lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): C[C@H]1NC(=O)c2cc(-c3c(F)ccc4c(=O)n(C)c(NC(C)(C)C)nc34)[nH]c21
InChIKey: GTKNYUJNLDGSDF-SECBINFHSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): 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 384-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 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 0.012 nM (pChEMBL 10.920000076293945)
Descriptors: MW 383.43, ALogP 3.08, HBA 5, HBD 3, PSA 91.81, QED 0.63
Compound (SMILES): O=C(c1ccc(F)cc1)N1CCN(c2nc(N3CCOCC3)ncc2Br)CC1
InChIKey: MLSMCXWBTQUJBM-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 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 450.31, ALogP 2.18, HBA 6, HBD 0, PSA 61.8, QED 0.71
Compound (SMILES): O=C(COc1cccc(NC2CCOCC2)n1)NCC(O)CN1CCc2ccccc2C1
InChIKey: XRVLKTZJAOAKJB-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 440.54, ALogP 1.59, HBA 7, HBD 3, PSA 95.95, QED 0.54
Compound (SMILES): CC(=O)N[C@@H](C)c1ccc(OC2CCN(c3ccc(OCOC(C)C)cc3)C2)cc1
InChIKey: SRXDTSMLMGICBZ-VEXWJQHLSA-N
Target: Acetyl-CoA carboxylase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O00763
Assay: Spectrophotometric 384 Well Assay: Assay reactions are then carried out in 384-well plates, with hACC2 in an appropriate dilution and at final assay concentrations (f.c.) of 100 mM Tris (pH 7.5), 10 mM trisodium citrate, 25 mM KHCO3, 10 mM MgCl2, 0.5 mg/ml BSA, 3.75 mM reduced L-glutathione, 15 U/ml lactate dehydrogenase, 0.5 mM phosphoenolpyruvate, 15 U/ml pyruvate kinase, compounds at different concentrations at final DMSO concentrations of 1%.The enzymatic reaction is then started by addition of a mixture of NADH, acetyl Coenzyme A (both 200 μM f.c.) and ATP (500 uM f.c.). The decrease of the optical density (slope S) is then determined at 25° C. at a wavelength of 340 nm over 15 minutes in a spectrophotometric reader.
Measurement: IC50 = 1210 nM (pChEMBL 5.920000076293945)
Descriptors: MW 412.53, ALogP 4.3, HBA 5, HBD 1, PSA 60.03, QED 0.62
Compound (SMILES): CC(=O)C1=C(C)N(c2cccc(C(F)(F)F)c2)C(=O)N(C)C1c1ccc(C#N)cc1C(=O)NCc1cn[nH]c1
InChIKey: HZWUPEDJPQPFBL-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: Materials: Human neutrophil elastase was purchased from Calbiochem (Cat. No.: 324681) and the elastase substrate MeOSuc-Ala-Ala-Pro-Val-AMC from Bachem (Cat. No.: I-1270). All other materials were of the highest grade commercially available.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 uL, of these compound dilutions were mixed with 10 uL 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 uL, substrate solution in assay buffer were added (250 uM 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. Each plate contains wells with a high value control (DMSO+enzyme+substrate) and wells with a low value control (DMSO+inactivated enzyme+substrate). IC50 values were estimated using a sigmoidal concentration response curve with variable slope.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 536.51, ALogP 4.71, HBA 5, HBD 2, PSA 122.19, QED 0.47
Compound (SMILES): Cc1cccc(Cc2n[nH]c3cc(O)c(C(=O)N(C)c4ccc(N(C)C)cc4)cc23)c1
InChIKey: MUTZUFZAIQEATK-UHFFFAOYSA-N
Target: Heat shock protein HSP 90-alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P07900
Assay: Recombinant Hsp90 [His-Tev-huHsp90-alpha (9-236)] was immobilized on a Biacore CM5 chip at 25 degrees C and a flow rate of 30 uL/min using amine coupling at pH 4.50 according to Biacore standard protocol. Hsp90 was applied at a concentration of 20 ug/mL with 50uM 17-DMAG.Data sets were processed and analyzed using the software Biacore 4000 Evaluation. Solvent corrected and double-referenced association and dissociation phase data were fitted to a simple 1:1 interaction model with mass transport limitations
Measurement: Kd = 6.872 nM (pChEMBL 8.15999984741211)
Descriptors: MW 414.51, ALogP 4.51, HBA 4, HBD 2, PSA 72.46, QED 0.5
Compound (SMILES): CC(C)(CNC(=O)c1ncc2cc(-c3ccc(F)cc3)ccc2c1O)C(=O)O
InChIKey: XPOYEEHRTKOESN-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 22870 nM (pChEMBL 4.639999866485596)
Descriptors: MW 382.39, ALogP 3.59, HBA 4, HBD 3, PSA 99.52, QED 0.63
Compound (SMILES): CC(C)Cc1cnc(N2CCC(c3nc4c(c(C5CCC(F)(F)CC5)c3[C@@H](F)c3ccc(C(F)(F)F)cc3)[C@@H](O)CC(C)(C)C4)CC2)nc1
InChIKey: RPCFSMBCYWIYSW-ZQAZVOLISA-N
Target: Cholesteryl ester transfer protein (SINGLE PROTEIN), Homo sapiens
UniProt: P11597
Assay: CETP Inhibition Assay: A recombinant human CETP protein (manufactured by Roar Biomedical Inc.; 4.5 ng), the acceptor lipoprotein described in (2) above (32.5 ug) and 5,5'-dithio-bis-(2-nitrobenzoic acid) (7 mM, 15 ul) were taken in a 96 well plate and the total amount of the mixture was adjusted to 48.5 ul with the PBS solution. The test compound [DMSO solution (concentration: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 uM; 1.5 ul] was added to each well and the mixture was incubated in a thermostatic bath at 37° C. for 60 minutes. The reconstituted HDL (50 ul) described in (1) above was added to each well and the mixture was reacted in a thermostatic bath at 37° C. for 60 minutes. The 96 well plate was moved onto ice and a precipitation reagent [a mixed solution of magnesium chloride (60 mM) and 0.1% dextran sulfate [1/1 (v/v)]; 15 ul] was added to each well, and then the mixture was allowed to stand on ice for 15 minutes.
