lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1nsc(-c2c(NC(=O)C3=C(C(=O)O)CCC3)sc(C3CC3)c2C)n1
InChIKey: PPVLCUXVOGWJMY-UHFFFAOYSA-N
Target: Fatty acid-binding protein, adipocyte (SINGLE PROTEIN), Homo sapiens
UniProt: P15090
Assay: Time Resolved-Fluorescence Energy Transfer (TR-FRET) Assay: Stock DMSO solutions (1.8 mM) of compounds were serially diluted 3-fold for ten concentrations with 100% DMSO (50 μM to 0.003 μM final compound concentration). 1 μl of these compound dilutions and 1 μl of Bodipy labeled fatty acid 4.5 μM in 100% DMSO (Bodipy FL C11, cat. no. D3862, Invitrogen) were sequentially pipetted in wells of 384-well black polypropylene plates (Thermo Matrix cat. no. 4344). FABP4 or FABP5 protein was then added (28 μl of 64 nM protein in 25 mM Tris pH 7.5, 0.4 mg/ml α-globulin, 1 mM DTT, 0.012% NP40, final protein concentration: 50 nM). Assay blanks contained ligand, but no protein. Neutral controls contained ligand, but no compound. After adding the detection reagent (Tb antiHis6 antibody, Columbia Biosciences, TB-110, 6 μl of a 24 nM Ab solution in 25 mM Tris pH 7.5, 0.4 mg/ml γ-globulin, final Tb antiHis6 Ab concentration: 4 nM), plates were spun one minute at 1000 rpm.
Measurement: IC50 = 22 nM (pChEMBL 7.659999847412109)
Descriptors: MW 389.5, ALogP 4.26, HBA 6, HBD 2, PSA 92.18, QED 0.8
Compound (SMILES): COC(=O)CCN/C=C1\C(=O)C(O)=C(C(C)C)c2cc(C)c(-c3c(C)cc4c(c3O)/C(=C/NCCC(=O)OC)C(=O)C(O)=C4C(C)C)c(O)c21
InChIKey: JELQEMUXDDRKOL-MPKYGIEOSA-N
Target: Apoptosis regulator Bcl-2 (SINGLE PROTEIN), Homo sapiens
UniProt: P10415
Assay: Inhibition of FAM-Bid binding to human BCL2 expressed in Escherichia coli BL21 after 30 mins by fluorescence polarization assay
Measurement: IC50 = 286 nM (pChEMBL 6.539999961853027)
Descriptors: MW 688.77, ALogP 5.38, HBA 12, HBD 6, PSA 191.72, QED 0.1
Compound (SMILES): Oc1cccc(O)c1-c1ccc(-c2cc3nc(O[C@@H]4CO[C@H]5[C@@H]4OC[C@H]5O)[nH]c3cc2Cl)cc1
InChIKey: MMOYHXCYJWHUCI-LUGTWXOSSA-N
Target: AMPK alpha2/beta2/gamma3 (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9UGI9
Assay: Activation of human recombinant AMPK alpha2/beta2/gamma3 expressed in African green monkey COS7 cells assessed as increase in biotinylated AAC (1 to 120 residues) peptide phosphorylation at Ser-79 residue measured after 60 mins by Alphascreen assay
Measurement: EC50 = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 480.9, ALogP 3.87, HBA 7, HBD 4, PSA 117.06, QED 0.35
Compound (SMILES): CCN(C)C(=O)c1cnn(C)c1C(=O)Nc1ccc2nc(N(C)C)nn2c1
InChIKey: CXCNRMXSFMDZLI-UHFFFAOYSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: Inhibition Assay: The human PDE10A full length assay was performed in 96-well micro titer plates. The reaction mixture of 50 ul contained 20 mM HEPES pH=7.5/10 mM MgCl2/0.05 mg/ml BSA (Sigma cat. # A-7906), 50 nM cGMP (Sigma, cat. # G6129) and 50 nM [3H]-cGMP (GE Healthcare, cat. # TRK392 S.A. 13.2 Ci/mmol), 3.75 ng/well PDE10A enzyme (Enzo Life Science, Lausen, Switzerland cat # SE-534) with or without a specific test compound. A range of concentrations of the potential inhibitor was used to generate data for calculating the concentration of inhibitor resulting in 50% of the effect (e.g. IC50, the concentration of the competitor inhibiting PDE10A activity by 50%). Non-specific activity was tested without the enzyme. The reaction was initiated by the addition of the substrate solution (cGMP and [3H]-cGMP) and allowed to progress for 20 minutes at room temperature. The reaction was terminated by adding 25 ul of YSi-SPA scintillation beads (GE Healthcare, cat. # RPNQ0150) in 18 mM zinc sulphate.
Measurement: IC50 = 33.5 nM (pChEMBL 7.46999979019165)
Descriptors: MW 370.42, ALogP 0.87, HBA 8, HBD 1, PSA 100.66, QED 0.72
Compound (SMILES): N#Cc1cc(-c2ccnc(Nc3cnn(CCN4CCOCC4)c3)n2)cnc1N1CC[C@H](F)C1
InChIKey: KFUBANNOQZGHJW-IBGZPJMESA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit epsilon (SINGLE PROTEIN), Homo sapiens
UniProt: Q14164
Assay: Time-Resolved Fluorescence-based Lanthascreen Assay: Kinase inhibition assays (10 μL) were performed at 20° C. in 384-well plate format. Compound IC50 values were determined at the apparent Km for ATP (20 μM) based on a radiometric assay (Invitrogen) using 8 or 10 point curves in duplicate. The final reaction conditions contained 400 nM fluorescein-IkBα substrate (DRHDSGLDSMKDE), 20 μM ATP, 2 nM or 8 nM IKKε or TBK1 kinase respectively, and 3% DMSO in kinase assay buffer consisting of 50 mM HEPES (pH 7.5), 10 mM MgCl, 1 mM EGTA, 0.01% Brij-35.Compound dilutions were prepared from 10 mM DMSO stocks by dilution into DMSO. Compound dilution series were further diluted in kinase assay buffer to give a 12% DMSO stock, the final concentration in the assay being 3% DMSO.The kinase phosphorylation assay was initiated by the addition of the kinase and the reaction was allowed to proceed for 1 h or 2.5 h for IKKε and TBK-1 kinase respectively.
