lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COCC(=O)Nc1cc(Oc2ccc(NC(=O)Nc3cc(C(C)(C)C)nn3-c3ccc(C)cc3)c3ccccc23)ccn1
InChIKey: AVUUHIYJPTWYNX-UHFFFAOYSA-N
Target: Tyrosine-protein kinase SYK (SINGLE PROTEIN), Homo sapiens
UniProt: P43405
Assay: c-Src and Syk Enzyme Inhibition: The inhibitory activities of compounds of the invention against c-Src and Syk enzymes (Invitrogen), were evaluated in a similar fashion to that described hereinabove. The relevant enzyme (3000 ng/mL or 2000 ng/mL respectively, 2.5 μL) was incubated with the test compound (either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL, 2.5 μL each) for 2 hr at RT. The FRET peptides (8 μM, 2.5 μL), and appropriate ATP solutions (2.5 μL, 800 μM for c-Src, and 60 μM ATP for Syk) were then added to the enzyme/compound mixtures and incubated for 1 hr. Development reagent (protease, 5 μL) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: Kd = 9 nM (pChEMBL 8.050000190734863)
Descriptors: MW 578.67, ALogP 7.05, HBA 7, HBD 3, PSA 119.4, QED 0.18
Compound (SMILES): Cc1c([C@@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-ZEQRLZLVSA-N
Target: Cytochrome P450 2C9 (SINGLE PROTEIN), Homo sapiens
UniProt: P11712
Assay: Reversible inhibition of human CYP2C9
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): COCC(=O)Nc1cc(Oc2ccc(NC(=O)Nc3cc(C(C)(C)C)nn3-c3ccc(C)cc3)c3ccccc23)ccn1
InChIKey: AVUUHIYJPTWYNX-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 alpha (SINGLE PROTEIN), Homo sapiens
UniProt: P49840
Assay: GSK 3α Enzyme Inhibition: The GSK3-α protein (500 ng/mL, 2.5 μL) was mixed with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, and ATP (40 μM, 2.5 μL) were then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 μL) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: Kd = 180 nM (pChEMBL 6.75)
Descriptors: MW 578.67, ALogP 7.05, HBA 7, HBD 3, PSA 119.4, QED 0.18
Compound (SMILES): Cc1c([C@@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-ZEQRLZLVSA-N
Target: Cytochrome P450 2D6 (SINGLE PROTEIN), Homo sapiens
UniProt: P10635
Assay: Reversible inhibition of human CYP2D6
Measurement: IC50 > 50000 nM (pChEMBL 4.301000118255615)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): COCC(=O)Nc1cc(Oc2ccc(NC(=O)Nc3cc(C(C)(C)C)nn3-c3ccc(C)cc3)c3ccccc23)ccn1
InChIKey: AVUUHIYJPTWYNX-UHFFFAOYSA-N
Target: Glycogen synthase kinase-3 beta (SINGLE PROTEIN), Homo sapiens
UniProt: P49841
Assay: GSK 3α Enzyme Inhibition: The GSK3-α protein (500 ng/mL, 2.5 μL) was mixed with the test compound (2.5 μL at either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL, or 0.004 μg/mL) for 2 hr at RT. The FRET peptide (8 μM, 2.5 μL), which is a phosphorylation target for GSK3α, and ATP (40 μM, 2.5 μL) were then added to the enzyme/compound mixture and the resulting mixture incubated for 1 hr. Development reagent (protease, 5 μL) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: Kd = 24 nM (pChEMBL 7.619999885559082)
Descriptors: MW 578.67, ALogP 7.05, HBA 7, HBD 3, PSA 119.4, QED 0.18
Compound (SMILES): Cc1c([C@@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-ZEQRLZLVSA-N
Target: Acetylcholinesterase (SINGLE PROTEIN), Homo sapiens
UniProt: P22303
Assay: Inhibition of ACES (unknown origin)
Measurement: IC50 = 9940 nM (pChEMBL 5)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): COCC(=O)Nc1cc(Oc2ccc(NC(=O)Nc3cc(C(C)(C)C)nn3-c3ccc(C)cc3)c3ccccc23)ccn1
InChIKey: AVUUHIYJPTWYNX-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 14 (SINGLE PROTEIN), Homo sapiens
UniProt: Q16539
Assay: p38 MAPKalpha Enzyme Inhibition: The inhibitory activities of test compounds against the p38 MAPKα isoform (MAPK14: Invitrogen), were evaluated indirectly by determining the level of activation phosphorylation of the down-stream molecule, MAPKAP-K2. The p38 MAPKα protein (80 ng/mL, 2.5 μL) was mixed with the test compound (2.5 μL of either 4 μg/mL, 0.4 μg/mL, 0.04 μg/mL or 0.004 μg/mL) for 2 hr at RT. The mix solution (2.5 μL) of the p38α inactive target MAPKAP-K2 (Invitrogen, 600 ng/mL) and FRET peptide (8 μM; a phosphorylation target for MAPKAP-K2) was then added and the kinase reaction was initiated by adding ATP (40 μM, 2.5 μL). The mixture was incubated for 1 hr at RT. Development reagent (protease, 5 μL) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific).