Measurement: IC50 = 54 nM (pChEMBL 7.269999980926514)
Descriptors: MW 688.8, ALogP 9.83, HBA 5, HBD 1, PSA 62.14, QED 0.25
Compound (SMILES): COc1cc2[nH]cc(C(=O)OCC34CCN(CC3)CC4)c2cc1OC
InChIKey: CRQRPHHTSUGZCI-UHFFFAOYSA-N
Target: Neuronal acetylcholine receptor subunit alpha-7 (SINGLE PROTEIN), Rattus norvegicus
UniProt: Q05941
Assay: Alpha-Bungarotoxin Binding Assay: Protocol 2: Studies were carried out using rat brain P2 membrane. All membrane was obtained from male Sprague-Dawley (SD) rats and collected on phosphate buffer. Protein concentration was determined using the bicinchoninic acid (BCA) protein assay reagent. In an α-Bungarotoxin binding assay, membrane was diluted with assay buffer (50 mM potassium phosphate, 1 mM pH 8.0 EDTA, 0.1 mM PMSF, 0.1% BSA). Test compounds were prepared in 100% DMSO solution. The starting concentration of the test compound was 3 μM, 8 points in 3 fold dilution. Assays were performed in a total volume of 200 μL containing [3H] α-Bungarotoxin (final concentration 0.4 nM), membrane suspension (80 μL containing 150 μg of membrane protein) and 20 μL of test compound. Non-specific binding (NSB) was determined with 1 μM α-Bungarotoxin. After 3 hours incubation (37° C.), the assay solution was transferred to a GF/B plate (Millipore). The assay was terminated by adding 100 μL ice-cold PBS dilution and then rapidly filtering over glass fiber filters presoaked in 0.5% polyethylenimine. The filters were washed 4 times with 200 μL/well of ice-cold PBS. They were then were transferred to the reading plate, and 250 μL of microscine 40 was added. The radioactivity trapped by the filters was determined by liquid scintillation counting MicroBeta.
Measurement: Ki = 2707 nM (pChEMBL 5.570000171661377)
Descriptors: MW 344.41, ALogP 2.83, HBA 5, HBD 1, PSA 63.79, QED 0.85
Compound (SMILES): CC(C)Oc1ccc(-c2nc(-c3cccc4c3CC[C@H]4N)no2)cc1C#N.Cl
InChIKey: VDVDVNSSLBNICM-GMUIIQOCSA-N
Target: Sphingosine 1-phosphate receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21453
Assay: Agonist activity at human S1P1 expressed in CRE-bla CHOK1 cells assessed as inhibition of forskolin-induced cAMP formation preincubated for 4 hrs followed by FRET-based beta-lactamase fluorescent substrate addition measured after 2 hrs
Measurement: EC50 = 0.17 nM (pChEMBL 9.770000457763672)
Descriptors: MW 360.42, ALogP 4.01, HBA 6, HBD 1, PSA 97.96, QED 0.75
Compound (SMILES): C[C@@H](Nc1ncnc2[nH]c(-c3ccc(OCCCCCC(=O)NO)cc3)cc12)c1ccccc1
InChIKey: CHPHUBQIYFZCCX-GOSISDBHSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of N-terminal GST-fused human EGFR (His672 to Ala1210 residues) expressed in baculovirus expression system using biotinylated PTP1B at Tyr66 as substrate preincubated for 5 mins followed by substrate addition measured after 30 mins by DELFIA
Measurement: IC50 <= 100 nM (pChEMBL 7)
Descriptors: MW 459.55, ALogP 5.24, HBA 6, HBD 4, PSA 112.16, QED 0.14
Compound (SMILES): FC(F)(F)c1ccc(-c2n[nH]c3ccc(-c4ncn[nH]4)cc23)cc1
InChIKey: PSLRFCGFAODRSP-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P45984
Assay: Inhibition of His6-tagged JNK2 (unknown origin) assessed as reduction in c-Jun phosphorylation using GST-c-Jun (1 to 79 residues) and [gamma32P]ATP incubated for 1 hr by scintillation counting assay
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 329.29, ALogP 4.03, HBA 3, HBD 2, PSA 70.25, QED 0.58
Compound (SMILES): COc1ccc(-c2cc(C#N)ccc2C2NC(=O)N(c3cccc(C(F)(F)F)c3)C3=C2C(=O)CC3)cn1
InChIKey: GHCLFPYSZJMPKS-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 = 21.8 nM (pChEMBL 7.659999847412109)
Descriptors: MW 504.47, ALogP 5.54, HBA 5, HBD 1, PSA 95.32, QED 0.5
Compound (SMILES): CC(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@H](C(=O)N[C@@H](CC(=O)O)C(=O)NCC(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(N)=O)C(C)C)C(C)C
InChIKey: GCNFMMYVZOHIKN-KQUOGIPMSA-N
Target: Tissue factor pathway inhibitor (SINGLE PROTEIN), Homo sapiens
UniProt: P10646
