lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1ccc(C(=O)Nc2ccc(CN3CCN(C)CC3)c(C(F)(F)F)c2)cc1C#Cn1cnc2cc(C(=O)O)ccc21
InChIKey: GBTVZRONPVRLHC-UHFFFAOYSA-N
Target: Tyrosine-protein kinase ABL1 (SINGLE PROTEIN), Homo sapiens
UniProt: P00519
Assay: Enzyme Assay: Kinase activity and inhibition was measured by established protocols (see e.g. Braunwalder et al., A Solid-Phase Assay for the Determination of Protein Tyrosine Kinase Activity of c-src Using Scintillating Microtitration Plates, Anal Biochem. 234, 1, 23-26). In such cases, the transfer of 33PO4 from ATP to the synthetic substrates poly(Glu, Tyr) 4:1 or poly(Arg, Ser) 3:1 attached to the bioactive surface of microtiter plates is taken as a measure of enzyme activity. After an incubation period, the amount of phosphate transferred is measured by first washing the plate with 0.5% phosphoric acid, adding liquid scintillant, and then counting in a liquid scintillation detector. The IC50 is determined by the concentration of compound that causes a 50% reduction in the amount of 33P incorporated onto the substrate bound to the plate.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 575.59, ALogP 4.92, HBA 6, HBD 2, PSA 90.7, QED 0.33
Compound (SMILES): COC(=O)Nc1ccc(C(=O)N[C@@H](CCN2CCC2)C(=O)N2CC[C@@]3(C2)OC(=O)Nc2ccc(Cl)cc23)cc1
InChIKey: ZQNYJUFTLDXIIN-CUNXSJBXSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Factor XIa Assay: The effectiveness of a compound of the present invention as an inhibitor of Coagulation Factor XIa can be determined using a relevant purified serine protease, and an appropriate synthetic substrate. The rate of hydrolysis of the chromogenic or fluorogenic substrate by the relevant serine protease was measured both in the absence and presence of compounds of the present invention. Assays were conducted at room temperature or at 37° C. Hydrolysis of the substrate resulted in release of amino trifluoromethylcoumarin (AFC), which was monitored spectrofluorometrically by measuring the increase in emission at 510 nm with excitation at 405 nm. A decrease in the rate of fluorescence change in the presence of inhibitor is indicative of enzyme inhibition. Such methods are known to one skilled in the art. The results of this assay are expressed as the inhibitory constant, Ki.Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 150 mM NaCl, 5 mM CaCl2, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 40 pM (Sekisui Diagnostics) and he synthetic substrate, Z-Gly-Pro-Arg-AFC, TFA salt (Sigma #C0980) at a concentration of 100 μM.Activity assays were performed by diluting a stock solution of substrate at least tenfold to a final concentration≤0.1 Km into a solution containing enzyme or enzyme equilibrated with inhibitor. Times required to achieve equilibration between enzyme and inhibitor were determined in control experiments. Initial velocities of product formation in the absence (Vo) or presence of inhibitor (Vi) were measured. Assuming competitive inhibition, and that unity is negligible compared Km/[S], [I]/e, and [I]/e (where [S], [I], and e respectively represent the total concentrations, of substrate, inhibitor and enzyme), the equilibrium constant (Ki) for dissociation of the inhibitor from the enzyme can be obtained from the dependence of Vo/Vi on [I] shown in the following equation. V o /V i=1+[I]/K iThe activities shown by this assay indicate that the compounds of the invention may be therapeutically useful for treating or preventing various cardiovascular and/or cerebrovascular thromboembolic conditions in patients suffering from unstable angina, acute coronary syndrome, refractory angina, myocardial infarction, transient ischemic attacks, atrial fibrillation, stroke such as thrombotic stroke or embolic stroke, venous thrombosis, coronary and cerebral arterial thrombosis, cerebral and pulmonary embolism, atherosclerosis, deep vein thrombosis, disseminated intravascular coagulation, and reocclusion or restenosis of recanalized vessels.
