lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1cn2c(-c3ccc(Cl)c(S(=O)(=O)NC4CCC(C)(O)CC4)c3)cnc2c(N)n1
InChIKey: XJBHDTPMQHNDGE-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 Perkin-Elmer (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 Sigma-Aldrich (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 449.96, ALogP 2.91, HBA 7, HBD 3, PSA 122.61, QED 0.56
Compound (SMILES): C[C@H](NC(=O)c1nc(C#N)cnc1NCc1ccc(-c2cnc(N)c(NCCN)n2)cc1)c1ccc(F)c(F)c1
InChIKey: ZCTFKPYOYKBIOT-HNNXBMFYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 544.57, ALogP 3.14, HBA 10, HBD 5, PSA 180.55, QED 0.2
Compound (SMILES): C=CC(=O)N1C[C@@H](n2nc(C#Cc3ccc4c(c3)ncn4C3CC3)c(C(N)=O)c2N)C[C@@H]1COC(F)F
InChIKey: BOAHBERXXMPZJR-DLBZAZTESA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: FGFR2 Kinase Assay: Small molecule inhibition of FGFR2 kinase activity was evaluated using a fluorescence-based microfluidic mobility shift assay. FGFR2 catalyzes the production of ADP from ATP during phosphoryl transfer to the substrate peptide, FLPeptide30 (5-FAM-KKKKEEIYFFFCONH2) (Perkin Elmer, 760430). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product were measured and the ratio of these values used to generate % conversion of substrate to product by the LabChip EZ reader (Perkin Elmer). Wild type FGFR2 (Carna Bioscience, 08-134) at 0.06 nM was prepared with 1.5 μM substrate, 10 mM MgCl and 100 μM ATP in a butler containing 50 nM HEPES, 1 mM EGTA, 0.01% Brij-35, 0.05% BSA, and 2 mM DTT prior to addition of compounds in DMSO and incubation for 80 minutes at room temperature. The reaction was terminated by addition of 0.5 MEDIA. IC50 values were calculated using the inhibition of conversion ratio using Dotniatics Knowledge Solutions Studies curve fitting (Dotmatics, Bishops Stortford, UK, CM23).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 509.52, ALogP 2.22, HBA 8, HBD 2, PSA 134.29, QED 0.37
Compound (SMILES): O=C1C2C=CC([C@@H](O)c3ccc(Br)cc3)N(Nc3ccc([N+](=O)[O-])cc3)[C@@H]2C(=O)N1c1ccccc1
InChIKey: XYHNBRXPLBTLKW-MXXWWZHRSA-N
Target: Bifunctional polynucleotide phosphatase/kinase (SINGLE PROTEIN), Homo sapiens
UniProt: Q96T60
Assay: Steady-State Fluorescence Assay: Kd values were measured by steady-state fluorescence spectroscopy, by following protein tryptophan fluorescence quenching as a function of ligand concentration, as previously described.
Measurement: Kd = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 549.38, ALogP 4.22, HBA 7, HBD 2, PSA 116.02, QED 0.2
Compound (SMILES): COc1cc(C(=O)N2CC3CCC2[C@@H]3N)cc2nc(-c3cc4ccc(-c5ccc(CO)c(C)c5)nc4n3CC3CC3)n(C)c12
InChIKey: HKEZNKGHOYLNSE-SGAJGPLNSA-N
Target: Protein-arginine deiminase type-4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM07
Assay: Rapid-Fire Mass Spectrum (RFMS) Assay: Compounds were solubilized in 100% DMSO to achieve a 10 mM compound concentration. Compound stock solutions were stored at RT. A series of dilutions were prepared in DMSO and mixed 8 times with 20 μL mixing volume. Final top concentration of compound in the assay is 50 μM. Final assay conditions were as follows:Reaction volume: 26 μlAssay buffer: 25 mM hepes, pH 7.5, 5 mM NaCl, 1 mM DTT, 0.2 mg/ml BSA, 0.01% CHAPS, 50 μM Calcium, and 5 μM TPENFinal concentrations: 5 nM hPAD4 enzyme, 250 μM BAEE, and 0.5% DMSOTotal incubation time: 30 mins compound and enzyme preincubation at 37° C., 90 min enzyme/substrate reaction, 30 min reaction with phenyl glyoxal at 37° C.Stop solution: 40 μl 5% TCA in ACN
Measurement: IC50 = 86 nM (pChEMBL 7.070000171661377)
Descriptors: MW 590.73, ALogP 5.04, HBA 8, HBD 2, PSA 111.43, QED 0.27
Compound (SMILES): COc1cccc2sc(C(=O)O)c(COc3c(F)cc(C(N)=O)cc3F)c12
