lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(=O)N[C@@H]1CCCC[C@H]1NC(=O)c1sc2nccc3c2c1NC(=O)N3c1ccc(Oc2ccccc2)cc1C
InChIKey: JTOYEYNFXIKFEC-FGZHOGPDSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Lanthascreen Binding Assay: A BTK kinase lanthascreen binding assay monitors compound binding to unphosphorylated-BTK kinase domain (UP-BTK), by competing with a fluorescent labeled tracer. UP-BTK, consisting of the kinase domain of non-phosphorylated BTK protein (389-659aa), was produced in a Baculovirus/insect cell expression system. Into a 384-well plate, 2 ng of GST-tagged human BTK (389-659aa) was incubated with compound, 50 nM of Tracer 236 and 2 nM anti-GST antibody for 60 minutes using an optimized Lanthascreen assay. After 60 minutes, plates were read at 340 nM and 615/665 nM in an Infinite F500 (Tecan).
Measurement: IC50 = 186 nM (pChEMBL 6.730000019073486)
Descriptors: MW 555.66, ALogP 6.26, HBA 6, HBD 3, PSA 112.66, QED 0.26
Compound (SMILES): O=C(Nc1ccc(Oc2ccnc3cc(OCCCN4CCCCC4)c4c(c23)OCCO4)cc1Cl)NC1CC1
InChIKey: FDJYUYTYXSFLSC-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Assay of Small Molecular Compounds for Inhibiting the Activity of VEGFR-2 Kinase: The assay is based on the LANCE TR-FRET technology of Perkin Elmer Inc., and the assay method is as follows:1. Dilution of compounds: a total of 11 concentrations were obtained using a 3-fold gradient dilution from the highest concentration of 2500 nM (the maximum final concentration of the drug used in this assay was 2500 nM, and the minimum final concentration was 0.042 nM).2. 2.5 μL of the gradient-diluted compounds were taken with a transfer pipette to a 384-well plate.3. Addition of enzyme: 5 μL of 2× VEGFR2 kinase solution (concentration was 0.5 nM) was taken with a transfer pipette to the corresponding reaction well of the 384-well plate, mixed well and pre-reacted at room temperature for 30 minutes.4. 2.5 μL 4× Ultra ULight-JAK-1 (Tyr1023) Peptide (concentration was 200 nM)/ATP (concentration was 40 μMM) mixture was taken with a transfer pipette to the corresponding reaction well of the 384-well plate.5. Negative control: 2.5 μL/well 4× substrate/ATP mixture and 7.5 μL 1× Kinase Assay Buffer were added to the wells of the 384-well plate.6. Positive control: 2.5 μL/well 4× substrate/ATP mixture, 2.5 μL/well 1× Kinase Assay Buffer containing 16% DMSO, and 5 μL/well 2× VEGFR2 kinase solution were added to the 384-well plate. The final concentration of DMSO in the reaction system was 4%.7. The mixture was mixed well and then centrifuged and reacted at room temperature in dark for 60 min.8. Termination of the enzymatic reaction: 5 μL of 4× stop solution was taken with a transfer pipette to the wells of the 384-well plate, mixed and then centrifuged, and reacted at room temperature for 5 min.9. Development of the reaction: 5 μL of 4× detection solution was taken with a transfer pipette to the wells of the 384-well plate for color development, and the mixture was mixed and then centrifuged and reacted at room temperature for 60 min.10. The 384-well plate was placed into the Envision plate reader and the signal was detected using the appropriate program.11. Analysis and processing of the raw data:The drug concentrations and the corresponding inhibition rates were input into GraphPad Prism5 for calculation, and the inhibition rate of the compounds were calculated as follows: inhibition rate (%)=(reading of positive well−reading of experimental well)/(reading of positive control well−reading of negative control well)×100%. Processing with GraphPad Prism5 software yielded the corresponding IC50 values (the concentration of the compound at which 50% of the highest inhibition of the enzyme is achieved).
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 553.06, ALogP 5.99, HBA 7, HBD 2, PSA 94.18, QED 0.31
Compound (SMILES): CSc1cccc2nc([C@@H]3CCCN3C(=O)c3nccnc3-c3cccc(C(F)(F)F)c3)[nH]c12
InChIKey: NZQJGWVKJLZJIZ-KRWDZBQOSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
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 = 282 nM (pChEMBL 6.550000190734863)
Descriptors: MW 483.52, ALogP 5.74, HBA 5, HBD 1, PSA 74.77, QED 0.37
Compound (SMILES): COC(=O)N1[C@H](C)C[C@H](N(Cc2ccc(OC)cc2)S(=O)(=O)N(C)C)[C@@H]1COC1CCC(OCc2ccccc2)CC1
InChIKey: KUZYNVMFJNXXJM-HVBDULTNSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Receptor Activity Assay: In a typical experiment, the OX1 and OX2 receptor agonist activity is determined in accordance with the following general experimental method. Chinese hamster ovary (CHO) cells expressing human OX1R and/or the human OX2R were grown in Iscove's modified DMEM containing glutaMAX®, 1% G418, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% heat-inactivated qualified fetal bovine serum (FBS). The OX2R cells were seeded at 10,000 cells/well/50 L and the OX1R cells were seeded at 20,000 cells/well/50 μL into 384-well white tissue culture plates (Greiner; cat #781080). All cell/media reagents were from GIBCO-Invitrogen Corp. The seeded cell plate(s) were incubated at 37° C. with 5% CO2 and 85% humidity for 20-24 hours. On the day of the assay, assay-ready compound plates were prepared using an acoustic liquid handler (ECHO; Labcyte), which dispensed sufficient volume of test compound stock (10 mM in DMSO) or 100% DMSO to prepare 10 point, ½-log dilutions in a final volume of 202.5 nL/well in all test wells of a 384-well diamond plate (Labcyte). Following completion of assay-ready plates, importantly, the next three steps were performed with minimal delay: 1) 20 μl of 1× stimulation buffer was added to the compound plate using a Multidrop Combi (small cassette, Thermo Fisher Scientific cat #24073290); 2) culture medium was removed from the cell plate using the Bluewasher plate washer (gentle spin; BlueCatBio); 3) 14 μl of compound/stimulation buffer mixture was added to the cell plate using a Bravo liquid handler (Agilent) prior to incubating cell plates at 37° C. with 5% CO2 and 85% humidity for 1 or 2 hours (OX1R and OX2R, respectively). During this incubation, IP-one detection reagents were prepared (38:1:1 lysis buffer:D2:AB-cryptate reagents). Six L of mixed detection reagents were added to the cell plate using a Multidrop Combi (small cassette, Thermo Fisher Scientific cat #24073290) and incubated 60 minutes at room temperature in the dark. Fluorescence signal was detected using an Envision plate reader (Perkin Elmer) [LANCE/DELFIA Dual Enh (Em: APC 665; Ex: Cy5 620)].
