lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): CCn1cc(-c2cc3c(cn2)cnn3-c2cccc(N3CCC(O)CC3)n2)cn1
InChIKey: SEWSVZNGUQAMFX-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Kinase Binding Assay: PIM-1, -2, and -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.PIM reactions were carried out in a final volume of 10 uL per well in a 384-well plate. A standard enzymatic reaction, initiated by the addition of 5 uL 2Ã¿Â¿ATP and test compound to 5 uL of 2x enzyme and FAM-peptide, contained 20 uM PIM1, 50 uM PIM2, or 55 uM PIM3, 1 uM FAM-peptide, and 10 uM ATP, in Reaction Buffer. After 90 minutes of incubation at room temperature, the phosphorylation reaction was stopped by the addition of 10 uL Termination Buffer. The product and substrate in each independent reaction were separated on a 12-sipper microfluidic chip (Caliper Life Sciences, Hopkinton, Mass.) run on a Caliper LC3000 (Caliper Life Sciences, Hopkinton, Mass.). The separation of product and substrate was optimized by choosing voltages and pressure using Caliper's Optimizer software (Hopkinton, Mass.). The separation conditions used a downstream voltage of -500V, an upstream voltage of -2150V, and a screening pressure of -1.2 psi. The product and substrate fluorophore were excited at 488 nm and detected at 530 nm. Substrate conversion was calculated from the electropherogram using HTS Well Analyzer software (Caliper Life Sciences, Hopkinton, Mass.). Ki values for the test compound were calculated.
Measurement: Ki = 28.3 nM (pChEMBL 7.550000190734863)
Descriptors: MW 389.46, ALogP 2.66, HBA 8, HBD 1, PSA 84.89, QED 0.58
Compound (SMILES): NC(=O)c1ccc2nc(C3C4Oc5ccc(Oc6ccnc7c6CCC(=O)N7)cc5C43)[nH]c2c1
InChIKey: ANQJLVLLYDBMBE-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Raf IC50 Assay: Compounds disclosed herein were tested against B-Raf (V600E) (PV3849, from Invitrogen) or C-Raf (Y340D/Y341D) (PV3805, from Invitrogen) in a time-resolved fluorescence energy transfer assay. The assay was carried out in reactions (10 uL) containing 0.0625 nM B-Raf or 0.5 nM C-Raf, 25 mM Tris pH7.4, 10 mM MgCl2, 0.5 mM EGTA, 0.5 mM Na3BO4, 5 mM beta-glycerophosphate, 0.01% Triton X-100, 2.5 mM DTT, 0.1% BSA, 0.1 mM ATP, 13.7 nM GST-tagged MEK1 (Full-length protein with K97R mutation, recombinant protein purified from bacterial expression system) and 0-5 uM compounds disclosed herein (final concentration of 1% DMSO). The enzyme was incubated with the compounds at room temperature for 60 minutes and the reactions were initiated by the addition of ATP and GST-MEK1. After incubating at room temperature for 60 minutes, an equal volume of stop buffer containing 25 mM Tris pH7.4, 400 mM KF, 50 mM EDTA, 0.01% BSA, 0.01% Triton X-100, 1 test of Eu3+ Cryptate-conjugated rabbit polyclonal antibody anti-Phospho MEK1/2 (Ser217/221) and 1 test of d2-conjugated mouse monoclonal antibody anti-glutathione S-transferase was added to stop the reactions. Plates were sealed and incubated at room temperature for 2 hours, and then the TR-FRET signals were read on BMG PHERAstar FS instrument. The IC50 for each compound was calculated by non linear regression by Graphpad Prism software.
Measurement: IC50 = 1 nM (pChEMBL 9)
Descriptors: MW 453.46, ALogP 3.38, HBA 6, HBD 3, PSA 132.22, QED 0.43
Compound (SMILES): CC(=O)Nc1cccc(O[C@H]2c3ccccc3C[C@@H]2N2CCCC2)c1
InChIKey: UBOGPGCPMSEBJS-SFTDATJTSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human NHE3 expressed in LAP1 cell assessed as intracellular pH recovery measured for 2 mins by CECF-AM dye based FLIPR assay
Measurement: IC50 = 438 nM (pChEMBL 6.360000133514404)
Descriptors: MW 336.43, ALogP 3.79, HBA 3, HBD 1, PSA 41.57, QED 0.92
Compound (SMILES): CC(C)(C)c1ccc(CCc2c(C(=O)O)[nH]c(=O)[nH]c2=O)cc1.NCCO
InChIKey: FOTUOOKSBVWMQM-UHFFFAOYSA-N
Target: Dihydroorotate dehydrogenase (quinone), mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: Q02127
Assay: Inhibition of human dihydroorotate dehydrogenase assessed as reduction in orotate production using L-dihydroorotate as substrate measured after 20 mins by spectrophotometric method
Measurement: IC50 = 16.8 nM (pChEMBL 7.78000020980835)
Descriptors: MW 316.36, ALogP 1.84, HBA 3, HBD 3, PSA 103.02, QED 0.8
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(S(=O)(=O)C(F)F)cc3)n2)CCC(Nc2ccccc2)CC1
InChIKey: UTAVQTXTWKRVCH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK1 (SINGLE PROTEIN), Homo sapiens
UniProt: P23458
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 0.05 nM (pChEMBL 10.300000190734863)
Descriptors: MW 528.59, ALogP 4.39, HBA 8, HBD 3, PSA 142.9, QED 0.37
Compound (SMILES): CCCC/C=C/c1ccc2c(NCc3ccc(NC(=O)c4ccc(F)cc4)cc3)nc(N(C)C)nc2c1
InChIKey: ZBNFPOPNAPVGTL-BQYQJAHWSA-N