Measurement: Kd = 1 nM (pChEMBL 9)
Descriptors: MW 578.67, ALogP 7.05, HBA 7, HBD 3, PSA 119.4, QED 0.18
Compound (SMILES): Cc1c([C@@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-ZEQRLZLVSA-N
Target: Somatostatin receptor type 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P30872
Assay: Inhibition of somatostatin receptor subtype 1 (unknown origin)
Measurement: IC50 = 2630 nM (pChEMBL 5.579999923706055)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): Cc1c([C@@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-ZEQRLZLVSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Inhibition of human SERT
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): Cc1c([C@@H](O)CN2CCN(C[C@H](O)c3ccc4c(c3C)COC4=O)CC2)ccc2c1COC2=O
InChIKey: OCKGFTQIICXDQW-ZEQRLZLVSA-N
Target: Sodium-dependent serotonin transporter (SINGLE PROTEIN), Homo sapiens
UniProt: P31645
Assay: Inhibition of [3H]-serotonin uptake at human SERT transfected in HEK293 cells
Measurement: IC50 = 2400 nM (pChEMBL 5.619999885559082)
Descriptors: MW 466.53, ALogP 2.03, HBA 8, HBD 2, PSA 99.54, QED 0.62
Compound (SMILES): C[C@@H]1Cn2cc(C(N)=O)nc2-c2cc(C#CC(C)(C)O)ccc2O1
InChIKey: FCLUEVDLZSWFCZ-LLVKDONJSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 insect cells using PIP2:3PS lipid substrate after 120 mins by ADP-Glo assay
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 325.37, ALogP 1.55, HBA 5, HBD 2, PSA 90.37, QED 0.78
Compound (SMILES): Cn1nccc1Cc1c(C(N)=O)nc2n1CCOc1cc(F)c(C#CC(C)(C)O)cc1-2
InChIKey: YEIUTWFMRCCWQY-UHFFFAOYSA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 insect cells using PIP2:3PS lipid substrate after 120 mins by ADP-Glo assay
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 423.45, ALogP 1.63, HBA 7, HBD 2, PSA 108.19, QED 0.62
Compound (SMILES): C[C@H]1Cn2cc(C(N)=O)nc2-c2cc(C#CC(C)(C)O)ccc2O1
InChIKey: FCLUEVDLZSWFCZ-NSHDSACASA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: P27986
Assay: Inhibition of N-terminal His6-tagged recombinant full-length human p110delta/untagged recombinant full length human p85alpha expressed in baculovirus infected Sf21 insect cells using PIP2:3PS lipid substrate after 120 mins by ADP-Glo assay
Measurement: Ki > 1000 nM (pChEMBL 6)
Descriptors: MW 325.37, ALogP 1.55, HBA 5, HBD 2, PSA 90.37, QED 0.78
Compound (SMILES): CC1CC(c2c(F)cccc2OCCO)N(Cc2ccc(OC(F)(F)F)cc2)C1=O
InChIKey: URRXRLRRPFREAS-UHFFFAOYSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: In Vitro Assay: Intracellular Calcium Measurements : In Vitro Assay: Intracellular Calcium Measurements:Chinese hamster ovary (CHO) cells expressing the human orexin-1 receptor and the human orexin-2 receptor, respectively, are grown in culture medium (Ham F-12 with L-Glutamine) containing 300 g/ml G418, 100 U/ml penicillin, 100 g/ml streptomycin and 10% heat inactivated fetal calf serum (FCS). The cells are seeded at 20,000 cells/well into 384-well black clear bottom sterile plates (Greiner). The seeded plates are incubated overnight at 37° C. in 5% CO2. Human orexin-A as an agonist is prepared as 1 mM stock solution in MeOH:water (1:1), diluted in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES for use in the assay at a final concentration of 3 nM.Antagonists are prepared as 10 mM stock solution in DMSO, then diluted in 384-well plates using DMSO followed by a transfer of the dilutions into in HBSS containing 0.1% bovine serum albumin (BSA), NaHCO3: 0.375 g/l and 20 mM HEPES. On the day of the assay, 50 μl of staining buffer (HBSS containing 1% FCS, 20 mM HEPES, NaHCO3: 0.375 g/l, 5 mM probenecid (Sigma) and 3 M of the fluorescent calcium indicator fluo-4 AM (1 mM stock solution in DMSO, containing 10% pluronic) is added to each well. The 384-well cell-plates are incubated for 50 min at 37° C. in 5% CO2 followed by equilibration at RT for 30 min before measurement.
Measurement: IC50 = 59 nM (pChEMBL 7.230000019073486)
Descriptors: MW 427.39, ALogP 4.21, HBA 4, HBD 1, PSA 59, QED 0.67
Compound (SMILES): Cn1c(=O)c2c(CC(=O)Nc3nc(-c4ccc(F)c(C(F)(F)F)c4F)cs3)c(Cl)oc2n(C)c1=O
InChIKey: ANPQBLBZMKRGJT-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Radiometric Calcium Assay: The inhibition of TRPA1 receptor activation was measured as inhibition of allylisothiocyanate (AITC) induced cellular uptake of radioactive calcium. Test compounds were dissolved in DMSO to prepare 10 mM stock solution and then diluted using plain medium with 0.1% BSA and 1.8 mM CaCl2 to get desired concentration. Final concentration of DMSO in the reaction was 0.5% (v/v). Human TRPA1 expressing CHO cells were grown in F-12 DMEM medium with 10% FBS, 1% penicillin-streptomycin solution, and 400 ug/ml of G-418. Cells were seeded 24 h prior to the assay in 96 well plates so as to get 50,000 cells per well on the day of experiment. Cells were treated with test compounds for 10 min followed by addition of AITC at a final concentration of 30 micromolar and 5 uCi/ml 45Ca+2 for 3 min at 25° C. Cells were washed and lysed using buffer containing 1% Triton X-100, 0.1% deoxycholate and 0.1% SDS. Radioactivity in the lysate was measured in Packard Top count.