Measurement: IC50 = 0.76 nM (pChEMBL 9.119999885559082)
Descriptors: MW 603.78, ALogP 4.45, HBA 7, HBD 0, PSA 97.85, QED 0.35
Compound (SMILES): Cc1ncc(COc2cc(F)cc(Oc3ccc(-c4nc(C5CC5)n5ccnc(N)c45)cc3)c2)cn1
InChIKey: OXEOQHZPVPJZHP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Kinase Inhibition Assay: Method A: Fluorescence polarization-based kinase assays were performed in 384 well-plate format using histidine tagged recombinant human full-length Bruton Agammaglobulinemia Tyrosine Kinase (Btk) and a modified protocol of the KinEASE FP Fluorescein Green Assay supplied from Millipore®. Kinase reaction were performed at room temperature for 60 minutes in presence of 250 μM substrate, 10 μM ATP and variable test article concentrations. The reaction was stopped with EDTA/kinease detection reagents. Phosphorylation of the substrate peptide was detected by fluorescence polarization measured with a Tecan 500 instrument.
Measurement: EC50 < 100 nM (pChEMBL 7)
Descriptors: MW 482.52, ALogP 5.46, HBA 8, HBD 1, PSA 100.45, QED 0.33
Compound (SMILES): C=CC(=O)Nc1cc(N2CCC(N(C)C3CC3)CC2)ccc1Nc1cc(N(C)C(=O)Nc2c(Cl)c(OC)cc(OC)c2Cl)ncn1
InChIKey: BESPBINHRRCJFV-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P21802
Assay: Inhibition Assay: The experimental procedure is briefly described as follows: the test compound was first dissolved in DMSO to prepare a stock solution, and then gradiently diluted with the buffer provided in the kit, and the final concentration of the test compound in the reaction system ranges from 10 μM to 0.1 nM. The concentration of ATP solution used in the test is the ATP Km concentration corresponding to each FGFR subtype measured in advance, and the ATP Km concentration corresponding to FGFR1-4 is 100 μM, 40 μM, 40 μM and 120 μM respectively. The reaction was carried out in a 384-well microplate, the compound and a certain amount of FGFR protein was firstly added to the well, and incubated at room temperature for 5-30 minutes, then the ATP solution and the biotinylated polypeptide substrate solution were added to the reaction solution, and incubated for 50 minutes with shaking at room temperature. Subsequently, an anti-phosphotyrosine antibody coupled with a europium compound and streptavidin coupled with the modified allophycocyanin XL665 were added to the reaction, and incubation was continued for 1 hour at room temperature with shaking. After the incubation ended, the fluorescence intensity values of the respective wells at an excitation wavelength of 304 nm and emission wavelengths of 620 nM and 665 nM were measured in a TF-FRET mode on a microplate reader.
Measurement: IC50 = 228 nM (pChEMBL 6.639999866485596)
Descriptors: MW 669.61, ALogP 6.4, HBA 9, HBD 3, PSA 124.19, QED 0.2
Compound (SMILES): CCc1nc2ccc(N3CCN4C(=O)OC[C@@H]4C3)nn2c1N(C)c1nc(-c2ccc(F)cc2)cs1
InChIKey: WKJBCVILBIFOCI-KRWDZBQOSA-N
Target: Autotaxin (SINGLE PROTEIN), Mus musculus
UniProt: Q9R1E6
Assay: Biochemical Assay: Compound IC50 values are determined in a hENPP2 (UniProtKB/SwissProt Sequence ref Q13822) biochemical assay using LPC as substrate.5 μL of a dilution series of compound, starting from 20 μM highest concentration, 1/5 dilution, is added to the wells. hENPP2 is used at a final concentration of 1 μg/mL or 3 μg/mL (it will be appreciated by the skilled person that the potency read out is independent of the enzyme concentration). The enzyme is diluted in 50 mM Tris-HCl pH 8.5, 500 mM NaCl, 5 mM KCl, 10 mM CaCl2, 0.1% fatty acid free BSA in a total volume of 10 μL. the reaction is started by the addition of 10 μL of 150 μM LPC (palmitoyl 16:0) diluted in the same buffer as described above and the mixture is incubated at 37° C. for 30 min. The reaction is terminated and choline quantified by the addition of a 25 μL of a mixture containing 0.6 U/mL of choline oxidase, 0.6 U/mL of peroxydase, 1.8 mM TOOS, 1.2 mM amino-antipyrine, 20 mM EGTA (stop-developer solution) diluted in the buffer described above. Luminescence is read on the Envision after an incubation of 30 min at room temperature (Excitation 555 nm, excitation light=70%).
