lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): O=C(O)c1cccc2nc(/C=C/c3cn4nccc(N5CCOCC5)c4n3)ccc12
InChIKey: FGUJIGJVSPNWLG-SNAWJCMRSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q9QYJ6
Assay: In Vitro Assay: Rat recombinant PDE10a (rPDE10a) was expressed in Sf9 cells using a recombinant rPDE10a baculovirus construct. Cells were harvested after 48 h of infection and the rPDE10a protein was purified by metal chelate chromatography on Ni-sepharose 6FF. Tested compounds were dissolved and diluted in 100% DMSO to a concentration 100 fold of the final concentration in the assay. Compound dilutions (0.4 uL) were added in 384 well plates to 20 uL of incubation buffer (50 mM Tris pH 7.8, 8.3 mM MgCl2, 1.7 mM EGTA). 10 uL of rPDE10a enzyme in incubation buffer was added and the reaction was started by addition of 10 uL substrate to a final concentration of 60 nM cAMP and 0.008 uCi3H-cAMP. The reaction was incubated for 60 min at rt. After incubation, the reaction was stopped with 20 uL of 17.8 mg/mL PDE SPA beads. After sedimentation of the beads during 30 min the radioactivity was measured in a Perkin Elmer Topcount scintillation counter.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 401.43, ALogP 2.98, HBA 7, HBD 1, PSA 92.85, QED 0.56
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)N/C(=C\c3ccccc3)C2=O)cn1
InChIKey: DJPPRYDDGMSXON-IUXPMGMMSA-N
Target: Taste receptor type 2 member 8 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYW2
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 340 nM (pChEMBL 6.46999979019165)
Descriptors: MW 363.38, ALogP 2.63, HBA 6, HBD 1, PSA 93.26, QED 0.57
Compound (SMILES): CCCc1ncc(Cc2ccc3oc4ccccc4c3c2)c2cc(OC)c(OC)cc12
InChIKey: SQBVSDMWUGZRBP-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Diaxonhit Phosphodiesterase Assay: The phosphodiesterase assay was developed using the LANCE cAMP kit (PerkinElmer). The assay buffer contained HBSS with 5 mM HEPES, 0.1% BSA, and 1.5 mM MgCl2, pH 7.4. PDE10A (BPS Bioscience) was used at 200 pg/well (with a specific activity of 3200 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min) and PDE4D3 (BPS Bioscience) was used at 100 pg/well (with a specific activity of 32713 pmole/min/ug with assay conditions: 10 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM MnCl2, 200 uM cAMP, 2.5 kU 5' nucleotidase, 37° C., 20 min). The Biotin-cAMP tracer, supplied in 10 mmol/L Tris-HCl buffered (pH 8.0) salt solution with 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1% bovine serum albumin (BSA), and 0.05% sodium azide, is used at a dilution of 1/375. The assay detection mixture contained the LANCE Eu-W8044 labeled streptavidin 1/2250 (supplied in 50 mmol/L Tris-HCl buffer).
Measurement: IC50 = 278 nM (pChEMBL 6.559999942779541)
Descriptors: MW 411.5, ALogP 6.69, HBA 4, HBD 0, PSA 44.49, QED 0.31
Compound (SMILES): CC(C)(O)Cc1cccc(N2CCC(CC#N)(n3cc(C(N)=O)c(Nc4ccc(F)cc4)n3)CC2)n1
InChIKey: YZUCDGVCEGCVNI-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 491.57, ALogP 3.48, HBA 8, HBD 3, PSA 133.09, QED 0.44
Compound (SMILES): O=S(=O)(c1cc(C(F)(F)F)cc(C(F)(F)F)c1)N1CCN(c2nc(Cl)ncc2Br)CC1
InChIKey: QZGAWXSHTXJXDY-UHFFFAOYSA-N
Target: Bcr/Abl fusion protein (CHIMERIC PROTEIN), Homo sapiens
UniProt: P11274
Assay: Inhibition of BRC/ABL1 (unknown origin) after 40 mins by ADP-Glo assay
Measurement: IC50 = 2250 nM (pChEMBL 5.650000095367432)
Descriptors: MW 553.71, ALogP 4.44, HBA 5, HBD 0, PSA 66.4, QED 0.42
Compound (SMILES): Cc1c(C(=O)Nc2c(C)n(C)n(C3CCCCC3)c2=O)noc1C1=CCC(C)(C)C1
InChIKey: RGRXGUWRVPJLEZ-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase SMURF1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HCE7