InChIKey: IQJXMNMTUZOOTE-UHFFFAOYSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: Surface Plasmon Resonance (SPR): Binding interactions of ligands with STING proteins were quantified using Surface Plasmon Resonance (SPR) with a minimally biotinylated STING protein immobilized on Streptavidin chip surface. In this manner highly active STING protein surfaces were obtained that were not compromised by a low pH required for amine coupling methods. Minimal biotinylation of purified huSTING protein was performed using a previously described methodology (Chabbra 2012). Briefly, approximately 20 nmol of recombinant STING protein in 1×TBS buffer (25 mM Tris-HCl, 150 mM NaCl, 5 mM DTT) was mixed with of EZ-LinkH Sulfo-NHS-LC-LC-Biotin (Thermofisher Scientific, cat #21338) at a molar ratio of 1 to 0.6 and then incubated on ice for 2 hours. To remove any unreacted biotin reagent, protein/biotin mixture was passed through a Superdex 75 (10/300 GL) column equilibrated with 10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM DTT, 5% (v/v) glycerol. A protein peak containing biotinylated huSTING protein was collected and stored in aliquots at −80° C. Streptavidin was simultaneously immobilized in all four channels of a CM5 sensor chip docked in a Biacore instrument (either Biacore S200 or Biacore T200, GE Healthcare) as described previously (Zender 2013). Minimally biotinylated STING protein was captured onto Streptavidin coated chip surface at 25° C. in SPR binding buffer (50 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM TCEP, 2% (v/v) DMSO) gradually injected in a single channel at a constant flow-rate of 2 μL/min until SPR response increases were no longer observed resulting in typical capture levels of 5000 to 7000 RU (1 RU=1 μg/mm2). To determine binding affinity, compound interaction with immobilized STING protein was analysed using dose-response experiments. All binding experiments were performed at 25° C. in SPR binding buffer. Fresh 10 mM DMSO solutions of compound were diluted directly into SPR binding buffer to a final concentration of 10 μM and then further diluted either 2-fold or 3-fold aiming for either a 5- or 7-point concentration series range. Each ligand concentration series was injected at a constant flow rate of 60 μL/min with a 90 second association and a 180 second dissociation time. Scrubber 2 (www.biologic.com.au) was utilized for data processing and analysis. Thus, SPR signals were referenced against the blank surface (streptavidin+D-biotin) and further corrected for DMSO refractive index change and then double-referenced using a buffer-blank injection (Papalia 2006). To determine binding affinities (KD values) from dose-response experiments, binding responses at equilibrium were fit to either a 1:1 steady state affinity or to 1:1 binding kinetic models (both available within Scrubber software).
Measurement: Kd = 22000 nM (pChEMBL 4.659999847412109)
Descriptors: MW 393.37, ALogP 3.56, HBA 5, HBD 2, PSA 98.85, QED 0.67
Compound (SMILES): Cc1ccc(S(=O)(=O)N2C(C)COCC2C)cc1-c1cnc2c(N)ncnn12
InChIKey: MQVRMOMFUDNZSN-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 Perkin-Elmer (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 Sigma-Aldrich (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 402.48, ALogP 1.48, HBA 8, HBD 1, PSA 115.71, QED 0.71
Compound (SMILES): CCn1nc(C(=O)NC[C@]2(O)CC[C@@H](S(C)(=O)=O)CC2)c(Cl)c1-c1ccc(CCCC(F)(F)F)cc1OC(F)F
InChIKey: KKQOBKSSIGTCFQ-ONEIWYMKSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Development, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells. Images were collected at each temperature, and the sum of the pixel intensity in a given area of the assay plate was recorded versus temperature.
Measurement: Kd = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 616.05, ALogP 5.16, HBA 7, HBD 2, PSA 110.52, QED 0.34
Compound (SMILES): N#Cc1cnc2ccc(-c3c(-c4ccc(F)cc4)ncn3C[C@@H](O)CO)cn12
InChIKey: KTBVMIBZFUFNME-QGZVFWFLSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assay: Assays for the compounds reported below were conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, anti-HIS detection antibody, a labeled small molecule probe (Kd=<100 nM; koff=<0.001 s−1) and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 0.05 mg/ml BSA). The reaction is incubated for 1 hour at room temperature and the HTRF signal was measured on an Envision plate reader (Ex: 340 nm; Em: 520 nm/495 nm). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay are 1 nM HIS-TGF-βR1 T204D or HIS-TGF-βR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule prode (at Kd) and 0.5% DMSO. Dose response curves were generated to determine the concentration required inhibiting 50% of kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations.
