lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CC(C)CN(Cc1ccc(N[C@H]2CCC[C@H](O)C2)cc1)S(=O)(=O)Cc1ccccc1
InChIKey: KKTDRUWPJURLEN-ZEQRLZLVSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In Vitro Assay: On day of the assay, 100 uL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20x required final concentration in DMSO (15 uL compound+30 uL DMSO). The 20x compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5x the final test concentration in 25% DMSO (10 uL compound+30 uL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 uL volume. For the assay, 10 uL of 5x compound stock solutions in 25% DMSO were added to the assay plate in duplicate.
Measurement: IC50 = 170 nM (pChEMBL 6.769999980926514)
Descriptors: MW 430.61, ALogP 4.39, HBA 4, HBD 2, PSA 69.64, QED 0.62
Compound (SMILES): CCCS(=O)(=O)c1ccc(-n2nc(C(N)=O)c3c2-c2cc(NC(=O)c4cc(N)ccc4Cl)ccc2CC3)cc1.O=C(O)C(F)(F)F
InChIKey: VFSAEBNCSUQEDS-UHFFFAOYSA-N
Target: Inhibitor of nuclear factor kappa-B kinase subunit beta (SINGLE PROTEIN), Homo sapiens
UniProt: O14920
Assay: Inhibition of human recombinant C-terminal FLAG-epitope-tagged IKK2 expressed in baculovirus using biotinylated IkappaBalpha peptide as substrate assessed as decrease in [gamma-33P]-ATP incorporation in substrate after 30 mins by scintillation counting method
Measurement: IC50 <= 1000 nM (pChEMBL 6)
Descriptors: MW 564.07, ALogP 4.41, HBA 7, HBD 3, PSA 150.17, QED 0.28
Compound (SMILES): Cc1cc2[nH]c(-c3ncsc3N3CCN(C(=O)Cn4cnc5cccnc54)[C@H](C)C3)nc2cc1Cl
InChIKey: FWCTVMMNOUKNDL-OAHLLOKOSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: FLIPR Assay: The bioactivity of compounds is tested in a fluorometric imaging plate reader (FLIPR: Molecular Devices) using engineered CHO-K1 cells expressing the human CXCR3A coupled to a G protein (Galpha(16)). Cells are plated the day prior to bioassay in F12 medium supplemented with 10% FBS and G418 and hygromycin antibiotics to maintain recombinant selection. At the day of bioassay, cells are washed and dye loaded for one hour with Fluo-4-AM (Invitrogen) in Hanks Balanced Salt Solution (Invitrogen), buffered with 20 mM Hepes at pH 7.4 and sodium bicarbonate (0.015%), containing 5 mM probenecid. This buffer, but lacking the dye and containing probenecid at a concentration of 2.5 nM, is also is used for washing steps (wash buffer); or lacking both dye and probenecid but supplemented with 0.1% BSA for compound dilution steps (dilution buffer). Cells are washed free of excess dye and 60 microliter of wash buffer is added. Stock solutions of test compounds are made up at a concentration of 10 mM in DMSO, and serially diluted in dilution buffer to concentrations required for inhibition dose response curves. After a 10 minute incubation period at 37° C., 10 microliters of each compound dilution are transferred from a compound plate to the plate containing the recombinant cells in the FLIPR instrument according to the manufacturer's instructions. Following basal readings, 10 microliter CXCL10 agonist at a concentration of 20 nM (from Peprotech) is added, again using the FLIPR instrument. Changes in fluorescence are monitored before and after addition of the test compounds.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 507.02, ALogP 4.13, HBA 8, HBD 1, PSA 95.83, QED 0.39
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)NCC(=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)NC(C)(C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)NC(C)(C)C(N)=O)C(C)C)C(C)C
InChIKey: ZJRRSKUAASKQPL-ZUWPZOMFSA-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 = 119 nM (pChEMBL 6.920000076293945)
Descriptors: MW 2316.65
Compound (SMILES): CC(OP(=O)(N[C@@H](C)C(=O)O)Oc1ccccc1)[C@H]1O[C@@H](n2ccc3c(N)ncnc32)[C@](C)(O)[C@@H]1O
InChIKey: AOEAHVTUHKMYIX-NFMQJHBNSA-N
Target: RNA-directed RNA polymerase (SINGLE PROTEIN), Hepacivirus hominis
UniProt: O39930
Assay: Polymerase Inhibition Assay: The ability of the nucleoside triphosphate derivatives to inhibit the enzymatic activity of the HCV NS5B RNA-dependent RNA polymerase was measured in a radiolabeled nucleotide incorporation assay modified from the method described in International Publication No. WO2002/057287. Briefly, 50 μL reactions containing 20 mM HEPES (pH 7.3), 7.5 mM DTT, 20 units/mL RNasIN, 1 μM each of ATP, GTP, UTP and CTP, 20 μCi/mL [33P]-CTP, 10 mM MgCl2, 60 mM NaCl, 100 μg/ml BSA, 0.021 μM DCoH heteropolymer RNA template and 5 nM NS5B (1b-BKΔ55) enzyme were incubated at room temperature for 1 hour. Assay was terminated by the addition of 500 mM EDTA (50 μL). The reaction mixture was transferred to a Millipore DE81 filter plate and the incorporation of labeled CTP was determined using Packard TopCount.