Target: TCF4/beta-catenin (PROTEIN COMPLEX), Homo sapiens
UniProt: P35222
Assay: Inhibition of TCF-4/beta catenin in human Tcf22C11 cells expressing Tcf-4/SV40 binding sites driving firefly luciferase reporter gene after 24 hrs by luminometer
Measurement: IC50 = 615 nM (pChEMBL 6.210000038146973)
Descriptors: MW 497.62, ALogP 6.9, HBA 5, HBD 2, PSA 70.15, QED 0.23
Compound (SMILES): CN1Cc2c(Cl)cc(Cl)cc2[C@H](c2cccc(S(=O)(=O)NCCOCCOCCNC(=O)CCC(CCC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)(CCC(=O)NCCOCCOCCNS(=O)(=O)c3cccc([C@@H]4CN(C)Cc5c(Cl)cc(Cl)cc54)c3)NC(=O)NC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO)c2)C1
InChIKey: MBBHFKPMGSMTNM-JTFILVBRSA-N
Target: Sodium/hydrogen exchanger 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P48764
Assay: Inhibition of human 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 = 53.7 nM (pChEMBL 7.269999980926514)
Descriptors: MW 1907.78
Compound (SMILES): COc1cc(N2CCOCC2)ccc1Nc1nc2c(c(Nc3ccc(F)c(F)c3)n1)C(C)(C)CN2
InChIKey: HCVKKXDEQZMTLX-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 482.54, ALogP 4.79, HBA 8, HBD 3, PSA 83.57, QED 0.46
Compound (SMILES): CC(=O)c1ccc(OCc2ccc(Oc3cccc(-c4nn[nH]n4)c3)cc2)c(C)c1O
InChIKey: QIADWIHDXWIEST-UHFFFAOYSA-N
Target: Cysteinyl leukotriene receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y271
Assay: Antagonist activity at human CysLT1 receptor expressed in Syrian hamster AV12-664 cells assessed as decrease in LTD4-induced intracellular calcium level pretreated followed by LTD4 addition by fluo-3 dye based FLIPR assay
Measurement: IC50 = 1430 nM (pChEMBL 5.840000152587891)
Descriptors: MW 416.44, ALogP 4.45, HBA 7, HBD 2, PSA 110.22, QED 0.43
Compound (SMILES): Cn1cc(-c2cccc(Nc3nn(C4(CC#N)CCN(C(=O)OCC(F)(F)F)CC4)cc3C(N)=O)c2)cn1
InChIKey: BNKQHKDVXKUESH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 21 nM (pChEMBL 7.679999828338623)
Descriptors: MW 530.51, ALogP 3.53, HBA 9, HBD 2, PSA 144.09, QED 0.48
Compound (SMILES): CN(c1cccc(CO)c1)c1nc(Nc2cc(N3CCOCC3)cc(S(C)(=O)=O)c2)ncc1F
InChIKey: VWEBEVMRPSIULB-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase PAK 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q13153
Assay: Inhibition of PAK1 (unknown origin) by ATP-kinaseGlo assay
Measurement: IC50 = 45 nM (pChEMBL 7.349999904632568)
Descriptors: MW 487.56, ALogP 2.86, HBA 9, HBD 2, PSA 107.89, QED 0.52
Compound (SMILES): CNc1nc2cc(C3(O)c4ccccc4C(=O)N3Cc3ccccc3)ccc2[nH]1
InChIKey: WCKJQTNVFAFOSR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 384.44, ALogP 3.45, HBA 4, HBD 3, PSA 81.25, QED 0.5
Compound (SMILES): Cc1cc(C)c(CNC(=O)c2cc(-c3ccc(CN4CCOCC4)cc3)cc3c2C(C)CN3C2CCOCC2)c(=O)[nH]1
InChIKey: AGFJSOIBLBIMSF-UHFFFAOYSA-N
Target: Histone-lysine N-methyltransferase EZH2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q15910
Assay: Inhibition of human wild type EZH2 using histone H3 as substrate after 1 hr in presence of 3H-SAM by filter paper detection analysis
Measurement: IC50 = 6980 nM (pChEMBL 5.159999847412109)
Descriptors: MW 570.73, ALogP 4.52, HBA 6, HBD 2, PSA 86.9, QED 0.43
Compound (SMILES): COC/C(CN(c1cc(OC)cc(OC)c1)c1ccc2ncc(-c3cnn(C)c3)nc2c1)=N/O
InChIKey: YIDCCLXYFLNYHA-OGLMXYFKSA-N
Target: Fibroblast growth factor receptor 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P22607
Assay: Inhibition of FGFR3 (unknown origin) transfected in mouse BA/F3 cells assessed as decrease in cell proliferation in presence of mouse IL3 after 24 hrs by Alamar blue assay
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 462.51, ALogP 3.66, HBA 10, HBD 1, PSA 107.12, QED 0.23
Compound (SMILES): N#CCN1CN(C2CCCCC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: ZKPOLKXRXJQNCH-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 309.37, ALogP 2.64, HBA 4, HBD 1, PSA 76.02, QED 0.87
Compound (SMILES): CS(=O)(=O)c1ccc(Nc2nn(C3(CC#N)CCC4(CC3)OCCO4)cc2C(N)=O)cc1
InChIKey: VDKBVSZYIVNJCX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 206 nM (pChEMBL 6.690000057220459)
Descriptors: MW 459.53, ALogP 2.06, HBA 9, HBD 2, PSA 149.33, QED 0.67
Compound (SMILES): COc1cc(/C=C/C(=O)CC(=O)/C=C/c2ccc(O)c(CO)c2)ccc1O
InChIKey: UAVJPMNEDOIHEI-YPCIICBESA-N
Target: Neutrophil collagenase (SINGLE PROTEIN), Homo sapiens
UniProt: P22894
Assay: In vitro MMP-8 Assay: The synthetic octapeptide substrate, containing the collagenase-susceptible glycine-isoleucine peptide bond, was incubated (37° C.) with commercially-available chromatrographically-purified human neutrophil collagenase (MMP-8) in the presence of 1 mM Ca++ and the tripeptide degradation fragment and undegraded substrate were separated and measured by HPLC (Waters Alliance 2695 System with a reverse phase C-18 column).
