lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COc1cc(C(=O)NCc2cccc(S(N)(=O)=O)c2)ccc1-c1cn[nH]c1
InChIKey: FTUKZMZRGJAOKF-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.
Measurement: IC50 = 0.99 nM (pChEMBL 9)
Descriptors: MW 386.43, ALogP 1.66, HBA 5, HBD 3, PSA 127.17, QED 0.59
Compound (SMILES): CC1CC2=NNC(=O)c3cccc4[nH]c(c2c34)C1
InChIKey: MLYYYJSVHUVZHH-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UGN5
Assay: PARP-2 and PARP-3 Enzymatic Assay: PARP-2 and PARP-3 enzymatic assays were conducted using commercial PARP-2/PARP-3 Chemiluminescent Assay Kit (BPS Biosciences) and the protocols with the kits. Briefly, histones were coated in a high binding plate first, and incubated with PARP-2 or PARP-3, and increasing concentrations of compounds for 0.5 h. Then, biotinylated NAD and activated DNA were added to the wells. The biotinylated PARylation product was measured by adding streptavidin-HRP and HRP substrates which produce chemiluminescence.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 239.28, ALogP 2.2, HBA 2, HBD 2, PSA 57.25, QED 0.73
Compound (SMILES): CO[C@H]1CC[C@]2(CC1)Cc1ccc(OCC3(C)CC3)cc1C21N=C(C)C(N)=N1
InChIKey: GSAVHMBMISQPPR-DRXONWLQSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Release Assay: SH-SY5Y cells were cultured in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids and cryopreserved and stored at -140 C. at a concentration of 7.5-9.5x106 cells per vial. Thaw cells and seed at a conc. of around 10000 cells/well in DMEM/F-12 with Glutamax, 10% FCS and 1% non-essential amino acids to a 384-well tissue culture treated plate, 1004, cell susp/well. The cell plates were then incubated for 7-24 h at 37 C., 5% CO2. The cell medium was removed, followed by addition of 30 uL compound diluted in DMEM/F-12 with Glutamax, 10% FCS, 1% non-essential amino acids and 1% PeSt to a final conc. of 1% DMSO. The compounds were incubated with the cells for 17 h (overnight) at 37 C., 5% CO2. Meso Scale Discovery (MSD) plates were used for the detection of sAPPbeta release. MSD sAPPbeta plates were blocked in 1% BSA in Tris wash buffer (40 uL/well) for 1 h on shake at r.t. and washed 1 time in Tris wash buffer (40 uL/well).
Measurement: IC50 = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 381.52, ALogP 3.98, HBA 5, HBD 1, PSA 69.2, QED 0.86
Compound (SMILES): Cc1ncc(C(=O)NCCN2CCCC2(C)C)cc1NC(=O)c1cnn2cc(-c3cnn(C)c3)ccc12
InChIKey: WUPFLUOYXIDHAV-UHFFFAOYSA-N
Target: Muscarinic acetylcholine receptor M1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11229
Assay: Inhibition of M1 receptor (unknown origin)
Measurement: IC50 = 5000 nM (pChEMBL 5.300000190734863)
Descriptors: MW 500.61, ALogP 3.29, HBA 8, HBD 2, PSA 109.45, QED 0.4
Compound (SMILES): Nc1ncnc(N2CCC(c3nc(-c4ccc(C(F)(F)F)c(F)c4)cn3CCN3CCCC3)CC2)c1C1CCC1
InChIKey: WLLLTIDXGZYUPF-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: P70S6K Enzyme Assay: P70S6K inhibitor compounds are diluted and plated in 96 well plates. A reaction mixture including the following components is then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) is mixed with 24 uM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 uM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction is incubated for 90 min at 25.degree. C., before the addition of 10 mM EDTA to stop the reaction. The proportion of substrate and product (phosphorylated) peptide is analysed on a Caliper Life Sciences Lab Chip 3000, using a pressure of -1.4 psi, and upstream and downstream voltages of -3000 and -700 respectively. Product peaks are resolved before substrate peaks on the resulting chromatograms.
Measurement: IC50 = 36 nM (pChEMBL 7.440000057220459)
Descriptors: MW 557.64, ALogP 5.83, HBA 7, HBD 1, PSA 76.1, QED 0.37
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2CCN(c3cc(OCC(=O)O)cc(-c4cccs4)c3)CC2)cc1
InChIKey: IANBGYNSZGCRRE-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Displacement of [3H]-PGD2 from PGD2 receptor in human platelet membranes after 2 hrs by micro beta scintillation counting analysis
Measurement: Ki = 520 nM (pChEMBL 6.28000020980835)
Descriptors: MW 516.64, ALogP 4.18, HBA 7, HBD 1, PSA 96.38, QED 0.46
Compound (SMILES): CN1CC2(C1)C(=N)N[C@](C)(c1cc(NC(=O)c3ccc(OC(F)F)cn3)ccc1F)CS2(=O)=O
InChIKey: ZNVPCWFQKHJDHK-FQEVSTJZSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3x the desired final concentration in 1Ã¿BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1xBACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 uM for 4 uM for autoBACE-2) prepared in 1xBACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 us delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values. IC50s are determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively. Similar IC50s are obtained when using raw RFU data. The Ki values are calculated from the IC50 using the Cheng-Prusoff equation.