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 463.52, ALogP 2.23, HBA 10, HBD 1, PSA 108.02, QED 0.56
Compound (SMILES): Oc1ccc(-n2cc(-c3ccc4cc(OCCOCCN5CCOCC5)ccc4n3)nn2)cc1F
InChIKey: GUBLWMMOACBZJA-UHFFFAOYSA-N
Target: Macrophage migration inhibitory factor (SINGLE PROTEIN), Homo sapiens
UniProt: P14174
Assay: Inhibition of Tautomerase Activity of Human MIF: Inhibition of the tautomerase activity of MIF was measured using 4-hydroxyphenyl pyruvic acid (HPP) as substrate, largely following previously reported protocols (Taylor et al., Biochemistry, 1999, 38, 7444-7452). HPP was dissolved in 0.5 M acetate buffer, pH 6.0 to a final concentration of 10 mM and incubated overnight at room temperature to allow equilibration of the keto and enol forms. MIF (6 μL) was premixed in 500 mM boric acid, pH 6.2 (142 μL) and transferred to a transparent U bottom 96-well plate to a final concentration of 50 nM MIF. At this concentration high signal-to-noise and linearity were observed after analysis of progress curves for enol production at different protein concentrations. Inhibitors were dissolved in DMSO to 10 mM and an initial screen was performed. For compounds that showed ca. 25% or greater inhibition at 10 μM, an inhibition constant, was measured.Compounds were placed into wells (2 μL) at 6 different concentrations and incubated for 30 minutes until the assay was started by addition of HPP (504) at two concentrations (1.0 and 2.5 mM). The negative control was MIF incubated with DMSO vehicle, which in all assays was 1% and did not influence tautomerase activity. MIF activity was monitored at 305 nm for formation of the borate-enol complex using an Infinite F500 plate reader (TECAN, Morrisville, N.C.) for 175 seconds. Calculation of initial velocities and the nonlinear regression analyses for the enzyme kinetics were repeated three times with the program Prism6 (GraphPad, La Jolla, Calif.).Data obtained for the Example compounds, obtained using the methods described in Example B, are provided in Table 3. Results are also included for a reference compound ISO-1 (Chang, K. F.; Al-Abed, Y. Bioorg. Med. Chem. Lett. 2006, 16, 3376-3379; Balachandran, S. et al. Bioorg. Med. Chem. Lett. 2009, 19, 4773-4776). X-ray crystal structures were obtained for complexes of 3a, 3b, and 3v (Examples 1, 2, and 21) with human MIF as described below in Example C, which confirm the binding of these inhibitors in the tautomerase active site.
Measurement: Ki = 150 nM (pChEMBL 6.820000171661377)
Descriptors: MW 479.51, ALogP 3.05, HBA 9, HBD 1, PSA 94.76, QED 0.37
Compound (SMILES): Cc1c(S(=O)(=O)NCc2cccc(C(F)(F)F)c2)cc(C(C)(C)C)n1CCCN1CCNCC1
InChIKey: CHBZBSIFZDPMTE-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: Ki = 3100 nM (pChEMBL 5.510000228881836)
Descriptors: MW 500.63, ALogP 3.89, HBA 5, HBD 2, PSA 66.37, QED 0.58
Compound (SMILES): CC(C)Oc1ccc(S(=O)(=O)N2CCC(Oc3ccccc3CCC(=O)O)CC2)cc1
InChIKey: KNYHEFOHOZZIHG-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q13258
Assay: Antagonist activity at PGD2 receptor in human platelets assessed as inhibition of PGD2-mediated cAMP accumulation preincubated for 5 mins followed by PGD2 addition measured after 2 mins by HTRF assay
Measurement: IC50 = 3000 nM (pChEMBL 5.519999980926514)
Descriptors: MW 447.55, ALogP 3.72, HBA 5, HBD 1, PSA 93.14, QED 0.63
Compound (SMILES): O=C(NC1CCCCC1)c1cccc(-c2n[nH]c3ccc(-c4nc[nH]n4)cc23)c1
InChIKey: DUXMGDMPXNIVPY-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 <= 500 nM (pChEMBL 6.301000118255615)
Descriptors: MW 386.46, ALogP 4.08, HBA 4, HBD 3, PSA 99.35, QED 0.49
Compound (SMILES): CCNC(=O)NC1=C(c2ccc(OCC(F)F)cc2)CC(c2ccc(OCCCC(F)(F)F)cc2)(C(F)(F)F)NC1=O
InChIKey: QBRPSHXYMWRQCJ-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 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 609.51, ALogP 6.06, HBA 4, HBD 3, PSA 88.69, QED 0.21
Compound (SMILES): CC(Nc1ncnc2[nH]cnc12)c1cc2ccccc2c(=O)n1C1CCN(C)CC1
InChIKey: COMRTFBDTCPYAT-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length p85alpha expressed in baculovirus infected Sf21 cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 1000 nM (pChEMBL 6)
Descriptors: MW 403.49, ALogP 3.11, HBA 7, HBD 2, PSA 91.73, QED 0.54
Compound (SMILES): CCn1nc(-c2cccc(-c3ccc(O)nc3)c2)cc(NC(=O)Nc2c(Cl)cncc2Cl)c1=O
InChIKey: AEZSONULEVONJD-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Inhibition of human recombinant PDE4B1 assessed as reduction in [3H]cAMP hydrolysis to [3H]AMP incubated for 60 mins by PDE-SPA assay
Measurement: IC50 = 0.38 nM (pChEMBL 9.420000076293945)
Descriptors: MW 497.34, ALogP 5.04, HBA 7, HBD 3, PSA 122.03, QED 0.36
Compound (SMILES): CCCCCCCCCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)B1O[C@@H]2C[C@@H]3C[C@@H](C3(C)C)[C@]2(C)O1)[C@H](C)O
InChIKey: DFHZYIDNBPWGQT-FDSSAQMGSA-N
Target: Signal peptidase I (SINGLE PROTEIN), Escherichia coli K-12