Measurement: IC50 = 14000 nM (pChEMBL 4.849999904632568)
Descriptors: MW 368.39, ALogP 2.85, HBA 6, HBD 3, PSA 104.06, QED 0.49
Compound (SMILES): NC1CCc2c(-c3ccc(C(F)(F)F)c(F)c3)cncc21
InChIKey: UEEOMMQKPLRCBM-UHFFFAOYSA-N
Target: Cytochrome P450 11B2, mitochondrial (SINGLE PROTEIN), Homo sapiens
UniProt: P19099
Assay: Cellular Enzyme Assay: G-402 cells expressing CYP11 constructs were established as described above and maintained in McCoy's 5a Medium Modified, ATCC Catalog No. 30-2007 containing 10% FCS and 400 ug/ml G418 (Geneticin) at 37° C. under an atmosphere of 5% CO2/95% air. Cellular enzyme assays were performed in DMEM/F12 medium containing 2.5% charcoal treated FCS and appropriate concentration of substrate (0.3-10 uM 11-Deoxycorticosterone, 11-Deoxycortisol or Corticosterone). For assaying enzymatic activity, cells were plated onto 96 well plates and incubated for 16 h. An aliquot of the supernatant is then transferred and analyzed for the concentration of the expected product (Aldosterone for CYP11B2; Cortisol for CYP11B1). The concentrations of these steroids can be determined using HTRF assays from CisBio analyzing either Aldosterone or Cortisol.
Measurement: EC50 = 54.7 nM (pChEMBL 7.260000228881836)
Descriptors: MW 296.27, ALogP 3.85, HBA 2, HBD 1, PSA 38.91, QED 0.81
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#Cc1ccc(F)cn1)c1nc2cccc(Cl)c2c(=O)n1-c1cccc(C(C)(C)C)c1
InChIKey: DMBSEXOESDZTMZ-SFHVURJKSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: PI3K isoforms were assayed under initial rate conditions in the presence of 25 mM Hepes (pH 7.4), and 2xKm ATP (100-300 uM), 10 uM PIP2, 5% glycerol, 5 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM dithiothreitol, 1% (v/v) DMSO at the following concentrations for each isoform: PI3K alpha, beta, delta, and gamma at 50 picomolar (pM) and PI3Kgamma at 2 nanomolar (nM). After an assay reaction time of 30 minutes at 25° C., reactions were terminated with a final concentration of 10 mM EDTA, 10 nM labeled-PIP3, and 35 nM Europium labeled GRP-1 detector protein before reading TR-FRET on an Envision plate reader (Ex: 340 nm; Em: 615/665 nm; 100 us delay and 500 us read window).
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 568.06, ALogP 5.82, HBA 8, HBD 2, PSA 111.61, QED 0.26
Compound (SMILES): Cc1cc(C#N)cnc1C(=O)Nc1cc(Cl)c(F)c([C@]2(C)C[C@@H](C(F)(F)F)OC(N)=N2)c1
InChIKey: ZBZPGJLTVUOKJO-LIRRHRJNSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: In Vitro BACE Cell-Based Assay: The cell-based assay measures inhibition or reduction of Aβ40 in conditioned medium of test compound treated cells expressing amyloid precursor protein. Cells stably expressing Amyloid Precursor Protein (APP) were plated at a density of 40K cells/well in 96 well plates (Costar). The cells were cultivated for 24 hours at 37° C. and 5% CO2 in DMEM supplemented with 10% FBS. The test compounds were then added to cells in 10-point dose response concentrations with the starting concentration being either 100 uM or 10 uM. The compounds were diluted from stock solutions in DMSO and the final DMSO concentration of the test compounds on cells was 0.1%. After 24 h of incubation with the test compounds the supernatant conditioned media was collected and the Aβ 40 levels were determined using a sandwich ELISA.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 469.83, ALogP 4.19, HBA 6, HBD 2, PSA 113.39, QED 0.65
Compound (SMILES): Cl.O=C(O)[C@]1(Cc2cc(Br)cc(Nc3nccs3)n2)CC[C@@H](Oc2cccc(Cl)c2F)CC1
InChIKey: HFKHTRIHTHFUPY-PGIBDOGXSA-N
Target: Aurora kinase B/Inner centromere protein (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9NQS7
Assay: Inhibition of human full-length N-terminal GST-tagged Aurora B/His-tagged INCENP (803 to 916 residues) using kemptide as substrate and [gamma33P]ATP after 45 mins by liquid scintillation counting method
Measurement: IC50 = 95 nM (pChEMBL 7.019999980926514)
Descriptors: MW 540.84, ALogP 6.47, HBA 6, HBD 2, PSA 84.34, QED 0.36
Compound (SMILES): Cc1cccc2cc(C(C)Nc3ncnc4[nH]cnc34)n(CCN3CCOCC3)c(=O)c12
InChIKey: CYQTVDMRYDCRGC-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit gamma isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P48736
Assay: Inhibition of recombinant human C-terminal His6-tagged p110gamma expressed in insect cells using phosphatidylinositol as substrate in presence of gamma-32P-ATP after 5 mins by thin layer chromatographic method
Measurement: IC50 < 10000 nM (pChEMBL 5)
Descriptors: MW 433.52, ALogP 2.48, HBA 8, HBD 2, PSA 100.96, QED 0.48
Compound (SMILES): O=C(Nc1ccc(Cl)c(S(=O)(=O)C2CCNCC2)c1O)Nc1cccc2c1OC(F)(F)O2
InChIKey: QBBZZDIQIBBIPR-UHFFFAOYSA-N
Target: C-X-C chemokine receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P25025