Measurement: IC50 > 1000 nM (pChEMBL 6)
Descriptors: MW 534.85, ALogP 4.09, HBA 8, HBD 1, PSA 99.13, QED 0.4
Compound (SMILES): CS(=O)(=O)N1CCCc2cccc(c2)Nc2ncc(C(F)(F)F)c(n2)NCc2cccnc21
InChIKey: BGNMLKFHDPQLLJ-UHFFFAOYSA-N
Target: Focal adhesion kinase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q05397
Assay: Inhibition of NH2-terminal His6-tagged FAK kinase domain (410 to 689 residues) (unknown origin) expressed in baculovirus infected sf9 cells using p(Glu/Tyr) as substrate in presence of ATP
Measurement: IC50 = 1.26 nM (pChEMBL 8.899999618530273)
Descriptors: MW 478.5, ALogP 3.96, HBA 7, HBD 2, PSA 100.11, QED 0.55
Compound (SMILES): C[C@H](CN1[C@@H]2CC[C@H]1C[C@@H](c1cccc(O)c1)C2)N(Cc1ccccc1)C(=O)C1CCCCC1
InChIKey: BADRCXSJUPOOOR-HLJLCZKJSA-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 460.66, ALogP 6.1, HBA 3, HBD 1, PSA 43.78, QED 0.54
Compound (SMILES): COc1cccc(OC)c1C(=O)N[C@H]1CCC[C@@H]1Nc1ncc(C(F)(F)F)cn1
InChIKey: VCKAHUHZSCQPDV-STQMWFEESA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: Fluorescent Imaging Plate Reader (FLIPR) Assay: Orexin antagonist activity was determined by measuring changes in intracellular calcium levels using a Ca2+ sensitive fluorescent dye. The changes in fluorescent signal were monitored by Fluorescent Imaging Plate Reader (FLIPR®) technology available from Molecular Devices, LLC, U.S.A. Orexin mediated increases in intracellular Ca2+ concentration were readily detected upon activation with orexin-A. Twenty-four hours prior to the assay, RBL-2H3 cells stably expressing either human orexin receptor 1 or human orexin receptor 2 were seeded in cell culture medium in black, clear-bottom 384-well plates (commercially available from Corning Inc., U.S.A.) and grown overnight at 37 °C., 5% CO2. On the day of the assay, cell culture media was removed and cells were loaded with Calcium 5 Dye (commercially sold by Molecular Devices, LLC, U.S.A.) for 1 hour at 37 °C., 5% CO2.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 410.4, ALogP 3.28, HBA 6, HBD 2, PSA 85.37, QED 0.76
Compound (SMILES): Cc1nnc2c(NC(C)C)nc(-c3nc4ccc(C(C)C)cc4n3[C@@H](C)c3ccccn3)cn12
InChIKey: BOANFFSJBALNEO-KRWDZBQOSA-N
Target: Histone H4 (SINGLE PROTEIN), Homo sapiens
UniProt: P62805
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 uM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15 nM GST-BRD4-BD1 protein (aa 44-168) or 150 nM GST-BRD4-BD2 (aa 333-460) and 15 nM biotinylated Acetyl-Histone H4 (Lys5, 8, 12, 16) peptide is prepared in assay buffer (50 mM HEPES pH=7.4; 25 mM NaCl; 0.05% Tween 20; 0.1% bovine serum albumin (BSA); 10 mM dithiothreitol (DTT)). 6 ul of the mix is added to the compound dilutions. Subsequently, 6 ul of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 ug/ml each) are added and the samples are incubated for 30 min at RT in the dark (shaking 300 rpm). Afterwards, the signal is measured in a PerkinElmer Envision HTS Multilabel Reader using the AlphaScreen protocol from PerkinElmer. Each plate contains negative controls where biotinylated Acetyl-Histone H4 peptide and GST-BRD4-BD1 or GST-BRD4-BD2 are left out and replaced by assay buffer.
Measurement: IC50 = 243 nM (pChEMBL 6.610000133514404)
Descriptors: MW 454.58, ALogP 5.4, HBA 8, HBD 1, PSA 85.82, QED 0.37
Compound (SMILES): COn1c(NC(C)c2ccc(C(F)(F)F)cc2)nc2c(c1=O)CN(C(=O)c1cnc3ccccc3c1)CC2
InChIKey: IVSFHOPYUVFWPS-UHFFFAOYSA-N
Target: Prolactin-releasing peptide receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P49683
Assay: Intracellular Calcium Mobilization Assay: HEK293 cells expressing human GPR10 were maintained in Dulbecco's Modified Eagles' medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin and 20 mM HEPES at 37° C. in a 5% CO2 incubator. GPR10-expressing HEK293 cells were placed in poly-D-lysine coated 96-well culture plates and cultured for 18-24 hr before the test at a density of 3×104 cells/well. The cells were incubated with 2.5 nM Fluo-8 for 1 hr at room temperature in Recording Buffer containing 1% bovine serum albumin, 0.01% pluronic F-127 and 20 mM HEPES, Hanks Balanced Buffered Saline, pH 7.4. The cells were incubated with test compounds for 15 min at room temperature, and then PrRP (the final concentration, 1 nM) was added into the medium. The changes in intracellular calcium-dependent fluorescence were monitored using a fluorescence imaging plate reader (FDSS3000, Hamamatsu Photonics K.K.). Fluo-8 fluorescence was measured with excitation at 490 nm and emission at 520 nm.