Measurement: EC50 = 4550 nM (pChEMBL 5.340000152587891)
Descriptors: MW 521.47, ALogP 1.68, HBA 11, HBD 5, PSA 191.28, QED 0.26
Compound (SMILES): O=C(Nc1ccc(C2CCCNC2)cc1)c1ccc(Cl)cc1
InChIKey: BOINLYJULUCZJQ-UHFFFAOYSA-N
Target: Trace amine-associated receptor 1 (SINGLE PROTEIN), Mus musculus
UniProt: Q923Y8
Assay: Radioligand Binding Assay: HEK-293 cells stably expressing mouse TAAR1 were maintained at 37° C. and 5% CO2 in DMEM high glucose medium, containing fetal calf serum (10%, heat inactivated for 30 min at 56° C.), penicillin/streptomycin (1%), and 375 ug/ml geneticin (Gibco). Cells were released from culture flasks using trypsin/EDTA, harvested, washed twice with ice-cold PBS (without Ca2+ and Mg2+), pelleted at 1,000 rpm for 5 min at 4° C., frozen and stored at -80° C. Frozen pellets were suspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 10 mM EDTA and homogenized with a Polytron (PT 6000, Kinematica) at 14,000 rpm for 20 s. The homogenate was centrifuged at 48,000Ã¿g for 30 min at 4° C. Subsequently, the supernatant was removed and discarded, and the pellet resuspended in 20 ml HEPES-NaOH (20 mM, pH 7.4) containing 0.1 mM EDTA using the Polytron (20 s at 14,000 rpm). This procedure was repeated and the final pellet resuspended in HEPES-NaOH containing 0.1 mM EDTA and homogenized using the Polytron. Typically, aliquots of 2 ml membrane portions were stored at -80° C. With each new membrane batch the dissociation constant (Kd) was determined via a saturation curve. The TAAR1 radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine (described in WO 2008/098857) was used at a concentration equal to the calculated Kd value, that was usually around 2.3 nM, resulting in the binding of approximately 0.2% of the radioligand and a specific binding representing approximately 85% of the total binding. Nonspecific binding was defined as the amount of 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine bound in the presence of 10 uM unlabeled ligand. All compounds were tested at a broad range of concentrations (10 pM to 10 uM) in duplicates. The test compounds (20 ul/well) were transferred into a 96 deep well plate (TreffLab), and 180 ul of HEPES-NaOH (20 mM, pH 7.4) containing MgCl2 (10 mM) and CaCl2 (2 mM) (binding buffer), 300 ul of the radioligand 3[H]â¿¿(S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine at a concentration of 3.3xKd in nM and 500 ul of the membranes (resuspended at 50 ug protein per ml) added. The 96 deep well plates were incubated for 1 hr at 4° C. Incubations were terminated by rapid filtration through Unifilter-96 plates (Packard Instrument Company) and glass filters GF/C (Perkin Elmer) presoaked for 1 hr in polyethylenimine (0.3%) and washed 3 times with 1 ml of cold binding buffer. After addition of 45 ul of Microscint 40 (PerkinElmer) the Unifilter-96 plate was sealed and after 1 hr the ratioactivity counted using a TopCount Microplate Scintillation Counter (Packard Instrument Company).
Measurement: Ki = 0.7 nM (pChEMBL 9.149999618530273)
Descriptors: MW 314.82, ALogP 4.06, HBA 2, HBD 2, PSA 41.13, QED 0.9
Compound (SMILES): Cc1nc(-c2ccccc2)[nH]c1-c1cnc(-c2ccccc2)nc1
InChIKey: CMUUNGLVTVUMPW-UHFFFAOYSA-N
Target: Hematopoietic prostaglandin D synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O60760
Assay: Fluorescence Polarization Binding Assay (FPBA): This assay essentially is available from Cayman Chemical Company as Catalog item #600007. The test data reported for the aforementioned Examples (H-PGDS FPBA IC.sub.50) were generated using a 96-well, instead of the 384-well, format.Detection analyte and H-PGDS-MBP fusion enzyme were incubated in the presence of reduced glutathione (5 mM) for 60-90 minutes at room temperature and FP was measured using a TECAN SAFIRE 2 plate reader equipped with absorbance, fluorescence, fluorescence polarization and FRET capabilities. Assays were performed in 96-well microtiter plates in 100 .mu.L of total sample volume. Excitation and emission wavelengths appropriate for the employed detection analyte were used.
Measurement: IC50 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 312.38, ALogP 4.51, HBA 3, HBD 1, PSA 54.46, QED 0.61
Compound (SMILES): O=C(NCCOCCOCCOCCNS(=O)(=O)c1ccc(O[C@H]2c3ccccc3C[C@@H]2N2CCCCC2)cc1)c1ccc(C(=O)NCCOCCOCCOCCNS(=O)(=O)c2ccc(O[C@H]3c4ccccc4C[C@@H]3N3CCCCC3)cc2)cc1
InChIKey: QXTNNWGFBDMUIX-YYHFWCTESA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 31.62 nM (pChEMBL 7.5)
Descriptors: MW 1225.54
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2C(c2ccc(S(=O)(=O)NCCOCCOCCOCCNC(=O)c3ccc(C(=O)NCCOCCOCCOCCNS(=O)(=O)c4ccc(C5CN(C)Cc6c(Cl)cc(Cl)cc65)cc4)cc3)cc2)C1
InChIKey: GFNIFQOPNUJPDY-UHFFFAOYSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3 expressed in opossum kidney cells assessed as reduction in Na-HEPES buffer-mediated pH recovery in presence of NEH1 inhibitor ethyl isopropyl amiloride by BCECF-AM dye based fluorescence assay
Measurement: IC50 = 39.81 nM (pChEMBL 7.400000095367432)
Descriptors: MW 1223.14
Compound (SMILES): CN1C(=O)CCC1c1cc2[nH]c(-c3ccccn3)cc2cc1Oc1ccc(CCC(=O)O)cc1
InChIKey: VJEURJGBLQRRLX-UHFFFAOYSA-N
Target: Hexokinase-4 (SINGLE PROTEIN), Homo sapiens
UniProt: P35557
Assay: Glucokinase-Activating Assay: To test the exemplified compounds, the following assay was employed. Recombinant human liver glucokinase was expressed as a FLAG fusion protein in E. coli, and purified on ANTIFLAG M2 AFFINITY GEL (Sigma). The assay was carried out at 30° C. in a 96-well plate. In the plate was distributed 69 ul each of assay buffer (25 mM Hepes Buffer: pH=7.2, 2 mM MgCl2, 1 mM ATP, 0.5 mM TNAD, 1 mM dithiothreitol), to which was added 1 ul of a DMSO solution of the compound or DMSO as control. Then, 20 ul of pre-ice-cooled enzyme mixture (FLAG-GK, 20 U/ml G6PDH) was distributed thereto, to which was added 10 ul of 25 mM glucose as substrate to initiate the reaction (final glucose concentration=2.5 mM). After starting the reaction, the absorbance at 405 nm was measured every 30 seconds for 10 minutes to evaluate the compound based on the initial increase for 5 minutes. FLAG-GK was added so that the increase of absorbance after 5 minutes fell between 0.05 to 0.1 in the presence of 1% DMSO. The OD values of the respective compounds were measured in the respective concentrations, wherein the OD value of DMSO as control is regarded as 100%. From the OD values at the respective concentrations, Emax (%, 2.5 mM Glu) and EC50 (nM, 2.5 mM Glu) were calculated and used as indicators of the GK activation capability of the compounds. According to the above assay, the GK activation capability of the exemplified compounds of the present invention was determined. The following table shows the results. Where different enantiomers of the same compound were tested, two numbers are provided. Where 3 numbers are provided, for example, Ex. #30, data for the racemic form is also shown.