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 493.57, ALogP 3.96, HBA 9, HBD 0, PSA 79.1, QED 0.42
Compound (SMILES): COc1ccc(C2CC2NCc2cn3c(n2)CN(CCCc2ccc(C(=O)NO)cc2)CC3)cc1
InChIKey: MADLLDQAZUTCQB-UHFFFAOYSA-N
Target: Protein deacetylase HDAC6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UBN7
Assay: Histone Deacetylase Assay : Histone deacetylase assay was done as per manufacturer's instructions. Briefly, assay buffer, 200 uM HDAC substrate (fluorogenic HDAC acetylated peptide substrate for class I HDACs (HDACs 1, 2, and 3) and class 2b HDACs (HDACs 6 and 10) and 1% BSA are taken as a master mix and aliquoted as 40 ul per well. Compounds (10×) were diluted in assay buffer and were added to respective wells of a black 96 well plate. HDAC6 human recombinant enzyme was thawed on ice and 5 μl (7 ng/ul) enzyme was added per well. The plate was incubated at 37° C. for 1 hour. Developer solution was then added (50 μl per well) and incubated at room temperature for 10 minutes. Fluorescence was measured at an excitation wave length of 350-380 nm and emission wavelength of 440-480 nm.
Measurement: IC50 = 28 nM (pChEMBL 7.550000190734863)
Descriptors: MW 475.59, ALogP 3.1, HBA 7, HBD 3, PSA 91.65, QED 0.31
Compound (SMILES): O=C1Nc2cn(C3CCOCC3)nc2C(=O)NCC(O)COc2ccc(=O)n(n2)Cc2cccc1c2
InChIKey: KNCBISXOBZEGTD-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P51617
Assay: Enzymatic Assay: IRAK1 is a human purified recombinant enzyme (His-TEV-IRAK1 (194-712)) In this assay, IRAK1 hydrolyses ATP and autophosphorylates. Measurement of IRAK-1 inhibition is performed in 384-well format based on luminescence assay (ADP-Glo Kinase Assay from Promega). Purified human recombinant IRAK1 (0.3 ug/ml) and serial diluted compounds in DMSO (range of concentration from 10 uM to 0.5 nM) or controls (1% DMSO) are incubated for 15 minutes at RT in assay buffer containing 50 mM Hepes pH 7.0, Fatty acid-free BSA 0.1%, Dithiothreitol (DTT) 2 mM, MgCl2 10 mM, EGTA 0.5 mM, Triton X-100 0.01%. The kinase reaction is then initiated by the addition of ATP at a concentration of 1 uM. After 2 hours of incubation at RT, the reaction is stopped and the unconsumed ATP depleted by the addition of ADP-Glo Reagent according to supplier instructions. After 40 minutes of incubation at RT, the Kinase Detection Reagent is then added to the assay plate according to supplier instructions. After 20 minutes of incubation at RT, the luminescence signal is measured with a luminometer (PerkinElmer Envision or equivalent reader).
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 494.51, ALogP 0.57, HBA 10, HBD 3, PSA 149.6, QED 0.44
Compound (SMILES): COc1ccc(C(=O)N[C@@H]2CCCN(c3cnc(C(N)=O)c(Nc4ccc(N5CCOCC5)nc4)n3)C2)cc1
InChIKey: AWMGSTACIVFGIG-HXUWFJFHSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk In Vitro Inhibitory Assay: Human Btk kinase (Genbank accession # NP_000052) was purified from insect cells as a full-length construct containing a N-terminal 6×-His tag. Btk kinase activity was determined using a radiometric filter binding assay. Measurements are performed in a low μL reaction volume 384-well assay plates. BTK enzyme (8 nM final in reaction), inhibitor (at requested doses), and 0.2 mg/mL peptide substrate (Poly-Glu-Tyr, 4:1 ratio) are incubated in a reaction buffer composed of 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, 1% DMSO for 15 min. followed by addition of 1 μM ATP to start the assay. Kinase reactions are carried out for 120 min. at room temperature. The reaction was stopped by spotting of reaction sample onto P81 cationic exchange paper (Whatman). Unbound phosphate was removed by extensive washing of filters in 0.75% Phosphoric acid. After subtraction of background derived from control reactions containing inactive enzyme (via addition of saturating EDTA), kinase activity data for each dose of compound tested was expressed as the percent of remaining kinase activity in test samples compared to vehicle (dimethyl sulfoxide) reactions.
Measurement: IC50 = 55 nM (pChEMBL 7.260000228881836)
Descriptors: MW 532.61, ALogP 1.96, HBA 10, HBD 3, PSA 147.83, QED 0.39
Compound (SMILES): C=C1C[C@@H](c2nc3cc(C(C)(C)C)ccc3[nH]2)N(C(=O)c2ccccc2-n2nccn2)C1
InChIKey: IXUDMSDRTJNPJS-QFIPXVFZSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
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 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 426.52, ALogP 4.58, HBA 5, HBD 1, PSA 79.7, QED 0.49
Compound (SMILES): CC(=O)N1CCc2c(c(Nc3cccc(C)c3)nn2CCC(N)=O)C1
InChIKey: GNSSLRMFFNVEAX-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaLisa Binding Assay: His/Flag epitope tagged BRD4 BD142-168 was cloned, expressed, and purified to. BRD4 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-1/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD4(BD1) (30 nM final) was combined with peptide (200 nM final) in 40 mM HEPES (pH 7.0), 40 mM NaCl, 1 mM DTT, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 1.2% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature Alpha streptavidin donor beads and AlphaLisa anti-Flag acceptor beads were added to a final concentration of 10 ug/mL each. After three hours equilibration plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Measurement: IC50 > 19740 nM (pChEMBL 4.704999923706055)
Descriptors: MW 341.42, ALogP 1.72, HBA 5, HBD 2, PSA 93.25, QED 0.87
Compound (SMILES): CS(=O)(=O)c1cccc(COc2ccc(Cl)cc2OC2CNC2)c1
InChIKey: OQTMFUPUVYSLQY-UHFFFAOYSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Binding Assay: Receptor binding was performed using membrane fractions prepared from the HEK-293 cell line recombinantly expressing rat 5-HT7 receptors (NCBI accession NM_022938). Compound affinity for the rat 5-HT7 receptor subtype was evaluated by competitive radioligand binding assays using 5-carboxamido[3H]tryptamine ([3H]5-CT) (Amersham Biosciences, cat. 90000403) detection. HitHunter cAMP assays are in-vitro based competitive immunoassays. The assay was performed on the HEK-293 cell line stably transfected with r5-HT7 receptor. Cells were pre-incubated with test compounds for 10 minutes. For antagonist testing, the cells were then challenged with 100 nM 5-CT for 20 minutes. Cells were then lysed and cAMP measured according to manufacturers protocol (Amersham, cat. NET791250UC) or [3H]mesulergine (Amersham, cat. TRK1041). The assay was performed on membranes prepared from HEK-293 cells stably transfected with h5-HT6. Following centrifugation, membranes were resuspended and incubated for 60 min at room temperature with 1.7 nM [3H]LSD in the presence of increasing concentration of test compounds. Nonspecific binding was defined in the presence of 10 μM clozapine (Tocris, cat. TRK1068). Homogenized HEK-293 membranes expressing the human SERT were incubated in 50 mM Tris-HCl (pH 7.5), 120 mM NaCl, 5 mM KCl with [3H]-citalopram (3 nM) with or without test compounds. Nonspecific binding was determined in the presence of 10 μM fluoxetine. Radioactivity readouts and Ki values were performed as previously described for r5-HT7.