Measurement: IC50 = 286 nM (pChEMBL 6.539999961853027)
Descriptors: MW 523.52, ALogP 4.24, HBA 7, HBD 1, PSA 89.35, QED 0.42
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(N(CCc2ccccc2)S(=O)(=O)CC)ccc1Oc1ccc(F)cc1F
InChIKey: OMHCRTBDAHRSBO-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay: Assay method C: Compound dilution series were prepared in DMSO via an approximately 3-fold serial dilution from one of the following: 0.047 mM to 0.78 nM or 5-fold serial dilution from Assay method A. Compound dilutions were added directly into white, low-volume assay plates (Perkin Elmer Proxiplate 384 Plus# 6008280) using a Labcyte Echo in conjunction with Labcyte Access and Thermo Multidrop CombinL robotics. Compounds were then suspended in eight microliters (μL) of assay buffer (20 mM Sodium Phosphate, pH 6.0, 50 mM NaCl, 1 mM Ethylenediaminetetraacetic acid disodium salt dihydrate, 0.01% Triton X-100, 1 mM DL-Dithiothreitol) containing His-tagged bromodomain, Europium-conjugated anti-His antibody (Invitrogen PV5596) and Alexa-647-conjugated probe. The final concentration of 1X assay mixture for assay methods A, B and C contains 2% DMSO, 8 nM His-tagged bromodomain, 1 nM Europium-conjugated anti-His-tag antibody and 100 nM or 30 nM probe (for BDI or BDII, respectively) and compound concentration in the range of: 49.02 μM-15.63 nM for method A, 9.19 μM 150 pM for method B, and 0.92 μM 15 pM for method C. After a one-hour equilibration at room temperature, TR-FRET ratios were determined using an Envision multilabel plate reader (Ex 340, Em 495/520).
Measurement: Ki = 47.8 nM (pChEMBL 7.320000171661377)
Descriptors: MW 568.64, ALogP 5.92, HBA 6, HBD 0, PSA 77.84, QED 0.22
Compound (SMILES): COc1ccc(F)cc1-c1ccnc2[nH]c(C3CCN(CCN4CCOCC4)CC3)cc12
InChIKey: DCGPJAOHTGLEOK-UHFFFAOYSA-N
Target: CDK9/cyclin T1 (PROTEIN COMPLEX), 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 directly added in 100% DMSO to white low volume assay plates (Perkin Elmer Proxiplate 6008289) using a Labcyte Echo acoustic dispenser. Assay reagents in serine/threonine kinase assay buffer containing 20 mM HEPES, 10 mM MgCl2, 100 mM Na3VO4, and 0.0075% Triton X-100. were added for final reaction mixture concentrations of 1000 μM ATP, 100 nM U-light MBP peptide (Perkin Elmer TRF0109M) and reaction initiated with 4 nM CDK9/Cyclin T1 (Carna Biosciences 04-110). The kinase reaction was carried out for 30 minutes 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 (PerkinElmer CR97-100). The reaction was equilibrated for 1 hour and the signal read in the Perkin Elmer Envision in TR-FRET mode (excitation at 320 nm and emission at 615/665 nm).
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 438.55, ALogP 3.89, HBA 5, HBD 1, PSA 53.62, QED 0.63
Compound (SMILES): CC1CN(c2cc(NC(=O)Nc3ccc(Oc4ccnc(NC(=O)C5CC5)c4)c(F)c3)cc(C(F)(F)F)c2)CCO1
InChIKey: HXTKTWLQDFJSIJ-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: Inhibition Assay: [Experimental method]: Use ADP-GLO™ assay kit from Promega to evaluate the inhibitory activity of compounds at B-Raf. [Instrument]: PerKinElmer's ENVISION plate reader. [Material]: Optiplate-384 well plate (PerKinElmer), kinase buffer (50 mM Hepes pH7.5, 1 mM EGTA, 2 mM DTT, 10 mM Mg2+, 0.05% BSA), B-Raf kinase (Millipore, catalogue #14-530-K), GST-MEK1 substrate (Carna, catalogue #07-141-10), Super pure ATP (Promega), ADP-GLO™ assay kit (Promega, catalogue # V9102), DMSO (Sigma, catalogue #34869), purified water (Millipore, type: Milli-Q). [Study conditions]: Mix B-Raf kinase (final concentration: 5 nM) and compound (DMSO final concentration: 0.25%) and preincubate at 30° C. for 20 min, then add ATP (final concentration: 6 μM) and reaction substrate (final concentration: 30 nM), React for 2 hrs at 30° C. After reaction, add ADP-GLO™ reagent, react for 40 min at room temperature, then add ADP-GLO™ detecting agent, react for 30 min at room temperature. After that, use ENVSION to measure the fluorescence.