Measurement: EC50 = 17.1 nM (pChEMBL 7.769999980926514)
Descriptors: MW 455.51, ALogP 5.33, HBA 4, HBD 2, PSA 95.52, QED 0.39
Compound (SMILES): CN(C)Cc1ccc(-c2c(-c3cccc(OC(F)(F)F)c3)cc3n2C(CCCl)CNC3=O)cc1
InChIKey: MEQUDKNTYPMAEZ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P11309
Assay: Trans-Phosphorylation Assay: The inhibitory activity of putative kinase inhibitors and the potency of selected compounds were determined using a trans-phosphorylation assay. Specific peptide or protein substrates were trans-phosphorylated by their specific ser-thr or tyr kinase in the presence of ATP traced with 33P-gamma-ATP, and in the presence of their own optimal buffer and cofactors.At the end of the phosphorylation reaction, more than 98% unlabeled ATP and radioactive ATP were captured by an excess of the ion exchange dowex resin; the resin then settled down to the bottom of the reaction plate by gravity. Supernatant was subsequently withdrawn and transferred into a counting plate, then evaluated by beta-counting. Kinase Buffer (KB):The buffer for PIM1 assay was composed of HEPES 50 mM, at pH 7.5, with 10 mM MgCl2, 1 mM DTT, 3 uM NaVO3, and 0.2 mg/mL BSA.Â  Full-length human PIM1 was expressed and purified as described in Bullock A N, et al., J. Biol. Chem. 2005, 280, 41675-82.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 491.94, ALogP 5.7, HBA 4, HBD 1, PSA 46.5, QED 0.43
Compound (SMILES): Cc1ccc(F)c(NC(=O)Nc2ccc(C3CN=C4c5ccccc5NC(=O)N4C3)c(C)c2)c1
InChIKey: CLVXURWUOYHVPU-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P35968
Assay: Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 μg/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 μL reaction volumes containing 2.7 μM 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 μl 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 μl 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 μl of O-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 μl of 2.5N H2SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 390 nM (pChEMBL 6.409999847412109)
Descriptors: MW 457.51, ALogP 5.48, HBA 3, HBD 3, PSA 85.83, QED 0.49
Compound (SMILES): Cc1c(C(=O)Nc2cccc(C(F)(F)F)c2)cc(-c2ccncc2)n1CCCN1CCOCC1
InChIKey: KRESADFNBWHBBX-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 = 780 nM (pChEMBL 6.110000133514404)
Descriptors: MW 472.51, ALogP 4.85, HBA 5, HBD 1, PSA 59.39, QED 0.53
Compound (SMILES): CCn1ncc2cc3nc(c21)CCCO[C@H](O)[C@H](c1ccccc1)NC(=O)N3
InChIKey: SPHRYCNMOJOQJP-HKUYNNGSSA-N
Target: Mitogen-activated protein kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28482
Assay: Inhibition of human activated ERK2 assessed as reduction in phosphorylation activity preincubated for 15 mins followed by addition of IMAP peptide as substrate and ATP measured after 60 mins by fluorescence polarization method
Measurement: IC50 = 24.3 nM (pChEMBL 7.610000133514404)
Descriptors: MW 381.44, ALogP 2.6, HBA 6, HBD 3, PSA 101.3, QED 0.63
Compound (SMILES): Cc1ccc(-c2noc(C3CC(F)(F)C3)n2)cc1NC(=O)c1cnc2ccccn12
InChIKey: YYJQXSJQDPOQPK-UHFFFAOYSA-N
Target: Mast/stem cell growth factor receptor Kit (SINGLE PROTEIN), Homo sapiens
UniProt: P10721
Assay: Cell Proliferation Assay: The compounds was tested for inhibition of SCF dependent proliferation using human Mo7e cells which endogenously express c-kit in a 384 well format. Three-fold serially diluted test compounds (Cmax=10 mM) were evaluated for their antiproliferative activity of Mo7e cells stimulated with human recombinant SCF. After 48 hours of incubation at 37° C., cell viability was measured by adding 25 uL of CellTiter Glo (Promega) to the cells and the luminescence was measured by a CLIPR CCD camera (Molecular Devices).