Measurement: Ki = 219 nM (pChEMBL 6.659999847412109)
Descriptors: MW 556.02, ALogP 3.4, HBA 7, HBD 3, PSA 129.31, QED 0.48
Compound (SMILES): C[C@@H]1OCC2(CCN(c3nc(N)c(-c4ccc(F)c(F)c4)c(=O)n3C)CC2)[C@@H]1N
InChIKey: BWZPUCYMMWVDMW-MEDUHNTESA-N
Target: Tyrosine-protein phosphatase non-receptor type 11 (SINGLE PROTEIN), Homo sapiens
UniProt: Q06124
Assay: SHP2 Allosteric Inhibition Assay: SHP2 is allosterically activated through binding of bis-tyrosyl-phosphorylated 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.More specifically, the phosphatase reactions were performed at room temperature in 384-well black polystyrene plate, flat bottom, low flange, non-binding surface (Corning, Cat#3575) using a final reaction volume of 25 μL and the following assay buffer conditions: 60 mM HEPES, pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% P-20, 5 mM DTT.The inhibition of SHP2 by compounds of the invention (concentrations varying from 0.003-100 μM) was monitored using an assay in which 0.5 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) (SEQ ID NO:1). After 30-60 minutes incubation at 25° C., the surrogate substrate DiFMUP (Invitrogen, cat# D6567) was added to the reaction and incubated at 25° C. for 30 minutes. The reaction was then carefully diluted by the addition of 5 μL of a 160 μM solution of bpV(Phen) (Enzo Life Sciences cat# ALX-270-204). The fluorescence signal was monitored using a microplate reader (Envision, Perki-Elmer) using excitation and emission wavelengths of 340 nm and 450 nm, respectively. The inhibitor dose response curves were analyzed using normalized IC50 regression curve fitting with control based normalization.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 405.45, ALogP 1.64, HBA 7, HBD 2, PSA 99.4, QED 0.79
Compound (SMILES): O=C(NC1CC1)c1nn2c(c1-c1ccc(Cl)cn1)OCCC2
InChIKey: ZQOTYNLKFJDUAT-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: cintillation Proximity Assay (SPA): The PDE4A3, PDE4B1, PDE4C1 and PDE4D3 assays use the Scintillation Proximity Assay (SPA) technology to measure the inhibition of human recombinant PDE4A3, PDE4B1, PDE4C1, and PDE4D3 enzyme activity by compounds in vitro. The PDE4A3, PDE4B1, PDE4C1, and PDE4D3 assays are run in parallel using identical parameters, except for the concentration of enzyme (80 pM PDE4A3, 40 pM PDE4B1, 40 pM PDE4C1 and 10 pM PDE4D3). The assays are performed in a 384-well format with 50 μL assay buffer (50 mM TRIS pH 7.5; 1.3 mM MgCl2; 0.01% Brij) containing enough PDE4A3, PDE4B1, PDE4C1, and PDE4D3 to convert 20% of substrate (1 μM cAMP consisting of 20 nM 3H-cAMP+980 μM cold cAMP) and a range of inhibitors. Reactions are incubated for 30 min at 25° C. The addition of 20 μL of 8 mg/mL yttrium silicate SPA beads (PerkinElmer) stops the reaction. The plates are sealed (TopSeal, PerkinElmer) and the beads are allowed to settle for 8 hrs, after which they are read on the TriLux MicroBeta overnight.
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 318.76, ALogP 2.27, HBA 5, HBD 1, PSA 69.04, QED 0.94
Compound (SMILES): O=C1NCCc2c1c(-c1ccc(F)cc1)c(-c1ccncc1)n2Cc1nnc(C2CC2)o1
InChIKey: RPTDKMGMYSAJGV-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y572
Assay: RIPK3 HTRF Kinase Assay: The assays were performed in black 1536-well plates. The final assay volume of 2 μl contained a mixture of a fluorescently labeled HTRF probe, Terbium (Tb) labeled anti-GST antibody, RIPK3, and test compounds in assay buffer (20 mM HEPES pH 7.4, 10 mM MgCl2, 0.015% Brij35, 4 mM DTT, and 50 μg/mL BSA). The reaction was incubated at room temperature for 60 min. and was analyzed on an Envision plate reader.Inhibition data were calculated by comparison to background control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay is RIPK3, 1 nM; HTRF probe, 23 nM; 0.2 nM Tb anti-GST; and DMSO, 0.5%. 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 seven concentrations, in singlet. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 7550 nM (pChEMBL 5.119999885559082)
Descriptors: MW 429.46, ALogP 3.95, HBA 6, HBD 1, PSA 85.84, QED 0.52
Compound (SMILES): Nc1[nH]c2c(N3CCCC3)ncnc2c1S(=O)(=O)c1ccccc1
InChIKey: KWNIVSQOHJDERJ-UHFFFAOYSA-N
Target: Free fatty acid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O15552
Assay: Ca-Flux Functional Assay: GPR43 agonist/PAM activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by FLIPR (manufactured by Molecular Devices). GPR43 mediated increases in intracellular Ca2+ concentration were readily detected upon activation with sodium acetate. Prior to the assay (24 hours), CHO-K1 Gα16 cells stably expressing human GPR43 were-seeded in cell culture medium in black, clear-bottom 384-well plates (Corning Inc) and grown overnight at 37° C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (Molecular Devices) diluted in HBSS containing 25 mM HEPES, 2.5 mM Probenecid, 0.1% BSA for 1 hour at 37° C., 5% CO2. 10 point half log concentration response curves of sodium acetate from 10 mM were conducted prior to the testing of compounds to calculate the sodium acetate concentration that produces 20% of the maximal response (EC20). Test compounds (at 10 point half log concentration response curves from 10 μM) were added in the presence of sodium acetate to achieve a final concentration that produces approximately 20% maximal response as calculated from the previous experiment. The changes in fluorescent signal were monitored by FLIPR upon addition of the compound/EC20 sodium acetate mix. The EC50 values were determined from ten point concentration response curves. Curves were generated using the average of two wells for each data point.
