccn1
InChIKey: RJOHXJHWCNMIRQ-UHFFFAOYSA-N
Target: Cytochrome P450 4F2 (SINGLE PROTEIN), Homo sapiens
UniProt: P78329
Assay: Inhibition Test for Each Compound of the Present Invention Against 20-HETE Producing Enzymes (CYP4F2 and CYP4A11): In the CYP4F2 inhibition test, the reaction solution containing each compound [final concentration of 50 mM, KPO4 (pH 7.4), 2.5 μM luciferine derivative, and 1 mM NADPH] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4F2 had been expressed. In the CYP4A11 inhibition test, the reaction solution containing each compound [final concentration of 100 mM, Tris-HCl (pH 7.5), 60 μM luciferine derivative, 1.3 mM NADP+, 3.3 mM Glucose 6-Phosphate, 3.3 mM MgCl2, and 0.4 U/mL Glucose 6-Phosphate dehydrogenase] was added to an Escherichia coli membrane fraction (100 μg/mL protein) in which human CYP4A11 had been expressed. Following this, the membrane fraction was left to stand at room temperature for 60 minutes to perform an enzymatic reaction. After the reaction, a luciferine detection reagent was added, and the luminescence value was measured using a plate reader. By using that value, the percent inhibition of 20-HETE producing enzyme (%) was calculated according to the equation described below, and the 50% inhibitory concentration (IC50 value) for each compound was calculated.
Measurement: IC50 = 2700 nM (pChEMBL 5.570000171661377)
Descriptors: MW 345.31, ALogP 2.48, HBA 5, HBD 2, PSA 106.78, QED 0.71
Compound (SMILES): C=C1C[C@@H](c2nc3ccc(Cl)c(C)c3[nH]2)N(C(=O)c2nc(C)sc2-c2ccc(F)c(Br)c2)C1
InChIKey: DIKCPRSWFGQRLD-IBGZPJMESA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Inhibition Assay: Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 μg/ml G418, 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 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2.Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH: water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 545.87, ALogP 7, HBA 4, HBD 1, PSA 61.88, QED 0.28