Measurement: Ki = 1679 nM (pChEMBL 5.78000020980835)
Descriptors: MW 367.85, ALogP 2.67, HBA 5, HBD 1, PSA 64.63, QED 0.85
Compound (SMILES): N#Cc1ccc(-c2cnc3ccc(-c4ccc(C(=O)NCCN5CCOCC5)cc4)cn23)cc1
InChIKey: XIQUTCDRLNPPCT-UHFFFAOYSA-N
Target: MAP kinase-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBH9
Assay: In Vitro Kinase Assay: MNK1 and MNK2 inhibitor activity was determined using recombinant kinase domains expressed in E. coli. MNK1 and MNK2 were expressed as GST fusion proteins and the GST tag was removed using PreScission protease. After concentration to 10-15 mg/ml the proteins were flash frozen in liquid nitrogen and stored at −80° C. MNK1 and MNK2 were activated using recombinant ERK2 which was activated using a constitutively active mutant of MEK1, both ERK2 and MEK1 were expressed in E. coli as N-terminally his tagged proteins. Recombinant ERK2 was activated by incubating 11.3 μM of the kinase with 1 μM MEK1 and 100 μM ATP. This reaction mixture was then used immediately for the activation of the MNKs. The activation of the MNK1 was performed by incubating 5.0 μM of MNK1 with 0.3 μM of activated ERK2 and 500 μM ATP at 30° C. for 6 hours. The activation of MNK2 was performed by incubating 50 μM of MNK2 with 3.0 μM of activated ERK2 and 500 μM ATP at 30° C. for 2 hours. The activated MNKs were stored at −20° C. until required for assay.
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 451.53, ALogP 3.6, HBA 6, HBD 1, PSA 82.66, QED 0.48
Compound (SMILES): C[C@@H]1C[C@@H](Nc2cc(Cl)ccn2)CN1C(=O)c1ccccc1-n1nccn1
InChIKey: FSNKYXWBSWTMEZ-UKRRQHHQSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: FLIPR Ca2+ Flux Assay: All reagents were from GIBCO-Invitrogen Corp. The seeded plates are incubated overnight at 37° C. and 5% CO2. Ala-6,12 human orexin-A as the agonist is prepared as a 1 mM stock solution in 1% bovine serum albumin (BSA) and diluted in assay buffer (HBSS containing 20 mM HEPES, 0.1% BSA and 2.5 mM probenecid, pH7.4) for use in the assay at a final concentration of 70pM. Test compounds are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates, first in DMSO, then assay buffer. On the day of the assay, cells are washed 3 times with 100 μl assay buffer and then incubated for 60 min (37° C., 5% CO2) in 60 μl assay buffer containing 1 μM Fluo-4AM ester, 0.02% pluronic acid, and 1% BSA. The dye loading solution is then aspirated and cells are washed 3 times with 100 μl assay buffer. 30 μl of that same buffer is left in each well. Within the Fluorescent Imaging Plate Reader (FLIPR, Molecular Devices), test compounds are added to the plate in a volume of 25 μl, incubated for 5 min and finally 25 μl of agonist is added. Fluorescence is measured for each well at 1 second intervals for 5 minutes and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 70 pM Ala-6,12 orexin-A with buffer in place of antagonist. For each antagonist, IC50 value (the concentration of compound needed to inhibit 50% of the agonist response) is determined. Alternatively, compound potency can be assessed by a radioligand binding assay (described in Bergman et. al. Bioorg. Med. Chem. Lett. 2008, 18, 1425-1430) in which the inhibition constant (Ki) is determined in membranes prepared from CHO cells expressing either the OX1 or OX2 receptor. The intrinsic orexin receptor antagonist activity of a compound which may be used in the present invention may be determined by these assays.