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 377.38, ALogP 2.23, HBA 7, HBD 2, PSA 99.37, QED 0.56
Compound (SMILES): O=C(Nc1cc2c(cc1N1CCOCC1)OC1(CCN(CCO)CC1)C2)c1cnn2cccnc12
InChIKey: NGPHJSKSCXRKST-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Biological Assay: Kinase activities were assayed using the Transcreener-Fluorecescence polarization platform (BelBrook Labs, Madison, Wis., USA) that measures amounts of the reaction product, ADP. The IRAK4 reaction conditions were optimized using an IRAK1-derived peptide (sequence H-KKARFSRFAGSSPSQSSMVAR) to provide a linear reaction rate over the course of a 90 min incubation, which resulted in 10-12% conversion of the starting ATP to ADP. Final IRAK4 assay conditions were 1.25 nM IRAK4; 125 uM ATP; 10 uM MgCl2; 125 uM peptide in reaction buffer (25 mM HEPES (pH7.4); 2 mM Dithiothreitol; 0.015% Brij-35; and 0.5% dimethyl sulfoxide. The IRAK1 activity was optimized similarly, yielding final assay conditions of 3 mM IRAK1; 62.5 uM ATP; 5 uM MgCl2, and 62.5 uM IRAK1 peptide in reaction buffer for 60 min.Assays of compounds for kinase inhibition were performed using inhibitors serially-diluted in dimethyl sulfoxide, which was accomplished with a LabCyte Echo 555 liquid dispenser. 384 well assay plates spotted with compound received 4 ul of a 2× substrate (ATP+peptide) mix in reaction buffer, followed by 4 ul of 2× enzyme diluted in reaction buffer. Reactions were halted at 60 (IRAK1) or 90 (IRAK4) min by addition of 6 ul of detection buffer, containing EDTA (40 nM final concentration), 0.95 ug of the ADP-binding antibody ADP2, ADP tracer (3 nM final concentration), and 25 uM HEPES. Following a 1 hr incubation, fluorescence polarization of the ADP2-antibody TRACER complex was read on a Tecan M1000 plate reader using a 635/20 excitation filter in combination with a 670/20 emission filter.
Measurement: Ki = 6.8 nM (pChEMBL 8.170000076293945)
Descriptors: MW 478.55, ALogP 1.58, HBA 9, HBD 2, PSA 104.46, QED 0.57
Compound (SMILES): CN(C(=O)Cc1cnccn1)C1CCN(c2cccc(-c3cc(-c4ccnn4C4CCOCC4)c4c(N)ncnn34)c2)C1
InChIKey: BREYVPNIZVQMGN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: ADP-Glo Format PI3K Assay: The ADP-Glo format PI3K assays were performed in Proxiplate 384-well plates (Perkin Elmer #6008280). The final assay volume was 2 μl prepared from 1 μl additions of enzyme/PIP2:PS lipid (Invitrogen #PV5100) mixture and 1 μl ATP (provided in kit, Promega #V9101) and test compounds in assay buffer (50 mM HEPES pH 7.5, 3 mM MgCl2, 100 mM NaCl, 0.5 mM EGTA, 2 mM DTT, 0.03% CHAPS). The reaction was initiated by the combination of enzyme/lipid, ATP, and test compounds. The reaction mixture was incubated at room temperature for 30 minutes (PI3K Alpha, Beta, Gamma) or 3 hours for PI3K Delta. ADP-Glo (2 μl), followed by Kinase Detection reagent (4 μl), were added to reactions following the initial incubation and allowed to incubate 40 minutes at room temperature. The reaction mixture was analyzed on the TOPCOUNT (Perkin Elmer). Inhibition data were calculated by comparison to no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of enzyme in the assays are PI3K Alpha [0.5 nM], PI3K Beta [2 nM], PI3K Gamma [20 nM], PI3K Delta [0.5 nM]. ATP final concentrations are as follows: for Alpha [10 μM], for Beta [12.5 μM], for Gamma [6.5 μM], for Delta [100 μM]. Lipid final concentration was the same for all enzymes, [25 μM]. Dose response curves were generated to determine the concentration required to inhibit 50% of activity. Compounds were dissolved at 0.12 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. The IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 578.68, ALogP 3.26, HBA 11, HBD 1, PSA 132.59, QED 0.31
Compound (SMILES): COc1cc(-c2cccc(-c3ccc4c(c3)OC(C#N)CO4)c2C)cc(F)c1CN
InChIKey: ANDWUJFPVFFWMA-UHFFFAOYSA-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 1 μg/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 0.3 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 (WNV)/0.05% TWEEN-20 for 10 min at 37° C. and the plates were washed 3 times with PBS/0.05% TWEEN-20. Streptavidin-HIRP 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.
Measurement: IC50 = 5500 nM (pChEMBL 5.260000228881836)
Descriptors: MW 404.44, ALogP 4.6, HBA 5, HBD 1, PSA 77.5, QED 0.69
Compound (SMILES): Cc1nc2c(ncn2[C@@H]2C[C@@H]3OP(=O)(S)O[C@H]4[C@H](F)[C@H](n5cnc6c(N)ncnc65)O[C@@H]4COP(=O)(S)O[C@@H]2[C@@H]3O)c(=O)[nH]1
InChIKey: CAQSVQKWQBDAOF-NJTHJTJJSA-N
Target: Stimulator of interferon genes protein (SINGLE PROTEIN), Homo sapiens
UniProt: Q86WV6
Assay: Surface Plasmon Resonance (SPR) Binding: Surface plasmon resonance (SPR) STING agonist binding studies were carried out using a Biacore T200 instrument (GE Healthcare) at 4° C. in a 150 mM KCl, 25 mM Hepes (pH 7.5), 1 mM TCEP, 2.5 mM MgCl2, 5% (v/v) glycerol, 0.005% (v/v) P20, 1% (v/v) DMSO running buffer. The recombinant protein immobilized on the streptavidin chip was either human WT or H232R STING. A truncated construct of STING was used in all studies. The STING constructs were comprised of residues 155-341 with both N- and C-terminal truncations; the N-terminal transmembrane domains were removed (1-154), as well as the C-terminal tail (342-379). A highly specific N-terminal biotinylation was achieved enzymatically with the E. coli biotin ligase (BirA) and inclusion of the high-affinity biotinylation peptide AviTag. A Carboxymethylated dextran pre-immobilized with streptavidin (series S Streptavidin CM5 Sensor Chip) was used to capture the biotinylated STING protein. Test compound injections were made at a flow rate of 100 μl per minute with a 60 second association time and variable dissociation time. A three-fold dilution series from a 10 μM starting concentration was used for all test compounds. Data analysis was performed using the BiacoreT200 data evaluation software package (GE Healthcare).