UniProt: P00803
Assay: Inhibition of full length Escherichia coli His6-tagged LepB expressed in Escherichia coli C43(DE3) using dabcyl-VEVGGTATAGAFSRPGLE-(EDANS) as substrate preincubated for 10 mins followed by substrate addition measured for 2 hrs by FRET assay
Measurement: IC50 = 48 nM (pChEMBL 7.320000171661377)
Descriptors: MW 760.78
Compound (SMILES): CC1(C)[C@H]2CC[C@@H](CCN3[C@@H]4CCC[C@H]3C[C@@H](n3c(=O)c(N5CC[C@H]5C(=O)O)nc5ccccc53)C4)[C@@H]1C2
InChIKey: OPNHKXQPNQNQOK-JEIKLXHSSA-N
Target: Opioid growth factor receptor-like protein 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5TC84
Assay: In Vitro ORL-1 Receptor Binding Assay: Radioligand binding assays (screening and dose-displacement) used 0.1 nM [3H]-nociceptin (NEN; 87.7 Ci/mmole) with 10-20 μg membrane protein in a final volume of 500 μL binding buffer (10 mM MgCl2, 1 mM EDTA, 5% DMSO, 50 mM HEPES, pH 7.4). Non-specific binding was determined in the presence of 10 nM unlabeled nociceptin (American Peptide Company). All reactions were performed in 96-deep well polypropylene plates for 1 h at about 25 °C. Binding reactions were terminated by rapid filtration onto 96-well Unifilter GF/C filter plates (Packard) presoaked in 0.5% polyethylenimine (Sigma-Aldrich). Harvesting was performed using a 96-well tissue harvester (Packard) followed by three filtration washes with 500 μL ice-cold binding buffer. Filter plates were subsequently dried at 50 °C. for 2-3 hours. Fifty μL/well scintillation cocktail (BetaScint; Wallac) was added and plates were counted in a Packard Top-Count for 1 min/well.
Measurement: Ki = 23.6 nM (pChEMBL 7.630000114440918)
Descriptors: MW 518.7, ALogP 5.08, HBA 6, HBD 1, PSA 78.67, QED 0.57
Compound (SMILES): CCN(Cc1ccncc1)c1ccc(C(=O)Nc2cc(-c3ccccc3)nn2C)nn1
InChIKey: MUOAPSYAPDUUDM-UHFFFAOYSA-N
Target: Autotaxin (SINGLE PROTEIN), Homo sapiens
UniProt: Q13822
Assay: FS-3 Assay: The FS-3 assay to identify ATX inhibitors was preformed as follows: 3 μl of standard inhibitor and test compounds were added to an assay plate. To each assay well, containing test compounds or standard, 24 μl of human Autotaxin enzyme (2 nM) was added. The assay plate was then centrifuged at 1000 rpm for 1 minute and allowed to incubate at 37° C. for 30 minutes. Following the incubation period each plate was read in a fluorescence plate reader (Spectra Max M5: excitation: 494 nm and emission: 520 nm) and IC50 values were derived from inhibition of FS-3 fluorescence.
Measurement: IC50 = 262 nM (pChEMBL 6.579999923706055)
Descriptors: MW 413.49, ALogP 3.55, HBA 7, HBD 1, PSA 88.83, QED 0.5
Compound (SMILES): O=S(=O)(Nc1nccs1)c1ccc2c(c1)OCCN2Cc1ccccc1
InChIKey: BSLPKPPAMRXZEA-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: In Vitro Assay: HEK 293 cells stably transfected with either Nav 1.7 or with Nav 1.5 were recorded in population patch-clamp mode with the IonWorks® Quattro automated electrophysiology system in accordance with the manufacturer's specifications (Molecular Devices, LLC, Sunnyvale, Calif.). Sodium channel currents were measured in response to a train of depolarizations that induced successively greater inactivation. Cells were held at −110 mV for three seconds (Nav 1.7) or half a second (Nav 1.5) from a holding voltage of −15 mV, then put through a series of 26 pulses of 150 msec duration to −20 mV at a frequency of 5 Hz. Cells were then left unclamped for a period of 3 to 8 minutes while a single concentration of test compound was added. Cells were then reclamped and put through the same voltage protocol. Current at the end of the 26th pulse to −20 mV was subtracted from the peak current evoked by the 26th pulse to −20 mV to correct for leak current.
Measurement: IC50 = 864 nM (pChEMBL 6.059999942779541)
Descriptors: MW 387.49, ALogP 3.34, HBA 6, HBD 1, PSA 71.53, QED 0.73
Compound (SMILES): O=C(Nc1cnn(S(=O)(=O)c2ccc(Cl)cc2F)c1)c1ccccn1
InChIKey: RPIJRXONNHCMCR-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14833
Assay: Glutamate Receptor Activity: The disclosed compounds and compositions can be evaluated for their ability to act as a potentiator of metabotropic glutamate receptor activity, in particular mGluR4 activity, by any suitable known methodology known in the art. For example, Chinese Hamster Ovary (CHO) cells transfected with human mGluR4 or HEK cells co-transfected with rat mGluR4 and the G-protein regulated Inwardly Rectifying Potassium channel (GIRK) were plated in clear bottom assay plates for assay in a Hamamatsu FDSS Fluorometric Plate Reader. The cells were loaded with either a Ca2+-sensitive fluorescent dye or the thallium responsive dye and the plates were washed and placed into a suitable kinetic plate reader. For human mGluR4 assays, a fluorescence baseline was established for 3-5 seconds, the disclosed compounds were then added to the cells, and the response in cells was measured.