Assay: Binding affinity to TFPI (unknown origin) incubated for 1.5 hrs using tracer peptide JBT0131 by screening ELISA
Measurement: IC50 = 47 nM (pChEMBL 7.329999923706055)
Descriptors: MW 2472.84
Compound (SMILES): C[C@]1(Nc2cnc(C(F)(F)F)cn2)CCC[C@@H]1NC(=O)c1c(Cl)cccc1-n1nccn1
InChIKey: GSWFYQGXQASRJV-LIRRHRJNSA-N
Target: Orexin/Hypocretin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: O43613
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 465.87, ALogP 3.88, HBA 7, HBD 2, PSA 97.62, QED 0.59
Compound (SMILES): Cc1noc(C)c1Cn1cc(N2C(=O)N(CCc3ccc(F)cc3)C(C)(C)C2=O)cn1
InChIKey: OSJWHQZSVAOWOU-UHFFFAOYSA-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 = 330 nM (pChEMBL 6.480000019073486)
Descriptors: MW 425.46, ALogP 3.47, HBA 6, HBD 0, PSA 84.47, QED 0.56
Compound (SMILES): Cn1c(=O)cc(NC2CCN(S(=O)(=O)C(F)(F)F)CC2)c2cc(C(c3ccc(Cl)cc3)c3ccc(Cl)cc3)ccc21
InChIKey: MIVBNSWKIKESQP-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 624.51, ALogP 6.75, HBA 5, HBD 1, PSA 71.41, QED 0.24
Compound (SMILES): Cc1nc(Cl)c(-c2cc(C(=O)N3CC(c4ccc(C#N)cc4)C3)c(C)cc2C)[nH]1
InChIKey: UROMHARKWFLKDW-UHFFFAOYSA-N
Target: Fatty acid synthase (SINGLE PROTEIN), Homo sapiens
UniProt: P49327
Assay: Biochemical Assay: The FASN enzyme was isolated from SKBr3 cells. SKBr3 is a human breast cancer cell-line with high levels of FASN expression. It is estimated that FASN comprises about 25% of the cytosolic proteins in this cell line. SKBr3 cells were homogenized in a dounce homogenizer then centrifuged for 15 minutes at 4° C. to remove particulate matter. The supernatant was then analyzed for protein content, diluted to the appropriate concentration, and used to measure FASN activity. The presence of FASN was confirmed by western blot analysis. A similar method for isolation of FASN from SKBr3 cells is described in Teresa, P. et al. (Clin. Cancer Res. 2009; 15(24), 7608-7615). FASN activity of the SKBr3 cell extract was determined by measuring either NADPH oxidation or the amount of thiol-containing coenzyme A (CoA) released during the fatty acid synthase reaction. The dye CPM (7-diethylamino-3-(4'-maleimidyl-phenyl)-4-methylcoumarin) contains a thiol reactive group that increases its fluorescence emission on reaction with the sulfhydryl group of CoA.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 404.9, ALogP 4.77, HBA 3, HBD 1, PSA 72.78, QED 0.68
Compound (SMILES): CCCCNC(=O)c1c(N)sc(-c2ccc(Cl)cc2)c1CCCC
InChIKey: VPKMHIKZKYMKJH-UHFFFAOYSA-N
Target: Polyunsaturated fatty acid lipoxygenase ALOX15 (SINGLE PROTEIN), Homo sapiens
UniProt: P16050
Assay: Inhibition of human 15-LOX-1 assessed as reduction in conversion of linoleic acid to 13(S)-HpODE incubated for 10 mins followed by linoleic acid addition by UV-absorbance analysis
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 364.94, ALogP 5.52, HBA 3, HBD 2, PSA 55.12, QED 0.61
Compound (SMILES): CSc1cccc(Nc2nc(=O)n(C(C)C)c(=O)n2Cc2ccc(C)cc2)c1
InChIKey: SERAWUWTGODSSL-UHFFFAOYSA-N
Target: P2X purinoceptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P56373
Assay: Inhibition Assay: Stably expressing cell line (C6BU-1 cell transfected with human P2X3 receptor gene (GenBank accession number Y07683)) was used. The cells were seeded in a 96-well microtiter plate at a concentration of 8000 cells/well and cultured in the medium (8.3% fetal bovine serum, 8.3% horse serum, 1% antibiotic and antifungal in DMEM) for one day at 37 C. under 5% carbon dioxide atmosphere. The medium was replaced with 4 uM Fluo-3-AM solution (pH 7.5) containing 20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, 10% BSA, and 0.08% Pluronic F-127, and incubated at 37° C. under 5% dioxide carbon atmosphere for one hour. The plate was washed with washing buffer (20 mM HEPES, 137 mM NaCl, 2.7 mM KCl, 0.9 mM MgCl2, 5.0 mM CaCl2, 5.6 mM D-glucose, 2.5 mM probenecid, pH 7.5), and each well was added with 40 uL of this buffer. The plate was placed in High-Throughput Screening System FDSS 3000 (Hamamatsu Photonics K.K.).