Assay: Displacement of [125I]IL8 from recombinant human CXCR2 expressed in CHO cell membranes measured after 1 hr by TopCount scintillation counting method
Measurement: IC50 < 30000 nM (pChEMBL 4.5229997634887695)
Descriptors: MW 489.88, ALogP 3.54, HBA 7, HBD 4, PSA 125.99, QED 0.48
Compound (SMILES): COc1cc(N2CCOCC2)ccc1Nc1nc2c(c(Nc3ccccc3S(=O)(=O)NC(C)(C)C)n1)C1(CC1)CN2
InChIKey: ILEIMULPPONSEI-UHFFFAOYSA-N
Target: Protein-tyrosine kinase 2-beta (SINGLE PROTEIN), Homo sapiens
UniProt: Q14289
Assay: Biochemical Assay: To measure inhibitory activity of FAK/PYK2 inhibitors, compounds were first prepare as a 1 mM stock in 100% DMSO and 3-fold serial dilution was performed in a 96-well plate (Corning, 3897) to generate 12 different concentration of 100× stock. A 5 μl of 100× stock of each concentration was added to wells containing 95 μl of 1× reaction buffer (40 mM Tris, pH7.5, mM MgCl2, 1 mM DTT and 1 mM CHAPS) to generate 5× stock. Then 2 μl of 5× stock of each concentration was added to a 384 wells-OptiPlate (PerkinElmer, 6007299). For FAK, 4 μl of 2.5 nM FAK, 1 μl of 8 μg/μl BSA and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 33.3 μM ATP. The reaction was allowed to proceed for 120 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). For PYK2, 5 μl of 2 nM PYK2 and 1.5 μl of 666 nM ULight-poly Glu, Ala, Tyr (1:1:1) peptide substrate, prepared in above reaction buffer, were added to each well. The reaction was initiated by adding 1.5 μl of 46.6 μM ATP. The reaction was allowed to proceed for 40 min before being quenched with 5 μl of 40 mM EDTA stop buffer prepared in 1×LANCE detection buffer (PerkinElmer, CR97-100). Upon quenching of the reaction, 5 μl of 8 nM anti-phosphotyrosine antibody was added to each well and incubated for 60 minutes.
Measurement: IC50 < 20 nM (pChEMBL 7.698999881744385)
Descriptors: MW 579.73, ALogP 4.34, HBA 10, HBD 4, PSA 129.74, QED 0.31
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCN(Cc2ccon2)CC1
InChIKey: WTYSYXMPFTVRTE-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: HTRF Biochemical Assay: The reactions employed a common peptide substrate, LCB-EQEDEPEGDYFEWLW-NH2 (in-house). The basic assay protocol is as follows: First, 250 nL of diluted compounds in DMSO were dispensed into the wells of a dry 384-well Black plate (Greiner #781076) using a Labcyte Echo 555 acoustic dispenser. Subsequent reagent additions employed an Agilent Bravo. Next, 18 uL of 1.11x enzyme and 1.11x substrate in 1x assay buffer (Invitrogen kinase buffer # PV3189, 2 mM DTT, 0.05% BSA) were added to the wells and shaken and then preincubated for 30 minutes at room temperature to allow compound binding to equilibrate. After equilibration, 2 uL of 10xATP in 1x assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at room temperature for 120 minutes. At the end of the incubation, 20 uL of 2x stop buffer (streptavidin-Dylight 650 (Thermo #84547B/100 mL), Eu-tagged pY20 antibody (Perkin Elmer #AD0067), EDTA, HEPES, and Triton) was added to quench the reaction.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 423.45, ALogP 2.76, HBA 8, HBD 2, PSA 126, QED 0.6
Compound (SMILES): CN1CN([C@H]2CC[C@H](CNCC(F)(F)F)CC2)c2c(cnc3[nH]ccc23)C1=O
InChIKey: PCSBZPSZMDJEPX-JOCQHMNTSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: The inhibitory activity of the compounds of the present invention against JAK was measured.The respective enzymes (JAK1, JAK2, JAK3 and Tyk2) were purchased from Carna Biosciences, Inc.As a substrate of the enzymes (hereinafter referred to as substrate), LANCE Ultra ULight-JAK-1 (Tyr1023) Peptide (manufactured by PerkinElmer Inc.) was used.As an antibody for detecting phosphorylation of the substrate, LANCE Ultra Europium-anti-phospho tyrosine antibody (PT66) (manufactured by PerkinElmer Inc.) was used.The other reagents were purchased from the following.Adenosine triphosphate (ATP): Sigma-Aldrich4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES): DOJINDO LABORATORIESGlycol ether diamine tetraacetic acid (EGTA): DOJINDO LABORATORIES Magnesium chloride (MgCl2): Wako Pure Chemical Industries, Ltd.Dithiothreitol (DTT): Wako Pure Chemical Industries, Ltd.Tween 20: Sigma-AldrichEthylenediaminetetraacetic acid (EDTA): DOJINDO LABORATORIESThe compounds of the present invention, the enzymes (JAK1, JAK2, JAK3 and Tyk2), the substrate and ATP were used for assay as diluted with assay buffer.Â  As the assay buffer, one having the following composition was used.Â  HEPES (pH7.5): 50 mM EGTA: 1 mM MgCl2: 10 mM DTT: 2 mM Tween 20: 0.01% (wt/wt). The dilute concentration and the addition amount on a well plate as described hereinafter were adjusted so that the following final concentrations were achieved on the well plate.