Measurement: Ki = 4.3 nM (pChEMBL 8.369999885559082)
Descriptors: MW 497.5, ALogP 1.97, HBA 7, HBD 3, PSA 124.48, QED 0.58
Compound (SMILES): Cc1nc(C(=O)N2CC[C@H]2c2nc(-c3ccccc3OC(F)(F)F)no2)c(-c2ccccc2)s1
InChIKey: PXTRDUIXBONJAT-INIZCTEOSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Intracellular Calcium 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 μM 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. Within the Fluorescent Imaging Plate Reader (FLIPR Tetra, Molecular Devices), antagonists are added to the plate in a volume of 10 μL/well, incubated for 120 min and finally 10 μL/well of agonist is added. Fluorescence is measured for each well at 1 second intervals, and the height of each fluorescence peak is compared to the height of the fluorescence peak induced by 3 nM orexin-A with vehicle in place of antagonist.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 486.48, ALogP 5.65, HBA 7, HBD 0, PSA 81.35, QED 0.36
Compound (SMILES): N#CC1(NC2CCN(C(=O)Cn3cc(NC(=O)c4cnn5cccnc45)c(-c4cc(Cl)ccc4OC(F)F)n3)CC2)CC1
InChIKey: VQTATMBZNMZWSV-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 0.75 nM (pChEMBL 9.119999885559082)
Descriptors: MW 610.03, ALogP 3.74, HBA 10, HBD 2, PSA 142.47, QED 0.29
Compound (SMILES): CCn1cc(C(=O)NCc2ccc(S(=O)(CC)=NC#N)cc2)c(=O)c(-c2cccc(C(F)F)c2)c1C
InChIKey: XVRYMSYJRGKUOF-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: The following buffers were used: Compound buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5; Assay buffer: 100 mM Tris, 500 mM NaCl, adjusted to pH 7.5, containing 0.01% BSA.Assay conditions: Test compounds were prediluted in DMSO and subsequently in compound buffer (5% DMSO final). 5 μL of these compound dilutions were mixed with 10 μl Neutrophil elastase (9 ng/ml in assay buffer) in a black 384 well OptiPlate (Perkin Elmer, Cat No.: 6007270) and incubated for 15 min at room temperature. Subsequently 10 μL substrate solution in assay buffer were added (250 μM final concentration) and the plates were incubated for 60 min at room temperature. After inactivation of the enzyme, fluorescence intensities were measured at 380 nm excitation and 460 nm emission wavelengths.
Measurement: IC50 = 2.2 nM (pChEMBL 8.65999984741211)
Descriptors: MW 512.58, ALogP 5.04, HBA 6, HBD 1, PSA 104.32, QED 0.43
Compound (SMILES): Cc1cc(C#N)cnc1C(=O)Nc1ccc(F)c([C@]2(C)CS(=O)(=O)C3(CNC3)C(=N)N2)c1
InChIKey: ATKAIMXQGZRDFH-FQEVSTJZSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: Time-Resolved Endpoint Proteolysis Assay: Inhibitor IC50s at purified human autoBACE-2 are determined in a time-resolved endpoint proteolysis assay that measures hydrolysis of the QSY7-EISEVNLDAEFC-Eu-amide FRET peptide substrate (BACE-HTRF assay). BACE-mediated hydrolysis of this peptide results in an increase in relative fluorescence (RFU) at 620 nm after excitation with 320 nm light. Inhibitor compounds, prepared at 3x the desired final concentration in 1Ã¿BACE assay buffer (20 mM sodium acetate pH 5.0, 10% glycerol, 0.1% Brij-35) supplemented with 7.5% DMSO are pre-incubated with an equal volume of autoBACE-2 enzyme diluted in 1xBACE assay buffer (final enzyme concentration 1 nM) in black 384-well NUNC plates for 30 minutes at 30° C. The assay is initiated by addition of an equal volume of the QSY7-EISEVNLDAEFC-Eu-amide substrate (200 nM final concentration, Km=8 uM for 4 uM for autoBACE-2) prepared in 1xBACE assay buffer supplemented with 7.5% DMSO and incubated for 90 minutes at 30° C. DMSO is present at 5% final concentration in the assay. Following laser excitation of sample wells at 320 nm, the fluorescence signal at 620 nm is collected for 400 ms following a 50 us delay on a RUBYstar HTRF plate reader (BMG Labtechnologies). Raw RFU data is normalized to maximum (1.0 nM BACE/DMSO) and minimum (no enzyme/DMSO) RFU values. IC50s are determined by nonlinear regression analysis (sigmoidal dose response, variable slope) of percent inhibition data with minimum and maximum values set to 0 and 100 percent respectively. Similar IC50s are obtained when using raw RFU data. The Ki values are calculated from the IC50 using the Cheng-Prusoff equation.