Measurement: IC50 = 231 nM (pChEMBL 6.639999866485596)
Descriptors: MW 396.52, ALogP 3.81, HBA 7, HBD 1, PSA 68.92, QED 0.64
Compound (SMILES): CC1CC(N2CC(CC#N)(n3cc(-c4ncnc5[nH]ccc45)cn3)C2)CCN1Cc1ccc(N(C)C)c(C#N)c1F
InChIKey: ISESDXMWPTZKRT-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Inhibition of human N-terminal His-tagged JAK1 catalytic domain (837 to 1142 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 > 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 552.66, ALogP 3.88, HBA 9, HBD 1, PSA 116.69, QED 0.37
Compound (SMILES): CCN(Cc1ccc(OCCn2c(=O)ccn(C)c2=O)c(OC)c1)C(CC(=O)O)c1ccc(Cl)cc1
InChIKey: PQTNFSYWNKZSBK-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Displacement of [125I]-CXCL10 from human recombinant CXCR3 transfected in Flp-In-CHO cell membranes after 60 mins by gamma counting method
Measurement: IC50 = 440 nM (pChEMBL 6.360000133514404)
Descriptors: MW 515.99, ALogP 3.33, HBA 8, HBD 1, PSA 103, QED 0.39
Compound (SMILES): C[C@H]1CNC(=O)c2cc3ccc(C(=O)Nc4cccnc4)cc3n21
InChIKey: NDCRHTQLZWHNTR-NSHDSACASA-N
Target: Ribosomal protein S6 kinase alpha-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15349
Assay: Kinase Glo Plus Assay: Human RSK2 protein, purchased from Invitrogen, is used to measure kinase activity utilizing Kinase Glo Plus (Promega) a homogeneous assay technology, which uses a luciferin-luciferase based ATP detection reagent to quantify residual ATP. The assay is performed using 0.75 nM His-RSK2, 0.75 .mu.M ATP and 1.0 uM S6 Kinase/RSK Substrate Peptide 1 (Upstate, catalog #12-124), in assay buffer consisting of 25 mM HEPES, pH 7.5, 10 mM MgCl2, 5 mM MnCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 uM Na3VO4, 0.5 mM DTT, and 1% DMSO. Solutions of test compounds at various concentrations are prepared by 1:3 fold serial dilution of a 1 mM solution of compound in DMSO. The DMSO solutions are further diluted with assay buffer to a final concentration of DMSO of 5%. The assay is performed in a 384 well, white, non-binding plate (Corning, catalogue #3574). Solutions of test compounds (10 uL) are transferred to a dry assay plate, followed by addition of 20 uL kinase.
Measurement: IC50 = 2300 nM (pChEMBL 5.639999866485596)
Descriptors: MW 320.35, ALogP 2.59, HBA 4, HBD 2, PSA 76.02, QED 0.76
Compound (SMILES): Brc1cc(CNc2ncc(Br)c(Nc3cc(C4CC4)[nH]n3)n2)on1
InChIKey: BEVWILIPUVPGIG-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) un-phosphorylated apo-enzyme form expressed in baculovirus based expression system using poly-EY peptide incubated for 2 hrs by luciferase-luciferin coupled chemiluminescent assay
Measurement: IC50 = 500 nM (pChEMBL 6.300000190734863)
Descriptors: MW 455.11, ALogP 3.95, HBA 7, HBD 3, PSA 104.55, QED 0.52
Compound (SMILES): Nc1ncnc2c1c(-c1ccc(Oc3ccccc3)cc1)nn2C1CCCN(C(=O)/C=C/CN2CCOCC2)C1
InChIKey: MADROAFGGPUYNI-WEVVVXLNSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: HotSpot kinase assay: IC50s were determined using the in vitro HotSpot kinase assay (purified enzymes, 33P-ATP, an appropriate substrate and 1 μM ATP.). For enzyme inhibition assays, compounds were tested in range of ten concentrations from 10 uM to 0.0005 uM using purified enzymes and the Hotspot kinase assay. Reaction conditions were 1 uM ATP, one hour incubation with inhibitor, and kinase activity detected using 33-ATP phosphorylation of an appropriately selected peptide substrate.