Measurement: IC50 = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 395.43, ALogP 3.12, HBA 4, HBD 2, PSA 64.26, QED 0.84
Compound (SMILES): CCCS(=O)(=O)Nc1ccc(F)c(C(=O)c2c[nH]c3ncc(-c4cnc(OCCCN(C)C)nc4)cc23)c1F
InChIKey: LZSFSMKLIQMQFG-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition of B-Raf V600E mutant (unknown origin) assessed as decrease in biotin-tagged MEK1 phosphorylation after 45 mins
Measurement: IC50 < 10 nM (pChEMBL 8)
Descriptors: MW 558.61, ALogP 4.01, HBA 8, HBD 2, PSA 130.17, QED 0.2
Compound (SMILES): COc1ccc(CN2CCC(c3ccc4nc(C(=O)N5CCN(Cc6ccc(C(F)(F)F)cc6)CC5)[nH]c4c3)CC2)cc1
InChIKey: DCOPJZULHMHERN-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42336
Assay: Cell Proliferation Inhibition Assay: In order to make human PIK3 CA mutation-positive breast cancer cell line MDA-MB-453 cells become 500 cells per well in a 384 well non-adhesive plate (Lipidure-Coat plate AT-384; manufactured by NOF Corporation), 36 μl/well was seeded in Leibovitz's L-15 medium including 10% fetal bovine serum (manufactured by Life technologies Corp.), followed by culturing at 37° C. overnight in the presence of CO2. The following day, the test compound (the test compound had 11 steps in a final concentration from 10000 nM to 0.1 nM), and DMSO which is a solvent for the test compound as a negative control were diluted with the medium, and 4 μl was added to cells. Thereafter, the cells were cultured for 4 days at 37° C. in the absence of CO2, and a cytometric reagent (CellTiter-Glo Luminescent Cell Viability assay manufactured by Promega Corporation) was added. The resultant product was stirred for 30 minutes, and then measurement was carried out using a luminescence measurement apparatus
Measurement: IC50 = 7.6 nM (pChEMBL 8.119999885559082)
Descriptors: MW 591.68, ALogP 5.93, HBA 5, HBD 1, PSA 64.7, QED 0.29
Compound (SMILES): COc1cccc(F)c1-c1c[nH]c2ncnc(N3CCOCC3)c12
InChIKey: UGVUXLJRIDUDEE-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Lantha Screen Technology Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat # PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: 3 uL of a working solution of substrate (233 nM LRRKtide, 117 uM ATP) prepared in assay buffer (50 mM HEEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to a low volume Greiner 384-well plate. The compound dose response was prepared by diluting compound to a top concentration of 3.16 mM in 100% DMSO and serial diluted by half-log in DMSO 11 times. Aliquots (3.5 uL) of the 100% DMSO dose response were mixed with 46.5 uL water.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 328.35, ALogP 2.61, HBA 5, HBD 1, PSA 63.27, QED 0.8
Compound (SMILES): CN[C@@H](C)C(=O)N[C@H](C(=O)N1CCc2cc(Cl)ccc2[C@@H]1C(=O)Nc1c(F)cccc1F)C(C)C
InChIKey: GAFFUNZAQFRLEV-PRPYNDMISA-N
Target: E3 ubiquitin-protein ligase XIAP (SINGLE PROTEIN), Homo sapiens
UniProt: P98170
Assay: TR-FRET Assay: The peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA ("Peptide A") was identified as a substrate for the TR-FRET assay by screening the 6× Histidine-tagged BIR2 domain and BIR3 domain of XIAP against a set of 29 peptides synthesized based on sequences reported by Sweeny et al. (Biochemistry, 2006, 45, 14740 14748). The peptides were labeled with the fluorescent tags FITC or TAMRA and Kd values were determined by fluorescence polarization assay. The sequence AVPIAQKSEK was identified as optimal for using in an assay. The peptide sequence was derivatized with biotin to provide AVPIAQKSEK-(Îµ-biotin)-OH 1:2 TFA as the substrate for the TR-FRET assay. Ten nanomolar of 6× Histidine-tagged BIR2 domain, corresponding to amino acids 124-240 of XIAP, or BIR3 domain, corresponding to amino acids 241-356 of XIAP, was mixed with 20 nM of the peptide AVPIAQKSEK-(ε-biotin)-OH 1:2 TFA, in the presence of 50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol (DTT) and 0.1 mg/mL bovine serum albumin (BSA). Following a 45 min. incubation at 37° C., Europium-Streptavidin and Allophycocyanin conjugated anti-Histidine antibody were added to a final concentration of 1.5 nM and 15 nM, respectively. Time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured 1 hour later at room temperature. Test compound potency was assessed at 10 serially diluted concentrations.