Measurement: Ki = 1.8 nM (pChEMBL 8.739999771118164)
Descriptors: MW 456.5, ALogP 1.21, HBA 7, HBD 4, PSA 147.83, QED 0.54
Compound (SMILES): O=C1[C@H](O)C[C@@H](c2cccc3c2OCCO3)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: XDKRISIFYVDHAI-JKSUJKDBSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements: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 g/ml G418, 100 U/ml penicillin, 100 g/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 = 5010 nM (pChEMBL 5.300000190734863)
Descriptors: MW 409.36, ALogP 3.19, HBA 5, HBD 1, PSA 68.23, QED 0.84
Compound (SMILES): Cc1cc(Nc2nccc(Oc3ccc(NC(=O)Nc4cc(C(C)(C)C)nn4-c4cccc(CN5CCCC5=O)c4)c4ccccc34)n2)cc2cn[nH]c12
InChIKey: KFJCYTXMHLMPLW-UHFFFAOYSA-N
Target: Proto-oncogene tyrosine-protein kinase Src (SINGLE PROTEIN), Homo sapiens
UniProt: P12931
Assay: c-Src and Syk Enzyme Inhibition Assay: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), are evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 uL) is incubated with the test compound (either 4 ug/mL, 0.4 ug/mL, 0.04 ug/mL, or 0.004 ug/mL, 2.5 uL each) for 2 hr at RT. The FRET peptides (8 uM, 2.5 uL), and appropriate ATP solutions (2.5 uL, 800 uM for c-Src, and 60 uM ATP for Syk) are then added to the enzymes/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 uL) is added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan Flash, ThermoFisher Scientific).
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 720.84, ALogP 8.6, HBA 9, HBD 4, PSA 154.98, QED 0.12
Compound (SMILES): CCOc1cc(N2CCN(C3CCN(CCS(C)(=O)=O)CC3)CC2)ccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: ZRBXIDVFBISSPM-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 835.98, ALogP 6.76, HBA 12, HBD 2, PSA 137.3, QED 0.13
Compound (SMILES): CN1CCN(c2cc(Nc3cc(C4CC4)[nH]n3)nc(NCc3cc(-c4cccs4)no3)n2)CC1
InChIKey: MAJRKJOXVGTAOY-UHFFFAOYSA-N
Target: Insulin-like growth factor 1 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P08069
Assay: Inhibition of human IGF1R kinase domain (M954 to C1367 residues) tri-phosphorylated enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 < 50 nM (pChEMBL 7.301000118255615)
Descriptors: MW 477.6, ALogP 3.9, HBA 10, HBD 3, PSA 111.03, QED 0.35
Compound (SMILES): Cc1cc(CN(CC2CCCC2)[C@@H](CC(=O)O)c2ccc(Cl)cc2)ccc1OCCN1C(=O)CCC1=O
InChIKey: LJWSTVOVBMILMB-VWLOTQADSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 527.06, ALogP 5.38, HBA 5, HBD 1, PSA 87.15, QED 0.37
Compound (SMILES): C[C@H](NC(=O)C(F)(F)F)[C@H](Oc1ccc2c(cnn2-c2ccc(F)cc2)c1)c1ccccc1
InChIKey: UITKQZZSZUEHEA-NYHFZMIOSA-N
Target: Glucocorticoid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04150
Assay: Binding affinity to GR (unknown origin) by FP assay
Measurement: IC50 = 2.7 nM (pChEMBL 8.569999694824219)
Descriptors: MW 457.43, ALogP 5.35, HBA 4, HBD 1, PSA 56.15, QED 0.4
Compound (SMILES): COCCNCc1cncc(Nc2nccc(-c3cnc(N4CC[C@H](F)C4)c(C#N)c3)n2)c1
InChIKey: UYYHTUSACVZJJW-IBGZPJMESA-N
Target: Serine/threonine-protein kinase TBK1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHD2
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 448.51, ALogP 2.83, HBA 9, HBD 2, PSA 111.88, QED 0.48
Compound (SMILES): CC(C)(C)NCc1cc(C(=O)N2CCC(N3CC(CC#N)(n4cc(-c5ncnc6[nH]ccc56)cn4)C3)CC2)nc(C(F)(F)F)n1.O=C(O)C(F)(F)F.O=C(O)C(F)(F)F.O=C(O)C(F)(F)F
InChIKey: XXJKNOLZTIXDFG-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Inhibition of human N-terminal His-tagged JAK2 catalytic domain (828 to 1132 residues) expressed in baculovirus infected insect cells assessed as decrease in phosphorylation of biotinylated peptide substrate after 1 hr in presence of 1 mM ATP by HTRF method
Measurement: IC50 > 10 nM (pChEMBL 8)
Descriptors: MW 621.67, ALogP 3.75, HBA 10, HBD 2, PSA 144.54, QED 0.32
Compound (SMILES): CCS(=O)(=O)Nc1cccc(Nc2ncnc3cc(OC)c(OC)cc23)c1
InChIKey: VYBZXECHOJSINA-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: In vitro Kinase Assay: In vitro kinase assays using LRRK WT and LRRK G2019S.