Measurement: IC50 = 1156 nM (pChEMBL 5.940000057220459)
Descriptors: MW 382.86, ALogP 3.03, HBA 6, HBD 1, PSA 75.94, QED 0.75
Compound (SMILES): C=CC(=O)N1CC(NC(=O)Cn2cc(C(=O)NCCC(=O)Nc3cccc4ccccc34)c3cc(Br)ccc32)C1
InChIKey: RXCGCKBMJWYLBH-UHFFFAOYSA-N
Target: GTPase KRas (SINGLE PROTEIN), Homo sapiens
UniProt: P01116
Assay: Coupled Nucleotide Exchange Assay (20 hours): Purified GDP-bound KRAS protein (aa 1-169), containing both G12C and C118A amino acid substitutions and an N-terminal His-tag, was pre-incubated in assay buffer (25 mM HEPES pH 7.4, 10 mM MgCl2, and 0.01% Triton X-100) with a compound dose-response titration for 20 hours. Following compound pre-incubation, purified SOS protein (aa 564-1049) and GTP (Roche 10106399001) were added to the assay wells and incubated for an additional 30 min (for 5 min compound pre-incubation) or 1 hour (for 2 hour compound pre-incubation). To determine the extent of inhibition of SOS-mediated nucleotide exchange, purified GST-tagged cRAF (aa 1-149), nickel chelate AlphaLISA acceptor beads (PerkinElmer AL108R), and AlphaScreen glutathione donor beads (PerkinElmer 6765302) were added to the assay wells and incubated for 10 minutes. The assay plates were then read on a PerkinElmer EnVision Multilabel Reader, using AlphaScreen® technology, and data were analyzed using a 4-parameter logistic model to calculate IC50 values.
Measurement: IC50 = 650 nM (pChEMBL 6.190000057220459)
Descriptors: MW 602.49, ALogP 3.83, HBA 5, HBD 3, PSA 112.54, QED 0.25
Compound (SMILES): CCC12CC(C3=C(CN4CCN(c5ccc(C(=O)NS(=O)(=O)c6ccc(NCC7CCOCC7)c([N+](=O)[O-])c6)c(Oc6cnc7[nH]ccc7c6)c5)CC4)CCC(C)(C)C3)(C1)C2
InChIKey: GEEUSUNAKHFNLU-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q07817
Assay: Binding Assay: Table 1: Binding to Bcl-2 proteins Bcl-2, and Bcl-XL was assessed using the Bcl2scan platform: T7 phage strains displaying BCL2 proteins were grown in parallel in 24-well blocks in an E. coli host derived from the BL21 strain. E. coli were grown to log-phase and infected with T7 phage from a frozen stock (multiplicity of infection=0.4) and incubated with shaking at 32° C. until lysis (90-150 minutes). The lysates were centrifuged (5,000×g) and filtered (0.2 μm) to remove cell debris. Streptavidin-coated magnetic beads were treated with biotinylated BIM peptide ligand for 30 minutes at room temperature to generate affinity resins for BCL2 assays. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific phage binding. Binding reactions were assembled by combining BCL2 proteins, liganded affinity beads, and test compounds in 1× binding buffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100× stocks in 100% DMSO. Kds were determined using an 11-point 3-fold compound dilution series with one DMSO control point. All compounds for Kd measurements were distributed by acoustic transfer in 100% DMSO. The compounds were then diluted directly into the assays such that the final concentration of DMSO was 0.9%. All reactions performed in polypropylene 384-well plates. Each was a final volume of 0.02 ml. The assay plates were incubated at room temperature with shaking for 1 hour and the affinity beads were washed with wash buffer (lx PBS, 0.05% Tween 20). The beads were then re-suspended in elution buffer (lx PBS, 0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 minutes.
Measurement: Kd > 100 nM (pChEMBL 7)
Descriptors: MW 852.07, ALogP 8.43, HBA 11, HBD 3, PSA 172.03, QED 0.06
Compound (SMILES): CC(C)c1c(-c2ccnc3n[nH]cc23)[nH]c2ccc(-c3nc(C(=O)NC4CCN(C(C)C)CC4)co3)cc12
InChIKey: GWPKJTMRXPJEDN-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: TLR7/8/9 Inhibition Reporter Assays: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κc Band AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Bue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications. The percent inhibition is determined as the % reduction in the HEK-Blue signal present in wells treated with agonist plus DMSO alone compared to wells treated with a known inhibitor.
Measurement: IC50 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 511.63, ALogP 5.49, HBA 6, HBD 3, PSA 115.73, QED 0.28
Compound (SMILES): CC(C)C[C@@H](CC(=O)NC[C@H](Cc1ccc2c(c1)oc(=O)n2COCC[Si](C)(C)C)N(C)C)c1ccccc1
InChIKey: VCPCOHDUSLEWMX-SVBPBHIXSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: DOR cAMP Agonist and Antagonist Assays: The dOR cAMP agonist and antagonist assays were conducted in a manner similar as described above for the mOR assays using CHO Tag-lite human dOR stable cell line from Cisbio (NCBI accession number: NM_000911.3, Bedford, Mass.).
Measurement: EC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 553.82
Compound (SMILES): COc1ccc(CN2CCN(c3ccc(-c4cc(OCC5CCC5)cn5ncc(C#N)c45)cn3)CC2)cn1
InChIKey: OFRJNHZOAKQYJV-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase receptor Ret (SINGLE PROTEIN), Homo sapiens
UniProt: P07949
Assay: RET Enzyme Assay: Compounds of Formula I were screened for their ability to inhibit wildtype and V804M mutant RET kinase using CisBio's HTRF® KinEASE¿-TK assay technology. Briefly, N-terminal GST tagged recombinant human RET cytoplasmic domain (aa 658-end) from Eurofins (0.25 nM RET; Catalog No. 14-570M) or N-terminal GST tagged recombinant human V804M mutant RET cytoplasmic domain (aa 658-end) from Millipore (0.25 nM enzyme; Catalog No. 14-760) was incubated with 250 nM TK-substrate biotin (CisBio, part of Catalog No. 62TK0PEC) and 1 mM ATP along with test compound in a buffer consisting of 25 mM HEPES pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO in a volume of 8 μL. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 30-minute incubation at 22° C., the reaction was quenched by adding 8 μL of quench solution containing 31.25 nM Sa-XL665 and 1×TK-ab-Cryptate in HTRF detection buffer (all from CisBio, part of Cat. No. 62TK0PEC). After a 1 hour incubation at 22° C., the extent of reaction was determined using a PerkinElmer EnVision multimode plate reader via HTRF dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using pre-quenched control reactions. The POC values were fit to a 4 parameter logistic curve, and the IC50 is defined as the concentration of inhibitor at which the POC equals 50 for the fitted curve.