Measurement: EC50 = 128 nM (pChEMBL 6.889999866485596)
Descriptors: MW 380.79, ALogP 2.56, HBA 6, HBD 1, PSA 93.95, QED 0.75
Compound (SMILES): Cc1nc(CC(=O)Nc2cc(C(=O)c3cn(C(C)C)c4ccncc34)ccn2)c(C2CC2)o1
InChIKey: KELCKDDHLORCJU-UHFFFAOYSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: HTRF Assay: Isolated TRK Enzyme assays use the HTRF KinEASE-TK kit (Cisbio Cat#62TK0PEJ) with recombinant His-tagged cytoplasmic domains of each TRK receptor sourced from Invitrogen (see table below). This activity-assay measures the phosphorylation of tyrosine residues within a substrate from the HTRF kit which has been validated by Cisbio for a variety of tyrosine kinases including the TRK receptors.Assay Details: Invitrogen Amino FAC FAC Assay ReactionTarget Cat# acids enzyme ATP TimeTRKA PV3144 aa 441-4 nM 40 uM 35 min (NTRK1) 796 TRKB PV3616 aa 526-1 nM 1.4 uM 40 min (NTRK2) 838 TRKC PV3617 aa 510- 10 nM 15 uM 30 min (NTRK3) 8250.5 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135 disclosed in WO2005/116035 of 150 uM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 uM.
Measurement: IC50 = 51.4 nM (pChEMBL 7.289999961853027)
Descriptors: MW 443.51, ALogP 4.6, HBA 7, HBD 1, PSA 102.91, QED 0.42
Compound (SMILES): COc1ccc(C2=NN(C3CCN(C(=O)CN4C(=O)CCC4=O)CC3)C(=O)C2(C)C)cc1OCC1CC1
InChIKey: WSNNMTCJCWWBKA-UHFFFAOYSA-N
Target: 3',5'-cyclic-AMP phosphodiesterase 4B (SINGLE PROTEIN), Homo sapiens
UniProt: Q07343
Assay: Scintillation Proximity Assay (SPA): PDE4B1 activity was inhibited by the compounds according to the invention in a modified SPA (scintillation proximity assay) test, supplied by Amersham Biosciences (see procedural instructions phosphodiesterase [3H]cAMP SPA enzyme assay, code TRKQ 7090), carried out in 96-well microtitre plates (MTP's). The test volume is 100 ul and contains 20 mM Tris buffer (pH 7.4), 0.1 mg/ml of BSA, 5 mM Mg2+, 0.5 uM cAMP (including about 50,000 cpm of [3H]cAMP), 1 ul of the respective substance dilution in DMSO and sufficient recombinant PDE (1000x g supernatant, see above) to ensure that 10-20% of the cAMP is converted under the said experimental conditions. The final concentration of DMSO in the assays (1% v/v) does not substantially affect the activity of the PDE investigated. After a preincubation of 5 min at 37° C., the reaction is started by adding the substrate (cAMP) and the assays are incubated for a further 15 min.
Measurement: IC50 = 70.8 nM (pChEMBL 7.150000095367432)
Descriptors: MW 510.59, ALogP 2.2, HBA 7, HBD 0, PSA 108.82, QED 0.5
Compound (SMILES): CC1(C)CC2=NN(CCO)C(=O)c3cccc4c3c2c(n4CCO)C1
InChIKey: OZDPEWPAHUWNKA-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: PARP-1 Enzymatic Assay: PARP-1 enzymatic assay was conducted using a method modified from HT F Homogeneous PARP Inhibition Assay Kit (Trevigen). 8.8 nM PARP-1 was pre-incubated with different concentrations of compounds in a buffer containing 100 mM Tris-HCl pH 8.0, 100 mM NaCl, 20 mM MgCl2, and 1% DMSO for 30 min at RT. The auto-PARylation reaction was initiated by addition of 500 nM NAD and 20 ng/ul activated DNA (Sigma) and incubated at RT for 40 min. The remaining NAD was detected by incubation with cycling assay solution containing 1% ethanol, 0.30 U/ml alcohol dehydrogenase, 25 uM resazurin, and 0.25 U/ml diaphorase for 50 min at RT. The concentration of NAD is proportional to the fluorescence signal at Ex 540 nm/Em 590 nm.
Measurement: IC50 = 1900 nM (pChEMBL 5.71999979019165)
Descriptors: MW 341.41, ALogP 1.76, HBA 5, HBD 2, PSA 78.06, QED 0.89
Compound (SMILES): NCc1ccc(Cl)cc1CNC(=O)[C@@H]1CCCN1C(=O)C1COc2ccccc21
InChIKey: RUWDHHHGUQTMCL-GGYWPGCISA-N
Target: Prothrombin (SINGLE PROTEIN), Homo sapiens
UniProt: P00734
Assay: In Vitro Assay: Relevant in vitro assays are referenced in Morrissette, et al., Bioorg. Med. Chem. Lett. 2004, 14, 4161-4164 and described in Lewis, et al. Thromb. Res. 1993, 70, 173 (assays of human α-thrombin and human trypsin), and Lewis, et al. Thromb. Haemostasis 1995, 74, 1107-1112. The assays were carried out at 25° C. in 0.05 M TRIS buffer pH 7.4, 0.15 M NaCl, 0.1% PEG. Trypsin assays also contained 1 mM CaC12. In assays wherein rates of hydrolysis of a p-nitroanilide (pna) substrate were determined, a Thermomax 96-well plate reader was used was used to measure (at 405 nm) the time dependent appearance of p-nitroaniline. sar-PR-pna was used to assay human α-thrombin (Km=125 μM) and bovine trypsin (Km=125 μM). p-Nitroanilide substrate concentration was determined from measurements of absorbance at 342 nm using an extinction coefficient of 8270 cm−1M−1.