Measurement: IC50 = 121 nM (pChEMBL 6.920000076293945)
Descriptors: MW 388.45, ALogP 3.15, HBA 7, HBD 2, PSA 102.44, QED 0.64
Compound (SMILES): CC(C)(C)c1cc(-c2cc(-c3ccc(C(=O)O)cc3)ccc2OCCCO)ccc1N1CCCC1
InChIKey: TZKPDARBQWSPHX-UHFFFAOYSA-N
Target: Retinoic acid receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P10826
Assay: Activation of RARbeta (unknown origin) expressed in human HeLa cells co-expressing ERE-beta-Glob-Luc-SV-Neo incubated for 18 hrs by luciferase reporter gene assay
Measurement: Kd = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 473.61, ALogP 6.38, HBA 4, HBD 2, PSA 70, QED 0.37
Compound (SMILES): CN(C)Cc1ccc(CSCCNc2ncccc2C#N)o1
InChIKey: XPQARXHJKZMDKV-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of equine serum BuChE using butyrylthiocholine as substrate preincubated for 8 mins followed by substrate addition by Ellman's method
Measurement: IC50 = 8360 nM (pChEMBL 5.079999923706055)
Descriptors: MW 316.43, ALogP 2.95, HBA 6, HBD 1, PSA 65.09, QED 0.76
Compound (SMILES): N=C(NC(=N)N1CCN(C(=N)NC(=N)Nc2ccc([N+](=O)[O-])cc2)CC1)Nc1ccc([N+](=O)[O-])cc1
InChIKey: SPDPQNRBXLDTDZ-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Non-competitive inhibition of Electrophorus electricus acetylcholinesterase in presence of varying acetylthiocholine iodide substrate level preincubated for 20 mins followed by substrate addition measured every 1 min for 30 mins
Measurement: Ki = 2960 nM (pChEMBL 5.53000020980835)
Descriptors: MW 496.49, ALogP 1.56, HBA 8, HBD 8, PSA 236.28, QED 0.13
Compound (SMILES): CCN(CC)CCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccc(CNC(=O)OC(C)(C)C)cc1)NC(=O)c1ccc(C(C)(C)C)cc1)C(=O)N[C@@H](CO)C(=O)OC
InChIKey: VLYHVKRJXDXUFJ-JYICSXSCSA-N
Target: Chromobox protein homolog 7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95931
Assay: Antagonist activity at recombinant human C-terminal His-tagged CBX7 chromodomain (8 to 62 residues) expressed in Escherichia coli Rosetta BL21(DE3)pLysS using biotin-labeled H3K9me3 measured after 30 mins by AlphaScreen assay
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 924.19, ALogP 4.03, HBA 11, HBD 7, PSA 233.6, QED 0.06
Compound (SMILES): O=C(NO)c1cnc(NC2(c3ccccc3)CCN(C(=O)Nc3c(F)cccc3F)CC2)nc1
InChIKey: IJOCXFUZXSAWMI-UHFFFAOYSA-N
Target: Histone deacetylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92769
Assay: HDAC Enzyme Assay: The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 uM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 uM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 uM (HDAC1), 10 uM (HDAC2), 17 uM (HDAC3) and 14 uM (HDAC6). Five ul of compounds and 20 ul of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five u1 of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 468.46, ALogP 3.51, HBA 6, HBD 4, PSA 119.48, QED 0.34
Compound (SMILES): CC(C)(C)c1cc(NC(=O)Nc2cnc(Oc3ccc(-c4cnc(N5CCCC5=O)s4)cc3)nc2)n(-c2ccc3c(c2)CCO3)n1
InChIKey: JECCWDHIEUBIRK-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Inhibition Assay: TrkA kinase-inhibiting activity in cell systems was measured using CHO-K1 cells expressing human TrkA and NFAT-bla (CellSenser TrkA-NFAT-bla CHO-K1 cells, Invitrogen).On the day before the assay, CellSenser TrkA-NFAT-bla CHO-K1 cells were suspended in an assay medium (Opti-MEM1 Reduced Serum Medium (Invitrogen) containing 0.5% dialysed fetal bovine serum (Invitrogen), 0.1 mM nonessential amino acids (Invitrogen), 1 mM sodium pyruvate (Invitrogen) and antibiotics (100 U/mL penicillin and 100 ug/mL streptomycin (Invitrogen)) and plated at a density of 2.4x104 cells/40 uL/well in a 96-well clear bottom plate (Corning, Catalogue No.: 3882). In some wells were added only the assay medium at 40 uL/well (Cell-free). On the day of the assay, 10 mM of the present compound (DMSO solution) was distributed in a 96-well plate (Costar, Catalogue No.: 3363) and serially diluted with DMSO with the geometrical ratio of 3. The serial dilutions were diluted with the assay medium to 100-fold to prepare a solution of the present compound with a 10-times concentration (DMSO concentration: 1%).