Assay: Inhibition of SMURF1 (unknown origin) in presence of E1/E2/Ub mix measured after 45 mins by europium-labeled streptavidin based TR-FRET assay
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 412.53, ALogP 4.75, HBA 6, HBD 1, PSA 82.06, QED 0.79
Compound (SMILES): CCNc1cc(O)c2c(c1)/C=C/C[C@@H]1OC(=O)O[C@@H]1C(=O)/C=C\[C@@H](C)[C@H](C)OC2=O
InChIKey: KUHHRXALBBVNHA-RKUKEFPASA-N
Target: Dual specificity mitogen-activated protein kinase kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q02750
Assay: Inhibition of MEK1 (unknown origin) using ERK2 as substrate after 40 mins by ELISA
Measurement: IC50 = 145 nM (pChEMBL 6.840000152587891)
Descriptors: MW 415.44, ALogP 3.45, HBA 8, HBD 2, PSA 111.16, QED 0.71
Compound (SMILES): COc1ccc(C2(C3CC3)NC(=N)N(C3CCOCC3)C2=O)cc1-c1cccc(C#N)c1
InChIKey: LRNWDZVTKKJAMY-UHFFFAOYSA-N
Target: Plasmepsin II (SINGLE PROTEIN), Plasmodium falciparum
UniProt: P46925
Assay: Enzyme Inhibition Assay : Plasmepsin-2 (PM-2; Plm II) and Plasmepsin (PM-4; Plm IV) expression and purification was performed following the published protocols (Istvan E S and Goldberg D E, 2005). The final purified protein was activated by diluting the protein to 0.3 mg/mL in activating buffer (0.1 M citrate pH 4.5, 0.1% Tween-20, 50 mM dithiothreitol) and incubated at room temperature for 40 min, then the activated enzyme was diluted in assay buffer (50 mM sodium acetate pH 4.7, 0.01% Tween-20). The enzymatic inhibition reaction was performed in 384 well plates with a total volume of 20 μl. 10 μl of diluted PM-2 or PM-4 enzyme was added to the 384-well plate except blank wells (blank wells add 10 μL of assay buffer) and 20 nL of serials of diluted 1000× compounds were added to the wells with 520 Echo® Liquid Handling System (Labcyte Inc.). 10 μL PM-2 peptide substrate (AnaSpec, Cat#, 62050) with assay buffer was then added to final concentration of 20 μM to start the reaction.
Measurement: IC50 = 51.7 nM (pChEMBL 7.289999961853027)
Descriptors: MW 430.51, ALogP 3.38, HBA 5, HBD 2, PSA 98.44, QED 0.76
Compound (SMILES): NC(=O)c1nn(-c2ccc(F)cc2)c2c1COc1ccc(NC(=O)c3ccncc3Cl)cc1-2
InChIKey: NJOHGLWBSXJNOZ-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus infected insect cells using biotinylated IkappaBalpha peptide as substrate in presence of [gamma-33P]-ATP after 30 mins by resin-based scintillation counting method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 463.86, ALogP 3.97, HBA 6, HBD 2, PSA 112.13, QED 0.48
Compound (SMILES): O=c1c2cc(O)ccc2ccn1CCCCN1CCN(c2cccc3sccc23)CC1
InChIKey: RHGQBJJYWPWFSG-UHFFFAOYSA-N
Target: D(2) dopamine receptor (SINGLE PROTEIN), Rattus norvegicus
UniProt: P61169
Assay: Dopamine D2 Receptor Binding Assay: The assay was performed according to the method by Kohler et al. (Kohler C, Hall H, Ogren S O and Gawell L, Specific in vitro and in vivo binding of 3H-raclopride. A potent substituted benzamide drug with high affinity for dopamine D-2 receptors in the rat brain. Biochem. Pharmacol., 1985; 34: 2251-2259). The binding assay was performed using 40 ul of the membrane specimen, 20 ul of [3H]-raclopride (final concentration 1 to 2 nM), 20 ul of a test drug and 50 mM Tris-hydrochloric acid buffer (containing 120 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) so that the total amount was 200 ul (final dimethylsulfoxide concentration 1%). The reaction was performed at room temperature for 1 hour and terminated by conducting suction filtration with a cell harvester on a glass fiber filter plate. The filter plate made of glass fiber was washed with 50 mM Tris-hydrochloric acid buffer (pH 7.4), and after dried, a microplate liquid scintillation cocktail was added.