Measurement: IC50 > 54800 nM (pChEMBL 4.261000156402588)
Descriptors: MW 506.98, ALogP 3.43, HBA 4, HBD 3, PSA 90.54, QED 0.54
Compound (SMILES): O[C@H]1[C@@H](O)C[C@@]2(n3cnc4c(NCc5cccc(OCCF)c5)nc(Cl)nc43)C[C@@H]12
InChIKey: ONBWPLMRNKCNOQ-AQXBJYLJSA-N
Target: Adenosine receptor A3 (SINGLE PROTEIN), Homo sapiens
UniProt: P0DMS8
Assay: Radioligand Binding Assay: Radioligand binding assays at A1, A2A, and A3ARs were performed according to the procedures described previously. Each tube in the binding assay contained 100 uL of membrane suspension (20 ug of protein), 50 pt of a stock solution of agonist radioligand, and 50 uL of increasing concentrations of the test ligands in Tris-HCl buffer (50 mM, pH 7.5) containing 10 mM MgCl2. Nonspecific binding was determined using a final concentration of 10 uM NECA, a non-specific agonist, diluted with the buffer.The mixtures were incubated at 25° C. for 60 min. Binding reactions were terminated by filtration through Whatman GF/B filters under a reduced pressure using a MT-24 cell harvester (Brandell, Gaithersburg, Md.). Filters were washed three times with 5 ml of 50 mM ice-cold Tris-HCl buffer (pH 7.5). The radioactive agonists [3H]R-PIA and [3H]CGS21680 were used for the A1 and A2AAR assays, respectively, while [125I]AB-MECA was used for the A3AR assay.
Measurement: Ki = 114 nM (pChEMBL 6.940000057220459)
Descriptors: MW 433.87, ALogP 2.28, HBA 8, HBD 3, PSA 105.32, QED 0.49
Compound (SMILES): CNCCN1CCC(CCc2ccccc2)CC1
InChIKey: VZZSAYLJCKTKMS-UHFFFAOYSA-N
Target: Protein arginine N-methyltransferase 6 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96LA8
Assay: Inhibition of human full length PRMT6 (1 to 375 residues) expressed in baculovirus expression system assessed as inhibition of methylation activity preincubated with protein followed by addition of biotin labeled histone H4 (1 to 24) peptide as substrate and [3H]-SAM measured after 3 hrs by scintillation proximity assay
Measurement: IC50 = 56 nM (pChEMBL 7.25)
Descriptors: MW 246.4, ALogP 2.55, HBA 2, HBD 1, PSA 15.27, QED 0.83
Compound (SMILES): COC(=O)[C@@H](Cc1cc2ccnc(N)c2cc1F)N1CC[C@H](NS(=O)(=O)c2cnc(N(C)C)c(Cl)c2)C1=O
InChIKey: MJYAEKTZMOJSNM-VQTJNVASSA-N
Target: Coagulation factor X (SINGLE PROTEIN), Homo sapiens
UniProt: P00742
Assay: Inhibition Assay: The compounds according to the invention are tested in a range of concentrations (10pM to 10 µM final) in assay buffer (50 mM TRIS, 100 mM NaCl, 0.1 % BSA, pH 7,5) with 0.1% maximal final DMSO concentration and deposited in a proportion of 25 µl per well, on 25 µL enzyme (human coagulation factor Xa: Enzyme Research Laboratories HFXa, final concentration 0.003Ul/ml). The reactive were mixed, centrifuged and incubated 10 minutes at 37°C in a 96 well microtiter plate. The enzyme reaction was started with 50 µL substrate (S-2765, Biogenic ref 821413 in a final concentration of 62.5µM final). The time course of the reaction was monitored at 405 nm in a microtiter plate reader (Tecan M200) for 20 minutes 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 = 3850 nM (pChEMBL 5.420000076293945)
Descriptors: MW 565.03, ALogP 1.73, HBA 9, HBD 2, PSA 147.82, QED 0.39
Compound (SMILES): CCN1CC(C)(C)Oc2nc(N3CCOC[C@@H]3C)nc(-c3ccc(NC(=O)Nc4ccon4)c(F)c3)c2C1=O
InChIKey: PHYRXMWTUDHGCP-HNNXBMFYSA-N
Target: Phosphatidylinositol 3-kinase regulatory subunit alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P27986
Assay: Enzyme Assay: The test uses a lucifeurerin/luciferase system to measure the concentration of ATP and its consumption during the enzymatic reaction. The test is performed in 96-well format (Corning/Costar 96 black flat-bottomed half-wells plate, ref. 3694) in a total volume of 30 μl. To 1 μl of inhibitor in 100% DMSO are added (final concentrations) 50 μM of the substrate PIP2 ((L-α-phosphatidyl-D-myoinositol 4,5-bisphosphate, Echelon 117P-4516-0500), 2 μM of ATP and 1.7 μg/ml of PI3Kα, (p110α/p85α, Invitrogen PV4788) in a buffer of Tris/HCl 50 mM pH 7.5, EGTA 1 mM, MgCl2 10 mM, Chaps 0.03%, 1 mM DTT). After 90 minutes, the reaction is quenched by adding 20 μl/well of KinaseGlo reagent (Promega V6713). After 10 minutes in the dark, the luminescence is read on the PHERAStar microplate reader (reading at 0.8 sec/well).