Measurement: EC50 = 37 nM (pChEMBL 7.429999828338623)
Descriptors: MW 343.41, ALogP 1.97, HBA 6, HBD 2, PSA 104.97, QED 0.75
Compound (SMILES): Fc1ccc(-c2ccccc2C2(Nc3ncc(-c4nnc(C(F)F)o4)cn3)CC2)cc1
InChIKey: HYTBUERZELYCOH-UHFFFAOYSA-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 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 423.4, ALogP 5.37, HBA 6, HBD 1, PSA 76.73, QED 0.45
Compound (SMILES): Cn1ccc(COc2ccc3nc(C4CCCC[C@@H]4C(=O)O)n(Cc4ccc(N5CCCCC5)cc4)c3c2)n1
InChIKey: COFJCDRSMCMUNE-GEVKEYJPSA-N
Target: Arachidonate 5-lipoxygenase-activating protein (SINGLE PROTEIN), Homo sapiens
UniProt: P20292
Assay: FLAP Binding Assay: The assay below is used to test the modulatory activity of compounds against FLAP. Human and mouse FLAP-encoding DNA was amplified by polymerase chain reaction and cloned into pFastBac1 (Invitrogen) with a NH2-terminal 6-His tag for expression in Spodoptera frugiperda (Sf-9) cells. FLAP-containing membranes were prepared as was a FITC-labeled FLAP modulator (3-(3-(tert-butylthio)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl)-2,2-dimethylpropanoic acid). The FLAP binding assay is performed in HTRF format (homogeneous time resolved fluorescence). FLAP-containing membranes (1 Î¼g/well final for human) are incubated in the presence of the HTRF ligand, [5-[({[2-(2-{3-[3-(tert-butylsulfanyl)-1-(4-chlorobenzyl)-5-(quinolin-2-ylmethoxy)-1H-indol-2-yl]-2,2-dimethylpropanoyl}hydrazino)-2-oxoethyl]sulfanyl}acetyl)amino]-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid] (25 nM final), a terbium labeled anti-His tag antibody (0.5 ng/well final, from Cisbio) and compounds. The reaction is allowed to proceed for two hours after which the plate is read on an Envision plate reader in HTRF mode.
Measurement: Ki = 32 nM (pChEMBL 7.5)
Descriptors: MW 527.67, ALogP 5.75, HBA 7, HBD 1, PSA 85.41, QED 0.31
Compound (SMILES): CN1CCN(c2ccn(-c3cc(-c4c(F)cccc4F)nc4c3C(=O)NC4)n2)CC1
InChIKey: CQBSIVNPMKWUIV-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: Caliper Assay: The caliper machine employs an off chip mobility shift assay to detect phosphorylated peptide substrates from kinase assays, using microfluidics technology. The assays are carried out at ATP concentration equivalent to the ATP Km, and at 1 mM ATP. Compounds are serially diluted in DMSO then further diluted in assay buffer (25 mM HEPES, pH 7.5, 0.01% Brij-35, 0.01% Triton, 0.5 mM EGTA). 5 ul of diluted compound was added into wells first, then 10 ul of enzyme mix was added into wells, followed by 10 uL of substrate mix (peptide and ATP in 10 mM MgCl2) to start reaction. Reaction was incubated at 28° C. for 25 min and then added 25 ul stop buffer (100 mM HEPES, 0.015% Brij-35, 50 mM EDTA), followed by reading with Caliper. JAK2 at 1 nM final concentration and TYK2 at 9.75 nM are from Carna, and substrates used are ATP at 20 and 16 uM, respectively. JAK2 assay uses peptide 22 and TYK2 uses peptide 30 (Caliper), each at 3 uM.
Measurement: Ki = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 410.43, ALogP 2.21, HBA 6, HBD 1, PSA 66.29, QED 0.72
Compound (SMILES): CCc1cc(O)c(F)cc1-c1ccc2c(-c3nc4c([nH]3)CCN(C[C@@H]3CCCN3)C4)n[nH]c2c1
InChIKey: VRNZXUFYZDRQPG-KRWDZBQOSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Biochemical JAK and Off-Target Kinase Assays: A panel of four LanthaScreen JAK biochemical assays (JAKL 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 1 h. ATP was subsequently added to initiate the kinase reactions in 10 μL total volume, with 1% DMSO. The final enzyme concentrations for JAKL 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 1 h 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.
Measurement: Ki = 0.55 nM (pChEMBL 9.260000228881836)
Descriptors: MW 460.56, ALogP 4.14, HBA 5, HBD 4, PSA 92.86, QED 0.36
Compound (SMILES): Cn1c(Nc2ccc(I)cc2F)c(C(=O)NOCCO)c2ncsc2c1=O
InChIKey: PBWDRHFDBJMELU-UHFFFAOYSA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: Enzymatic Assay: Compounds were diluted from DMSO stocks into 1× buffer (20 mM MOPS, PH 7.4, 5 mM MgCl2, 0.5 mM MnCl2, 100 uM Sodium Orthovanadate, 0.01% Triton X-100, 1 mM DTT). A typical reaction assay contained 0.01 nanomoles MEK1 kinase, 0.01 nanomoles ATP, 10 nanograms substrate. The screening assay essentially comprised four additions. 2 ul of diluted compounds were dispensed to 384 well white assay plates. 6 ul of kinase-substrate cocktail was then added to each well. 2 ul 5×ATP was subsequently added to each well to start the reaction. A top seal was applied and the plate was incubated at 22 degree avoiding light for 60 minutes. Finally, 10 ul of the Kinase Glo plus reagent was added to each well to stop the reaction. Incubated at room temperature and avoid light for ten minutes. The top seal was removed and the plate was counted by the EnVision 2104 multi labeled plate reader (PerkinElmer) with a standard luminescent program.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 504.28, ALogP 2.14, HBA 8, HBD 3, PSA 105.48, QED 0.27
Compound (SMILES): CC(C(=O)N1C[C@H](F)[C@H](NC(=O)c2cc(-c3cc(C(F)(F)F)c4c(N)ncnn34)cc(F)c2CF)C1)C(F)(F)F
InChIKey: MYAYIXVQACDODP-BZRNNEMISA-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 = 7100 nM (pChEMBL 5.150000095367432)
Descriptors: MW 582.43, ALogP 4.08, HBA 6, HBD 2, PSA 105.62, QED 0.44
Compound (SMILES): CC1(NC(=O)c2cc(NC(=O)Cc3cc(Cl)ccc3O)ccn2)CC1
InChIKey: AGOBMKMEYFKNBY-UHFFFAOYSA-N
Target: Anoctamin-1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5XXA6
Assay: Automated Whole-Cell Patch Clamp Assay to Detect TMEM16A Activity in Recombinant Cells: Compound activity was quantified by measuring the increase in current upon compound addition and expressing this as a percentage increase of baseline TMEM16A current level. Percentage increases in current were determined for each concentration and the data plotted as a function of concentration using either the Qpatch software or Graphpad Prism v6.05 providing the concentration which gave 50% of its maximal effect (EC50) and maximum efficacy (percentage of baseline increase). The method of calculating the results is illustrated in FIG. 1, which shows an example trace from the Qpatch TMEM16A assay. In FIG. 1, IBL equals baseline current, I[#1] equals the peak current during test compound concentration 1 incubation period and so on.