Measurement: IC50 = 748 nM (pChEMBL 6.130000114440918)
Descriptors: MW 509.61, ALogP 4.17, HBA 9, HBD 0, PSA 91.81, QED 0.35
Compound (SMILES): Cn1nc(C2CCN(S(=O)(=O)c3ccc(C(N)=O)cc3)CC2)c2cc(C(c3ccc(Cl)cc3)c3ccc(Cl)cc3)ccc21
InChIKey: VKDBYVJPZIZHOD-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: Biological Assay: CB1 and CB2 receptors are Gi-coupled GPCR. Activation of CB1 and CB2 receptors results in a decrease in cAMP production. An inverse agonist of the CB1 or CB2 receptor results in the opposite effect, an increase of cAMP production. The principle of this assay is based on HTRF® technology (Homogeneous Time-Resolved Fluorescence). The method is a competitive immunoassay between native cAMP produced by cells and the cAMP labeled with the fluorophore d2. The tracer binding is quantified by a Mab anti-cAMP labeled with Eu3+TBP-NHS Cryptate (supplied as part of the assay kit). The specific signal (i.e. energy transfer) is inversely proportional to the concentration of cAMP in the standard or sample.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 633.6, ALogP 6.73, HBA 5, HBD 1, PSA 98.29, QED 0.2
Compound (SMILES): CC(=O)N1CCn2c(C3CC3)nc(N3CCCc4cc(-c5cnn(C6CC6)c5)ccc43)c2C1
InChIKey: UJQNZVLNIIKZKF-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: BRD4 AlphaScreen Assay: The binding of 2.5 nM of BRD4(49-170) to 10 nM biotin-H4(1-21) Ac-K5/8/12/16 (AnaSpec. 64989) was assessed following the same procedure described for the CBP assay. The standard dose response curves were fitted by Genedata Screener software using the variable-slope model:Signal=Signalnegative control+(SignalDMSO control−Signalnegative control)/(1+(IC50/Dose){circumflex over ( )}Hill slope).
Measurement: IC50 = 14329 nM (pChEMBL 4.840000152587891)
Descriptors: MW 442.57, ALogP 4.41, HBA 6, HBD 0, PSA 59.19, QED 0.6
Compound (SMILES): Cc1cc(-c2[nH]c3ccc(C4CCN(C5CCN(C(C)C)CC5)CC4)cc3c2C)ccn1
InChIKey: GKENUMJCKKDCSN-UHFFFAOYSA-N
Target: Toll-like receptor 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NR97
Assay: Inhibition Reporter Assay: HEK-Blue-cells (Invivogen) overexpressing human TLR7, TLR8 or TLR9 receptors were used for screening inhibitors of these receptors using an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-β minimal promoter fused to five NF-κB and AP-1-binding sites. Briefly, cells are seeded into Greiner 384 well plates (15000 cells per well for TLR7, 20,000 for TLR8 and 25,000 for TLR9) and then treated with test compounds in DMSO to yield a final dose response concentration range of 0.05 nM-50 μM. After a 30 minute compound pre-treatment at room temperature, the cells are then stimulated with a TLR7 ligand (gardiquimod at a final concentration of 7.5 μM), TLR8 ligand (R848 at a final concentration of 15.9 μM) or TLR9 ligand (ODN2006 at a final concentration of 5 nM) to activate NF-κB and AP-1 which induce the production of SEAP. After a 22 hour incubation at 37° C., 5% CO2, SEAP levels are determined with the addition of HEK-Blue Detection reagent (Invivogen), a cell culture medium that allows for detection of SEAP, according to manufacturer's specifications.
Measurement: IC50 = 47.2 nM (pChEMBL 7.329999923706055)
Descriptors: MW 430.64, ALogP 5.9, HBA 3, HBD 1, PSA 35.16, QED 0.56
Compound (SMILES): Cn1cnc2ncn(Cc3nc([C@@H]4CO[C@@H](c5cccc(Cl)c5)C4)no3)c(=O)c21
InChIKey: ZSBRAOYPHXSBBJ-GXTWGEPZSA-N
Target: Transient receptor potential cation channel subfamily A member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O75762
Assay: FLIPR Assay (90 minutes): The IC50 (effective concentration) of compounds on the human TRPA1 channel was determined using a FLIPR Tetra instrument. CHO cells expressing TRPA1 were plated into 384-well plates, incubated overnight at 37° C., and loaded with BD calcium indicator dye for 1 hr at 37° C. followed by 90 minutes at room temperature. The assay buffer was Hank's Balanced Salt Solution (HBSS) containing 20 mM HEPES (pH readjusted to 7.4) along with 0.02% BSA.Following dye load and plate cool down, compounds were added to the cells using FLIPR Tetra. Plates were then incubated with compounds for 10 minutes or 90 minutes at room temperature prior to adding agonist. Following this incubation, about an EC80 concentration of cinnamaldehyde (75) was added to active the channels and block of cinnamaldehyde induced calcium influx was measured.The IC50 results were fit with a standard Hill function, keeping the Hill coefficient (n) fixed to 1.5. Fixing the Hill coefficient will generally reduce variability of the IC50 determination. The IC50 results were individually examined to make sure the MIN and MAX points were set correctly prior to validation of the results.