Measurement: IC50 = 561 nM (pChEMBL 6.25)
Descriptors: MW 539.57, ALogP 3.77, HBA 9, HBD 2, PSA 134.95, QED 0.5
Compound (SMILES): N#CCC1(n2cc(-c3ncnc4[nH]ccc34)cn2)CN(C2CCN(S(=O)(=O)C3CCCC3)CC2)C1
InChIKey: MVGIEAHSBOJVAK-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: In Vitro JAK Kinase Assay: Compounds herein were tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (a.a. 837-1142), Jak2 (a.a. 828-1132) and Jak3 (a.a. 781-1124) with an N-terminal His tag were expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). IC50s of compounds were measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1 mM IC50 measurements, ATP concentration in the reactions was 1 mM. Reactions were carried out at room temperature for 1 hr and then stopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeled antibody took place for 40 minutes and HTRF signal was measured on a Fusion plate reader (Perkin Elmer, Boston, Mass.).
Measurement: IC50 < 5 nM (pChEMBL 8.300999641418457)
Descriptors: MW 494.63, ALogP 2.48, HBA 8, HBD 1, PSA 123.8, QED 0.56
Compound (SMILES): O=C(NCC(c1cnc(C2CC2)nc1)N1CCC(F)(F)CC1)c1c(Cl)cccc1Cl
InChIKey: AJGMVGBVXCRDTI-UHFFFAOYSA-N
Target: P2X purinoceptor 7 (SINGLE PROTEIN), Homo sapiens
UniProt: Q99572
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Briefly, 293-human or mouse P2X7 stable cells were incubated in sucrose buffer, pH 7.4 [KCl (5 mM), NaH2PO42H2O (9.6 mM), HEPES (25 mM), sucrose (280 mM), glucose (5 mM), CaCl2 (0.5 mM), and probenecid (0.1425 g in 3 mL 1N NaOH was added for 500 mL solution)] in 384-well plates. 93-rat P2X7 stable cells were incubated in HHPB (pH 7.4) [consisting of Hank's BSS (1X); HEPES (pH 7.4) (20 mM) (Sigma); probenecid (0.710 g/5 mL 1N NaOH) (Sigma); and BSA (0.05%) (Roche) which was added after the pH had been adjusted] in 384-well plates. Fluo-4 NW dye mix (Molecular Probes, Inc., Eugene, Oreg., USA) was prepared in buffer (see manufacturer's instructions). Cell plates were removed from the 37° C. incubator, the media discarded and then 30 μL of dye was added to each well. Plates were placed in the 37° C., non-CO2 incubator for 30 minutes and then room temperature for 30 minutes. Two sets of drug plates were prepared: A) Mixtures of compound plus agonist were prepared as follows, in order to determine close response: BzATP: 11 point ½ log, diluted in buffer, starting from 1 mM. Testing compounds: 11 point ½ log, diluted in 2% DMSO buffer starting from 10 μM. B) Agonist only mixture was prepared with BzATP at a single concentration in buffer (concentration determined by dose response). Compound mixtures (A) were added to assay plates containing cells and placed at room temperature for 30 minutes, then BzATP (B) was added. Fluorescence was read using the Tetra FLIPR® (Molecular Devices, Inc., Sunnyvale, Calif., USA) and IC50 values were calculated by standard methods to determine antagonist activity.
Measurement: IC50 = 5.3 nM (pChEMBL 8.279999732971191)
Descriptors: MW 455.34, ALogP 4.86, HBA 4, HBD 1, PSA 58.12, QED 0.68
Compound (SMILES): O=C(Nc1cn(CC(=O)N2CCC(N3CCCC3)CC2)nc1-c1cc(Cl)ccc1OC(F)F)c1cnn2cccnc12
InChIKey: ZNCDEAFFPCDSRS-UHFFFAOYSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: JAK Enzyme Assay: The activity of the isolated recombinant JAK1 and JAK2 kinase domain was measured by monitoring phosphorylation of a peptide derived from JAK3 (Val-Ala-Leu-Val-Asp-Gly-Tyr-Phe-Arg-Leu-Thr-Thr, fluorescently labeled on the N-terminus with 5-carboxyfluorescein) using the Caliper LabChip® technology (Caliper Life Sciences, Hopkinton, Mass.). To determine inhibition constants (Ki), compounds were diluted serially in DMSO and added to 50 μL kinase reactions containing purified enzyme (1.5 nM JAK1, or 0.2 nM JAK2), 100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 1.5 μM peptide substrate, ATP (25 μM), 10 mM MgCl2, 4 mM DTT at a final DMSO concentration of 2%. Reactions were incubated at 22° C. in 384-well polypropylene microtiter plates for 30 minutes and then stopped by addition of 25 μL of an EDTA containing solution (100 mM HEPES buffer (pH 7.2), 0.015% Brij-35, 150 mM EDTA), resulting in a final EDTA concentration of 50 mM. After termination of the kinase reaction, the proportion of phosphorylated product was determined as a fraction of total peptide substrate using the Caliper LabChip® 3000 according to the manufacturer's specifications.