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 636.74, ALogP 6.58, HBA 10, HBD 2, PSA 136.39, QED 0.2
Compound (SMILES): O=C(CC(c1ccccc1)c1ccccc1)N1CCN(C(c2ccccc2)c2ccccc2)C(=O)C1
InChIKey: XWJCWOICXJFXAF-UHFFFAOYSA-N
Target: Voltage-dependent N-type calcium channel subunit alpha-1B (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q02294
Assay: Antagonist activity at rat alpha1B N-type Ca2+ channel expressed in HEK293 cells assessed as 0.2 Hz current frequency dependent channel blocking for 2 mins in presence of Ba2+ by whole-cell patch clamp assay
Measurement: IC50 = 680 nM (pChEMBL 6.170000076293945)
Descriptors: MW 474.6, ALogP 5.67, HBA 2, HBD 0, PSA 40.62, QED 0.35
Compound (SMILES): Cc1nnc2c(NC(C)C)nc(-c3nc4ccc(C5CCOCC5)cc4n3Cc3ccccn3)cn12
InChIKey: FRMBPERTEITNRY-UHFFFAOYSA-N
Target: Histone H4 (SINGLE PROTEIN), Homo sapiens
UniProt: P62805
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 uM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 ul of the mix is added to the compound dilutions. Subsequently, 6 ul of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 ug/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 482.59, ALogP 4.61, HBA 9, HBD 1, PSA 95.05, QED 0.38
Compound (SMILES): N#Cc1cnc(NC(=O)N2CCCc3cc(CO)c(C=O)nc32)cc1O[C@H]1CCOC1
InChIKey: JDSIDNMVOWOKBD-INIZCTEOSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Mus musculus
UniProt: Q03142
Assay: Inhibition of FGFR4 in mouse BAF3 cells assessed as decrease in FGFR4 phosphorylation incubated for 40 mins
Measurement: IC50 = 14 nM (pChEMBL 7.849999904632568)
Descriptors: MW 423.43, ALogP 1.81, HBA 8, HBD 2, PSA 137.67, QED 0.69
Compound (SMILES): COc1cc(OC)cc(N(C[C@H](O)C(F)(F)F)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1
InChIKey: INWHLZUNIJSPHA-QFIPXVFZSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 473.46, ALogP 4.11, HBA 8, HBD 1, PSA 85.53, QED 0.43
Compound (SMILES): CNc1nnc(-c2ccc3[nH]cc(-c4cccc(OC(F)(F)F)c4)c3c2)s1
InChIKey: UNZGSSHQOSZPRP-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 390.39, ALogP 5.29, HBA 5, HBD 2, PSA 62.83, QED 0.5
Compound (SMILES): O=c1ccc2ccc(OCCCN3CCCN(c4cccc(Cl)c4Cl)CC3)cc2[nH]1
InChIKey: WXHPNHJKGIFCOC-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P14416
Assay: cAMP GloSensor Assay: HEK293T cells co-expressing the cAMP biosensor GloSensor-22F (Promega) and hD2 receptors were seeded (10,000 cells/20 ul/well) into white, clear-bottom, tissue culture plates in HBSS, 10% FBS, 20 mM HEPES, pH 7.4. After a one- to two-hour recovery, cells were treated with 10 ul of 3x test or reference drug prepared in HBSS, 10% FBS, 20 mM HEPES, pH 7.4. After 30 minutes, cells were challenged with 10 ul of 1,200 nM (4x) isoproterenol in 8% (4x) GloSensor reagent. Luminescence per well per second was read on a Wallac TriLux microbeta plate counter. Data were normalized to the isoproterenol response (100%) and the maximal quinpirole-induced inhibition thereof (0%) and regressed using the sigmoidal dose-response function built into GraphPad Prism 4.0. Notably, HEK293T cells expressing the GloSensor-22F alone (no hD2) were assayed in parallel and displayed no inhibition of isoproterenol-stimulated cAMP.