Measurement: IC50 = 7 nM (pChEMBL 8.149999618530273)
Descriptors: MW 409.4, ALogP 4.46, HBA 6, HBD 1, PSA 85.32, QED 0.54
Compound (SMILES): COc1ccc(NC(=O)NCc2ccc(C(=O)NO)cc2)cc1
InChIKey: ZXGFCHWWLPADDY-UHFFFAOYSA-N
Target: Histone deacetylase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13547
Assay: Inhibition Assay: HDAC inhibition assays were performed by Reaction Biology Corp. (Malvern, Pa.) using isolated human, recombinant full-length HDAC1 and -6 from a baculovirus expression system in Sf9 cells. An acetylated fluorogenic peptide, RHKKAc, derived from residues 379-382 of p53 was used as substrate. The reaction buffer was made up of 50 mM Tris-HCl pH 8.0, 127 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/mL BSA, and a final concentration of 1% DMSO. Compounds were delivered in DMSO and delivered to enzyme mixture with preincubation of 5-10 min followed by substrate addition and incubation for 2 h at 30° C. Trichostatin A and developer were added to quench the reaction and generate fluorescence, respectively. Dose-response curves were generated starting at 30 μM compound with three-fold serial dilutions to generate a 10-dose plot.
Measurement: IC50 = 1060 nM (pChEMBL 5.96999979019165)
Descriptors: MW 315.33, ALogP 2.14, HBA 4, HBD 4, PSA 99.69, QED 0.5
Compound (SMILES): COc1ccc(CC(=O)O)cc1-c1ccc(F)c2c1CN(C(=O)CCc1c(C)[nH]c3ccccc13)CC2
InChIKey: KMEOLLKMVHOVGE-UHFFFAOYSA-N
Target: Prostaglandin D2 receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Y4
Assay: Radioligand Displacement Assay: Binding assay was performed in a final assay volume of 250 ul. First, 25 ul of test compound, previously diluted in Binding-Buffer (Binding-Buffer: 50 mM Tris-Base, 100 mM NaCl, 1 mM EDTA, 0.1% BSA (protease free), 0.01% NaN3, 10 mM MnCl2, pH 7.0) was placed into each well. After addition of 75 ul Binding-Buffer, 50 ul of the radioligand 3H-PGD2 (at 2.5 nM (220.000 dpm/well) from ANAWA ART0662) was added to each well. Binding assay was started by addition of 100 ul CRTH2 membrane fragments, reaching a final concentration of 20 ug/well. For non-specific binding, PGD2 was added to the reaction mixture to 10 mM final concentration. This assay mix was incubated for 90 minutes at room temperature and then filtered through a GF/C filter 96-well plate which was pre-soaked for 3 hours in 0.5% polyethyleneimine (PEI). The filter-wells were washed three times with ice cold Binding-Buffer. Then, 40 ul of Microscint-40 (Packard) was added to each well and the retained radioactivity quantified in a Topcount (Packard).
Measurement: IC50 = 8 nM (pChEMBL 8.100000381469727)
Descriptors: MW 500.57, ALogP 5.44, HBA 3, HBD 2, PSA 82.63, QED 0.35
Compound (SMILES): Cc1cc2ccc1[C@@H](C)COC(=O)Nc1ccc(S(=O)(=O)C3CC3)c(c1)CN(C)C(=O)[C@@H]2Nc1cc(F)c2c(N)nccc2c1
InChIKey: DVTJAXWYFLZLAI-WWOZWPLTSA-N
Target: Kallikrein-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P06870
Assay: Inhibition of human HK1 using H-D-Val-Leu-Arg-AFC as substrate assessed as release of AFC after 10 to 120 mins by spectrofluorimetric method
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 631.73, ALogP 5.68, HBA 8, HBD 3, PSA 143.72, QED 0.26
Compound (SMILES): CN1Cc2c(-c3ccc(S(C)(=O)=O)nc3)[nH]c3nccc(c23)-c2cc(F)ccc21
InChIKey: CQTIMMHXSUPUFQ-UHFFFAOYSA-N
Target: Cyclin-dependent kinase 9 (SINGLE PROTEIN), Homo sapiens
UniProt: P50750
Assay: LANCE ULight TR-FRET Kinase Assay : CDK9 enzyme activities were measured using LANCE ULight TR-FRET kinase assay reagents (PerkinElmer, Waltham, Mass.). Compounds were diluted in 100% DMSO then diluted with 1:10 in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. Equal volume of the compound dilutions was added to a final reaction mixture containing LANCE detection buffer (PerkinElmer CR97-100), 100 nM ULight MBP (PerkinElmer TRF0109M), 1000 μM ATP, and CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 1 hour before addition of stopping buffer to a final of 20 mM EDTA and 0.5 nM of LANCE Ultra Europium anti-phospho-MBP antibody (PerkinElmer TRF0201M) in LANCE detection buffer. The reaction was incubated for 1 hour and the signal was read in Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 110 nM (pChEMBL 6.960000038146973)
Descriptors: MW 408.46, ALogP 3.78, HBA 5, HBD 1, PSA 78.95, QED 0.55
Compound (SMILES): CCCCCCCCCC/N=C1/SC[C@H]2[C@H](O)[C@@H](O)[C@H](CO)N12