Measurement: Ki = 9.2 nM (pChEMBL 8.039999961853027)
Descriptors: MW 413.91, ALogP 2.58, HBA 4, HBD 2, PSA 84.66, QED 0.79
Compound (SMILES): FC(F)(F)c1c(CN2CCN(c3ccc(Cl)cc3Cl)CC2)ccn2c(CC3CC3)nnc12
InChIKey: NKFVYUUZXLQBGE-UHFFFAOYSA-N
Target: Metabotropic glutamate receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q14416
Assay: Positive allosteric modulation of human mGlu2 receptor expressed in CHO cell membranes assessed as potentiation of glutamate-induced effect incubated for 30 mins prior to [35S]GTPgamma addition measured after 30 mins by scintillation counting method
Measurement: EC50 = 17 nM (pChEMBL 7.769999980926514)
Descriptors: MW 484.35, ALogP 5.33, HBA 5, HBD 0, PSA 36.67, QED 0.49
Compound (SMILES): Cc1noc(C)c1-c1ccc2c(c1)C(N[C@@H](C)CO)(c1ccccc1)C(=O)N2
InChIKey: JQGLKQWMMRCIIG-JHAYDQECSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: The assay was carried out in binding mixtures of the bromodomain protein, 0-10 μM compounds and tetra-acetylated histone peptide (SGRGAC.KGGAC.KGLGAC.KGGAAC.KRHGSGSK-biotin) in buffer containing 25 mM HEPES pH 7.5, 100 mM NaCl, 0.1% BSA, 0.05% CHAPS, and detection reagents. The detection reagents, including streptavidin-labeled Tb cryptate and XL665-labeled anti-6×His antibody, were added after binding equilibrium was achieved for protein, compound and peptide. Upon further incubation for 1 hr, the TR-FRET signals were recorded on a BMG PHERAstar FS instrument.
Measurement: IC50 = 860 nM (pChEMBL 6.070000171661377)
Descriptors: MW 377.44, ALogP 3.12, HBA 5, HBD 3, PSA 87.39, QED 0.64
Compound (SMILES): Fc1ccc(-c2cc3c(-c4cccc(N5CCNCC5)n4)n[nH]c3cn2)cn1
InChIKey: BINWPFGNFSEKRS-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 0.796 nM (pChEMBL 9.100000381469727)
Descriptors: MW 375.41, ALogP 2.63, HBA 6, HBD 2, PSA 82.62, QED 0.54
Compound (SMILES): COc1ccc(C(=O)NCCCCCCNC(=O)N2Cc3ccccc3C2)c(OC)c1
InChIKey: ZGNOKQHCKZAPKM-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 = 2.65 nM (pChEMBL 8.579999923706055)
Descriptors: MW 425.53, ALogP 3.72, HBA 4, HBD 2, PSA 79.9, QED 0.57
Compound (SMILES): Cc1nc(CS(=O)(=O)CC[C@@H]2OCC[C@@]3(S(=O)(=O)c4ccc(Cl)cc4)c4c(F)ccc(F)c4OCC23)no1
InChIKey: ZYQZOZIQPXMFMN-OUSZHMKWSA-N
Target: Amyloid-beta precursor protein (SINGLE PROTEIN), Homo sapiens
UniProt: P05067
Assay: Electrochemiluminescence (ECL)-based Immunoassay: To measure γ-secretase activity, membranes were incubated at 37° C. for 1 h in 50 μL of buffer containing 20 mM Hepes (pH 7.0) and 2 mM EDTA. At the end of the incubation, Aβ 40 and Aβ 42 were measured using an electrochemiluminescence (ECL)-based immunoassay. Aβ 40 was identified with antibody pairs TAG-G2-10 and biotin-W02, while Aβ 42 was identified with TAG-G2-11 and biotin-4G8. The ECL signal was measured using an ECL-M8 instrument (IGEN International, Inc.) according to the manufacturer's instructions.
Measurement: IC50 = 5.8 nM (pChEMBL 8.239999771118164)
Descriptors: MW 589.04, ALogP 3.78, HBA 9, HBD 0, PSA 125.66, QED 0.41
Compound (SMILES): C#CC(O)(c1ccc2nc(OC)c(Cc3ccc(C(F)(F)F)cc3)c(Cl)c2c1)c1cnc(C)n1C
InChIKey: ACZQKZSCRWXAGR-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: ThermoFluor® Assay: ThermoFluor® experiments were carried out using instruments owned by Janssen Research and Discovery, L.L.C. through its acquisition of 3-Dimensional Pharmaceuticals, Inc. 1,8-ANS (Invitrogen) was used as a fluorescent dye. Protein and compound solutions are dispensed into black 384-well polypropylene PCR microplates (Abgene) and overlayed with silicone oil (1 μL, Fluka, type DC 200) to prevent evaporation. Bar-coded assay plates are robotically loaded onto a thermostatically controlled PCR-type thermal block and then heated at a typical ramp-rate of 1° C./min for all experiments. Fluorescence was measured by continuous illumination with UV light (Hamamatsu LC6) supplied via fiber optic and filtered through a band-pass filter (380-400 nm; >6 OD cutoff). Fluorescence emission of the entire 384-well plate was detected by measuring light intensity using a CCD camera (Sensys, Roper Scientific) filtered to detect 500±25 nm, resulting in simultaneous and independent readings of all 384 wells.Â  Reference wells contained RORγt without compounds, and the assay conditions were as follows: 0.065 mg/mL RORγt, 60 μM 1,8-ANS, 100 mM Hepes, pH 7.0, 10 mM NaCl, 2.5 mM GSH, 0.002% Tween-20.