Measurement: Kd = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 691.56, ALogP 1.65, HBA 17, HBD 5, PSA 233.71, QED 0.15
Compound (SMILES): CC(C)c1c(-c2cnco2)[nH]c2ncnn2c1=O
InChIKey: PXKLLMFFDSNTMI-UHFFFAOYSA-N
Target: Lysine-specific demethylase 5B (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGL1
Assay: TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) Assay: In this assay, the potency (IC50) of each compound was determined from a ten point (1:3 serial dilution; final compound concentration range in assay from 100000 nM to 5.08 nM) titration curve using the following outlined procedure. To each well of a white Greiner 1536 Lumitrac 1536 well-plate, 50 nL of compound (100 fold dilution in final assay volume of 5 μL) was dispensed, followed by the addition of 4 μL of 1× assay buffer (50 mM Hepes 7.3, 0.5 mM TCEP, 0.005% Brij-35, 0.02% BSA, 50 μM Na-L-Ascorbate and 2 μM AmFe(II)Sulfate) containing 5 nM of Full-length KDM5B enzyme (recombinant protein from baculovirus-transfected Sf21 cells: full-length KDM5B; MW=176.825 kDa). Following a 30 minutes compound and enzyme incubation in a humidified chamber, each reaction was initiated by the addition of 1 μL 1× assay buffer containing 50 nM biotinylated H3K4Me3 peptide, and 500 nM α-ketoglutarate. The final reaction in each well of 5 μL consists of 5 nM KDM5B, 500 nM biotinylated-peptide, and 500 nM α-ketoglutarate. De-methylation reactions were allowed to proceed for 60 minutes. Reactions were immediately quenched by the addition of 5 uL of 2× Lance Detection Buffer (PerkinElmer) with 0.5 mM EDTA and 2 nM of Eu-anti-H3K4Me1-2 antibody, and 30 nM of Streptavidin-conjugated Dylight 650 detection reagents. After 60 minutes incubation with detection reagents, reaction plates were read on a PerkinElmer EnVision plate reader using standard TR-FRET protocol. Briefly, excitation of donor molecules (Eu-chelate-anti-H3K4Me1-2-antibody) with a laser light source at 337 nm produces energy that can be transferred to Dylight-650 acceptor molecules if this donor:acceptor pair is within close proximity. Fluorescence intensity at both 665 nm (acceptor) and 615 nm (donor) are measured and a TR-FRET ratio calculated for each well (acceptor intensity/donor intensity).
Measurement: IC50 = 193 nM (pChEMBL 6.710000038146973)
Descriptors: MW 245.24, ALogP 1.2, HBA 6, HBD 1, PSA 89.08, QED 0.73
Compound (SMILES): C=C1C[C@@H](c2nc3cc(OC)ccc3[nH]2)N(C(=O)c2nc(C3CC3)sc2-c2ccccc2)C1
InChIKey: XVEAOCPGIRMOIJ-NRFANRHFSA-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 = 206 nM (pChEMBL 6.690000057220459)
Descriptors: MW 456.57, ALogP 5.72, HBA 5, HBD 1, PSA 71.11, QED 0.39
Compound (SMILES): C[C@]12CCC(=O)C=C1CC[C@@H]1[C@@H]2[C@@H](O)C[C@@]2(C)[C@H]1CC[C@]2(OC(=O)C1CCCC1)C(=O)CO
InChIKey: YZWQCWSVBKUHRP-KJKDQFDUSA-N
Target: Mineralocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08235
Assay: Mineralocorticoid Receptor Transactivation Potencies for Cortisol and 17-ester Derivatives: Mineralocorticoid receptor (MR) activation potency was assessed using a HEK 293T cell line containing the UAS-bla reporter (UAS-bla HEK 293T CELLSENSOR®). This cell line was stably cotransfected with an expression construct containing β-lactamase cDNA under control of the GAL4 Upstream Activator Sequence (UAS) and another expression construct encoding for the fusion protein GAL4(DBD)-MR(LBD). Results for one experiment performed in duplicate for 9 compounds and the control compound, aldosterone, in agonist mode are summarized in Table 4. All assays were performed as 10-point dose responses using a half log-fold dilution series starting with a maximum compound concentration of 100 nM. The compounds were incubated for 16 hours. The activation of the fusion protein GAL4(DBD)-MR(LBD) leads to expression of the reporter β-lactamase which is detected by the conversion of a FRET substrate in a ratiometric assay format. This functional assay allows for measurement of receptor agonism by compounds and can be used to determine compound potency and selectivity. Assay reproducibility was determined by calculating Z′ values for untreated versus maximum stimulation. The Z′ value was greater than 0.6, indicating good reproducibility of the assay format.