Measurement: Ki = 1.2 nM (pChEMBL 8.920000076293945)
Descriptors: MW 433.58, ALogP 4.52, HBA 6, HBD 1, PSA 48.71, QED 0.45
Compound (SMILES): COc1c(F)cccc1-c1noc([C@@H]2CCN2C(=O)c2cc(C)ccc2-n2nccn2)n1
InChIKey: LBWZEGBRYDWCLD-SFHVURJKSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
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 = 641 nM (pChEMBL 6.190000057220459)
Descriptors: MW 434.43, ALogP 3.36, HBA 8, HBD 0, PSA 99.17, QED 0.47
Compound (SMILES): CCN1C(=O)Cc2c(CN3C(=O)[C@@H](NC(=O)[C@H](C)NC)COc4ccccc43)cccc21
InChIKey: FJKGNKQMJJKBRW-YJBOKZPZSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 1121 nM (pChEMBL 5.949999809265137)
Descriptors: MW 436.51, ALogP 1.61, HBA 5, HBD 2, PSA 90.98, QED 0.72
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H]1COc2c(Br)cccc2N(Cc2c(C)ccc3ccccc23)C1=O
InChIKey: XPHDLVAVZMWLKT-KKSFZXQISA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: Ten nanomolar of 6x Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(8-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-C1, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 = 124 nM (pChEMBL 6.909999847412109)
Descriptors: MW 496.41, ALogP 3.93, HBA 4, HBD 2, PSA 70.67, QED 0.56
Compound (SMILES): NC(=O)c1ccc(NCCCc2ccccc2)c2cccnc12
InChIKey: GOIIQXABHXILGU-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° 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 > 1000 nM (pChEMBL 6)
Descriptors: MW 305.38, ALogP 3.38, HBA 3, HBD 2, PSA 68.01, QED 0.69
Compound (SMILES): NS(=O)(=O)c1ccccc1NS(=O)(=O)c1ccc(Oc2ccc(Cl)cc2)cc1
InChIKey: ABBGTBRNVJKHDD-UHFFFAOYSA-N
Target: Prostaglandin E synthase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9H7Z7
Assay: Biochemical Assay: A solution of test compound was added to a diluted microsome preparation containing human mPGES-1 and pre-incubated for 15 minutes in potassium phosphate buffer pH 6.8 with cofactor glutathione (GSH). Corresponding solutions without test compound were used as positive controls, and corresponding solutions without test compound and without microsomes were used as negative controls. The enzymatic reaction was then started by addition of the substrate PGH2 in an organic solution (dry acetonitrile).The typical reaction conditions of the enzymatic reaction were thus: Test compound: ranging from 60 uM to 0.002 uM, or zero in positive and negative controls; potassium phosphate buffer pH 6.8: 50 mM; GSH: 2.5 mM; mPGES-1-containing microsomes: 2 ug/mL (sample and positive controls) or 0 ug/mL (negative control); PGH2: 10.8 uM; Acetonitrile: 7.7% (v/v); DMSO: 0.6% (v/v). The reaction was stopped after one minute by adding an acidic solution (pH 1.9) of ferric chloride.
Measurement: IC50 = 3700 nM (pChEMBL 5.429999828338623)
Descriptors: MW 438.91, ALogP 3.58, HBA 5, HBD 2, PSA 115.56, QED 0.61
Compound (SMILES): CC(C)Nc1nc2c(nc1N1CCC(C(=O)c3cc(Cl)ccc3F)CC1)CN(C(=O)N(C)C)CC2
InChIKey: JOAGWBROEFGAGV-UHFFFAOYSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.1): This cell based assay measures the ability of compounds to inhibit the constitutive cAMP activity of GPR6 receptor expressed in CHO-K1 cells. CHO cells were stably expressed with GPR6 receptor, whose expression is controlled by a tetracycline inducable element. The cells were cultured in medium containing F12K, 10% FBS, 1% PennStrep, 200 ug/mL Hygromycin. GPR6 receptor expression was induced for 20 hrs with 1 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 250-500 cells per well in half-volume black clear bottom plates (Costar) and place in an incubator (37° C, 5% C(O)2) for 20 hours prior to cAMP assays.Culture media was removed from cells and they were washed with 50 uL of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHC(O)3 1.26 mg/mL, D-glucose 1.8 mg/mL, NaCl 7 mg/mL; pH=7.4). Compounds suspended in DMSO were diluted in Ringer's Buffer.