Measurement: IC50 = 5.6 nM (pChEMBL 8.25)
Descriptors: MW 539.64, ALogP 3.92, HBA 9, HBD 1, PSA 111.63, QED 0.35
Compound (SMILES): COc1ccc(OC2CCN(c3nc4c(nc3NC(C)C)CN(C)CC4)CC2)c(F)c1
InChIKey: DFMRQAQETRMYOT-UHFFFAOYSA-N
Target: G-protein coupled receptor 6 (SINGLE PROTEIN), Homo sapiens
UniProt: P46095
Assay: GPR6 In Vitro Assay (A.2): 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 2 ug/ml doxycycline (sigma D9891) in growth media. After addition of doxycycline cells were plated at a density of 450-750 cells per well in 96-well half-volume black tissue culture plates (Costar) and placed in an incubator (37°, 5% CO2) for 20 hours prior to cAMP assays. Culture media was removed from cells and they were washed with 50 uL/well of Ringer's Buffer (MgCl2 0.047 mg/mL, NaH2PO4 0.18 mg/mL, Na2HPO4 0.1 mg/mL, KCl 0.34 mg/mL, NaHCO3 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 = 0.1402 nM (pChEMBL 9.850000381469727)
Descriptors: MW 429.54, ALogP 3.48, HBA 7, HBD 1, PSA 62.75, QED 0.75
Compound (SMILES): COc1n[nH]c2cc(NC(=O)NC(C)c3ccc(F)c(F)c3)nc(CO)c12
InChIKey: UWXLKWRIOZBVAH-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 377.35, ALogP 2.62, HBA 5, HBD 4, PSA 112.16, QED 0.55
Compound (SMILES): CCN1CCCC1CNc1nc(N2CCOCC2)[nH]c(=O)c1-c1ccc2ccccc2n1
InChIKey: FBXXLGYLCDDZBQ-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: IMAP technology (Molecular Devices): A 20 ul reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 uM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 ul of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LJL BioSystems).
Measurement: IC50 = 1600 nM (pChEMBL 5.800000190734863)
Descriptors: MW 434.54, ALogP 2.72, HBA 7, HBD 2, PSA 86.38, QED 0.62
Compound (SMILES): NC(=O)c1ccc(N2CC[C@@](N)(C(=O)Nc3ccc(F)c(C(F)(F)F)c3)C2)c2cncnc12
InChIKey: XGDOTBLDLALKSZ-FQEVSTJZSA-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 = 520 nM (pChEMBL 6.28000020980835)
Descriptors: MW 462.41, ALogP 2.43, HBA 6, HBD 3, PSA 127.23, QED 0.51
Compound (SMILES): Cc1cc(N)nc(C)c1CNC(=O)c1cnn(Cc2ccc3ccn(C)c3c2)c1
InChIKey: MHLALIQKGDASOX-UHFFFAOYSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In-vitro Plasma Kallikrein Inhibition: Enzymatic reactions were conducted in â¿¿assay bufferâ¿¿, comprising 50 mM Hepes/NaOH at pH 7.8, 150 mM NaCl, 1 mM EDTA and 0.05% (w/v) CHAPS. For the determination of IC50 values, the assays were performed at room temperature in 384-well plates with a total assay volume of 25.25 ul per well. The test compound was dissolved in 90% (v/v) DMSO/water. For the assays, 250 nL of the 90% (v/v) DMSO/water solution or compound solution were added per well, followed by the addition of 12.5 ul protease solution (protease in assay buffer). The final assay concentration of the human plasma kallikrein was nominally 25 pM, the 11 compound concentrations in the dilution series were in the range form 1 nM to 100 uM. After 1 hour of pre-incubation at room temperature, the reactions were started by the addition of 12.5 ul substrate solution (in assay buffer, final assay concentration was 0.5 uM). After the addition of the substrate solution, the final DMSO concentration in the assay was 0.9% (v/v).
Measurement: IC50 = 46 nM (pChEMBL 7.340000152587891)
Descriptors: MW 388.48, ALogP 2.95, HBA 6, HBD 2, PSA 90.76, QED 0.55
Compound (SMILES): C=Cc1c(N)ncnc1N1CCN(C(CN)c2ccc(C(F)(F)F)cc2)CC1
InChIKey: CRRPJTULCQOFOC-UHFFFAOYSA-N
Target: Ribosomal protein S6 kinase beta-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23443
Assay: Enzyme Assay: P70S6K inhibitor compounds were diluted and plated in 96 well plates. A reaction mixture including the following components was then added to the compound plate to initiate the enzyme reaction; P70S6K (3 nM, T412E mutant, Millipore) was mixed with 24 μM ATP in an assay buffer containing 100 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM DTT, 0.015% Brij and 1 μM of the substrate peptide FITC-AHA-AKRRRLSSLRA-OH (derived from the S6 ribosomal protein sequence, FITC=fluorescein isothiocyanate, AHA=6-aminohexanoic acid). The reaction was incubated for 90 min at 25° C., before the addition of 10 mM EDTA to stop the reaction.