Measurement: EC50 > 100 nM (pChEMBL 7)
Descriptors: MW 458.6, ALogP 3.52, HBA 6, HBD 2, PSA 100.9, QED 0.63
Compound (SMILES): CS(=O)(=O)CC(CO)(OC[C@H]1O[C@@H](n2ncc3c(NC4CCCC4)nc(Cl)nc32)[C@H](O)[C@@H]1O)P(=O)(O)O
InChIKey: NEWFMKFQQZZAGW-RXUYWNQFSA-N
Target: 5'-nucleotidase (SINGLE PROTEIN), Homo sapiens
UniProt: P21589
Assay: Biochemical Assay: Assay Reaction ConditionsAssay Volume: 70 μlReaction Volume: 50 μlCD73: 0.3208 nMAMP: 15 μMAssay Buffer: 25 mM Tris-HCL, pH 7.4, 0.01% Brij-35, 0.01% BSA, 5 mM MgCl2Assay Procedure:Used 384 clear plate.Made dose titration of testing compounds in assay buffer, 10 points ½ log titrations in duplicates starting at 100 μM.Added 25 μl of CD73 to each well for a final concentration of 320 pM.Incubated at RT for 15 min.Added 25 μl of AMP to each well for a final concentration of 15 μM.Incubated at RT for 10 min.Added 10 μl of Malachite Green Reagent A, incubate at RT for 10 min.Added 10 of Malachite Green Reagent B, incubate at RT for 45 min.Read the Absorbance on Envision plate reader using excitation filter: Cy5 620 nM.The ability of the compounds disclosed herein to inhibit CD73 activity was quantified and the respective IC50 values were determined.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 585.96, ALogP -0.62, HBA 13, HBD 6, PSA 226.45, QED 0.15
Compound (SMILES): C=CC(=O)NC1C=C(c2ncnc3[nH]cc(-c4cccs4)c23)CCC1
InChIKey: VAQSCHBMTIJOBO-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Kinase-Inhibiting Activity Assay: JAK 1: Among materials for the measurement of this inhibiting activity, a substrate peptide and a kinase protein were acquired as follows. As such a substrate peptide, a substrate peptide for QSS Assist JAK1-MSA assay kit (Carna Biosciences, Inc.) was purchased. As such a kinase protein, a purified recombinant human JAK1 protein (Carna Biosciences, Inc.) was purchased.The method for measuring the inhibiting activity is as follows. First, the compounds of the present invention were each dissolved in dimethyl sulfoxide (DMSO), and a serial dilution was then prepared using DMSO. Subsequently, a serial dilution solution of the compound (the final concentration of DMSO upon a kinase reaction: 5.0%) or DMSO (final concentration: 5.0%) was mixed with a solution comprising the substrate peptide (final concentration: 1 μM), magnesium chloride (final concentration: 5 mM) and ATP (final concentration: 75 μM) in a buffer for kinase reaction (20 mM HEPES (pH 7.5), 2 mM dithiothreitol and 0.01% Triton X-100). Thereafter, a JAK1 protein was further added to the mixed solution, and the obtained mixture was then incubated at 25° C. for 120 minutes to carry out a kinase reaction. To the reaction solution, EDTA was added to a final concentration of 30 mM, so as to terminate the reaction. Finally, using LabChip EZ Reader II (Perkin Elmer Corp.), an unphosphorylated substrate peptide (S) and a phosphorylated peptide (P) were subjected to microchannel capillary electrophoresis, so that the two peptides were separated from each other and were then detected.
Measurement: IC50 = 3200 nM (pChEMBL 5.5)
Descriptors: MW 350.45, ALogP 3.92, HBA 4, HBD 2, PSA 70.67, QED 0.7
Compound (SMILES): CCCCc1nc(=O)c(S(=O)(=O)c2ccc(-c3ccc(C(N)=O)cc3F)cc2)c(O)n1[C@@H](CC)c1cccc(C#N)c1
InChIKey: XVNJRHHQNWXKET-SANMLTNESA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: Intracellular cAMP Accumulation Assay: HEK293 cells stably expressing human APJ receptor were used to assess the activity of compounds. Cultured cells were detached and resuspended in the cAMP Homogeneous Time-Resolved Fluorescence (HTRF) assay buffer (Cisbio cat; #62AM4PEJ). The assay was performed in 384-well assay plates (Perkin-Elmer; cat #6008289) according to assay protocol provided by the manufacturer. Serial dilutions of a compound together with assay buffer containing 0.2 nM IBMX and 2 μM forskolin were added to each well containing 5,000 cells and incubated for 30 minutes at room temperature. Subsequently, cAMP D2 reagent was added in the lysis buffer followed by the EuK antibody (Cisbio; cat #62AM4PEJ) and incubated for 60 min. The fluorescence emission ratio was measured using fluorometer. The intracellular cAMP concentrations (compound-stimulated inhibition of forskolin-mediated cAMP production) were calculated by extrapolation from a standard curve using known cAMP concentrations. The EC50 values were obtained by fitting the data to a sigmoidal concentration-response curve with variable slope. The maximal achievable inhibition of forskolin-induced cAMP levels (Ymax) for each compound was expressed as relative percentage of inhibition attained using pyroglutamated apelin-13 ((Pyrl)apelin-13) peptide, which was set to 100%.