InChIKey: KZFUVJOWYFCJRT-FSKGAIIPSA-N
Target: Beta-galactosidase (SINGLE PROTEIN), Bos taurus
UniProt: Q58D55
Assay: Inhibition of bovine liver beta-galactosidase using p-nitrophenyl-glycoside as substrate by spectrophotometric method
Measurement: IC50 = 36000 nM (pChEMBL 4.440000057220459)
Descriptors: MW 344.52, ALogP 2, HBA 5, HBD 3, PSA 76.29, QED 0.53
Compound (SMILES): NC(=O)CNC(=O)c1cc(NC(=O)c2cc(-c3ccc(F)cn3)c(Cl)cc2Cl)n(-c2ccccc2)n1
InChIKey: KVRMIHAPDKYOIT-UHFFFAOYSA-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 = 64.1 nM (pChEMBL 7.190000057220459)
Descriptors: MW 527.34, ALogP 3.85, HBA 6, HBD 3, PSA 132, QED 0.34
Compound (SMILES): CS(=O)(=O)Nc1ccc(S(=O)(=O)N(CCCCC(F)(F)F)c2sc3ccccc3c2Cl)cc1
InChIKey: CAGBSCUPLVJWCJ-UHFFFAOYSA-N
Target: Transient receptor potential M8 protein (SINGLE PROTEIN), Canis lupus familiaris
UniProt: Q1A7N1
Assay: Functional Assay: The functional activity of compounds of the formula (I) was determined by measuring changes in intracellular calcium concentration using a Ca2+-sensitive fluorescent dye. The changes in fluorescent signal were monitored by a fluorescence plate reader, either a FLIPR (Molecular Devices) or FDSS (Hamamatsu). Increases in intracellular Ca2+ concentration were readily detected upon activation with icilin.At 24 hrs prior to assay, HEK293 cells stably expressing canine TRPM8 were seeded in culture medium in black wall, clear-base poly-D-lysine coated 384-well plates (BD Biosciences, NJ, USA) and grown overnight in 5% CO2 at 37° C. On assay day, growth media was removed and cells were loaded with Calcium 3 Dye (Molecular Devices) for 35 min at 37° C., under 5% CO2 and then for 25 min at room temperature and atmosphere. Subsequently, cells were tested for agonist-induced increases in intracellular Ca2+ levels using FLIPR or FDSS. Cells were challenged with a compound of the Formula (I) (at varying concentrations) and intracellular Ca2+ was measured for 5 min prior to the addition of icilin to all wells to achieve a final concentration that produces approximately an 80% maximal response. EC50 or IC50 values for compounds of the present invention were determined from eight-point dose-response studies.
Measurement: IC50 = 57.9 nM (pChEMBL 7.239999771118164)
Descriptors: MW 541.04, ALogP 5.85, HBA 5, HBD 1, PSA 83.55, QED 0.34
Compound (SMILES): CCN(CC)CCC(=O)N1CCC(c2cc(NC(=O)c3cnn4cc(Br)cnc34)n(-c3ccc(C)cc3)n2)CC1
InChIKey: RUQWXDGDBYGUMS-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 < 100 nM (pChEMBL 7)
Descriptors: MW 607.56, ALogP 4.68, HBA 8, HBD 1, PSA 100.66, QED 0.3
Compound (SMILES): O=C(C(O)C1([C@H]2COC[C@@H](C3CC3)N2S(=O)(=O)c2ccc(Cl)cc2)CC1)N1CC2CCC(C1)N2
InChIKey: XAEYUPWUZQUEBV-CJYVTADDSA-N
Target: Cytochrome P450 3A4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08684
Assay: Inhibition of recombinant human CYP3A4 expressed in baculovirus infected insect cells using fluorescent substrate
Measurement: IC50 > 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 510.06, ALogP 1.61, HBA 6, HBD 2, PSA 99.18, QED 0.6
Compound (SMILES): CNC(C)C(=O)Nc1cc(-c2ncnc3c(C)nn(C)c23)cc(C#Cc2ccc3nc(C)ccc3c2)n1
InChIKey: ARTUOGQVGICVCP-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: XIAP BIR3 & cIAP1 BIR3 Binding Assays (DELFIA): BIR3 domains (10 nM) were incubated with SMAC peptide (10 nM) in assay buffer (50 mM Tris, 120 mM NaCl, 0.1% BSA, 1 mM DTT, 0.05% TritonX100) for one hour at room temperature in the presence of inhibitory compounds. The assay mixture was transferred to a strepatvidin coated plate and incubated for one hour at room temperature to allow binding of the biotinylated peptide and associated BIR3 domains to the plate. After several washing steps Eu labeled anti-GST antibody (e.g. Perkin Elmer DELFIA Eu-N1-antiGST AD0250) was added to detect BIR3 domain-SMAC peptide interactions according to Perkin Elmer's instructions. Briefly, the antibody was added (dilution 1:5000 in Perkin Elmer DELFIA Assay Buffer 2013-01) and incubated for one hour. After 3 washing steps using Delfia Washing Buffer (Perkin Elmer DELFIA Wash 2013-05), Enhancement Solution (Perkin Elmer Enhancement Asolution 2013-02) was added and incubation continued for 10 minutes. Time resolved Europium fluorescence was measured in a Wallac Victor using Standard assay settings.