Measurement: IC50 = 22.5 nM (pChEMBL 7.650000095367432)
Descriptors: MW 573.55, ALogP 6.25, HBA 6, HBD 3, PSA 104.82, QED 0.29
Compound (SMILES): CNCCn1cc(-c2ccnc(C(F)(F)F)c2)nc1C1CCN(c2ncnc(N)c2C#N)CC1
InChIKey: HQPYWCQKQNPHGR-UHFFFAOYSA-N
Target: RAC-alpha serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P31749
Assay: AKT Enzyme Assay: A TTP Mosquito liquid handling instrument is used to place 125 nl of the appropriate concentration of inhibitor in 100% DMSO (for a dose response curve calculation) into each well of a 384-well plate. To this reaction components are added to a final volume of 12.5 .mu.l: 0.1 ng/.mu.l His-AKT (Full Length), (Invitrogen, Part # P2999, Lot #641228C). 160 uM ATP (Fluka, 02055) 1 mM DTT (Sigma, D0632) 1 mM MgCl2 (Sigma, M1028) 1 .mu.M substrate peptide (sequence FITC-AHA-GRPRTSSFAEG-NH2), synthesized by Tufts Peptide Synthesis service. 100 mM HEPES pH 7.5 (Calbiochem, 391338)0.015% Brij-35 (Sigma, B4184)The reaction is incubated for 90 min at 25 C, and then stopped by the addition of 70 .mu.l of Stop buffer (100 mM HEPES pH 7.5, 0.015% Brij-35, 10 mM EDTA (Sigma, E7889)).The plate is read on a Caliper LC 3000 in an Off-Chip mobility shift assay format, using the following parameters for a 12-sipper chip: screening pressure -2.3 psi, upstream voltage -500, and downstream voltage -3000.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 471.49, ALogP 2.81, HBA 9, HBD 2, PSA 121.57, QED 0.56
Compound (SMILES): C[C@H](Nc1ncnc(N)c1C#CC(C)(C)O)c1nc2cccc(Cl)c2c(=O)n1-c1ccccc1
InChIKey: SSANTSALJYXTJW-HNNXBMFYSA-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 474.95, ALogP 3.71, HBA 8, HBD 3, PSA 118.95, QED 0.39
Compound (SMILES): Cc1c(C(=O)Nc2c(C)n(C)n(C3CCCCC3)c2=O)noc1C1CCC(C)CO1
InChIKey: SUECNHKSTARKJF-UHFFFAOYSA-N
Target: E3 ubiquitin-protein ligase SMURF1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HCE7
Assay: Inhibition of SMURF1 (unknown origin) in presence of E1/E2/Ub mix measured after 45 mins by europium-labeled streptavidin based TR-FRET assay
Measurement: IC50 = 570 nM (pChEMBL 6.239999771118164)
Descriptors: MW 416.52, ALogP 4.04, HBA 7, HBD 1, PSA 91.29, QED 0.81
Compound (SMILES): CS(=O)(=O)NC(=O)c1cc(/C=C/c2cn3nc(Cl)cc(N4CCOCC4)c3n2)nc2ccccc12
InChIKey: BWAVUGUTXVMGRZ-VOTSOKGWSA-N
Target: cAMP and cAMP-inhibited cGMP 3',5'-cyclic phosphodiesterase 10A (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y233
Assay: In Vitro Assay: Human recombinant PDE10A2 was expressed in Sf9 cells, using a recombinant baculovirus construct containing the full length sequence containing a 6Ã¿His sequence following the start Met to allow metal affinity purification of the recombinant protein. Cells were harvested and the phosphodiesterase protein was purified by metal chelate chromatography on Ni-sepharose 6FF.The affinity of the compounds of Formula (I) for phophodiesterases (PDE) was measured by a scintillation proximity assay (SPA). PDE Yttrium Silicate SPA beads allow PDE activity to be measured by direct binding of the primary phosphate groups of non-cyclic AMP or GMP to the beads via a complex iron chelation mechanism. The amount of bound tritiated product ([3H]-AMP) is measured by liquid scintillation counting.The compounds were dissolved and diluted in 100% DMSO in polystyrene plates to a concentration of 100-fold the final concentration in the assay. Human PDE10A enzyme solution (10 uL) was added.
Measurement: IC50 < 1 nM (pChEMBL 9)
Descriptors: MW 512.98, ALogP 2.63, HBA 9, HBD 1, PSA 118.79, QED 0.43
Compound (SMILES): C[C@H](c1nc(Cl)cc(N2C(=O)OC[C@@H]2[C@@H](C)O)n1)c1cc(F)c(-c2cnn(C)c2)cc1F
InChIKey: DHUAMXPDUGZPMC-WVQJBOLRSA-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 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 463.87, ALogP 3.67, HBA 7, HBD 1, PSA 93.37, QED 0.58
Compound (SMILES): COc1cc(N2CCN(C(C)=O)CC2)ccc1Nc1ncc(C(F)(F)F)c(Nc2ccc(N3CCN(CCO)CC3)cc2OC)n1
InChIKey: OZLURHFFMFKQPX-UHFFFAOYSA-N
Target: ALK tyrosine kinase receptor (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UM73
Assay: Homogeneous Time-resolved fluorescence (HTRF) Assay: In order to measure the inhibitory activity against ALK, a Grainer 96-well round type bottom plate was added with the compounds (2 μl) prepared in Examples 1 to 89, and mixed with ALK enzyme (1 ul) and a peptide substrate (2 μl) with biotin attached thereto for 15 minutes and cultured thereafter. Here, ATP solution (5 μl) was added thereto and kinase reaction was performed at room temperature for 30 minutes. XL 665 (5 μl), to which Streptavidin dissolved in ethylenediaminetetraacetic acid solution was attached, anti-phosphotyrosine antibody (5 μl), to which europium (Eu3+) was attached, were added to a reaction solution to stop the reaction, cultured for 1 hour, and analyzed using Homogeneous Time-resolved fluorescence (HTRF, Cisbio). The result was read by a Wallac Envision 2103 device at the wavelength range of 615/665 nm.