Measurement: EC50 = 13 nM (pChEMBL 7.889999866485596)
Descriptors: MW 446.38, ALogP 4.82, HBA 4, HBD 1, PSA 48.57, QED 0.55
Compound (SMILES): CC(C)CCN1C[C@@H](C)N(C(=O)CCc2cccnc2)[C@@H](C)c2ccccc21
InChIKey: SKLATZHLNGJXPX-UXHICEINSA-N
Target: Voltage-dependent T-type calcium channel subunit alpha-1H (SINGLE PROTEIN), Homo sapiens
UniProt: O95180
Assay: Channel blocking activity at recombinant human CaV 3.2 channel expressed in HEK293 cells assessed as inhibition of Ca2+ flux preincubated for 3 mins followed by CaCl2 addition by fluo-4 dye-based FLIPR assay
Measurement: IC50 = 62 nM (pChEMBL 7.210000038146973)
Descriptors: MW 379.55, ALogP 4.86, HBA 3, HBD 0, PSA 36.44, QED 0.72
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(CC2CCC2)C(=O)[C@@H](O)CO)c1.O=C(O)C(F)(F)F
InChIKey: BDEXIXJJZXJWDS-HBTDVHQBSA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 429.56, ALogP 1.48, HBA 5, HBD 3, PSA 107.1, QED 0.55
Compound (SMILES): COc1ccc(C(=O)CCC(=O)Nc2cccc(Cc3n[nH]c(=O)c4c3CCCC4)c2)cc1
InChIKey: AMCZIPHXRVMECL-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP Enzyme Inhibition Assay: PARP1 assay was conducted in PARP assay buffer containing 50 mM Tris pH 8.0, 1 mM DTT, 4 mM MgCl2. PARP reactions contained 1.5 uM [3H]-NAD+ (1.6 uCi/mmol), 200 nM biotinylated histone H1, 200 nM sIDNA, and 1 nM PARP enzyme. Auto reactions utilizing SPA bead-based detection were carried out in 100 uL volumes in white 96 well plates. Reactions were initiated by adding 50 ul of 2xNAD+ substrate mixture to 50 uL of 2x enzyme mixture containing PARP and DNA. These reactions were terminated by the addition of 150 uL of 1.5 mM benzamide (~1000-fold over its IC50). 170 uL of the stopped reaction mixtures were transferred to streptavidin Flash Plates, incubated for 1 hour, and counted using a TopCount microplate scintillation counter.
Measurement: Ki = 1.9 nM (pChEMBL 8.720000267028809)
Descriptors: MW 445.52, ALogP 3.85, HBA 5, HBD 2, PSA 101.15, QED 0.51
Compound (SMILES): OC(COc1cccc(-c2cccc3c2oc2ccccc23)c1)CN1CCc2ccccc2C1
InChIKey: BTQXPNBTNKWTGB-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 5 (SINGLE PROTEIN), Homo sapiens
UniProt: O14744
Assay: Inhibition of PRMT5 in human Z138 cells assessed as decrease in di-methyl arginine levels after 96 hrs by Western blot method
Measurement: EC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 449.55, ALogP 6.05, HBA 4, HBD 1, PSA 45.84, QED 0.34
Compound (SMILES): Cc1cc(-c2nnc(-c3ccc4c(c3)CC(N)(CO)CC4)o2)ccc1OC(C)C
InChIKey: STYOWZCUSFFFIJ-UHFFFAOYSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: O08530
Assay: β-Arrestin Assay: Assay procedures: OCC culture solution was preheated in water tank at 37° C. Cells were taken out from liquor nitrogen tank, left on the dry-ice, and dissolved in water tank at 37° C. 0.5 ml OCC culture solution was added and blended slowly. The cells were diluted in 11.5 mL OCC culture solution, and 100 μL cell suspension (containing 8333 cells) was added to each well of the 96-well plate. The 96-well plate was incubated for 8 hours under 5% CO2 at 37° C., and 10 μL test compound dissolved in DMSO was added in; 10 μL DMSO was used as negative control. After the plate was incubated for 90 min at 37° C., 55 μL detection solution was added to each well. Then the results were read out by EnVision after the cell plate was incubated for 90 min at room temperature.
Measurement: EC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 393.49, ALogP 3.68, HBA 6, HBD 2, PSA 94.4, QED 0.69
Compound (SMILES): COc1ccc(CNc2nnc(-c3ccc4[nH]cc(-c5cncc(C6CC6)n5)c4c3)o2)cc1
InChIKey: WINYVBSIMZFCQW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 211.59 nM (pChEMBL 6.670000076293945)
Descriptors: MW 438.49, ALogP 5.17, HBA 7, HBD 2, PSA 101.75, QED 0.36
Compound (SMILES): COC[C@](N)(CSCCNC(=N)N)C(=O)O.Cl.Cl
InChIKey: HJBXSBPYGJZJML-JZGIKJSDSA-N
Target: Nitric oxide synthase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P29475
Assay: Inhibition of human neuronal constitutive NOS expressed in Sf9 insect cells assessed as reduction in conversion of L-[2,3-3H]-arginine to L-[2,3-3H]-citrulline incubated for 15 mins by liquid scintillation counting method
Measurement: IC50 = 58000 nM (pChEMBL 4.239999771118164)
Descriptors: MW 250.32, ALogP -1.37, HBA 5, HBD 5, PSA 134.45, QED 0.2
Compound (SMILES): Cc1c(CN2CCN(C)CC2)cccc1Nc1nccc(-c2c(-c3cccc(C(=O)Nc4c(F)cccc4F)c3)nc3ccccn23)n1
InChIKey: ZDAMHYQVVDUIAD-UHFFFAOYSA-N
Target: Insulin receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P06213
Assay: Inhibition of human GST-rTEV-tagged insulin receptor (979 to 1382 residues) expressed in baculovirus infected insect Sf9 cells assessed as decrease in biotin-aminohexyl-AEEEEYMMMMAKKKK-NH2 phosphorylation after 1 hr by fluorometer
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 644.73, ALogP 6.79, HBA 8, HBD 2, PSA 90.69, QED 0.19
Compound (SMILES): Cc1cc(N2CC3CN(C(=O)c4ccccc4-n4nccn4)CC3C2)nc(C(F)(F)F)n1
InChIKey: CGKPQJZFNDMIHC-UHFFFAOYSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Biological Assay: The in vitro affinity of the compounds for the human orexin-1 and orexin-2 receptors was determined by competitive radioligand binding using [3H]SB SB674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Langmead et al., British Journal of Pharmacology 2004, 141:340-346) and [3H]EMPA (N-ethyl-2[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl acetamide) (Malherbe et al., British Journal of Pharmacology, 2009, 156(8), 1326-1341), respectively. The in vitro functional antagonism of the compounds on the human orexin-1 and orexin-2 receptors was determined using fluorometric imaging plate reader (FLIPR) based calcium assays.