Measurement: IC50 = 131 nM (pChEMBL 6.880000114440918)
Descriptors: MW 490.57, ALogP 3.54, HBA 8, HBD 2, PSA 110.51, QED 0.37
Compound (SMILES): CCN1CC(NC(=O)c2ccc3cc(Oc4ccc(C(F)(F)F)cn4)ccc3n2)CC1=O
InChIKey: PJWLKPFEAXQJNQ-UHFFFAOYSA-N
Target: Sodium channel protein type 9 subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: Q15858
Assay: FRET-Based Membrane Potential Assay: Two days prior to the experiment, frozen HEK293 cells stably expressing recombinant human Nav1.7 were quickly thawed and plated at 25,000 cells/well in growth medium [DMEM (Invitrogen #11965) with 10% HI FBS (Invitrogen #10082), 2 mM glutamine, 100 units/mL penicillin, 0.1 mg/mL streptomycin (PSG, Sigma #G1146), and 500 ug/mL Geneticin (Invitrogen #10131)] in black-walled, clear-bottom 384-well poly-D-lysine-coated assay plates (Greiner Bio-One, Frickenhausen, Germany) and incubated in a humidified 5% CO2 incubator at 37° C. On the day of the assay, medium was removed by aspiration, and cells were washed with assay buffer [HBSS (Invitrogen, Carlsbad, Calif.) containing 20 mM HEPES (Invitrogen, Carlsbad, Calif.)]. After washing, 30 uL assay buffer containing the fluorescent voltage-sensor probe CC2-DMPE (Invitrogen, Carlsbad, Calif.) at 20 uM and 0.01% pluronic F-127 (Invitrogen, Carlsbad, Calif.) was added to the cells. Cells were incubated for 40 minutes at room temperature in the dark. Following the incubation, the cells were washed and 30 uL assay buffer containing 2.5 uM DiSBAC2(3) substrate (Invitrogen, Carlsbad, Calif.) and 0.5 mM VABSC-1 (Invitrogen, Carlsbad, Calif.) was added to the cells. The cells were incubated for 90 minutes at room temperature in the dark. Fluorescence readings were made using a FLIPR TETRA (Molecular Devices, Sunnyvale Calif.) equipped with voltage-sensor probe optics. At the start of each experiment the optimal (EC80) concentration of depolarizing agent (veratridine) was determined by testing a dilution curve of assay buffer containing veratridine (Sigma-Aldrich, St. Louis, Mo.) and 1 mg/mL scorpion venom (SVqq, from Leiurus quinquestriatus; Sigma-Aldrich, St. Louis, Mo.). Compounds were dissolved in dimethyl sulfoxide, and 8-point, 1:3 dilution concentration-response curves were prepared in duplicate in dimethyl sulfoxide, followed by preparation of 0.8 uL/well daughter plates of the dilutions. Test compounds in the daughter plate were diluted to (Ë¿3x) solutions in assay buffer immediately before assaying. Using the FLIPR TETRA, 20 uL of the (3x) compound solutions were first added to the cells, then 20 uL of depolarizing solution (3xEC80 veratridine+SVqq) were added 3 minutes later to activate the channel. Changes in fluorescence were measured at wavelengths of 440-480 nm and 565-625 nm over the course of the experimental run. Membrane depolarization was expressed as a ratio of the maximum F440-480 nm/F565-625 nm reading above average baseline F440-480 nm/F565-625 nm reading. IC50 values were calculated from curve fits of the ratio data using a four-parameter logistic Hill equation (Accelrys Assay Explorer 3.3 Client, Accelrys, San Diego, Calif.) with percent inhibition plotted against compound concentration.
Measurement: IC50 = 325 nM (pChEMBL 6.489999771118164)
Descriptors: MW 444.41, ALogP 3.79, HBA 5, HBD 1, PSA 84.42, QED 0.65
Compound (SMILES): Cc1noc(C)c1CN1CC2CN(CCS(=O)(=O)N(Cc3ccc(C#N)cc3)Cc3c(C)noc3C)CC(C1)O2
InChIKey: KBHAJPOEOUVFQK-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG expressed in CHO cells incubated for 10 mins after 3 hrs incubation with Rb+-load buffer by atomic absorption spectrometry based Rb+ efflux assay
Measurement: IC50 < 31622.78 nM (pChEMBL 4.5)
Descriptors: MW 568.7, ALogP 2.69, HBA 10, HBD 0, PSA 128.94, QED 0.36
Compound (SMILES): C[C@H](Nc1nccc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1cnc(OCC(F)(F)F)c(Cl)c1
InChIKey: SRPTYBWYKYVGBT-OPQQBVKSSA-N
Target: Isocitrate dehydrogenase [NADP] cytoplasmic (SINGLE PROTEIN), Homo sapiens
UniProt: O75874
Assay: LC-MS Detection of 2-HG Assay: The biochemical reactions were performed at room temperature in 384-well Greiner flat-bottom plates (Costar, Cat. No. 781201) using a final reaction volume of 30 μL and the following assay buffer conditions: 50 mM HEPES pH 7.4, 10 mM MgCl2, 50 mM KCl, 1 mM DTT, 0.02% BSA, 5 uM NADPH and 100 uM alpha-KG. The final reaction mixture contained 3.3% DMSO and inhibitors with concentrations ranging 0.02-50 μM. The IDH1 enzyme was used at a final concentration of 0.25 nM. Following 45 minutes incubation, the reaction mixtures were quenched by the addition of 10 μL of 16% formic acid containing 800 nM of 5-carbon labeled 13C-2-HG). The protein was then precipitated by the addition of 2.5 volumes of acetonitrile followed by centrifugation (3000×g, minutes). The concentration of 2-HG in the resulting supernatants was measured by LC-MS (see below). LC-MS method. Reaction mixture supernatants were submitted to chromatographic separation on a BiobasicAX column (2.1 mm×20 mm, 5 μm particle, Thermo Scientific Inc.). The chromatographic mobile phases were A) 25 mM ammonium biocarbonate and B) acetonitrile (0.1% ammonium hydroxide). Nicotinamide was eluted at 1 ml/min using a 85-5% B gradient over 0.9 minutes (Agilent 1200SL LC system, Thermofisher LX-4 autosampler) and analyzed by multiple reaction monitoring (MRM) on a API4000 QTrap mass spectrometer (ABSciex, Framingham, Mass.) in the positive electrospray ionization (ESI+) mode. The mass transition for 2-HG and 13C-2-HG were 147→129 and 152→134, respectively.