Measurement: IC50 = 11 nM (pChEMBL 7.960000038146973)
Descriptors: MW 644.7, ALogP 3.78, HBA 11, HBD 3, PSA 118.56, QED 0.3
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccc(F)cc3)n2)CCC(=O)CC1
InChIKey: SFVVXJIMLBNPTH-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 = 39 nM (pChEMBL 7.409999847412109)
Descriptors: MW 355.37, ALogP 2.62, HBA 6, HBD 2, PSA 113.8, QED 0.86
Compound (SMILES): C[C@@H]1CN(c2sc(C(F)(F)F)nc2-c2nc3ccc(CO)cc3[nH]2)CCN1C(=O)Cn1cnc2cccnc21
InChIKey: XRUVZBDFYNULNQ-CQSZACIVSA-N
Target: C-X-C chemokine receptor type 3 (SINGLE PROTEIN), Homo sapiens
UniProt: P49682
Assay: Antagonist activity at human recombinant CXCR3 expressed in CHO-K1 cells measured after 10 mins in presence of CXCL10 by FLIPR analysis
Measurement: IC50 = 4.6 nM (pChEMBL 8.34000015258789)
Descriptors: MW 556.57, ALogP 3.68, HBA 9, HBD 2, PSA 116.06, QED 0.34
Compound (SMILES): Cc1ncc(C2CCN(C(=O)OC3C4CC5CC3CC(C(N)=O)(C5)C4)C2)cn1
InChIKey: TZPRKTMMSOWSCM-UHFFFAOYSA-N
Target: 11-beta-hydroxysteroid dehydrogenase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P28845
Assay: Scintillation Proximity Assay: Inhibition of microsomal preparations of 11.beta.-HSDl by compounds of the present invention, as described essentially previously (K. Solly, SS Mundt, HJ Zokian, GJ Ding, A. Hermanowski-Vosatka, B. Strulovici, and W. Zheng, High-Throughput Screening of 11-Beta-Hydroxyseroid Dehydrogenase Type 1 in Scintillation Proximity Assay Format. Assay Drug Dev Technol 3 (2005) 377-384) was measured. All reactions were carried out in a clear flexible PET Microbeta plates 96-well at room temperature (PerkinElmer). Assay, substrate solution (50mM HEPES, pH7.4,100mM KCl, 5mM NaCl, 2mM MgCl 2, 2mM NADPH and 160nM [3 H] cortisone (1Ci / mmol)) a 49ul and distribution, and starting from the 0.1mM on to start by combining the semi-log (half-log) multiple of the incremental test compound 1uL in DMSO previously diluted (8 points). After pre-incubation of 10 minutes, incubated human 11-HSD1 overexpress the enzyme containing CHO cells isolated from microsomes solution 50uL.
Measurement: IC50 > 100 nM (pChEMBL 7)
Descriptors: MW 384.48, ALogP 2.39, HBA 5, HBD 1, PSA 98.41, QED 0.86
Compound (SMILES): COc1cccc2c1ncc1c(=O)n(-c3cccs3)c(=O)n(C3CCCC3)c12
InChIKey: WEWXZCSTWDNTEE-UHFFFAOYSA-N
Target: Lipopolysaccharide-induced tumor necrosis factor-alpha factor (SINGLE PROTEIN), Homo sapiens
UniProt: Q99732
Assay: TNF inhibition assay: Samples were tested for TNF as per the manufacturer's instructions (TNF ELISA; R&D Systems, Cat #DY210). The samples were serially diluted with PBS+1% BSA to the appropriate dilution condition for the cell prep, typically a 1:4 or 1:6 dilution.
Measurement: IC50 = 410 nM (pChEMBL 6.389999866485596)
Descriptors: MW 393.47, ALogP 3.89, HBA 7, HBD 0, PSA 66.12, QED 0.5
Compound (SMILES): CN(c1ccccc1)c1c(Cl)nc2ccc(C(O)(c3cccnc3)c3cccc(Cl)c3)cc2c1Cl
InChIKey: CXISRSBZHJUAJP-UHFFFAOYSA-N
Target: Nuclear receptor ROR-gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P51449
Assay: RORγt Reporter Assay: The reporter assay was performed by transiently transfecting HEK293T cells with 5 μg of pBIND-RORγt LBD or pBIND-RORγt LBD-AF2 and 5 μg pGL4.31 (Promega Cat no. C935A) using Fugene 6 (Invitrogen Cat no. E2691) at a 1:6 ratio of DNA: Fugene 6 in a T-75 flask in which cells were at least 80% confluent. Twenty four hours after bulk transfection, cells were plated into 96-well plates at 50,000 cells/well in phenol-red free DMEM containing 5% Lipid Reduced FCS and Pen/Strep. Six hours after plating, cells were treated with compounds for 24 hours. Media was removed and cells were lysed with 50 μL 1x Glo Lysis Buffer (Promega). Dual Glo Luciferase Reagent (50 μL/well) was then added and firefly luciferase luminescence was read on an Envision after a ten minute incubation. Finally, Stop and Glo reagent (50 μL/well) was added and renilla luciferase luminescence was read on an Envision after a ten minute incubation.