Measurement: EC50 = 180 nM (pChEMBL 6.75)
Descriptors: MW 359.81, ALogP 2.9, HBA 4, HBD 3, PSA 91.32, QED 0.77
Compound (SMILES): O=c1[nH]c(CSC2CCOCC2)nc2cc(OC[C@H]3CNC[C@H]3F)cc(F)c12
InChIKey: MHEFUQNSWJQRNC-IAQYHMDHSA-N
Target: Protein mono-ADP-ribosyltransferase PARP14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q460N5
Assay: Enzymatic Inhibition Assay: Enzymatic inhibition of PARP14 was measured using a dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) monitoring the auto-modification of PARP14 by biotinylated nicotinamide adenine dinucleotide (biotin-NAD). 1 μL of a dose response curve of each test compound was spotted in 384-well nickel-coated white microplates (Thermo) using a Mosquito (TTP Labtech). Reactions were performed in a 50 μL volume by adding 40 μL of PARP14 in assay buffer (20 mM HEPES pH==8, 100 mM NaCl, 0.1% bovine serum albumin, 2 mM DTT and 0.002%0 Tween20), incubating with test compound at 25° C. for 30 min, then adding 10 μL of biotin-NAD (Biolog). The final concentrations of PARP14 and biotin-NAD are 50 nM and 3 μM, respectively. Reactions proceeded at 25° C. for 3 h, then were quenched with 5 μL of 10 mM unmodified nicotinamide adenine dinucleotide (Sigma-Aldrich). The quenched reactions were washed 3 times with 100 μL of TBST wash buffer (50 mM Tris-HCl, 150 mM NaCl and 0.1% Tween20). Next, to the washed and dried plate was added 25 μL of DELFIA Europium-N1 streptavidin (Perkin Elmer) diluted in DELFIA assay buffer (Perkin Elmer). After a 30 min incubation at 25° C., the plate was washed 5 times with TBST wash buffer. Finally, 25 μL of DELFIA enhancement solution was added. After a 5 min incubation the plate was read on an Envision platereader equipped with a LANCE/DELFIA top mirror (Perkin Elmer) using excitation of 340 nm and emission of 615 nm to measure the amount of Europium present in each well, informing on the amount of biotin-NAD that was transferred in the automodification reaction.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 411.47, ALogP 2.41, HBA 6, HBD 2, PSA 76.24, QED 0.76
Compound (SMILES): C=C1C[C@@H](c2nc3cc(OC)ccc3[nH]2)N(C(=O)c2ccccc2-c2ccc(C)cc2)C1
InChIKey: KAXREJIDRGZNPT-VWLOTQADSA-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 = 298 nM (pChEMBL 6.53000020980835)
Descriptors: MW 423.52, ALogP 5.69, HBA 3, HBD 1, PSA 58.22, QED 0.43
Compound (SMILES): COCCC(C(=O)Nc1ccc(C(N)=O)c(F)c1)n1cc2c(cc1=O)-c1cc(Cl)ccc1CC(CF)NC2=O
InChIKey: TUSKZVWLXAIZDG-UHFFFAOYSA-N
Target: Coagulation factor XI (SINGLE PROTEIN), Homo sapiens
UniProt: P03951
Assay: Measurement of FXIa Inhibition: The factor XIa inhibition of the substances according to the invention is determined using a biochemical test system which utilizes the reaction of a peptidic factor XIa substrate to determine the enzymatic activity of human factor XIa. Here, factor XIa cleaves from the peptic factor XIa substrate the C-terminal aminomethylcoumarin (AMC), the fluorescence of which is measured. The determinations are carried out in microtitre plates.Test substances are dissolved in dimethyl sulphoxide and serially diluted in dimethyl sulphoxide (3000 μM to 0.0078 μM; resulting final concentrations in the test: 50 μM to 0.00013 μM). 1 μl of the diluted substance solutions is placed into each of the wells of white microtitre plates from Greiner (384 wells). 20 μl of assay buffer (50 mM of Tris/HCl pH 7.4; 100 mM of sodium chloride; 5 mM of calcium chloride; 0.1% of bovine serum albumin) and 20 μl of factor XIa from Kordia (0.45 nM in assay buffer) are then added successively. After 15 min of incubation, the enzyme reaction is started by addition of 20 μl of the factor XIa substrate Boc-Glu(OBzl)-Ala-Arg-AMC dissolved in assay buffer (10 μM in assay buffer) from Bachem, the mixture is incubated at room temperature (22° C.) for 30 min and fluorescence is then measured (excitation: 360 nm, emission: 460 nm). The measured emissions of the test batches with test substance are compared to those of control batches without test substance (only dimethyl sulphoxide instead of test substance in dimethyl sulphoxide), and IC50 values are calculated from the concentration/activity relationships.