Measurement: IC50 = 265 nM (pChEMBL 6.579999923706055)
Descriptors: MW 461.83, ALogP 3.35, HBA 8, HBD 2, PSA 109.7, QED 0.65
Compound (SMILES): N#CCCCCCCNc1c2c(nc3ccccc13)CCCC2
InChIKey: IXXXENXGUZOIDA-UHFFFAOYSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Electrophorus electricus
UniProt: O42275
Assay: Inhibition of electric eel AChE using acetylthiocholine iodide as substrate preincubated for 20 mins followed by substrate addition measured after 5 mins by spectrophotometric analysis
Measurement: IC50 = 53.9 nM (pChEMBL 7.269999980926514)
Descriptors: MW 307.44, ALogP 5, HBA 3, HBD 1, PSA 48.71, QED 0.73
Compound (SMILES): C(=C/c1n[nH]c2cc(-c3ccncc3)ccc12)\c1ccc2ccccc2c1
InChIKey: RWDBMDCJBPONOC-UXBLZVDNSA-N
Target: Serine/threonine-protein kinase Chk1 (SINGLE PROTEIN), Homo sapiens
UniProt: O14757
Assay: Inhibition of full length human C-terminal 6His-tagged CHK1 expressed in baculovirus/insect expression system assessed as reduction in NADH oxidation using Syntide2 as substrate by pyruvate kinase/lactate dehydrogenase coupled assay
Measurement: Ki = 1300 nM (pChEMBL 5.889999866485596)
Descriptors: MW 347.42, ALogP 5.95, HBA 2, HBD 1, PSA 41.57, QED 0.44
Compound (SMILES): O=C1COc2cc(NC[C@H]3CC[C@H](c4nnn5cnc6[nH]ccc6c45)CC3)ccc2N1
InChIKey: VHUPRAWJGIQSPZ-HDJSIYSDSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 417.47, ALogP 3.32, HBA 7, HBD 3, PSA 109.23, QED 0.47
Compound (SMILES): CN(CC(=O)NCCOCCOCCNC(=O)NCCOCCOCCOCCOc1cccc(-c2nc(NC(=N)N)nc3ccc(Cl)cc23)c1)CC(=O)NCCOCCOCCNC(=O)NCCOCCOCCOCCOc1cccc(-c2nc(NC(=N)N)nc3ccc(Cl)cc23)c1
InChIKey: DPHPLWGDZGMANI-UHFFFAOYSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Rattus norvegicus
UniProt: P26433
Assay: Inhibition of rat NHE3-mediated sodium-dependant hydrogen ion antiport in Opossum kidney cells preincubated in NH4Cl-HEPES buffer containing BCECF-AM dye followed by washout with ammonium free HEPES and subsequent addition of compound in presence of NHE1 antagonist EIPA by fluorescence assay
Measurement: IC50 = 31.62 nM (pChEMBL 7.5)
Descriptors: MW 1437.45
Compound (SMILES): CCNc1n[nH]c2cc(NC(=O)NCc3c(C)cccc3Cl)ncc12
InChIKey: UCFNJCPBORQIOX-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. The final reaction in each well of 25 uL consists of 0.5 nM hERK2, 900 nM unlabeled peptide, 80 nM labeled-peptide, and 30 uM ATP.
Measurement: IC50 = 11.3 nM (pChEMBL 7.949999809265137)
Descriptors: MW 358.83, ALogP 3.67, HBA 4, HBD 4, PSA 94.73, QED 0.56
Compound (SMILES): C[C@H]1C[C@H]2CSC(N)=N[C@@]2(c2nc(NC(=O)c3ccc(=O)n(C)c3)cs2)CO1
InChIKey: IECQITLUAYZASQ-RSNWOKGLSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: BACE1 Cell-Free Assay: The substrate is Biotin-GLTNIKTEEISEISY^EVEFR-C[Oregon Green]KK-OH. The BACE1 enzyme is affinity purified material from conditioned media of CHO-K1 cells that have been transfected with a soluble BACE construct (BACE1deltaTM96His). Compounds are incubated in a ½ log dose response curve from a top concentration of 100 μM with BACE1 enzyme and the biotinylated fluorescent peptide in 384-well black plates (Thermo Scientific #4318). BACE1 is at a final concentration of 0.1 nM with a final concentration of peptide substrate of 150 nM in a reaction volume of 30 μL assay buffer [100 mM sodium acetate, pH 4.5 (brought to pH with acetic acid), and 0.001% Tween-20]. Plates are covered and incubated for 3 hours at 37 °C. The reaction is stopped with the addition of 30 μL of 1.5 μM Streptavidin (Pierce, #21125). After a 10 minute incubation at room temperature, plates are read on a PerkinElmer EnVision for fluorescence polarization (Ex485 nm/Em530 nm).
Measurement: IC50 = 1410 nM (pChEMBL 5.849999904632568)
Descriptors: MW 419.53, ALogP 1.78, HBA 9, HBD 2, PSA 111.6, QED 0.78
Compound (SMILES): Cn1cnc(S(=O)(=O)NCCN2CC3CN(CCCOc4ccc(C#N)cc4)CC(C2)O3)c1
InChIKey: IUXXPERGRRBARE-UHFFFAOYSA-N
Target: Voltage-gated inwardly rectifying potassium channel KCNH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q12809
Assay: Inhibition of human ERG expressed in CHO cells incubated for 10 mins after 3 hrs incubation with Rb+-load buffer by atomic absorption spectrometry based Rb+ efflux assay
Measurement: IC50 < 31622.78 nM (pChEMBL 4.5)
Descriptors: MW 474.59, ALogP 0.42, HBA 9, HBD 1, PSA 112.72, QED 0.49
Compound (SMILES): NCc1ccc(CNC(=O)[C@H](Cc2ccccc2)NC(=O)[C@@H](CC2CCCCC2)NCC(N)=O)cc1
InChIKey: AVERXCDVOOTDJJ-RPBOFIJWSA-N
Target: Plasma kallikrein (SINGLE PROTEIN), Homo sapiens
UniProt: P03952
Assay: In Vitro Inhibition Assay: Plasma kallikrein inhibitory activity in vitro was determined using standard published methods (see e.g. Johansen et al., Int. J. Tiss. Reac. 1986, 8, 185; Shori et al., Biochem. Pharmacol., 1992, 43, 1209; Stiirzebecher et al., Biol. Chem. Hoppe-Seyler, 1992, 373, 1025). Human plasma kallikrein (Protogen) was incubated at 37° C. with the fluorogenic substrate H-DPro-Phe-Arg-AFC and various concentrations of the test compound. Residual enzyme activity (initial rate of reaction) was determined by measuring the change in optical absorbance at 410 nm and the IC50 value for the test compound was determined.