Measurement: Ki = 354 nM (pChEMBL 6.449999809265137)
Descriptors: MW 443.43, ALogP 2.59, HBA 7, HBD 0, PSA 80.04, QED 0.62
Compound (SMILES): CCN(c1cc(Cl)cc(C(=O)NCc2c(C)cc(C)[nH]c2=O)c1C)C1CCC(C)(C)CC1
InChIKey: MGZOOCDTTGHJQB-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition Assay: Test compounds were serially diluted 3-fold in DMSO in a 10 point-curve and 1 uL was spotted into a 384-well microplate in duplicate using a Platemate Plus equipped with 384-channel head (Thermo Scientific). The final top concentration of test compounds in the assay was 10 uM. Positive control (100% inhibition standard) was 1 mM final concentration of SAH and negative control (0% inhibition standard) contained 1 uL of DMSO. Test compounds were then incubated for 30 minutes with 40 uL per well of wild-type EZH2 (final concentration was 4 nM), Y641F EZH2 (final concentration was 0.1 nM) and A677G and A687V EZH2 (for each, final concentration was 2nM) and peptide in 1x assay buffer (20 mM BICINE pH =7.6, 1 mM DTT, 0.002% TWEEN 20 (generic: polysorbate 20), 0.005% BSG). For the wild-type EZH2 and A677G EZH2 assays, biotinylated peptide H3:21-44 with unmethylated K27 was present at a final concentration of 200 nM, while in the A687V EZH2 assay, biotinylated peptide H3.
Measurement: IC50 = 29.3 nM (pChEMBL 7.53000020980835)
Descriptors: MW 458.05, ALogP 5.68, HBA 3, HBD 2, PSA 65.2, QED 0.59
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CC4CCC(C3)O4)nc(-c3ccc(NC(=O)Nc4ccc(OC)nc4)c(F)c3)c2C1=O
InChIKey: AFBRBTWTGWGUPW-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase mTOR (SINGLE PROTEIN), Homo sapiens
UniProt: P42345
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The test detects the phosphorylation of the substrate GFP-4EBP1 using a terbium-labelled antibody that is specific for the phospho-Thr46 epitope. Binding of the Tb-antibody to the phosphorylated GFP peptide allows the transfer of energy from Tb, excited at 340 nm, to GFP with an increase in the fluorescence emission of the GFP at 520 nm. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 ul. To 1 ul of inhibitor in 100% DMSO are added (final concentrations) 400 nM of GFP-4EBP1 (Invitrogen PV4759), 8 uM of ATP, 200 ng/ml of enzyme (human recombinant aa mTOR 1360-2549, Invitrogen PV4753) in a buffer of HEPES 50 mM pH 7.5, EGTA 1 mM, MnCl2 10 mM, BSA 0.1 mg/ml, glycerol 0.5%, NV-10 0.1 mg/ml, DTT 2 mM. The mixture is incubated for 30 minutes at room temperature. 10 ul of solution are taken up and the reaction is quenched by adding 10 ul of Tb-antibody solution (Invitrogen PV4757)/EDTA.
Measurement: IC50 = 61 nM (pChEMBL 7.210000038146973)
Descriptors: MW 591.64, ALogP 4.33, HBA 9, HBD 2, PSA 131.04, QED 0.43
Compound (SMILES): COc1cccc2cc([C@H](C)NC(=O)c3c(N)nn4cccnc34)n(-c3ccccc3)c(=O)c12
InChIKey: XOOLORDUKXZNOY-HNNXBMFYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of human recombinant full length N-terminal His-tagged PI3Kgamma expressed in Sf9 insect cells using diC8PIP2 as substrate incubated for 15 mins followed by substrate addition measured after 2 hr by ADP-Glo luminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 454.49, ALogP 3.12, HBA 8, HBD 2, PSA 116.54, QED 0.42
Compound (SMILES): COC(=O)Nc1ccc2c(c1)C(=O)NCCCC[C@H](N1CCC(c3c(F)ccc(Cl)c3F)=CC1=O)c1cc-2ccn1
InChIKey: WLTAHYDRUHDZHH-VWLOTQADSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Assay: Plasma kallikrein determinations were made in 0.1 M sodium phosphate buffer at a pH of 7.5 containing 0.1-0.2 M sodium chloride and 0.5% PEG 8000. Determinations were made using purified human plasma kallikrein (Enzyme Research Laboratories) at a final assay concentration of 200 pM and the synthetic substrate S-2302 (H-(D)-Pro-Phe-Arg-pNA; CHROMOGENIX®) at a concentration of 0.00008-0.0004 M.