Measurement: IC50 = 19 nM (pChEMBL 7.71999979019165)
Descriptors: MW 392.43, ALogP 2.54, HBA 6, HBD 2, PSA 84.3, QED 0.81
Compound (SMILES): COc1ccc(CN2C(=O)N(c3cnn(Cc4c(C)noc4C)c3)C(=O)C23CC3)cc1O
InChIKey: IRCZSPSICVPEKJ-UHFFFAOYSA-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 = 20 nM (pChEMBL 7.699999809265137)
Descriptors: MW 437.46, ALogP 2.75, HBA 8, HBD 1, PSA 113.93, QED 0.59
Compound (SMILES): Cn1c(=O)n(CC(C)(C)C)c2ccc(C3=CC4CCC3CN4C(=O)c3ncco3)nc21
InChIKey: GMULSUZDWRYCMT-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Fluorescence Laser Imaging Plate Reader (FLIPR) Based Assay: The compounds of the present invention may be tested in a fluorescence laser imaging plate reader (FLIPR) based assay. This assay is a common functional assay to monitor Ca2+ mobilization in whole cells expressing recombinant receptor coupled with a promiscuous G-protein. CHO dhfr-cells stably expressing recombinant human mGluR2 and Gα16 loaded with Fluo-4 AM (Invitrogen, Carlsbad Calif.) are treated with dose responses of compounds and the Ca2+ response is monitored on a FLIPR384 (Molecular Devices, Sunnydale Calif.) for agonist activity. The potentiation response is monitored after a subsequent addition of an EC20 concentration of glutamate (900 nM).
Measurement: EC50 = 22.79 nM (pChEMBL 7.639999866485596)
Descriptors: MW 421.5, ALogP 3.09, HBA 7, HBD 0, PSA 86.16, QED 0.65
Compound (SMILES): COCc1ccc(C2=C(c3nn[nH]n3)C(=O)NC(c3ccc(OCCCC(F)(F)F)cc3)(C(F)(F)F)C2)cc1
InChIKey: AWKCANMQAVTPER-UHFFFAOYSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: MGAT LCMS Assay: The MGAT enzyme reactions were performed in CORNING Falcon 96-well Polypropylene plates, in a total volume of 60 uL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 uM 2-oleoylglycerol, 15 uM oleoyl-Coenzyme A and 0.0013 ug/uL Human or Mouse MGAT-2 or 0.0026 ug/uL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 uL of ice cold methanol containing 1 ug/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50x2.1 mm column, was used for the chromatography of enzyme products.
Measurement: IC50 = 89 nM (pChEMBL 7.050000190734863)
Descriptors: MW 555.48, ALogP 4.96, HBA 6, HBD 2, PSA 102.02, QED 0.29
Compound (SMILES): Cc1c(-c2cccc(C(F)(F)F)c2)c(=O)c(C(=O)NCc2ccc(S(C)(=O)=O)cn2)cn1CC(C)C
InChIKey: UVBOGTQCKVSVCZ-UHFFFAOYSA-N
Target: Neutrophil elastase (SINGLE PROTEIN), Homo sapiens
UniProt: P08246
Assay: Human Neutrophil Elastase Assay: 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. 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 Î¼L 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 = 30 nM (pChEMBL 7.519999980926514)
Descriptors: MW 521.56, ALogP 4.23, HBA 6, HBD 1, PSA 98.13, QED 0.5
Compound (SMILES): Cc1ccc(-n2nc(C3CN(S(=O)(=O)C4CC4)C3)cc2NC(=O)c2cnn3cccnc23)cc1
InChIKey: DDEVBDDPCNILDX-UHFFFAOYSA-N
Target: Interleukin-1 receptor-associated kinase 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NWZ3
Assay: Kinase Assay: The kinase activity of IRAK-4 is determined by its ability to catalyze the phosphorylation of a fluorescent polypeptide substrate. The extent of phosphorylation is measured using the IMAP technology (Molecular Devices) where the phosphorylated fluorescent substrate binds to the large M(III)-based nanoparticles which reduces the rotational speed of the substrate and thus increases its fluorescent polarization (FP). 20 μl reaction mixture contains 10 mM TriHCl, pH 7.2, 0.5 nM GST tagged IRAK-4 (SignalChem), 100 nM fluorescent peptide substrate (RP7030, Molecular Devices), 100 μM ATP, 1 mM DTT, 1 mM MgCl2, and 0.01% Tween 20. The reaction is initiated by the addition of ATP. After incubation for 30 minutes at 25° C., 60 μl of Progressive IMAP Reagent (Molecular Devices) is added to stop the reaction. Change in RP7030's FP is determined by a FP reader (Analyst HT, LH, BioSystems).