Measurement: IC50 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 558.97, ALogP 3.25, HBA 6, HBD 3, PSA 132.52, QED 0.39
Compound (SMILES): CNC(=O)C1CC(NC(=O)N2Cc3nc(NC4CCOCC4)ncc3C2(C)C)CN(Cc2cccc(Cl)c2)C1
InChIKey: MTPOIINKMCMSED-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Activated ERK2 Kinase Assay: Compound potency against activated ERK2 was determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R- [NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay was carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 μM ATP (all concentrations are final in the reaction) in a total volume of 10.25 μL. A 16-point, half-log dilution series of compounds at 41× final concentration was used for generating IC50 curves. Compound dilution series were prepared in 100% DMSO. ERK2 was preincubated with compounds for 30 minutes at ambient temperature. Reaction was initiated by addition of a substrate cocktail of ERKtide peptide and ATP and was allowed to proceed for 2-3 hours at ambient temperature. Reaction was terminated by addition of 10 μL of a 2× stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 μg/mL of AlphaScreen Protein A Acceptor Beads, 10 μg/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 μg/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions were read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively.
Measurement: IC50 = 9192 nM (pChEMBL 5.039999961853027)
Descriptors: MW 556.11, ALogP 3.12, HBA 7, HBD 3, PSA 111.72, QED 0.5
Compound (SMILES): CC(C)(O)c1ccc2c(c1)C(=O)N(Cc1ccc(Cl)cc1)[C@@]2(OCC1(C#N)CC1)c1ccc(Cl)cc1
InChIKey: CHHGWWCMCGHFMJ-GDLZYMKVSA-N
Target: E3 ubiquitin-protein ligase Mdm2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q00987
Assay: MDM2-p53 Interaction Using a 96-Well Plate Binding Assay (ELISA): The ELISA assay was performed in streptavidin coated plates which were preincubated with 200 μl per well of 1 μg ml−1 biotinylated IP3 peptide. The plates were ready to use for MDM2 binding after washing the plate with PBS.Compounds and control solutions in DMSO aliquoted in 96-well plates were pre-incubated in a final 2.5-5% (v/v) DMSO concentration at room temperature (for example 20° C.) for 20 min with 190 μl aliquots of optimized concentrations of in vitro translated MDM2, before transfer of the MDM2-compound mixture to the b-IP3 streptavidin plates, and incubation at 4° C. for 90 min. After washing three times with PBS to remove unbound MDM2, each well was incubated at 20° C. for 1 hour with a TBS-Tween (50 mM Tris pH7.5; 150 mM NaCl; 0.05% Tween 20 nonionic detergent) buffered solution of primary mouse monoclonal anti-MDM2 antibody (Ab-5, Calbiochem, used at a 1/10000 or 1/200 dilution depending on the antibody stock solution used), then washed three times with TBS-Tween before incubation for 45 mins at 20° C. with a TBS-Tween buffered solution of a goat-anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (used at 1/20000 or 1/2000 depending on the antibody stock solution). The unbound secondary antibody was removed by washing three times with TBS-Tween. The bound HRP activity was measured by enhanced chemiluminescence (ECL, Amersham Biosciences) using the oxidation of the diacylhydrazide substrate, luminol, to generate a quantifiable light signal. The percentage of MDM2 inhibition at a given concentration is calculated as the [1−(RLU detected in the compound treated sampleRLU negative DMSO control)÷(RLU of DMSO positive and negative controls)]×100 or as the (RLU detected in the compound treated sample÷RLU of DMSO controls)×100. The IC50 was calculated using a plot of % MDM2 inhibition vs concentration and is the average of two or three independent experiments.