Measurement: IC50 = 4300 nM (pChEMBL 5.369999885559082)
Descriptors: MW 493.65, ALogP 1.9, HBA 5, HBD 5, PSA 139.34, QED 0.29
Compound (SMILES): NC1=N[C@](CF)(c2cccc(Nc3nccc4cc(Cl)cnc34)c2)C[C@@H](C(F)(F)F)O1
InChIKey: WUZUAEBAYWXFPN-HNAYVOBHSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro Enzymatic BACE FRET Assay: The assay buffer used in this screen is 0.05 M acetate, pH 4.2, 10% DMSO final, 100 uM genapol (which is a nonionic detergent, below its Critical Micelle Concentration). The Beta Secretase enzyme (0.2 nM) is pre-incubated for one hour with inhibitors, typically in about 1 uL of DMSO according to a serial dilution, are added thereto. This assay is effectively started by the addition of FRET substrate (50 nM) and the combination is incubated for one hour. The FRET assay is terminated with by addition of Tris buffer, which raises the pH to neutrality, and the fluorescence is determined. The FRET substrate is a peptide with commercially available fluorophore and quencher, on opposite sides of the BACE cleavage site. Proteolytic cleavage of the FRET substrate releases quenching of fluorescence (excitation 488 nm and emission 425 nm).
Measurement: IC50 = 67 nM (pChEMBL 7.170000076293945)
Descriptors: MW 453.83, ALogP 4.86, HBA 6, HBD 2, PSA 85.42, QED 0.55
Compound (SMILES): COc1ccc(CNc2nc(N3CCC4(CC3)CC4)ncc2C(=O)NCc2ncccn2)cc1Cl
InChIKey: BMDHRXZKXUVMEA-UHFFFAOYSA-N
Target: cGMP-specific 3',5'-cyclic phosphodiesterase (SINGLE PROTEIN), Homo sapiens
UniProt: O76074
Assay: Caliper Mobility-Shift Assay: To a 96-well plate, 20 μL, of 10 μM FL-cGMP as the substrate was added; then 1 μL, of solution of the compound in DMSO or DMSO solution without compound was added, and then 29 μL of 1.38 ng/μL PDE-5A enzyme buffer (100 mM Hepes pH 7.5, 5 mM MgCl2, 0.002% Brij-35) was added. The maximum final concentration of the compound was 10 μM. The plate was incubated at 30° C. for 1 h, and 20 μL of 70 μM EDTA was then added to stop the reaction. 26 μL of solution of each well was transferred to a 384-well plate.
Measurement: IC50 = 26.76 nM (pChEMBL 7.570000171661377)
Descriptors: MW 494, ALogP 3.85, HBA 8, HBD 2, PSA 105.16, QED 0.49
Compound (SMILES): CC(C)CN(c1cccc(Cl)c1)S(=O)(=O)c1cccc(-c2ccc(S(C)(=O)=O)cc2)c1
InChIKey: HHBCTRPETODNQC-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: In vitro RORc Ligand Binding Assay: On day of the assay, 100 μL of 0.05% CHAPS (in deionized H2O) was added to all wells of the GFB Unifilter plate and allowed soak for 1 h. A wash buffer of 50 mM HEPES (pH 7.4), 150 mM NaCl, and 5 mM MgCl2 was prepared to wash the filter plate. To prepare an assay buffer, BSA was added to the wash buffer to reach 0.01% and DTT was added to reach 1 mM. For IC50 mode, 10 mM compound stocks were serially diluted in DMSO with DMSO to give 20× required final concentration in DMSO (15 μL compound + 30 μL DMSO). The 20× compound stocks were diluted in DMSO with Assay Buffer 4-fold to reach 5× the final test concentration in 25% DMSO (10 μL compound + 30 μL Assay Buffer). Solutions were mixed by aspiration several times with a pipette set on 50 μL volume. For the assay, 10 μL of 5× compound stock solutions in 25% DMSO were added to the assay plate in duplicate. Assay plates were 96-well polypropylene V-bottom plates. 10 μL of 5× compound in 25% DMSO/75% Assay Buffer was added to Test wells. 10 μL of 25% DMSO/75% Assay Buffer was added to Total Binding or No Receptor wells. 10 μL of 5 μM 25-hydroxycholesterol in 25% DMSO/75% Assay Buffer was added to NSB wells. 20 μL of 15 nM 25-[3H]hydroxycholesterol prepared in Assay Buffer was added to all wells. 20 μL of 1.5 ug/mL RORc receptor was added to wells (or 40 μL Assay Buffer to No R wells). Following addition to the wells, the plates were incubated 3 h at 25 °C. Final concentrations were as follows: 50 mM HEPES buffer (pH 7.4); 150 mM NaCl; 1 mM DTT; 5 mM MgCl2; 0.01% BSA; 5% DMSO; 0.6 ug/mL RORc receptor; 6 nM 25-[3H]hydroxycholesterol. For NSB wells, 1 μM 25-hydroxycholesterol was also present.
Measurement: IC50 = 82 nM (pChEMBL 7.090000152587891)
Descriptors: MW 478.04, ALogP 5.26, HBA 4, HBD 0, PSA 71.52, QED 0.46