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 412.84, ALogP 2.46, HBA 9, HBD 0, PSA 100.86, QED 0.51
Compound (SMILES): C=CC(=O)N1CCCN(C(=O)c2cc(Sc3cnc(NC(=O)C(C)C)s3)c(C)cc2OC)CC1
InChIKey: PUQQULIZFUPRHD-UHFFFAOYSA-N
Target: Tyrosine-protein kinase TXK (SINGLE PROTEIN), Homo sapiens
UniProt: P42681
Assay: In Vitro Assay: The potency of inhibition is measured by binding affinity.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 502.66, ALogP 4.07, HBA 7, HBD 1, PSA 91.84, QED 0.57
Compound (SMILES): CN[C@@H]1CC[C@@H](Oc2cncc(OC)c2-c2cc(Nc3cnc(C#N)cn3)n[nH]2)C1
InChIKey: BVLLVNWURWCCQW-TZMCWYRMSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: hERG Assay: The test compounds were added to cultured hERG (human Ether-a-go-go Related Gene) gene stably expressing CHO cell line cells to achieve 0.27 to 100 μM. The hERG current under electric potential stimulation was measured using Qube384 (Sophion Bioscience) to calculate the concentration at which each test compound suppresses 50% hERG current (IC50 value; μM).
Measurement: IC50 = 8700 nM (pChEMBL 5.059999942779541)
Descriptors: MW 406.45, ALogP 2.4, HBA 9, HBD 3, PSA 133.66, QED 0.54
Compound (SMILES): Cc1ncc(-c2ccc(C3=NNC(=O)OC3)cc2C(F)(F)F)cc1F
InChIKey: HFHRODZMIPNVBF-UHFFFAOYSA-N
Target: cGMP-inhibited 3',5'-cyclic phosphodiesterase 3B (SINGLE PROTEIN), Homo sapiens
UniProt: Q13370
Assay: PDE3B Enzyme Inhibition: The commercially available 3H-cAMP Scintillation Proximity Assay (SPA, Perkin Elmer) system was used for enzyme inhibition studies. For the determination of the in vitro effect of example compounds on the PDE3B reactions 2 μl of the respective example compound solution in DMSO (serial dilutions) were placed in wells of microtiter plates (Isoplate-96/200W; Perkin Elmer). 50 μl of a dilution of PDE3B cell extract from Sf9 cells overexpressing human full length PDE3B (SB Drug Discovery, UK) in buffer A (50 mM Tris/HCl pH 7.5, 8.3 mM MgCl2, 1.7 mM EDTA, 0.2% BSA) was added. The dilution of the PDE3B cell extract was chosen such that the reaction kinetics was linear and less than 70% of the substrate was consumed (typical dilution 1:6000). The reaction was started by addition of 50 μl (0.025 μCi) of 1:2000 in buffer A w/o BSA diluted substrate [8-3H]adenosine 3′, 5′-cyclic phosphate (1 μCi/μl; Perkin Elmer). After incubation at room temperature for 60 min, the reaction was stopped by addition of 25 μl of a suspension containing 18 mg/ml yttrium scintillation proximity beads (Perkin Elmer) in water. The microtiter plates were sealed and measured in a Microbeta scintillation counter (PerkinElmer Wallac). IC50 values were determined from sigmoidal curves by plotting percentage PDE3B activity vs log compound concentration.
Measurement: IC50 = 240 nM (pChEMBL 6.619999885559082)
Descriptors: MW 353.28, ALogP 3.66, HBA 4, HBD 1, PSA 63.58, QED 0.84
Compound (SMILES): CCCCCCNc1nc(Nc2cnn(CC)c2)ncc1C(N)=O
InChIKey: AJZUEWKISNZIHN-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK3 (SINGLE PROTEIN), Homo sapiens
UniProt: P52333
Assay: Kinase Activity Assay: JAK3 (the final concentration was 0.36 nM); ATP (the final concentration was 10 μM); ULight-labeled JAK-1 (Tyr1023) Peptide (the final concentration was 50 nM); the enzymatic reaction time was 1 hour. The maximum final concentration of compounds was 2.5 μM. After a 3-fold gradient dilution, 11 concentrations were obtained, with the minimum final concentration of 0.042 nM. The final DMSO concentration was 1%.
Measurement: IC50 = 3.06 nM (pChEMBL 8.510000228881836)
Descriptors: MW 331.42, ALogP 2.53, HBA 7, HBD 3, PSA 110.75, QED 0.58
Compound (SMILES): CC(C)(O)Cn1cc(C(=O)Nc2ccc(-c3n[nH]c(=O)c4ccccc34)cc2)cn1
InChIKey: OCUISYNOIPSGBD-UHFFFAOYSA-N
Target: Rho-associated protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O75116
Assay: In Vitro Assay: The effectiveness of compounds of the present invention as ROCK inhibitors can be determined in a 30 μL assay containing 20 mM HEPES, pH 7.5, 20 mM MgCl2, 0.015% Brij-35, 4 mM DTT, 5 μM ATP and 1.5 μM peptide substrate (FITC-AHA-AKRRRLSSLRA-OH). Compounds were dissolved in DMSO so that the final concentration of DMSO was <2%, and the reaction was initiated with Rho kinase variants. After incubation, the reaction was terminated by the addition of EDTA and the phosphorylated and non-phosphorylated peptides separated using a LABCHIP® 3000 Reader (Caliper Life Sciences). Controls consisted of assays that did not contain compound, and backgrounds consisted of assays that contained enzyme and substrate but had EDTA from the beginning of the reaction to inhibit kinase activity. Compounds were tested in dose-response format, and the inhibition of kinase activity was calculated at each concentration of compound. The inhibition data were fit using a curve-fitting program to determine the IC50; i.e., the concentration of compound required to inhibit 50% of kinase activity.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 403.44, ALogP 2.81, HBA 6, HBD 3, PSA 112.9, QED 0.47
Compound (SMILES): C=CC(=O)N1CC[C@@H](c2nc(-c3ccc(Oc4cccc(OC)c4F)cc3)c3cncc(OCC)n23)C1