Measurement: Kd = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 499.92, ALogP 5.42, HBA 5, HBD 1, PSA 60.17, QED 0.37
Compound (SMILES): N#Cc1ccc(-c2[nH]c(=O)c3cc(F)ccc3c2OCCN)cc1
InChIKey: VUECMVWDRZENTP-UHFFFAOYSA-N
Target: Poly [ADP-ribose] polymerase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P09874
Assay: Biochemical Assay: The identification of compounds capable of binding several PARP proteins is carried out through a screening method including the steps of  a) providing a reaction mixture containing:  b) comparing the polarization signal generated in the absence of the test compound with the one generated in the presence of different concentrations of the test compound, and  c) evaluating the ability of the test compound to displace the compound of formula (IP) as defined above indicated from a decreased fluorescence polarization level.The polarization signal can be measured, e.g., by a plate reader such as the Saphire2 (Tecan).
Measurement: Kd = 10 nM (pChEMBL 8)
Descriptors: MW 323.33, ALogP 2.54, HBA 4, HBD 2, PSA 91.9, QED 0.77
Compound (SMILES): CN(C)c1ccc(S(=O)(=O)N[C@H]2CCN([C@H](Cc3cc4ccnc(N)c4cc3C(F)(F)F)C(=O)O)C2=O)cn1
InChIKey: SNMOFBUTBIIIQV-RBUKOAKNSA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (4.9 nM to 5 µM final) and deposited in a proportion of 25 µl per well. After the deposition of 50 µl of Spectrozyme 229 substrate (American Diagnostica), at the final concentration of 625 µM, the reaction is triggered by the addition of 25 µl of FIXa (human factor IXa supplied by Enzyme Research Laboratories (ERL)), at the final concentration of 2.5 U/ml. Reading is carried out at 405 nm for 15 min at 37°C. The percentage of inhibition of enzymatic activity (expressed as maximum rate of cleavage of the substrate) is calculated with respect to the enzymatic activity in the absence of inhibitor.
Measurement: IC50 = 69 nM (pChEMBL 7.159999847412109)
Descriptors: MW 566.56, ALogP 1.87, HBA 8, HBD 3, PSA 158.82, QED 0.37
Compound (SMILES): CN1CCCC1CCNC(=O)c1cc(-c2cc(NC3CC3)n3ncc(/C=C4\NC(=O)NC4=O)c3n2)cs1
InChIKey: MXCSLCGRMKRTEN-OCKHKDLRSA-N
Target: Casein kinase II subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: P67870
Assay: Inhibition Assay: 5 uM ATP: Test compounds dissolved and diluted in DMSO (2 μl) were added to a reaction mixture comprising 10 μl of 5× Reaction Buffer (40 mM MOPS pH 7.0, 5 mM EDTA), 10 μl of recombinant human PIM2 solution (4 ng PIM-2 dissolved in dilution buffer (20 mM MOPS pH 7.0; EDTA 1 mM; 5% Glycerol; 0.01% Brij 35; 0.1%; 0.1% 2-mercaptoethanol; 1 mg/ml BSA)) and 8 ul of water. Reactions were initiated by the addition of 10 ul of ATP Solution (49% (15 mM MgCl2; 5 uM ATP) 1% ([γ-33P]ATP: Stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer)) and 10 ul of substrate peptide solution (RSRSSYPAGT (SEQ ID NO.: 6), dissolved in water at a concentration of 1 mM), Reactions were maintained for 10 min at 30° C. The reactions were quenched with 100 ul of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore, MSPH-N6B-50). After washing each well 4 times with 0.75% Phosphoric acid, scintillation fluid (20 uL) was added to each well and
Measurement: IC50 = 654 nM (pChEMBL 6.179999828338623)
Descriptors: MW 520.62, ALogP 2.43, HBA 9, HBD 4, PSA 132.76, QED 0.26
Compound (SMILES): O=C(NC1CCN(c2ccccn2)CC1)c1nn(-c2ccc(Cl)cc2Cl)c2c1CCC/C2=C\c1ccc(Cl)cc1
InChIKey: SJTBCCQSQGIGSA-DYTRJAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: Inverse agonist activity at human cannabinoid receptor 1 expressed in HEK293 cells assessed as increase in forskolin-induced cAMP production preincubated with cells followed by forskolin stimulation measured after 30 mins in presence of D2-labeled cAMP and Eu3+-TBP-NHS cryptate by HTRF assay
Measurement: EC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 592.96, ALogP 7.5, HBA 5, HBD 1, PSA 63.05, QED 0.26
Compound (SMILES): CCOC(=O)c1cnc2ccc(F)cc2c1N1CCN(c2ncccc2C#N)CC1
InChIKey: SVLZNUGVKFIWNW-UHFFFAOYSA-N
Target: Cholinesterase (SINGLE PROTEIN), Equus caballus
UniProt: P81908
Assay: Inhibition of equine serum BuChE preincubated for 5 mins followed by addition of butyrylthiocholine iodide as substrate measured after 2 mins by Ellman's method
Measurement: IC50 = 1230 nM (pChEMBL 5.909999847412109)
Descriptors: MW 405.43, ALogP 3.14, HBA 7, HBD 0, PSA 82.35, QED 0.62
Compound (SMILES): O=C(N[C@H]1CC[C@@H](c2ccccc2)CC1)C1=C(O)C(=O)N(CCC(F)(F)C(F)(F)F)C1
InChIKey: UVBDTAFEQRSACL-GASCZTMLSA-N
Target: Endothelial lipase (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5X9
Assay: Enzymatic Assay: Endothelial lipase activity was measured using a fluorescent substrate, A10070, (Invitrogen, CA) doped into an artificial vesicle containing DMPG (Avanti Polar Lipids) as the excipient. Vesicles were prepared by combining 285 uL of 1 mMDMPG in a 1:1 mixture of MeOH and CHCl3 with 15 uL of 1 mM A10070 in a 1:1 mixture of MeOH and CHCl3. The mixture was dried under nitrogen and resuspended in 150 uL of 50 mM HEPES pH 8.0 buffer containing 100 mM NaCl and 0.2 mM EDTA. The sample was allowed to sit at rt for 15 min and then was sonicated 3x4 mins on ice with a Branson Sonicator using duty cycle 1. This preparation provides vesicles with a mole fraction of 0.05 for the FRET substrate.The enzymatic assay was measured using white, opaque 96-well half area plates. Each well contained 60 uL of assay buffer (50 mM HEPES pH 8.0, 50 mM NaCl and 1 mM CaCl2) and 2 ul of a DMSO solution containing compound of interest. Conditioned media obtained from HT-1080 cells, which were transformed by RAGE technology (Athersys) to overexpress endogenous EL, was added and the reaction was allowed to incubate for 20 min at 37deg; C. with gentle agitation. The reaction was started by the addition of 20 uL of a 1:4 dilution of vesicles. The final total reaction volume was 100 uL. The reaction rates were measured on a Gemini plate reader with an excitation wavelength of 488 nm and a emission of 530 nm. Readings were taken every 20 seconds for 10 min with agitation between each reading. The slope of the linear portion of the readout was used to calculate the rate of the reaction.