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 521.44, ALogP 6.4, HBA 4, HBD 1, PSA 73.56, QED 0.39
Compound (SMILES): CC(=O)Nc1cc(Nc2nc(-c3ccc(C(F)(F)F)cn3)nn3cccc23)ccn1
InChIKey: JFKKWOYQCVEYMK-UHFFFAOYSA-N
Target: TGF-beta receptor type-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P36897
Assay: Biological Assays: Assays are conducted in 1536-well plates and 2 mL reactions are prepared from addition of HIS-TGFβR1 T204D or HIS-TGFβR2 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-TGFβR1 T204D or HIS-TGFβR2 WT, 0.2 nM anti-HIS detection antibody, labeled small molecule probe (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. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 413.36, ALogP 3.91, HBA 7, HBD 2, PSA 97.1, QED 0.53
Compound (SMILES): COc1ncccc1CC(=O)Nc1cc([C@H]2CC[C@@H](OC(=O)NC(C)(C)C)C2)[nH]n1
InChIKey: VIJFNCBXICGJBO-DZGCQCFKSA-N
Target: Cyclin-dependent kinase 2/cyclin E1 (PROTEIN COMPLEX), Homo sapiens
UniProt: P24864
Assay: Biochemical Assay: The purpose of CDK2/Cyclin E1 assay is to evaluate the inhibition (% inhibition, Kiapp and Ki values) of small molecule inhibitors by using a fluorescence-based microfluidic mobility shift assay. CDK2/Cyclin E1 full length catalyzes the production of ADP from ATP that accompanies the phosphoryl transfer to the substrate peptide FL-Peptide-18 (5-FAM-QSPKKG-CONH2, CPC Scientific, Sunnyvale, Calif.) (SEQ ID NO:1). The mobility shift assay electrophoretically separates the fluorescently labeled peptides (substrate and phosphorylated product) following the kinase reaction. Both substrate and product are measured and the ratio of these values is used to generate % conversion of substrate to product by the LabChip EZ Reader. Wild-type CDK2/wild-type full length Cyclin E1 enzyme complex was produced in-house (baculoviral expression, LJIC-2080/LJIC-2103) and phosphorylated by CDK7/Cyclin H1/Matl enzyme complex with CDK2:CDK7 ratio of 50:1 (concentration mg/mL) in the presence of 10 mM MgCl2 and 5 mM ATP at room temperature for one hour. Typical reaction solutions (50 μL final reaction volume) contained 2% DMSO (±inhibitor), 4 mM MgCl2, 1 mM DTT, 150 μM ATP (ATP Km=67.4 μM), 0.005% Tween-20, 3 μM FL-Peptide-18, and 0.36 nM (catalytically competent active site) phosphorylated wild-type full length CDK2/Cyclin E1 enzyme complex in 25 mM HEPES buffer at pH 7.15. The assay was initiated with the addition of ATP, following a fifteen minutes pre-incubation of enzyme and inhibitor at room temperature in the reaction mixture. The reaction was stopped after 45 minutes at room temperature by the addition of 50 μL of 80 mM EDTA.
Measurement: Ki = 0.73 nM (pChEMBL 9.140000343322754)
Descriptors: MW 415.49, ALogP 3.16, HBA 6, HBD 3, PSA 118.23, QED 0.67
Compound (SMILES): CN(C(N)=O)c1ccc(Nc2ncc(C(N)=O)c(NC3CC3)n2)cc1
InChIKey: PTUIOQXDADQRPN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: In Vitro Assay: SYK tyrosine phosphorylation activity is measured using the LANCE Technology developed by Perkin Elmer Life and Analytical Sciences (Boston, Mass.). LANCE refers to homogeneous time resolved fluorometry applications using techniques such as time-resolved fluorescence resonance energy transfer assay (TR-FRET) (see generally for procedures in Perkin Elmer Application NoteHow to Optimize a Tyrosine Kinase Assay Using Time Resolved Fluorescence-Based LANCE Detection, wwww.perkinelmer.com/lifesciences). The assay principle involves detection of a phosphorylated substrate using energy transfer from a phosphospecific europium-labeled antibody to streptavidin-allophycocyanin as an acceptor.To test the ability of candidate molecules to inhibit SYK tyrosine phosphorylation activity, molecules are reconstituted in 30% DMSO and serially diluted 1:3 with the final dilution containing DMSO in the absence of the candidate molecule. The final DMSO concentration in the assay is 3%. Kinase assays are performed as a two part reaction. The first reaction is a kinase reaction and which comprises of a candidate molecule, full length active recombinant SYK enzyme (Millipore, CA) and biotin-labeled SYK-specific substrate biotin-DEEDYESP-OH. The second reaction involves termination of the kinase reaction and the simultaneous addition of the detection reagents-europium-labeled anti-phosphotyrosine reagent (Eu-W1024-PY100, Perkin Elmer, Boston, Mass.) and Streptavidin-Allophycocyanin detection reagent (SA-APC, Prozyme, CA). The kinase reaction is performed in a black U-bottom 96-well microtitre plate. The final reaction volume is 50 μL and contains a final concentration of 1 nM active SYK enzyme, 550 nM SYK-substrate, and 100 μM ATP diluted in a buffer containing 50 mM Tris pH 7.5, 5 mM MgCl2, and 1 mM DTT. The reaction is allowed to proceed for 1 hour at room temperature. The quench buffer contains 100 mM Tris pH 7.5, 300 mM NaCl2, 20 mM EDTA, 0.02% Brij35, and 0.5% BSA. The detection reagents are added to the reaction mixture at the following dilutions1:500 for Eu-W1024-PY100 and 1:250 for SA-APC. The kinase reaction is terminated by the addition of 50 μL quench buffer containing the detection reagents. The detection is allowed to proceed for 1 hr at room temperature.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 341.38, ALogP 1.41, HBA 6, HBD 4, PSA 139.26, QED 0.63
Compound (SMILES): CCCN(CCC)c1ccc(CCc2ccc(CC(=O)NN)cc2)cn1
InChIKey: UFSBHRLNFDWGAE-UHFFFAOYSA-N
Target: Amine oxidase [copper-containing] 3 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16853
Assay: Inhibitory Assay: The compounds of the present invention obtained in Production Examples were examined for the inhibitory effect on human and rat VAP-1 enzyme (SSAO) by the following method.The VAP-1 enzyme (SSAO) activity in both human and rat was measured by a radiochemical-enzyme assay using 14C-benzylamine as an artificial substrate. Human or rat VAP-1 was cloned from the cDNA library and expressed in a cell. The cell extract was preincubated with a test compound solution (final concentration 1×10−7-1×10−10 mol/L) at room temperature for 20 minutes. Then, 14C-benzylamine (final concentration 1×10−5 mol/L) was added, and the mixture was incubated at a final volume of 200 μL at 37° C. for 2 hours. The enzyme reaction was stopped by addition of 2 mol/L (200 μL) citric acid. The oxidation product was extracted with 1 mL toluene/ethyl acetate (1:1), and the radioactivity thereof was measured by a liquid scintillation counter.