Measurement: Ki = 2.45 nM (pChEMBL 8.609999656677246)
Descriptors: MW 599.04, ALogP 4.19, HBA 9, HBD 1, PSA 109.89, QED 0.32
Compound (SMILES): c1cc2[nH]cc(-c3cncc(C4CC4)n3)c2cc1-c1nnc(NC2CC2)o1
InChIKey: GHJCZGQHMLEEKQ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Enzyme Assay: The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 96-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction.Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 10-point dosing curve having a high dose of 1 uM. A reference compound was included on each assay plate in order to validate that plate; on
Measurement: IC50 = 7.641 nM (pChEMBL 8.119999885559082)
Descriptors: MW 358.41, ALogP 4.13, HBA 6, HBD 2, PSA 92.52, QED 0.56
Compound (SMILES): CN[C@H]1CC[C@H](n2c(C(C)(C)C)cc(C(=O)Nc3cccc(C(F)(F)F)c3)c2C)CC1
InChIKey: CIWCSKGAUSXEJQ-UAPYVXQJSA-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: IC50 = 5600 nM (pChEMBL 5.25)
Descriptors: MW 435.53, ALogP 6.07, HBA 3, HBD 2, PSA 46.06, QED 0.61
Compound (SMILES): CC1(N)CCN(c2cccnc2NC(=O)c2nc(-c3ncccc3Cl)cnc2N)CC1
InChIKey: MFDKBLCTDGSTAK-UHFFFAOYSA-N
Target: Protein kinase C theta type (SINGLE PROTEIN), Homo sapiens
UniProt: Q04759
Assay: In Vitro Inhibition Activity Assay: The compounds of formula I were tested for their activity on different PKC isoforms according to a published method (D. Geiges et al. Biochem. Pharmacol. 1997; 53:865-875) The assay is performed in a 96-well polypropylene microtiterplate (Costar 3794) that has been previously siliconized with Sigmacote (Sigma SL-2). The reaction mixture (50 μL) contains 10 μL of the relevant PKC isozyme together with 25 μL of the PKC inhibitor compound and 15 μL of a mix solution that contains 200 μg/mL protamine sulfate, 10 mM Mg(NO3)2, 10 μM ATP (Boehringer 519987) and 3750 Bq of 33P-ATP (Hartmann Analytic SFC301, 110 TBq/mmol) in 20 mM Tris-buffer pH 7.4+0.1% BSA. Incubation was performed for 15 minutes at 32° C. in a microtiterplate shaking incubator (Biolabo Scientific Instruments). Reaction was stopped by adding 10 μl of 0.5 M Na2EDTA, pH 7.4. 50 μl of mixture are pipetted onto a pre-wetted phosphocellulose paper (Whatmann 3698-915) under gentle pressure.
Measurement: IC50 = 0.9 nM (pChEMBL 9.050000190734863)
Descriptors: MW 438.92, ALogP 2.74, HBA 8, HBD 3, PSA 135.94, QED 0.57
Compound (SMILES): COc1ccc2c3c1O[C@H]1[C@@]4(OC)CC[C@@]5(C[C@@H]4COCc4cccc(C)c4)[C@@H](C2)N(CC2CC2)CC[C@]315
InChIKey: UBANXCKYJYETIO-SROIDHQSSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: Binding assay of certain compounds of this invention to the opioid receptor was determine using radioligand binding assay (screening and dose-displacement).
Measurement: Ki = 0.15 nM (pChEMBL 9.819999694824219)
Descriptors: MW 515.69, ALogP 5.44, HBA 5, HBD 0, PSA 40.16, QED 0.47
Compound (SMILES): COc1cc(F)cc(-c2nn(C3CCc4ccccc4C3)c3ncnc(N)c23)c1
InChIKey: BAKCQTPYFUNEFJ-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit delta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: O00329
Assay: Inhibition of PI3Kdelta (unknown origin) using biotinylated PIP2 as substrate in presence of streptavidin-APC by FRET assay
Measurement: IC50 = 1340 nM (pChEMBL 5.869999885559082)
Descriptors: MW 389.43, ALogP 3.95, HBA 6, HBD 1, PSA 78.85, QED 0.57
Compound (SMILES): CN(C)c1cc(NC(=O)[C@@H]2CCC3CN2C(=O)N3OS(=O)(=O)O)ccn1
InChIKey: DTWNRDKVLZLZTB-DTIOYNMSSA-N
Target: Beta-lactamase (SINGLE PROTEIN), Pseudomonas aeruginosa
UniProt: Q8RL90
Assay: Spectrophotometric Assay: Enzyme activities were measured in the presence of the test inhibitor in spectrophotometric assay.
Measurement: IC50 = 1.6 nM (pChEMBL 8.800000190734863)
Descriptors: MW 385.4, ALogP 0.09, HBA 7, HBD 2, PSA 132.38, QED 0.68
Compound (SMILES): CC(=O)N(CCN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)Cc1c(F)cccc1F
InChIKey: RAZYOIFBXMSUEN-ACDBMABISA-N
Target: Delta-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P41143
Assay: Displacement of [3H]-diprenorphine from human delta opioid receptor expressed in cell membranes
Measurement: Ki = 100 nM (pChEMBL 7)
Descriptors: MW 414.5, ALogP 4.43, HBA 3, HBD 1, PSA 43.78, QED 0.76