Measurement: Ki = 214 nM (pChEMBL 6.670000076293945)
Descriptors: MW 581.02, ALogP 6.13, HBA 5, HBD 2, PSA 100.63, QED 0.36
Compound (SMILES): CN1C(=O)C2(N=C1N)c1cc(-c3cccnc3F)ccc1CC21CCc2ccccc2CC1
InChIKey: DAWZTCXCSMUINX-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE Enzyme Assay (Ex.435): For each compound being tested, the BACE activity was monitored in a fluorescence quenching assay (FRET) using the ectodomain of BACE (aa 1-454) fused to a myc-his tag and secreted from HEK293/BACEect. cells into OptiMEM (Invitrogen) as enzyme source and a substrate peptide derived from the APP-Swedish mutation which possesses a Cy3-fluorophore at the N-terminus and a Cy5Q-quencher at the C-terminus (Cy3-SEVNLDAEFK-Cy5Q-NH2; Amersham). The substrate was dissolved at 1 mg/mL in DMSO.The assay was performed in the presence of 5 ul OptiMEM (supernatant collected over 24 hours and cleared from cellular debris by centrifugation) containing the ectodomain of BACE, 25 ul water containing the desired concentration of test compound and 1% DMSO, substrate peptide, 20 mM NaOAc, pH 4.4 and 0.04% Triton-X100 in a total assay volume of 50 ul in a 384 well plate. In general, 25 ul of compound dilution were given to the plate followed by the addition of 10 ul of BACE.
Measurement: IC50 = 19.1 nM (pChEMBL 7.71999979019165)
Descriptors: MW 440.52, ALogP 3.99, HBA 4, HBD 1, PSA 71.58, QED 0.58
Compound (SMILES): O=C(Nc1nc2ccccc2s1)c1cccc2c1CN(c1ccc(-c3ccc4c(c3)CCN(Cc3ccccn3)C4)c(C(=O)O)n1)CC2
InChIKey: KYJMLYBOYHSDKK-UHFFFAOYSA-N
Target: Bcl-2-like protein 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q64373
Assay: FL5.12 Cellular Assay: The efficacy of the compounds of Formula (I) can also be determined in cell-based killing assays using a variety of cell lines and mouse tumor models. For example, their activity on cell viability can be assessed on a panel of cultured tumorigenic and non-tumorigenic cell lines, as well as primary mouse or human cell populations. In one exemplary set of conditions, mouse FL5.12 cells transfected with Bcl-XL were cultured under standard conditions in RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3). For assaying the compound activity, the cells were exchanged into an IL-3-depleted deprivation media, which was identical to the growth media except for the absence of FBS and WEHI-3B conditional media, for 2 days. Then the cells were exchanged to 3% FBS assay media (RPMI with 2 mM glutamine, 1% 100 mM sodium pyruvate, 2% 1 M HEPES, 4 uL/L of beta;-mercaptoethanol, 1% penicillin-streptomycin, 10% FBS, and 10% WEHI-3B conditioned media (for IL-3).
Measurement: EC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 652.78, ALogP 6.82, HBA 8, HBD 2, PSA 111.55, QED 0.19
Compound (SMILES): O=C(NN1CCCCC1)c1cccc(OCC2CC2)n1
InChIKey: SMKUAWDANSQUQK-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.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.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 275.35, ALogP 2, HBA 4, HBD 1, PSA 54.46, QED 0.89
Compound (SMILES): O=C(Nc1ccc(C#CC2CC2)c(F)c1)c1cc2nc(Nc3c(F)cccc3Cl)[nH]c2c2c1OCCO2
InChIKey: CSOLVYOAIFCMHY-UHFFFAOYSA-N
Target: Prostaglandin E synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O14684
Assay: Inhibition of human recombinant mPGES-1 expressed in CHO cells assessed as reduction in conversion of PGH2 to PGE2 incubated for 10 mins followed by substrate addition measured after 1 min in presence of GSH
Measurement: IC50 = 3.56 nM (pChEMBL 8.449999809265137)
Descriptors: MW 520.92, ALogP 6.02, HBA 5, HBD 3, PSA 88.27, QED 0.29
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4ccccc4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: JHEVLALGAHRXET-BUVWRSTLSA-N
Target: Kappa-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41145
Assay: Binding Assay: Binding assay of certain compounds of this invention to the opioid receptor was determine using radioligand binding assay (screening and dose-displacement).
Measurement: Ki = 0.12 nM (pChEMBL 9.920000076293945)
Descriptors: MW 501.67, ALogP 5.14, HBA 5, HBD 0, PSA 40.16, QED 0.5