Measurement: IC50 = 100 nM (pChEMBL 7)
Descriptors: MW 520.85, ALogP 7.64, HBA 4, HBD 1, PSA 49.25, QED 0.24
Compound (SMILES): c1ccc(CCN2CCN(c3cc(N4CCCC4)nc(N4CCCC4)n3)CC2)cc1
InChIKey: GRTICURLEQIPRO-UHFFFAOYSA-N
Target: Caspase-1 (SINGLE PROTEIN), Homo sapiens
UniProt: P29466
Assay: Inhibition of human recombinant N-terminal His-tagged DTT-activated caspase-1 expressed in Escherichia coli using Ac-WEHD-AMC as substrate preincubated for 30 mins followed by substrate addition by fluorescence assay
Measurement: Ki = 16 nM (pChEMBL 7.800000190734863)
Descriptors: MW 406.58, ALogP 3.04, HBA 6, HBD 0, PSA 38.74, QED 0.73
Compound (SMILES): N#CCC1(n2cc(C(N)=O)c(Nc3ccnc(F)c3)n2)CCN(Cc2cccnc2)CC1
InChIKey: UYOSVQOOBXCODU-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 = 5 nM (pChEMBL 8.300000190734863)
Descriptors: MW 434.48, ALogP 2.56, HBA 8, HBD 2, PSA 125.75, QED 0.55
Compound (SMILES): Cc1ccccc1[C@H](CC(=O)O)NC(=O)c1nc(C#N)c2cc(Oc3ccccc3)ccc2c1O
InChIKey: GIOAJCVNRUNIID-QFIPXVFZSA-N
Target: Egl nine homolog 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9GZT9
Assay: HIF-PH Assay: Ketoglutaric acid α-[1-14C]-sodium salt, alpha-ketoglutaric acid sodium salt, and HPLC purified peptide may be obtained from commercial sources, e.g., Perkin-Elmer (Wellesley Mass.), Sigma-Aldrich, and SynPep Corp. (Dublin Calif.), respectively. Peptides for use in the assay may be fragments of HIFα as described above or as disclosed in International Publication WO 2005/118836, incorporated by reference herein. HIF-pH, e.g., PHD1 (EGLN2) or PHD2 (EGLN1), can be expressed in, e.g., insect Hi5 cells, and partially purified, e.g., through a SP ion exchange chromatography column. Enzyme activity is determined by capturing 14CO2 using an assay described by Kivirikko and Myllyla (1982, Methods Enzymol. 82:245-304). Assay reactions contain 50 mM HEPES (pH 7.4), 100 μM α-ketoglutaric acid sodium salt, 0.30 mCi/mL ketoglutaric acid α-[1-14C]-sodium salt, 40 μM FeSO4, 1 mM ascorbate, 1541.8 units/mL Catalase, with or without 50 μM peptide substrate and various concentrations of compound of the invention. Reactions are initiated by addition of HIF-PH enzyme.
Measurement: IC50 = 45740 nM (pChEMBL 4.340000152587891)
Descriptors: MW 467.48, ALogP 4.86, HBA 6, HBD 3, PSA 132.54, QED 0.35
Compound (SMILES): CCC#CC[C@H](C)[C@H](O)/C=C/[C@H]1CC(F)(F)C(=O)N1CCCc1ccc(C(=O)O)s1
InChIKey: UMYXRXWDPHAKGZ-XLJJWPCRSA-N
Target: Prostaglandin E2 receptor EP4 subtype (SINGLE PROTEIN), Homo sapiens
UniProt: P35408
Assay: Radioligand Binding Assay: Assay Volume and Format:200 μl in 96-well plateCell membrane homogenates (20 μg protein) are incubated for 120 min at 22° C. with 0.5 nM [3H]PGE2 in the absence or presence of the test compound in a buffer containing 10 mM MES/KOH (pH 6.0), 10 mM MgCl2 and 1 mM EDTA.Nonspecific binding is determined in the presence of 10 M PGE2.Following incubation, the samples are filtered rapidly under vacuum through glass fiber filters (GF/B, Packard) presoaked with 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). The filters are dried then counted for radioactivity in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard).
Measurement: IC50 = 3.3 nM (pChEMBL 8.479999542236328)
Descriptors: MW 439.52, ALogP 3.97, HBA 4, HBD 2, PSA 77.84, QED 0.45
Compound (SMILES): O=C1COc2ccc(C3(O)c4ccccc4C(=O)N3Cc3cccc(I)c3)cc2N1
InChIKey: IVRIHMKFJQECLE-UHFFFAOYSA-N
Target: RAF proto-oncogene serine/threonine-protein kinase (SINGLE PROTEIN), Homo sapiens
UniProt: P04049
Assay: Inhibition of c-Raf1 (unknown origin) pre-incubated for 30 mins by luciferase-luciferin-coupled chemiluminescence assay
Measurement: IC50 = 1000 nM (pChEMBL 6)
Descriptors: MW 512.3, ALogP 3.47, HBA 4, HBD 2, PSA 78.87, QED 0.53
Compound (SMILES): CCC(CC)(NC(=O)c1cccc(-c2ccc(Cl)cc2)n1)C(=O)NC
InChIKey: YQBCNOJHSPFHPI-UHFFFAOYSA-N
Target: Cannabinoid receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P21554
Assay: cAMP Assay: CHO cells expressing human CB1 or CB2 receptors are seeded 17-24 hours prior to the experiment 50.000 cells per well in a black 96 well plate with flat clear bottom (Corning Costar #3904) in DMEM (Invitrogen No. 31331), 1.times.HT supplement, with 10% fetal calf serum and incubated at 5% CO2 and 37° C. in a humidified incubator. The growth medium was exchanged with Krebs Ringer Bicarbonate buffer with 1 mM IBMX and incubated at 30.degree. C. for 30 min Compounds were added to a final assay volume of 100 ul and incubated for 30 min at 30° C. Using the cAMP-Nano-TRF detection kit the assay (Roche Diagnostics) was stopped by the addition of 50 ul lysis reagent (Tris, NaCl, 1.5% Triton X100, 2.5% NP40, 10% NaN.sub.3) and 50 ul detection solutions (20 uM mAb Alexa700-cAMP 1:1, and 48 uM Ruthenium-2-AHA-cAMP) and shaken for 2 h at room temperature. The time-resolved energy transfer is measured by a TRF reader (Evotec Technologies GmbH), equipped with a ND:YAG laser as excitation source. The plate is measured twice with the excitation at 355 nm and at the emission with a delay of 100 ns and a gate of 100 ns, total exposure time 10 s at 730 (bandwidth 30 nm) or 645 nm (bandwidth 75 nm), respectively. The FRET signal is calculated.