Measurement: EC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 588.66, ALogP 4.9, HBA 8, HBD 2, PSA 156.14, QED 0.27
Compound (SMILES): CC1(C(=O)N2CCC(O)(Cn3cnc4c(cc(C#N)n4-c4ccccc4)c3=O)CC2)CC1
InChIKey: SACXMRDWUOQUAA-UHFFFAOYSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q93009
Assay: Fluorescence Polarisation (FP) Homogeneous Assay: USP7 activity was monitored in a fluorescence polarisation (FP) homogeneous assay using the isopeptide ubiquitin-Lys-TAMRA substrate (U-558, Boston Biochem). Full-length USP7 was purchased from Boston Biochem (His6-USP7FL, E-519). Unless otherwise stated, all other reagents were purchased from Sigma-Aldrich. Enzymatic reactions were conducted in black flat-bottom polystyrene 384-well plates (Nunc) and 15 μL total volume. USP7 (2.5 nM, 5 μL) was incubated in assay buffer (50 mM HEPES (pH 7.2), 150 mM NaCl, 0.5 mM EDTA, 5 mM DTT, 0.05% BSA (w/v), 0.05% CHAPS) in the presence or absence of inhibitor (5 μl). Inhibitors were stored as 10 mM DMSO stocks in an inert environment (low humidity, dark, low oxygen, room temperature) using a Storage Pod System and serial dilutions were prepared in buffer just prior to the assay (from 200 to 0.1 μM, 8 dp curve). Following incubation at room temperature for 30 min, the enzymatic reactions were initiated by dispensing the Ub substrate (250 nM, 5 μL). FP was measured every 15 min over a period of 1.5 h (within the linear range of the assay) using a Synergy 4 plate reader (BioTek) exciting at 530 nm and measuring the amount of parallel and perpendicular light at 575 nm.
Measurement: IC50 < 250 nM (pChEMBL 6.6020002365112305)
Descriptors: MW 431.5, ALogP 2.21, HBA 7, HBD 1, PSA 104.15, QED 0.68
Compound (SMILES): CCn1nc(C(O)c2cc(-c3ncnc4cc(N5CCOCC5)ccc34)c(F)cc2Cl)ccc1=O
InChIKey: ROTFCYSSIJCDLR-UHFFFAOYSA-N
Target: DNA-dependent protein kinase catalytic subunit (SINGLE PROTEIN), Homo sapiens
UniProt: P78527
Assay: Biochemical Assay: The kinase assay was carried out in streptavidin-coated 348-well microtitre flashplates. To this end, 1.5 ug of DNA-PK/protein complex and 100 ng of biotinylated substrate, such as, for example, PESQEAFADLWKK-biotin-NH2 (biotin-DNA-PK peptide), were incubated for 90 min at room temperature in a total volume of 36.5 ul (34.25 mM HEPES/KOH; 7.85 mM Tris HCl; 68.5 mM KCl; 5 uM ATP; 6.85 mM MgCl2; 0.5 mM EDTA; 0.14 mM EGTA; 0.69 mM DTT; pH 7.4) with 500 ng of DNA from calf thymus, 0.1 uCi of 33P-ATP and 1.8% of DMSO per well with and without the test compound. The reaction was stopped using 50 ul/well of 200 mM EDTA. After incubation for a further 30 min at room temperature, the liquid was removed. Each well was washed three times with 100 ul of 0.9% saline solution. A nonspecific reaction (blank value) was determined using 10 uM of an innate kinase inhibitor. The radioactivity measurement was carried out using a TopCount. IC50 values were calculated in RS1.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 495.94, ALogP 3.58, HBA 8, HBD 1, PSA 93.37, QED 0.45
Compound (SMILES): Cc1cc(C2CCOCC2)n2nc(NCc3ccnc(C(F)(F)F)c3)nc2n1
InChIKey: GECQOSSUNZKRNN-UHFFFAOYSA-N
Target: cGMP-dependent 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O00408
Assay: IMAP TR-FRET-based phosphodiesterase assay: An IMAP TR-FRET-based phosphodiesterase assay was developed using the PDE2A isoform. IMAP technology is based on high-affinity binding of phosphate by immobilized metal (MIII) coordination complexes on nanoparticles. The IMAP binding reagent recognizes phosphate groups on AMP or GMP generated from cAMP or cGMP in a PDE reaction. Cyclic nucleotides that carry a phosphodiester bond and not a free phosphate are not recognized by the binding reagent. The time resolved fluorescence resonance energy transfer (TR-FRET) is afforded by a Terbium (Tb)-Donor pre-bound to the nanoparticles. FRET occurs when the fluorescent-labeled AMP or GMP product of a PDE reaction binds and comes into close proximity to the Tb-Donor complex. Due to the long lifetime of Tb fluorescence, detection can be run in time-resolved mode to reduce or eliminate interference from auto-fluorescent compounds.The IMAP TR-FRET PDE2A assay was performed in 1536-well white plates. A total of 250 pg per well of FLAG-tagged PDE2A1 (amino acids 2-941) was dispensed in 2.5 μL IMAP assay buffer consisting of 10 mM Tris pH 7.2, 10 mM MgCl2, 1 mM DTT, and 0.1% fatty acid free BSA. 30 nL of compound was then added from 1 mM stocks in DMSO using a Kalypsys Pintool. Plates were incubated for 5 min at room temperature before dispensing 1.5 μL of 533 nM FAM-cAMP substrate for a final concentration of 200 nM. Following a brief centrifugation, plates were incubated for 30 min at room temperature. The assay was terminated by adding 5 μL IMAP binding reagent Tb complex to each well which was prepared according to manufacturer's recommendations (Molecular Devices). Plates were incubated an additional 90 minutes at room temperature and read on a Viewlux plate reader. All compounds were solvated at a concentration of 10 mM in DMSO and tested in 11-point half-log dose-response. Curve fitting and IC50 values were determined using a standard four parameter fit.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 392.39, ALogP 3.35, HBA 7, HBD 1, PSA 77.23, QED 0.73