Measurement: IC50 = 0.0249 nM (pChEMBL 10.600000381469727)
Descriptors: MW 503.02, ALogP 4.23, HBA 6, HBD 1, PSA 81.67, QED 0.62
Compound (SMILES): CC(=O)c1ccc(OCc2ccc(F)cc2)cc1O
InChIKey: RFHGZHJAFVVQFU-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibition of recombinant human MAO-B using p-tyramine as substrate assessed as decrease in H2O2 production incubated for 15 mins by fluorimetric method
Measurement: IC50 = 28.42 nM (pChEMBL 7.550000190734863)
Descriptors: MW 260.26, ALogP 3.31, HBA 3, HBD 1, PSA 46.53, QED 0.86
Compound (SMILES): Cc1c(-c2ccc(Cl)cc2)sc2c1c(=O)n(CCCO)c(=O)n2C
InChIKey: WQRGHSSPNYUZEW-UHFFFAOYSA-N
Target: Short transient receptor potential channel 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UL62
Assay: High Throughput Screening Assay: The commercially available HEK293/TREx line (Invitrogen) was stably transfected with a TRPC5 construct and screened by conventional calcium imaging to find clones with TRPC5 expression following stimulation with 1 μg/ml tetracycline. These cells were maintained in the growth medium recommended by the manufacturer supplemented with 100 μg/ml hygromycin to promote retention of the TRPC5 construct. After growing to near confluency, cells were plated at a density of Ë¿35,000 cells/well in 384 well CellBind plates (Corning) in the presence of 1 μg/ml tetracycline, and allowed to grow for 20-30 hrs. A nearly confluent monolayer resulted. Cells were then loaded with Ca2+ dye: Fura-2/AM or Fluo4/AM was added to the wells to a final concentration of 4 μM or 0.5 μM, respectively, and incubated for 80 min or 60 min, respectively, at room temperature. Supernatant was then removed from the cells by inverting plates with a sharp flick, and 25 μl Hank's Balanced Salt Solution (HBSS; 0.185 g/l D-glucose, 0.9767 g/l MgS04 (anhydrous), 0.4 g/l KCl, 0.06 g/l KH2PO4 (anhydrous), 0.35 g/l NaHCO3, 8.0 g/l NaCl, and 0.04788 g/l Na2HPO4 (anhydrous); pH 7.4) was then added to each well. Following Ë¿0.5 hour for recovery from loading, cells were assayed using the Hamamatsu FDSS 6000 system, which permitted illumination alternately at 340 nm and 380 nm for Fura-2 experiments, or at 485 nm for Fluo4 experiments. Frames were acquired at a rate of 0.2 Hz. During the assay, the plates were continuously vortexed, with pipette mixing of wells following addition of each reagent. For the screening assay, 26 μl of a diluted compound stock (at 50 μM) was added to each well for 2 minutes following the collection of a short (4 frame) baseline. 13 μl 62 mM high-Ca2+Ringer solution (4.17 ml of normal ringer (with 2 mM Ca2+) plus 5.83 ml of isotonic calcium ringer (105 mM Ca2+; in this ringer all sodium has been replaced with calcium)) was then added to each well, achieving a final concentration of 14 mM Ca2+ and 10 μM test compound. Data was collected for Ë¿3 minutes following addition of high Ca2+Ringer, where the fluorescent intensity (for Fluo4) and the F340/F380 ratio (for Fura-2) were proportional to the [Ca2+]i Negative controls consisted of HEK293/TREx TRPC5 cells exposed to high Ca2+ solution, but no compound. Positive control conditions consisted of addition of 2-APB, a promiscuous blocker of TRPC5 and other channels, to columns 23 and 24 of the plates, to a final concentration of 200 μM. The Fluo4 cell-based fluorescence assay was used to determine the intracellular Ca2+ concentration in the presence of varying drug concentration. Final concentrations of compounds tested were 20 μM, 6.667 μM, 2.222 μM, 0.741 μM, 0.247 μM, 0.082 μM, and 0.027 μM. Compounds were tested in triplicate at all concentrations.
Measurement: IC50 = 168 nM (pChEMBL 6.78000020980835)
Descriptors: MW 364.85, ALogP 2.77, HBA 6, HBD 1, PSA 64.23, QED 0.77
Compound (SMILES): COc1cc(F)c(C)cc1-c1c[nH]c2ncc(-c3cccc(NC(=O)/C=C/CN(C)C)c3)cc12
InChIKey: ABNHJJMMVSPRKX-RMKNXTFCSA-N
Target: Tyrosine-protein kinase ITK/TSK (SINGLE PROTEIN), Homo sapiens
UniProt: Q08881
Assay: In Vitro Inhibition Assay: ITK and JAK3 Kinase assay procedures:Enzyme was incubated with substrate peptide in reaction buffer in the presence and absence of test compounds or Staurosporine. All additions were done on ice, followed by the addition of ATP mix. Wells were uniformly mixed using an Eppendorff plate shaker and incubated at 30° C. for 20 min, and stopped by the addition of 5 μL of 3% phosphoric acid. Volume was increased to 100 μL by adding 0.8% phosphoric acid which was then transferred to PC filter mats (Millipore), pre-equilibrated with 70% ethanol and water. Plates were washed thrice with 100 μL 0.8% phosphoric acid and dried for an hour at 60° C. 100 μL scintillation fluid was added into each well and reading taken in Perkin Elmer TOPCOUNT beta counter. The data analysis was performed by averaging the duplicate top count readings for each standard, negative, positive control (enzyme control) and samples and subtracting the average negative control from each reading which results in corrected values.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 458.54, ALogP 5.41, HBA 4, HBD 2, PSA 70.25, QED 0.36