Measurement: IC50 = 7169 nM (pChEMBL 5.139999866485596)
Descriptors: MW 446.42, ALogP 4.07, HBA 3, HBD 2, PSA 69.64, QED 0.65
Compound (SMILES): CNC(=O)c1c(-c2ccc(F)cc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(c3)-c3nc5cccnc5n3CO4)cc12
InChIKey: GLVHBWLXRHGZJD-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1a (isolate H) (HCV)
UniProt: P27958
Assay: ELISA-Based Assay: Measurement of inhibition by compounds was performed using the HCV replicon system. Several different replicons encoding different HCV genotypes or mutations were used. In addition, potency measurements were made using different formats of the replicon assay, including different ways of measurements and different plating formats. See Jan M. Vrolijk et al., A replicons-based bioassay for the measurement of interferons in patients with chronic hepatitis C, 110 J. VIROLOGICAL METHODS 201 (2003); Steven S. Carroll et al., Inhibition of Hepatitis C Virus RNA Replication by 2'-Modified Nucleoside Analogs, 278(14) J. BIOLOGICAL CHEMISTRY 11979 (2003). However, the underlying principles are common to all of these determinations, and are outlined below.Stable neomycin phosphotransferase encoding replicons-harboring cell lines were used, so all cell lines were maintained under G418 selection prior to the assay. Potency was determined using a cell ELISA assay with an antibody to the replicons encoded NS3/4a protease. See Caterina Trozzi et al., In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor, 77(6) J. Virol. 3669 (2003).
Measurement: IC50 = 2.4 nM (pChEMBL 8.619999885559082)
Descriptors: MW 597.63, ALogP 5.42, HBA 8, HBD 1, PSA 119.56, QED 0.28
Compound (SMILES): CC(C)CC(=O)N1CC[C@H](Oc2ccc(NC(=O)N3Cc4ccncc4C3)cc2)C1
InChIKey: BIZKTPKQPKRZMM-NRFANRHFSA-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 = 11.8 nM (pChEMBL 7.929999828338623)
Descriptors: MW 408.5, ALogP 3.66, HBA 4, HBD 1, PSA 74.77, QED 0.82
Compound (SMILES): CCn1cc(-c2cc3c(-c4cc(C)cc(N5CCCNCC5)n4)n[nH]c3cn2)cn1
InChIKey: FTCGBSBOZWGTEI-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: PIM Kinase Binding Activity Assay: PIM-1, PIM-2, and PIM-3 enzymes were generated as fusion proteins expressed in bacteria and purified by IMAC column chromatography (Sun, X., Chiu, J. F., and He, Q. Y. (2005) Expert Rev. Proteomics, 2:649-657). A fluorescent-labeled Pim-specific peptide substrate, was custom synthesized by American Peptide Company (Sunnyvale, Calif.). Reaction Buffer contained 10 mM HEPES, pH 7.2, 10 mM MgCl2, 0.01% Tween 20, 2 mM DTT. Termination Buffer contained 190 mM HEPES, pH 7.2, 0.015% Brij-35, 0.2% Coating Reagent 3 (Caliper Life Sciences, Hopkinton, Mass.), 20 mM EDTA. Separation Buffer contained 100 mM HEPES, pH 7.2, 0.015% Brij-35, 0.1% Coating Reagent 3, 1:200 Coating Reagent 8 (Caliper Life Sciences, Hopkinton, Mass.), 10 mM EDTA and 5% DMSO.
Measurement: Ki = 0.049 nM (pChEMBL 10.3100004196167)
Descriptors: MW 402.51, ALogP 3.01, HBA 7, HBD 2, PSA 87.55, QED 0.55
Compound (SMILES): CN(C)CCCNc1ncc(Br)c(Nc2cc(C3CC3)n[nH]2)n1
InChIKey: QEWXLASRORBCGI-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 >= 5000 nM (pChEMBL 5.301000118255615)
Descriptors: MW 380.29, ALogP 2.95, HBA 6, HBD 3, PSA 81.76, QED 0.61
Compound (SMILES): CC1CCC[C@H](N2CCC(c3c(Br)ccc(Cl)c3F)=CC2=O)c2cc(ccn2)-c2ccc(C(N)=O)cc2NC1=O
InChIKey: GZIWAJRNQSUBEG-JIKORUOASA-N
Target: Coagulation factor IX (SINGLE PROTEIN), Homo sapiens
UniProt: P00740
Assay: In Vitro Assay: Factor XIa determinations were made in 50 mM HEPES buffer at pH 7.4 containing 145 mM NaCl, 5 mM KCl, and 0.1% PEG 8000 (polyethylene glycol; JT Baker or Fisher Scientific). Determinations were made using purified human Factor XIa at a final concentration of 25-200 pM (Haematologic Technologies) and the synthetic substrate S-2366 (pyroGlu-Pro-Arg-pNA; CHROMOGENIX® or AnaSpec) at a concentration of 0.0002-0.001 M.