Measurement: IC50 = 20.5 nM (pChEMBL 7.690000057220459)
Descriptors: MW 354.5, ALogP 3.03, HBA 4, HBD 2, PSA 71.25, QED 0.39
Compound (SMILES): O=C(Nc1cc2c(cn1)cnn2S(=O)(=O)c1ccc(F)cc1)c1c(C(F)(F)F)cccc1C(F)(F)F
InChIKey: KNSUHTLXOPCPBS-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORgamma-Ligand Binding Domain TR-FRET Assay Protocol I: Recombinant, HIS-tagged RORγ-LBD was expressed in SF9 cells using a baculovirus expression system. Cells were lysed and the lysate was used as a source for RORγ-LBD for the assay. A 1:80 dilution of RORγ-LBD lysate in assay buffer (25 mM HEPES pH 7.0, 100 mM NaCl, 0.01% Tween, 0.1% BSA) was prepared and 5 μL was added to each well (RORγ-LBD final concentration 3 nM). Control wells received lysate from SF9 cells not expressing RORγ-LBD.Compounds to be tested were diluted to 100× final test concentration in DMSO and further diluted to 4× final test concentration using assay buffer to provide the test compound mixture. An aliquot (5 μL) of the test compound mixture was added to each well.A 4× stock of biotinylated-LXXLL peptide from SRC1-2 (Biotin-CPSSHSSLTERHKILHRLLQEGSPS) was prepared in assay buffer and a 5 μL aliquot added to each well (450 nM final concentration). A 4× solution of europium tagged anti-HIS antibody (2 nM final concentration) and APC conjugated streptavidin (60 nM final concentration) were prepared and a 5 μL aliquot added to each well.The final assay mixture was incubated for 4 hours to overnight, and the fluorescence signal was measured on an Envision plate reader: (Excitation filter=340 nm; APC emission=665 nm; Europium emission=615 nm; dichroic mirror=D400/D630; delay time=100 μs, integration time=200 μs).EC50 values for test compounds were calculated from the quotient of the fluorescence signal at 665 nm divided by the fluorescence signal at 615 nm using GraphPad Prism software.
Measurement: EC50 = 2409 nM (pChEMBL 5.619999885559082)
Descriptors: MW 532.4, ALogP 5.1, HBA 6, HBD 1, PSA 93.95, QED 0.37
Compound (SMILES): S=C=NCCN1CCC(Cc2ccccc2)CC1
InChIKey: GRCDPQIAFVDINA-UHFFFAOYSA-N
Target: N-acylethanolamine-hydrolyzing acid amidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q02083
Assay: Inhibition Assay: In order to have an assay method more conducive to high-throughput screening than those published for measuring the NAE hydrolyzing activity of NAAA, we developed the fluorogenic PEA analog N-(4-methyl coumarin)palmitamide (PAMCA), which is hydrolyzed to fluorescent 7-amino-4-methyl coumarin (AMC) and palmitic acid. For three point concentration inhibition assays with hNAAA the following procedure is used. Purified activated NAAA (final concentration of 0.25 μg/mL) is incubated in assay buffer (100 mM citrate-phosphate buffer, pH 4.5, 3 mM DTT, 0.1% Triton X-100, 0.05% BSA, and 150 mM NaCl) made up to a total volume of 180 μL, followed by addition of the compound dissolved in 10 μL DMSO (along with DMSO neat for the control sample) with the final concentrations for each compound of 100, 10, and 1 μM, in triplicate on a 96 well plate. These samples are allowed to incubate for 15 min at room temperature and then 10 μL of a PAMCA stock solution in DMSO (final PAMCA concentration [5 μM]) was added. After 5 minutes of agitation on a shaking plate, the reaction is allowed to proceed at 37° C. for 120 minutes, with fluorescence readings taken every 10 minutes at a wavelength of 460 nm (using an excitation wavelength of 360 nm) on a Synergy HT Plate Reader using Gen5 software from Bio-Tek. The enzyme activity is calculated by converting the relative fluorescence units to AMC formed, using a standard curve of AMC. For compounds that inhibit hNAAA in range IC50 <1 μM full inhibition curves using eight different concentrations of inhibitor (8 point assay) are generated. The assay procedure used is the same as the three point assay. For ostensibly covalent compounds (as observed by a significant decrease in the slope of a plot of fluorescence vs. time in three point screen) samples are allowed to incubate for 2 hours at 37° C., instead of 15 minutes, before addition of 10 μL of a PAMCA stock solution in DMSO for a final PAMCA concentration of 5 μM. After 5 minutes of agitation on a shaking plate, the reaction is allowed to proceed at 37° C. for 120 minutes Inhibition constants are calculated using pro Fit software (Quantum Soft, Uetikon am See, Switzerland) and a Levenberg-Marquardt algorithm.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 260.41, ALogP 3.04, HBA 3, HBD 0, PSA 15.6, QED 0.6
Compound (SMILES): Cc1nc(C)c(-c2ccncc2N(C)C(=O)c2cc(C(F)(F)F)cc(C(F)(F)F)c2)s1
InChIKey: MCGICVDEOBVJBV-UHFFFAOYSA-N
Target: G-protein coupled bile acid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8TDU6
Assay: cAMP Assay: CHO-dhfr(minus) cells expressing human GPBAR1 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), lx 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 μl and incubated for 30 min at 30° C. The assay was stopped by the addition of 50 μl lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN3) and 50 detection solutions (20 μM mAb Alexa700-cAMP 1:1, and 48 μM Ruthenium-2-AHA-cAMP) and shaked for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH, Hamburg Germany), 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 measured signal at 730 nm has to be corrected for the ruthenium background, the direct excitation of Alexa and the buffer control. 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 μM to 0.13 nM cAMP.