Compound (SMILES): CN1CCN(Cc2ccc(-c3cn4c5nc(ncc35)NCCCCCCCCC(O)CCC4)cc2)CC1
InChIKey: DTHWSLYDAOOPOE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase Mer (SINGLE PROTEIN), Homo sapiens
UniProt: Q12866
Assay: Inhibition of human MerTK kinase domain (1585 to 3000 residues) expressed in HEK293 cells co-expressing rat EGFR LBD assessed as inhibition of EGF-stimulated MerTK phosphorylation preincubated for 30 mins followed by EGF stimulation for 15 mins by ELISA
Measurement: EC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 504.72, ALogP 5.14, HBA 7, HBD 2, PSA 69.45, QED 0.51
Compound (SMILES): COc1cccc2c1ncc1c(=O)n(-c3cccc(Cl)c3)c(=O)n(C3CCCCC3)c12
InChIKey: BOWZLOCTDYTWKK-UHFFFAOYSA-N
Target: Tumor necrosis factor (SINGLE PROTEIN), Homo sapiens
UniProt: P01375
Assay: ELISA Assay: Add 100 μL of sample or standards in Reagent Diluent, or an appropriate diluent, per well. Cover with an adhesive strip and incubate 2 hours at room temperature. Repeat the aspiration/wash as in step 2 of Plate Preparation. Add 100 μL of the Detection Antibody, diluted in Reagent Diluent, to each well. Cover with a new adhesive strip and incubate 2 hours at room temperature. Repeat the aspiration/wash as in step 2 of Plate Preparation. Add 100 μL of the working dilution of Streptavidin-HRP to each well. Cover the plate and incubate for 20 minutes at room temperature. Avoid placing the plate in direct light. Repeat the aspiration/wash as in step 2. Add 100 μL of Substrate Solution to each well. Incubate for 20 minutes at room temperature. Avoid placing the plate in direct light. Add 50 μL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing. Determine the optical density of each well immediately, using a microplate reader set to 450 nm. If wavelength correction is available, set to 540 nm or 570 nm. If wavelength correction is not available, subtract readings at 540 nm or 570 nm from the readings at 450 nm. This subtraction will correct for optical imperfections in the plate. Readings made directly at 450 nm without correction may be higher and less accurate.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 435.91, ALogP 4.87, HBA 6, HBD 0, PSA 66.12, QED 0.43
Compound (SMILES): Nc1cn2nc(C#Cc3ccc(NC(=O)Nc4cc(C(F)(F)F)ccc4F)cc3)ccc2n1
InChIKey: BHKGZILBWHHNDQ-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: PDGFRbeta Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T.
Measurement: IC50 = 73 nM (pChEMBL 7.139999866485596)
Descriptors: MW 454.39, ALogP 4.51, HBA 5, HBD 3, PSA 97.34, QED 0.31
Compound (SMILES): Cc1cc(C(=O)NC[C@H]2CC[C@H](Oc3ccnc4ccccc34)CC2)no1
InChIKey: ZXIJUQZCPPJBSO-WKILWMFISA-N
Target: Androgen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P10275
Assay: Transactivation Assay: For determining the androgen receptor-dependent transcription, a cellular assay system was used, consisting of PC-3 cells (Kaighn et al., Invest. Urol. 17: 16-23, 1979), which express the human androgen receptor stably and recombinantly (full length, wild-type form, see Swiss-Prot Acc. No. P10275, Entry Version 159, Sequence Version 2). In addition, these PC3 cells contain a stably integrated reporter gene plasmid, which is based on the commercially available plasmid pGL4.14 (#E6691, Promega Corporation, Madison, Wis., USA) and contains the luciferase gene from the American firefly (Photinus pyralis) under the control of the MMTV promoter (Cato et al., EMBO J. 6: 363-368, 1987). These cells were propagated in routine cell culture at 37° C. and 5% CO2 in a medium containing 90% RPMI 1640 (Invitrogen GmbH, Darmstadt, Germany), 100 U penicillin, 100 μg/ml streptomycin (Invitrogen), 4 mM L-glutamine (Invitrogen), 10% fetal calf serum (FCS Serum Gold, PAA Laboratories GmbH, Cölbe, Germany), 600 μg/ml Geneticin (G418-sulphate, Invitrogen) and 10 μg/ml puromycin (Sigma Aldrich GmbH, Germany). For carrying out the transactivation assays, approx. 1000 cells per well were plated out in a 384-well cell culture plate in a medium that contained activated charcoal-treated calf serum (FCS Serum Gold, PAA Laboratories) at a concentration of 5% (v/v). The test substances were added in a concentration series from 5.12×10^−12 to 1×10^−5 M in the presence of 1×10^−1° R1881 (methyltrienolone). The test plates were incubated overnight at 37° C. and 5% CO2. After 16 hours, 15 μl of Steady Glo Lysis and Detection reagent (E2550, Promega Corporation, Madison, Wis., USA) was added per well and the luminescence was read in a Topcount Luminometer (PerkinElmer, Waltham, Mass., USA) for 4 seconds per well. The luminescence values obtained were normalized, wherein 100% corresponded to the effect of the unstimulated control (without R1881), and 0% corresponded to the effect of the stimulated control (R1881 plus DMSO instead of test substance).