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 477.55, ALogP 2.37, HBA 8, HBD 1, PSA 114.49, QED 0.46
Compound (SMILES): O=C(Nc1cccc(OCC(O)CN2CCc3ccccc3C2)c1)c1cncnc1
InChIKey: COGWFSGRMVMLNG-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 1001 nM (pChEMBL 6)
Descriptors: MW 404.47, ALogP 2.53, HBA 6, HBD 2, PSA 87.58, QED 0.63
Compound (SMILES): N#Cc1nc(C(=O)NCC(C(=O)O)C2CC2)c(O)c2ccc(Oc3ccccc3F)cc12
InChIKey: WRHGNOBRJVLDQS-UHFFFAOYSA-N
Target: Prolyl hydroxylase EGLN2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96KS0
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 750 nM (pChEMBL 6.119999885559082)
Descriptors: MW 435.41, ALogP 3.58, HBA 6, HBD 3, PSA 132.54, QED 0.52
Compound (SMILES): CCC(C)CNC(=O)c1ccc(NC(=O)N2Cc3ccccc3C2)cc1
InChIKey: ZLOZQNWQYQWSHJ-UHFFFAOYSA-N
Target: Nicotinamide phosphoribosyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: P43490
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Binding Assay : Probe 1: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-1251667.0. 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 = 28.4 nM (pChEMBL 7.550000190734863)
Descriptors: MW 351.45, ALogP 4.01, HBA 2, HBD 2, PSA 61.44, QED 0.85
Compound (SMILES): CC1CN(C(=O)c2cc(Cc3n[nH]c(=O)c4ccccc34)ccc2F)C(Cc2ccccc2)c2nnc(C(F)(F)F)n21
InChIKey: NONXRXUOTXVDIP-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: ELISA Assay: The HTb-PARP1 positive clones were obtained using the full-length PARP1 plasmid, through PCR amplification, enzyme digestion, ligation, and transformation into DH5a. The plasmids were extracted and determined by enzyme cleavage, and then transformed into DH10Bac. Bacmid/PARP is determined by PCR and sequencing. TNI was transfected, the viruses were collected, and cells were lysed. PARP1 protein was purified by affinity chromatography and determined by Western blotting. A plate was coated by substrate histone, NAD+ and DNA, as well as expressed PARP1 enzyme, was placed into 96-well plate reaction system. Various reaction conditions were optimized and ultimately determined.
Measurement: IC50 = 310 nM (pChEMBL 6.510000228881836)
Descriptors: MW 576.55, ALogP 5.26, HBA 6, HBD 1, PSA 96.77, QED 0.29
Compound (SMILES): CN1CCC[C@H]1C(=O)N1CC[C@@]2(C)c3cccc(O)c3C[C@@H]1C2(C)C
InChIKey: XVVLUMSQAZOVPZ-CDXJDZJCSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: Homogeneous Time-Resolved Fluorescence (HTRF): In vitro inhibition of 11beta -HSD1 by test compounds was determined with HTRF (Homogeneous Time-Resolved Fluorescence) technology (cisbio international, France) detecting cortisol generated from cortisterone by human liver microsomes. Briefly, compounds were incubated for 1 hour at 37 C. in Tris buffer (20 mM tris, 5 mM EDTA, pH 6.0) containing NADPH (200 uM) and cortisone (80 nM). Cortisol generated in the reaction is then detected with a competitive immunoassay, involving two HTRF conjugates: cortisol linked to XL665 and anti-cortisol antibody labeled with Europium cryptate. The incubation period for detection reaction was typically 2 hours. The amount of cortisol is determined by reading the time-resolved fluorescence of the wells (Ex 320/75 nm; Em 615/8.5 nm and 665/7.5 nm). The ratio of the two emission signals is then calculated (Em665*10000/Em615).