Compound (SMILES): N#C[C@H]1CCCC[C@@H]1n1nc(Nc2ccc3c(c2)CC(C(=O)O)C3)c2c(=O)[nH]ccc21
InChIKey: PTPJMVHMKNLZSY-ZRFIFAGDSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical HTRF Assay Protocol: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format biochemical assay. The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 μL of 1.11× enzyme and 1.11× substrate in 1× assay buffer (Invitrogen kinase buffer #PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 μL of 2× stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction. Plates were shaken and centrifuged and then incubated 60 minutes at room temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading. After normalization to untreated controls, the percent inhibition of the HTRF signal at each compound concentration was calculated.
Measurement: IC50 = 470 nM (pChEMBL 6.329999923706055)
Descriptors: MW 417.47, ALogP 3.52, HBA 6, HBD 3, PSA 123.8, QED 0.6
Compound (SMILES): Nc1cnc(-c2ccc(CNc3ncc(C(F)(F)F)cc3C(=O)Nc3ccc(F)cc3)cc2)cn1
InChIKey: SVKHYPWNJQZSGR-UHFFFAOYSA-N
Target: 3-phosphoinositide-dependent protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O15530
Assay: PDK1 kinase assay: PDK1 (amino acids 51-359) and AKT2 (amino acids 140-467 fused to PIFtide, amino acids EEQEMFRDFDYIADW) were expressed as N-terminally tagged GST fusion proteins in insect cells and purified to homogeneity. Enzyme activity was determined in a coupled PDK1/AKT/FAM-crosstide assay and phosphorylation of FAM-crosstide was determined by standard IMAP protocol (Molecular Devices). For inhibition studies, compounds were titrated 4-fold in DMSO and diluted 40-fold into assay buffer (10 mM Tris HCl pH7.2; 10 mM MgCl2; 0.01% Triton X-100; 1 mM DTT) containing PDK1, AKT2, and FAM-crosstide (final concentrations: 0.75 nM PDK1, 10 nM unphosphorylated AKT2, and 100 nM crosstide substrate). The kinase reaction was initiated by adding ATP to a final concentration of 40 μM of PDK1 and incubated at 25° C. for 30 min. To detect assay product, the kinase reaction was combined with Progressive Binding Solution (1:600 Progressive Binding Reagent, 50% Buffer A, 50% Buffer B, Molecular Devices) in a 1:3 ratio. The mixture was incubated for 2 hours at 25° C. and the plate was scanned on an Analyst AD with excitation at 485 nm and emission at 530 nm. The fluorescence polarization value P is defined by the equation below. The value mP is generated by multiplying the P value for each reaction well by a factor of 1000. The value ΔmP for each well is the mP value for that well minus the mP value for the average negative control. P=(Fpar−Fperp)/(Fpar+Fperp)  Eq.:Where par is fluorescence intensity parallel to the excitation plane; and perp is fluorescence intensity perpendicular to the excitation plane. ΔmP values were plotted as a function of compound concentration and the data were analyzed with a 4-parameter fit using GraphPad Prism software.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 482.44, ALogP 5.14, HBA 6, HBD 3, PSA 105.82, QED 0.33
Compound (SMILES): Cc1cc(-c2cc(Cl)ccc2OCCn2c(C)nc3nc(C(F)(F)F)c(N4CCC4)nc3c2=O)c2scc(C(=O)O)c2n1
InChIKey: KMPHXPFOGPLPFM-UHFFFAOYSA-N
Target: Eukaryotic translation initiation factor 4E-binding protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13541
Assay: Fluorescence Polarization Assay: eIF4E protein was pre-incubated with EDA-m7GDP-ATTO-550 in FPB at 2xconcentrations for 5 minutes prior to the addition of compounds. Compounds (100x) were prepared using 3-fold serial dilutions in 100% DMSO, and subsequently diluted 1:50 in FPB to produce 2x stocks. 50 ul of 2x eIF4E/EDA-m7GDP-ATTO-550 were transferred to the wells of a 96-well half-area black flat bottom polystyrene plate. 50 ul of 2x test compound were added, and binding reactions were allowed to equilibrate for 30 minutes at room temperature while protected from light. The fluorescent polarization signal was detected using a Victor 2 multi-label counter (Perkin Elmer) and the concentration necessary to achieve inhibition of eIF4E/EDA-m7GDP-ATTO-550 binding by 50% (IC50) was calculated using data from an 8-point compound dilution series.