Measurement: IC50 = 210 nM (pChEMBL 6.679999828338623)
Descriptors: MW 365.43, ALogP 3.9, HBA 5, HBD 1, PSA 77.25, QED 0.74
Compound (SMILES): CC(=O)Nc1c(N(Cc2ccc(F)c(C(F)(F)F)c2)S(=O)(=O)c2ccccc2)sc2ccccc12
InChIKey: YULVNNLAGJFFOO-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 522.55, ALogP 6.41, HBA 4, HBD 1, PSA 66.48, QED 0.3
Compound (SMILES): CCCCN(CCCC)C(=O)c1cc(C)n(-c2ccc(N[S+]([O-])c3cccc(Cl)c3Cl)cc2C(=O)N2Cc3ccccc3C[C@H]2CO)n1
InChIKey: OMUKBBNSBJRBRG-SAUCOOFJSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Surface plasmon resonance (SPR): Methods for Determining IC50s.The present method includes utility of a Surface plasmon resonance (SPR)- based biosensor (Biacore GE Healthcare, Uppsala, Sweden) to characterize BCL-2 inhibitors.Biacore utilizes the phenomenon of surface plasmon resonance (SPR) to detect and measure binding interactions. In a typical Biacore experiment, one of the interacting molecules (ligand) is immobilized on a flexible dextran matrix while the interacting partner (analyte) is allowed to flow across that surface. A binding interaction results in an increase in mass on the sensor surface and a corresponding direct change in the refractive index of the medium in the vicinity of the sensor surface. Changes in refractive index or signal are recorded in resonance units (R.U.) Signal changes due to association and dissociation of complexes are monitored in a non-invasive manner, continuously and in real-time, the results of which are reported in the form of a sensorgram.
Measurement: IC50 = 493.55 nM (pChEMBL 6.309999942779541)
Descriptors: MW 710.73, ALogP 7.22, HBA 7, HBD 2, PSA 113.76, QED 0.14
Compound (SMILES): Cc1nc([C@H](C)O)n(-c2cc(C3C[C@H]3c3nc4ccccc4n3C)nc(C(F)(F)F)n2)n1
InChIKey: PIPXXCIAJLPPBJ-RDWQBYKPSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Fluorescence Polarization Assay: The fluorescence polarization assay for cyclic nucleotide phosphodiesterases was performed using an IMAP® FP kit supplied by Molecular Devices, Sunnyvale, Calif. (product # R8139). IMAP® technology has been applied previously to phosphodiesterase assays (Huang, W., et al., J. Biomol Screen, 2002, 7: 215). Assays were performed at room temperature in 384-well microtiter plates with an incubation volume of 20.2 μL. Solutions of test compounds were prepared in DMSO and serially diluted with DMSO to yield 8 μL of each of 10 solutions differing by 3-fold in concentration, at 32 serial dilutions per plate. A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 nL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/1600 dilution from aliquots; sufficient to produce 20% substrate conversion) and a separate solution of FAM-labeled cAMP PDE from Molecular Devices (product # R7506), at a final concentration of 50 nM are made in the assay buffer (10 mM Tris HCl, pH 7.2, 10 mM MgCl2, 0.05% NaN3 0.01% Tween-20, and 1 mM DTT). The enzyme and the substrate are then added to the assay plates in two consecutive additions of 10 μL, and then shaken to mix. The reaction is allowed to proceed at room temperature for 30 minutes. A binding solution is then made from the kit components, comprised of 80% Solution A, 20% Solution B and binding reagent at a volume of 1/600 the total binding solution. The enzymatic reaction is stopped by addition of 60 μL of the binding solution to each well of the assay plates and the plates are sealed and shaken for 10 seconds. The plate was incubated at room temperature for at least one hour prior to determining the fluorescence polarization (FP). The parallel and perpendicular fluorescence of each well of the plate was measured using a Perkin Elmer EnVision™ plate reader (Waltham, Mass.).