Measurement: IC50 = 6036 nM (pChEMBL 5.21999979019165)
Descriptors: MW 342.48, ALogP 2.93, HBA 3, HBD 1, PSA 43.78, QED 0.85
Compound (SMILES): CN1C[C@@H](c2cnc(N)c(-c3ccc(C(=O)N[C@H](CO)c4cccc(Cl)c4)c(F)c3)c2)CC1=O
InChIKey: ULFXLAIDIKUMBB-HTAPYJJXSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Kinase Assay (New): Compound potency against activated ERK2 is determined using a kinase assay that measures ERK2-catalyzed phosphorylation of biotinylated ERKtide peptide substrate ([Biotin]-AHA-K-R-E-L-V-E-P-L-T-P-S-G-E-A-P-N-Q-A-L-L-R-[NH2], the peptide sequence derived from EGF receptor: SEQ ID NO:1). The assay is carried out in 50 mM HEPES [pH 7.5], 5 mM MgCl2, 1 mM DTT, 0.01% Tween-20, 0.05% BSA using 0.25 nM ERK2, 200 nM ERKtide peptide and 35 uM ATP (all concentrations are final in the reaction) in a total volume of 10.25 uL. A 16-point, half-log dilution series of compounds at 41x final concentration is used for generating IC50 curves. Compound dilution series are prepared in 100% DMSO. ERK2 is preincubated with compounds for 30 minutes at ambient temperature. Reaction is initiated by addition of a substrate cocktail of ERKtide peptide and ATP and is allowed to proceed for 2-3 hours at ambient temperature. Reaction is terminated by addition of 10 uL of a 2x stop buffer consisting of 100 mM Tris-Cl [pH 7.5], 25 mM EDTA, 0.01% Tween 20, 10 ug/mL of AlphaScreen Protein A Acceptor Beads, 10 ug/mL of Streptavidin Donor Beads (PerkinElmer, Waltham, Mass.), and 1.4 ug/mL phospho-EGF Receptor (Thr669) antibody (Cat #3056, Cell Signaling Technology, Danvers, Mass.). Terminated reactions are read, after overnight incubation in the dark, on an EnVision Multilabel Plate Reader (PerkinElmer, Waltham, Mass.), with excitation and emission wavelengths set to 680 nm and 570 nm, respectively. IC50 values are determined using a four-parameter fit.
Measurement: IC50 = 5.32 nM (pChEMBL 8.270000457763672)
Descriptors: MW 482.94, ALogP 3.53, HBA 5, HBD 3, PSA 108.55, QED 0.5
Compound (SMILES): Cc1ccc(C2=C(C(=O)NS(=O)(=O)c3ccccc3)C(=O)N[C@@](c3ccc(OCCCC(F)(F)F)cc3)(C(F)(F)F)C2)cc1
InChIKey: UUBTZSFRZTYOLM-NDEPHWFRSA-N
Target: Alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase (SINGLE PROTEIN), Homo sapiens
UniProt: Q10469
Assay: LCMS Assay: The MGAT enzyme reactions were performed in Corning FALCON® 96-well Polypropylene plates, in a total volume of 60 μL of 50 mM Potassium Phosphate buffer pH 7.4, containing a final concentration of 100 μM 2-oleoylglycerol, 15 μM oleoyl-Coenzyme A and 0.0013 μg/μL Human or Mouse MGAT-2 or 0.0026 μg/μL Rat recombinant MGAT-2 membranes expressed in Sf9 cells. Assay plates were run through a fully automated robotics system and shaken for 5 seconds every minute for a total 10 minutes. The reactions were then quenched with 120 μL of ice cold methanol containing 1 μg/mL 1,2-distearoyl-rac-glycerol as the internal standard. Plates were shaken for 2 minutes and spun down to remove protein precipitation. After the spin, samples were transferred to LC/MS compatible PCR plates. For LC/MS analysis, a ThermoFisher Surveyor pump, utilizing a Waters Symmetry C8, 50×2.1 mm column, was used for the chromatography of enzyme products. The buffer system consists of 0.1% formic acid in water with a mobile phase consisting 0.1% formic acid in methanol. The shallow gradient is 90-100% mobile phase in 0.2 min with a total run time of 2.3 min. The first 0.5 minutes of each injection was diverted to waste to eliminate the presence of Phosphate buffer in the enzymatic reaction. The column was run at 0.6 mL/min and a temperature of 65° C.
Measurement: IC50 = 84 nM (pChEMBL 7.079999923706055)
Descriptors: MW 640.6, ALogP 5.95, HBA 5, HBD 2, PSA 101.57, QED 0.17
Compound (SMILES): CNC(=O)C(NC(=O)Nc1cc2[nH]nc(OC)c2cn1)c1ccc(F)cc1
InChIKey: XDJVZARKNNAANW-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: IMAP-FP Assay: Activated ERK2 activity was determined in an IMAP-FP assay (Molecular Devices). Using this assay format, the potency (IC50) of each compound was determined from a 10 point (1:3 serial dilution, 3 uM starting compound concentration) titration curve using the following outlined procedure. To each well of a black Corning 384-well plate (Corning Catalog #3575), 7.5 nL of compound (3333 fold dilution in final assay volume of 25 uL) was dispensed, followed by the addition of 15 uL of kinase buffer (tween containing kinase buffer, Molecular Devices) containing 0.0364 ng/mL (0.833 nM) of phosphorylated active hERK2 enzyme. Following a 15 minute compound & enzyme incubation, each reaction was initiated by the addition of 10 uL kinase buffer containing 2.45 uM ERK2 IMAP substrate peptides (2.25 uM-unlabeled IPTTPITTTYFFFK-COOH and 200 nM-labeled IPTTPITTTYFFFK-SFAM (5-carboxyfluorescein)-COOH), and 75 uM ATP.
Measurement: IC50 = 276.8 nM (pChEMBL 6.559999942779541)
Descriptors: MW 372.36, ALogP 1.71, HBA 5, HBD 4, PSA 121.03, QED 0.54