Measurement: IC50 = 45.1 nM (pChEMBL 7.349999904632568)
Descriptors: MW 631.04, ALogP 5.74, HBA 10, HBD 1, PSA 123.33, QED 0.24
Compound (SMILES): Clc1ccc(OC2CNC2)cc1Cl
InChIKey: MRTKDTHRKQRAKJ-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Radioligand Binding Assays: IC50 Values for Example 1 and the Reference Compound 3-(3,4-Dichlorophenoxy)Azetidine Hydrochloric Acid Salt Employing Radioligand Binding Assays Using Cells Expressing Human Transporters and ReceptorsIn another binding study employing the same assays for the three monoamine transporters as described herein above, one of the compounds of the invention (Example 1A) was tested at different concentrations in order to obtain dose-response curves. Furthermore, the above mentioned Reference Compound was tested in the same assays at different concentrations and the IC50 values of both of the compounds are presented in Table 6.
Measurement: IC50 = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 218.08, ALogP 2.34, HBA 2, HBD 1, PSA 21.26, QED 0.82
Compound (SMILES): O=C(O)CCN1CCC2(CC1)COc1cc(C3CC3c3c(Cl)cccc3Cl)ccc12
InChIKey: WQAYCIIYJFRSRL-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H228
Assay: In Vitro Functional Activity (Agonism) on Human S1P5 Receptors: The CHO-human-S1P5-Aeqorin assay was bought from Euroscreen, Brussels (Euroscreen, Technical dossier, Human Lysophospholid S1P5 (Edg8) receptor, DNA clone and CHO AequoScreen™ recombinant cell-line, catalog no: ES-593-A, September 2006). Human-S1P5-Aequorin cells express mitochondrial targeted apo-Aequorin. Cells have to be loaded with coelanterazine, in order to reconstitute active Aequorin. After binding of agonists to the human S1P5 receptor the intracellular calcium concentration increases and binding of calcium to the apo-Aequorin/coelenterazine complex leads to an oxidation reaction of coelenterazine, which results in the production of apo-Aequorin, coelenteramide, CO2 and light (□max 469 nm). This luminescent response is dependent on the agonist concentration. Luminescence is measured using the MicroBeta Jet (Perkin Elmer). Agonistic effects of compounds are expressed as pEC50. Compounds were tested at a 10 points half log concentration range, and 3 independent experiments were performed in single point's measurements.
Measurement: EC50 = 79.4 nM (pChEMBL 7.099999904632568)
Descriptors: MW 446.37, ALogP 5.47, HBA 3, HBD 1, PSA 49.77, QED 0.66
Compound (SMILES): COc1cc(Oc2ccnc(Nc3ccnc(C(C)(C)O)c3)c2)n(C2CCOCC2)n1
InChIKey: QFFVJNUXWALVAU-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: TGFβR1 Kinase Inhibition Assay: TGFβR1 kinase assay was performed according to the instruction manual of the ADP-Glo™ Kinase Assay kit provided by Promega. Prepare 1× Kinase buffer (50 mM Tris pH7.5, 0.1% BSA, 10 mM MgCl<sub>2</sub>, 1 mM DTT). Before activation reaction was started, Compounds were dissolved in DMSO and make 100× solution with 3-fold serial dilution for a total of 10 concentrations. Transfer 50 nL compounds to 384-well plate according to plate map using the automated liquid handler. Prepare enzyme mix containing 2× enzyme mix containing 40 nM TGFβR1 with 1× Kinase buffer, add 2.5 μL enzyme mix to 384-well plate and pre-incubate with compounds at RT for 10 minutes. Prepare 2× substrate mix containing 5.4 μM ATP 1× Kinase buffer and add 2.5 μL substrate mix to 384-well plate, react at RT for 120 min. Add 5 μL ADP-Glo Reagent to terminate the kinase reaction and deplete the remaining ATP, incubate at RT for 60 minutes. Add 10 μL Kinase Detection Reagent to convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferin reaction, incubate at RT for 30 minutes. Collect luminescence data with Envision.
Measurement: IC50 = 126 nM (pChEMBL 6.900000095367432)
Descriptors: MW 425.49, ALogP 3.8, HBA 9, HBD 2, PSA 103.55, QED 0.59