Measurement: Ki = 2.88 nM (pChEMBL 8.539999961853027)
Descriptors: MW 625.93, ALogP 6.52, HBA 4, HBD 2, PSA 105.39, QED 0.32
Compound (SMILES): Cc1nc(N2CC(c3nc4ccccc4n3CCO)C2)cc(-n2nc(C)nc2C)n1
InChIKey: UTEHRXCFEXDRMZ-UHFFFAOYSA-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. 100% inhibition is determined using a known PDE10 inhibitor, which can be any compound that is present at 5,000 times its Ki value in the assay described as follows, such as papaverine (see Siuciak, et al. Neuropharmacology (2006) 51:386-396; Becker, et al. Behav Brain Res (2008) 186(2):155-60; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3):785-795), 2-{4-[pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]phenoxymethyl}quinoline succinic acid or 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]quinoline succinic acid (see Schmidt, et al. J Pharmacol Exp Ther (2008) 325:681-690; Threlfell, et al., J Pharmacol Exp Ther (2009) 328(3): 785-795). 0% of inhibition is determined by using DMSO (1% final concentrations). A Labcyte Echo 555 (Labcyte, Sunnyvale, Calif.) is used to dispense 200 mL from each well of the titration plate to the 384 well assay plate. A solution of enzyme (1/7000 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 is added to the assay plates by the addition of 10 μL of enzyme solution to each well, shaken to mix and incubated at room temperature for 60 minutes. The substrate is then added to the assay plates by the addition of 10 μÎ¼L of substrate solution to each well, shaken to mix, and incubated at room temperature for 60 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 lit 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).
Measurement: Ki = 6.6 nM (pChEMBL 8.180000305175781)
Descriptors: MW 404.48, ALogP 1.93, HBA 9, HBD 1, PSA 97.78, QED 0.54
Compound (SMILES): C[C@H](N)CNC(=O)c1cc(NC(=O)c2cc(-c3ncccc3F)c(Cl)cc2Cl)n(-c2ccccc2)n1
InChIKey: JEYFYFWJSFFCIS-AWEZNQCLSA-N
Target: High affinity nerve growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P04629
Assay: Cell Based Assay: TRKA co-expressed with p75: The assays are based upon DiscoveRx's proprietary Enzyme Fragment Complementation (EFC) technology. In the case of the TRK cell lines, the enzyme acceptor (EA) protein is fused to a SH2 protein and the TRK receptor of interest has been tagged with a Prolink tag. Cryopreserved PathHunter cells are used from either in-house produced batches or bulk batches bought directly from DiscoveRx. Cryopreserved cells are resuscitated, spun 1000 rpm for 4 min to remove freezing media, and resuspended in MEM+0.5% horse serum (both Invitrogen) to 5e^5 cells/ml. The cells are then plated using a Multidrop into Greiner white tissue culture treated plates at 20 μl/well and incubated for 24 h at 37 °C., 5% CO2, high humidity. On the day of the assay, the cell plates are allowed to cool to room temperature for 30 min prior to the assay. 4 mM stock solutions of test compounds are prepared and serially diluted in 100% DMSO. A standard curve using the compound of Example 135, WO2005/116035 at a top concentration of 150 μM is also prepared on each test plate. High percentage effect (HPE) is defined by 150 μM of the compound of Example 135, WO2005/116035 and 0% effect (ZPE) is defined by 100% DMSO. Plates containing 1 μl of serially diluted compound, standard and HPE/ZPE are diluted 1/66 in assay buffer (PBS minus Ca2+, minus Mg2+ with 0.05% pluronic F127) using a Wellmate. Using a Platemate Plus, 5 μl of 1/66 diluted test compounds is then transferred to the cell plate and allowed to reach equilibrium by incubating for 30 min at room temperature before addition of agonist stimulus: 10 μl/well of 2 nM (0.571 nM FAC) of the cognate neurotrophin (Peprotech) diluted in agonist buffer (HBSS with 0.25% BSA). Final assay concentration of the test compounds is 8.66 μM, (the compound of Example 135, WO2005/116035 FAC is 0.325 μM). The plates are left at room temperature for a further 2 hours before addition of 10 μl of the DiscoveRx PathHunter detection reagent (made up by adding 1 part Galacton Star, 5 parts Emerald II and 19 parts Cell Assay Buffer as per the manufacturer's instructions). After reagent addition, plates are covered and incubated at room temperature for 60 minutes. Luminescence signal is read using an Envision.
Measurement: IC50 = 92.8 nM (pChEMBL 7.03000020980835)
Descriptors: MW 527.39, ALogP 4.71, HBA 6, HBD 3, PSA 114.93, QED 0.32
Compound (SMILES): COC(=O)CCCCS(C)(=O)=NC(=O)c1cnc(N)c(C#Cc2cccc(NC(=O)c3ccc(OC)c(OC)c3)c2)c1
InChIKey: RDOPCBYTZLJSSR-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: VEGFR2 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. 100 ul of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate diluted 1:10,000 in PBS-T was added to the wells for 30 minutes. Following a 2 mls per well wash with PBS-Tween-20, 100 ul of 0-Phenylenediamine Dihydrochloride in phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7-10 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 ul of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 15 nM (pChEMBL 7.820000171661377)
Descriptors: MW 592.67, ALogP 3.91, HBA 9, HBD 2, PSA 159.27, QED 0.2