Measurement: Ki = 2.8 nM (pChEMBL 8.550000190734863)
Descriptors: MW 443.43, ALogP 3.6, HBA 8, HBD 1, PSA 94.54, QED 0.52
Compound (SMILES): O=C(Nc1cn(Cc2cn(C3CCN(C4CCOCC4)CC3)nn2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: JWZWPTJBLNSRPJ-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
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 = 0.45 nM (pChEMBL 9.350000381469727)
Descriptors: MW 653.09, ALogP 4.56, HBA 12, HBD 1, PSA 129.52, QED 0.24
Compound (SMILES): COc1cc(OC)cc(N(CCNC(C)C)c2ccc3ncc(-c4cnn(C)c4)nc3c2)c1.Cl
InChIKey: QAAOEMXHEWVIBB-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11362
Assay: Inhibition of human FGFR1 using Btn-Flt3 as substrate after 60 mins by TR-FRET assay
Measurement: IC50 = 104.71 nM (pChEMBL 6.980000019073486)
Descriptors: MW 446.56, ALogP 4.18, HBA 8, HBD 1, PSA 77.33, QED 0.41
Compound (SMILES): Cc1ccc(NC(=O)c2ccnc(C(C)(C)C#N)c2)cc1-c1ccnc(N2C[C@H](CN(C)C)OC[C@@H]2C)c1
InChIKey: STAZWCVPUNIJEO-OFVILXPXSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 30.65 nM (pChEMBL 7.510000228881836)
Descriptors: MW 512.66, ALogP 4.66, HBA 7, HBD 1, PSA 94.38, QED 0.49
Compound (SMILES): NC1CCC(NC2CC2c2ccccc2F)CC1
InChIKey: XYAZRGFFUVDRRE-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] A (SINGLE PROTEIN), Homo sapiens
UniProt: P21397
Assay: Fluorescence-Based (Inhibitor)-Screening Assay: In order to monitor MAO enzymatic activities and/or their inhibition rate by inhibitor(s) of interest, a fluorescence-based (inhibitor)-screening assay was set up. 3-(2-Aminophenyl)-3-oxopropanamine (kynuramine dihydrobromide, Sigma Aldrich), a non fluorescent compound was chosen as a substrate. Kynuramine is a non-specific substrate for both MAO-A and MAO-B activities. While undergoing oxidative deamination by MAO activities, kynuramine is converted into 4-hydroxyquinoline (4-HQ), a resulting fluorescent product.The monoamine oxidase activity was estimated by measuring the conversion of kynuramine into 4-hydroxyquinoline. Assays were conducted in 96-well black plates with clear bottom (Corning) in a final volume of 100 uL. The assay buffer was 100 mM HEPES, pH 7.5. Each experiment was performed in duplicate within the same experiment.Briefly, a fixed amount of MAO (0.25 ug for MAO-A and 0.5 ug for MAO-B) was incubated on ice for 15 minutes in the reaction buffer, in the absence and/or in the presence of at least eight 3-fold serial dilutions each. Clorgyline and Deprenyl (Sigma Aldrich) was used as a control for specific inhibition of MAO-A and MAO-B respectively. After leaving the enzyme(s) interacting with the inhibitor, KM of kynuramine was added to each reaction for MAO-B and MAO-A assay respectively, and the reaction was left for 1 hour at 37° C. in the dark. The oxidative deamination of the substrate was stopped by adding 50 uL of NaOH 2N. The conversion of kynuramine to 4-hydroxyquinoline, was monitored by fluorescence (excitation at 320 nm, emission at 360 nm) using a microplate reader (Infinite 200, Tecan). Arbitrary units were used to measure levels of fluorescence produced in the absence and/or in the presence of inhibitor. The maximum of oxidative deamination activity was obtained by measuring the amount of 4-hydroxyquinoline formed from kynuramine deamination in the absence of inhibitor and corrected for background fluorescence in the absence of MAO enzymes. The IC50 values of each inhibitor were calculated with GraphPad Prism Software.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 248.34, ALogP 2.54, HBA 2, HBD 2, PSA 38.05, QED 0.86
Compound (SMILES): Cc1nc2ccc(NS(=O)(=O)c3ccc4c(c3)n(C)c(=O)c(=O)n4C)cc2nc1C
InChIKey: QNZGCWZGBMSSDR-UHFFFAOYSA-N
Target: Taste receptor type 2 member 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NYV8
Assay: Fluorescence Polarization Assays: In another embodiment, Fluorescence Polarization ("FP") based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low picomolar to micromolar levels.