Measurement: EC50 = 1470 nM (pChEMBL 5.829999923706055)
Descriptors: MW 459.42, ALogP 6.14, HBA 4, HBD 0, PSA 46.09, QED 0.44
Compound (SMILES): COc1cc2nc(N)n3nc(C4CCC(C)N(c5cnn(C6CCOCC6)c5C)C4)nc3c2cc1F
InChIKey: WUNVGZMQDISFMR-UHFFFAOYSA-N
Target: Adenosine receptor A2a (SINGLE PROTEIN), Homo sapiens
UniProt: P29274
Assay: Measurement of A2a Binding Affinity Using Radioligand Binding: 148 μL (5 μg/mL) membranes (Perkin Elmer, Cat. No. RBHA2aM400UA) and 2 μL compounds of the invention to be tested (test compound) were transferred to individual wells of a 96-well polypropylene assay plate and incubated for 15 to 30 minutes at room temperature. [3H] SCH58261 ((7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine)) was diluted in assay buffer (50 mM Tris pH 7.4, 10 mM MgCl2, 0.005% Tween20) to a concentration of 4 nM and 50 μL transferred to each well of the assay plate. To define total and non-specific binding, wells containing 1% DMSO and 1 μM ZM241385 (Tocris Bioscience, Cat. No. 1036) respectively, were also included. The assay plate was incubated at room temperature for 60 minutes with agitation. Using a FilterMate Harvester® (Perkin Elmer), the contents of the assay plate were filtered through a UniFilter-96® PEI coated plate (Perkin Elmer Cat. No. 6005274 or 6005277). Filtering was achieved by aspirating the contents of the assay plate for 5 seconds, then washing and aspirating the contents three times with ice-cooled wash buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) and allowing the vacuum manifold to dry the plate for 30 seconds. The filter plate was incubated for at least 1 hour at 55° C., and allowed to dry. The bottom of the filter plate was sealed with backing tape. 40 μL Ultima Gold (Perkin Elmer, Cat. No. 6013329) was added to each well of the filter plate and the top of the plate was sealed with TopSeal-A PLUS® clear plate seal (Perkin Elmer, Cat. No. 6050185). The plate was incubated for at least 20 minutes, and then the amount of radioactivity remaining in each well was determined using a TopCount® (Perkin Elmer) scintillation counter. After normalization to total and non-specific binding, the percent effect at each compound concentration was calculated. The plot of percent effect versus the log of compound concentration was analyzed electronically using a 4-parameter logistic fit based on the Levenberg-Marquardt algorithm to generate IC50 values.
Measurement: IC50 = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 494.58, ALogP 3.64, HBA 10, HBD 1, PSA 108.62, QED 0.46
Compound (SMILES): COc1cccc(OC)c1-n1c(NS(=O)(=O)[C@@H](C)[C@H](C)c2ncc(Cl)cn2)nnc1[C@H]1CC1(F)F
InChIKey: BRLJJMQPIYYAQS-SGMGOOAPSA-N
Target: Apelin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35414
Assay: [35S]GTPgammaS Binding: The human APJ receptor was cloned by polymerase chain reaction and the gene encoding the receptor was subcloned in pFLAG-CMV-3 expression vector (Sigma, Saint Louis, Mo. USA) in-house at Amgen. A GTPγS binding assay was performed on membranes prepared from CHO cells stably expressing human APJ receptor. The optimum experimental conditions for the concentrations of GDP, MgCl2, and NaCl in the assay buffer were initially determined. The assay was performed in 9 μL assay buffer [20 mM HEPES, pH 7.5, 5 mM MgCl2, 100 mM NaCl and 0.1% (w/v) BSA], 1 μL of diluted test compound (starting with 0.75 mM, 2-fold serial dilution with DMSO, total 22 points), 10 μL of 18 μM GDP (final concentration of 3 μM GDP), 20 μL of 0.25 μg/mL membrane protein expressing human APJ receptor captured with WGA PS beads (final concentration of 5 μg per well), and 20 μL of 0.3 nM [35S]GTPγS (final concentration is 0.1 nM [35S]GTPγS)(Perkin Elmer Life and Analytical Sciences, Waltham USA). One column of the plate was 1 μL of DMSO as background and another column of the plate was 1 μL of 180 μM Pyr-Apelin-13 which was used as control at a final concentration of 3 μM. Incubation was at RT for 90 min and the microplate was read using a ViewLux ultra HTS Microplate Imager (PerkinElmer, Inc.). All the results presented are means of several independent experiments and analyzed by non-linear regression methods using the commercially available program Prism (GraphPad, San Diego, Calif.) providing the EC50 values.
Measurement: EC50 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 528.97, ALogP 3.78, HBA 9, HBD 1, PSA 121.12, QED 0.45