InChIKey: MTZGKIUVTHAEHA-LJQANCHMSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Btk Kinase Assay: Btk kinase activity was determined using a homogenous time resolved fluorescence (HTRF) methodology. Measurements were performed in a reaction volume of 15 μL using 384-well assay plates. Kinase enzyme, inhibitor, ATP and 1 M peptide substrate were incubated in a reaction buffer compose of Hepes 50 mM (pH7.0), NaN3 0.02%, BSA 0.01%, Orthocanadate 0.1 mM. After one hour, the kinase reaction was quenched by the addition of Ep-labeled antibody and XL-665 in 1× Detection buffer containing 60 mM EDTA (Cisbio), and the mixture was allowed to incubate for one hour. The HTRF signal was measured on a multimode plate reader (EnVision® Multilabel Reader, Perkin Elmer) with an excitation wavelength (λEx) of 330 nm and detection wavelengths (λEm) of 615 and 665 nm. Activity was determined by the ratio of the fluorescence at 665 nm to that at 615 nm. For each compound, enzyme activity as measured at various concentrations of compound, Negative control reactions were performed in the absence of inhibitor in two replicates and eight no enzyme controls were used to determine baseline fluorescence levels
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 502.55, ALogP 5.24, HBA 7, HBD 0, PSA 78.19, QED 0.31
Compound (SMILES): CS(=O)(=O)c1cccc(N2CCN(Cc3ccccc3)C(c3ccccc3)C2)c1
InChIKey: JJJWPNKCBQSHHN-UHFFFAOYSA-N
Target: Oxysterols receptor LXR-beta (SINGLE PROTEIN), Homo sapiens
UniProt: P55055
Assay: Human LXRalpha and LXRbeta Coactivator Recruitment FRET Assay: This assay is based on the ability of the LXR-LBDs (LXRα and LXRβ) to recruit and interact with a co-activator peptide. This assay was run in agonist mode, to characterize compounds of the invention that exhibit LXR agonist activity, and in antagonist mode to characterize compounds of the invention having LXR antagonist activity and which cause a concentration-dependent release of the co-activator peptide in the presence of the full LXR agonist T0901317. The interaction between glutathione S-transferase (GST)-tagged, recombinant human LXRα-LBD or LXRβ-LBD and fluorescein-conjugated co-activator peptide SRC2-3 (Cat# PV4588, Life Technologies, Grand Island, N.Y.) was measured via time resolved-fluorescence resonance energy transfer (TR-FRET). The assay was performed in 96-well half-area black Opti-plates (Cat# 3686, Corning, Lowell, Mass.) in 20 mM TRIS-HCl buffer, pH 8, containing 150 mM NaCl and 5 mM DDT in a total volume of 80 μL. Test compounds were dissolved in DMSO, and semi-log (3.162×) serial dilutions were also prepared in DMSO, as 40× solutions.
Measurement: EC50 = 192 nM (pChEMBL 6.71999979019165)
Descriptors: MW 406.55, ALogP 4.15, HBA 4, HBD 0, PSA 40.62, QED 0.64
Compound (SMILES): CCNc1cc2c(cn1)c(-c1cnc(C)nc1)nn2C1CCC(Oc2nccc(C(F)(F)F)c2F)CC1
InChIKey: KVQWIKMHQTWKMP-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Enzyme Assay: MER: Compounds of Formula I were screened for their ability to inhibit MER kinase using Invitrogen's LanthaScreen Eu Kinase Binding technology. His-tagged recombinant human MER cytoplasmic domain (5 nM) was incubated with 20 nM Alexa-Fluor® Tracer 236 (PR9078A), 2 nM biotinylated anti-His (Cat. No. M4408), and 2 nM europium-labeled Streptavidin (Cat. No. PV5899) along with test compound in a buffer consisting of 25 mM HEPES, pH 7.4, 10 mM MgCl2, 0.01% Triton X-100, and 2% DMSO. Compounds were typically prepared in a threefold serial dilution in DMSO and added to the assay to give the appropriate final concentration. After a 60-minute incubation at 22° C., the reaction was measured using a PerkinElmer EnVision multimode plate reader via TR-FRET dual wavelength detection, and the percent of control (POC) was calculated using a ratiometric emission factor. 100 POC was determined using no test compounds and 0 POC was determined using a concentration of control compound that completely inhibits the enzyme. The POC values are fit to a 4 parameter logistic curve and the IC50 value is point where the curve crosses 50 POC.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 515.52, ALogP 5.74, HBA 8, HBD 1, PSA 90.64, QED 0.33
Compound (SMILES): CC(C)C[C@H](N)[C@](O)(Cc1cc(OCc2ccccc2)ccn1)C(=O)O
InChIKey: QCELINXSUWBJTD-AZUAARDMSA-N
Target: Leucyl-cystinyl aminopeptidase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UIQ6
Assay: Inhibition of Human P-LAP (hP-LAP) Assay: HEK293 cells forced to transiently express hP-LAP (J Biol Chem 1996; 271: 56-61) were prepared by lipofection, homogenized, and then subjected to ultracentrifugation at 100,000×g for 30 minutes. Microsomes containing hP-LAP were thereby prepared. The microsomes (with a total protein content of 0.5 to 1.5 μg/well) were mixed with a solvent (DMSO; final concentration: 0.1%) or with each test compound (common ratio: 3; maximum concentration: 10 μM). AVP was then added to the solution into a final concentration of 25 μM, and the resulting solution was allowed to react for one hour at 37° C. An aqueous TFA solution was then added to the solution (final concentration: 1%) to stop the enzymatic reaction. Residual AVP was then determined by mass spectrometry (MALDI-MS). Based on the results, IC50 values (nM), i.e. concentrations required for 50% inhibition of decrease in AVP level in the solvent control group, of the individual test compounds were calculated by logistic regression to evaluate inhibition of human P-LAP (hP-LAP) activity. The results are shown in Tables 1 and 2 and indicate that the example compounds effectively inhibit AVP degradation by hP-LAP.
Measurement: IC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 358.44, ALogP 2.39, HBA 5, HBD 3, PSA 105.67, QED 0.64