Measurement: IC50 = 6332 nM (pChEMBL 5.199999809265137)
Descriptors: MW 425.47, ALogP 1.6, HBA 8, HBD 1, PSA 115.95, QED 0.5
Compound (SMILES): N#Cc1c(N)nc(N)nc1N[C@H](c1nc2cccc(Cl)c2c(=O)n1-c1cncc(F)c1)C1CC1
InChIKey: JYKDGFHMXCXLAC-KRWDZBQOSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Time-Resolved Fluorescence Resonance Energy transfer (TR-FRET) Assay: Class I PI3K isoforms were expressed and purified as heterodimeric recombinant proteins. All assay reagents and buffers for the TR-FRET assay were purchased from Millipore. PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2× Km ATP (75-500 μM), 2 μM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoforms: PI3Kα, PI3Kβ, and PI3Kδ between 25 and 50 pM, and PI3Kγ at 2 nM. After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 10lps delay and 500 μs read window).
Measurement: IC50 = 2 nM (pChEMBL 8.699999809265137)
Descriptors: MW 477.89, ALogP 2.96, HBA 10, HBD 3, PSA 161.42, QED 0.39
Compound (SMILES): O=C(OCc1ccccc1)C1CC(NC(=O)N2Cc3ccncc3C2)C1
InChIKey: TUYQXSMRRRESJP-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 2:The assay was carried out in 18 uL low volume plates (Owens Corning) in reaction buffer (50 mM HEPES (NaOH), pH 7.5, 100 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% Glycerol) using 6.8 nM recombinant, human, C-terminally-His tagged NAMPT, 1 nM Tb-anti-His antibody (Invitrogen, Cat #PV5895), and 200 nM probe (Oregon Green 488-conjugated APO0866; A-1287128.0. Plates were covered, and reactions were carried out for 2-3 hours. Plates were read with Envision (Laser Lantha low volume protocol) after 2 to 3 hours. Excitation was carried out at 337 nm, and the ratio of emission of Oregon Green (520 nm) to terbium (492 nm) was determined and used to calculate IC50 values of test compounds.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 351.41, ALogP 2.63, HBA 4, HBD 1, PSA 71.53, QED 0.86
Compound (SMILES): Cc1noc(C(=O)Nc2csc([C@]34CO[C@@H](C)C[C@H]3CSC(N)=N4)n2)n1
InChIKey: AQIUHWLUHZEXFK-UHCFIKNESA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37 °C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 98 nM (pChEMBL 7.010000228881836)
Descriptors: MW 394.48, ALogP 1.77, HBA 10, HBD 2, PSA 128.52, QED 0.81
Compound (SMILES): Cc1ncc2ccc(C(=O)N3CCC[C@H]3c3ccc(Cl)cc3)cn12
InChIKey: UBAOTUBXUAQBKH-SFHVURJKSA-N
Target: CDK8/Cyclin C (PROTEIN COMPLEX), Homo sapiens
UniProt: P24863
Assay: Inhibition of Alexa647 tracer binding to full length recombinant human His-tagged CDK8/cyclin C expressed in Baculovirus expression system preincubated for 20 mins followed by tracer addition and incubated in dark for 60 mins by FRET-based Lanthascreen assay
Measurement: IC50 = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 339.83, ALogP 4.27, HBA 3, HBD 0, PSA 37.61, QED 0.7
Compound (SMILES): Cc1ccc(S(=O)(=O)N[C@H]2CC[C@H](CCN3CCN(c4nsc5ccccc45)CC3)CC2)cc1
InChIKey: SOMWRASUKKDBSH-HZCBDIJESA-N
Target: D(3) dopamine receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35462
Assay: Displacement of [3H]methyl-spiperone from recombinant human D3 receptor expressed in CHO cell membranes after 60 mins by scintillation counting method
Measurement: Ki = 0.062 nM (pChEMBL 10.210000038146973)
Descriptors: MW 498.72, ALogP 4.65, HBA 6, HBD 1, PSA 65.54, QED 0.51
Compound (SMILES): NS(=O)(=O)c1ccc(SCc2cccc(-c3ccccc3)c2)s1
InChIKey: CWDPZVCRDBASOZ-UHFFFAOYSA-N
Target: Carbonic anhydrase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P00918
Assay: Inhibition of human recombinant cytosolic carbonic anhydrase 2 preincubated for 10 mins prior to testing by stopped-flow CO2 hydration assay
Measurement: Ki = 316 nM (pChEMBL 6.5)
Descriptors: MW 361.51, ALogP 4.35, HBA 4, HBD 1, PSA 60.16, QED 0.69
Compound (SMILES): COc1cc(F)ccc1[C@@H]1C[C@H](F)C(=O)N1Cc1ccc(OC(F)(F)F)cc1
InChIKey: JPVQRTHHGCIQAP-HOTGVXAUSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
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 = 1376 nM (pChEMBL 5.860000133514404)
Descriptors: MW 401.33, ALogP 4.54, HBA 3, HBD 0, PSA 38.77, QED 0.69