Measurement: EC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 359.86, ALogP 3.44, HBA 3, HBD 2, PSA 71.09, QED 0.83
Compound (SMILES): COc1cc(/C=N/NC(=O)CCn2cc(C)c3c(C)cccc32)cc(Cl)c1OC
InChIKey: YLQQSBKVRVVRGE-WYMPLXKRSA-N
Target: Transient receptor potential cation channel subfamily M member 5 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9NZQ8
Assay: Inhibition of human TRPM5 transfected in HEK293 cells assessed as inhibition of KCl-stimulated membrane potential by FLIPR assay
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 413.91, ALogP 4.47, HBA 5, HBD 1, PSA 64.85, QED 0.46
Compound (SMILES): Fc1cccc(-c2ncc(-c3cnc(-c4cccnc4)[nH]3)cn2)c1
InChIKey: ATIJGZNOVFHFBW-UHFFFAOYSA-N
Target: Hematopoietic prostaglandin D synthase (SINGLE PROTEIN), Homo sapiens
UniProt: O60760
Assay: Enzyme Immunoassay (EIA)): The assay is carried out by the following steps: 1. Inhibitor screening is performed in 100 mM Tris-HCl, pH 8.0 containing 1 mM GSH, 1 mM MgCl.sub.2, 4% inhibitor/DMSO, 40 .mu.M PGH.sub.2 and 25 ng of PGDS in a total volume of 125 .mu.L. 2. A reaction mixture containing 100 mM Tris-HCl, pH 8.0, 1 mM GSH, 1 mM MgCl.sub.2, 25 ng H-PGDS and 4% inhibitor or DMSO (uninhibited reaction) is preincubated at 25.degree. C. for 10 minutes. 3. Reactions are initiated using 5 .mu.L of PGH.sub.2 and quenched every 15 seconds for one minute. Reactions are quenched by taking 10 .mu.L of reaction mixture and adding to 490 .mu.L of 100 mM phosphate buffer containing 0.1% BSA, 400 mM NaCl, 1 mM EDTA, 20 mM FeCl.sub.2, 10% 1N HCl, and 0.01% azide to prevent the non-enzymatic formation of PGD.sub.2 from PGH.sub.2. The FeCl.sub.2 serves the purpose of reducing PGH.sub.2 to 12-HHT. (Quench buffer is kept on ice at all times). 4. Quenched samples (5 .mu.L) are further diluted 100 fold in 495 .mu.L of 100 mM.
Measurement: IC50 = 41 nM (pChEMBL 7.389999866485596)
Descriptors: MW 317.33, ALogP 3.73, HBA 4, HBD 1, PSA 67.35, QED 0.62
Compound (SMILES): CC(C)[C@H]1CNC(=O)c2cc(-c3cccc(OC(F)(F)F)c3)c(-c3ccc(CN)cc3)n21
InChIKey: KREARTJFHWAQOG-OAQYLSRUSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
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 PIM2 assay was composed of HEPES 50 mM, at pH 7.5, with 1 mM MgCl2, 1 mM DTT, 3 uM Na3VO4, and 0.2 mg/mL BSA.Â  Full-length human PIM2 was expressed and purified as described in Fedorov O, et al., PNAS 2007 104, 51, 20523-28.
Measurement: IC50 = 173 nM (pChEMBL 6.760000228881836)
Descriptors: MW 443.47, ALogP 5.12, HBA 4, HBD 2, PSA 69.28, QED 0.58
Compound (SMILES): Cc1cc(-c2ccnc3[nH]cc(C#N)c23)ccc1CN(C)C
InChIKey: MGZVMXARJNUNDE-UHFFFAOYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: Homogeneous Time Resolved-fluorescence Resonance Energy Transfer (TR-FRET) Assay: LRRK2 kinase activity is measured using a LanthaScreen kinase activity assay available from Invitrogen (Life Technologies Corporation). The assay is a homogeneous time resolved-fluorescence resonance energy transfer (TR-FRET) assay that measures phosphorylation of a fluorescein-labelled peptide substrate (fluorescein-LRRKtide, fluorescein-GAGRLGRDKYKTLRQIRQ) (SEQ ID NO 1) as a result of LRRK2 kinase activity. The phosphorylated peptide is recognized by a terbium-labelled phospho-specific anti-LRRKtide antibody and, subsequently, the phosphorylated LRRKtide can be quantified by the extent of TR-FRET between the terbium donor and fluorescein acceptor.The LRRK2 kinase is obtained from Invitrogen (Life Technologies Corporation) and comprises residues 970 to 2527 of the full length human wild-type LRRK2 kinase or a similar sequence with the G2019S mutation. As discussed above, this mutation increases the kinase activity relative to the wild-type. The kinase reactions are performed in a 20 uL volume in 384-well plates. The kinase reaction buffer consists of 50 mM Tris pH 8.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, and 2 mM DTT.Â  In the assay, 1 nM LRRK2 WT or 250 pM LRRK2 G2019S kinase is incubated with the test compound (typically at 0 to 30 uM) for 30 minutes before the kinase reaction is initiated by addition of 1.3 mM ATP and 0.4 uM fluorescein-LRRKtide. The reaction mixture (20 ul total volume) is incubated for 2 hours at 30° C. before the reaction is terminated by addition of 10 mM EDTA and 1 nM terbium-labelled anti-phospho-LRRKtide antibody (final volume 20 ul). The mixture is further incubated for 30 minutes at RT. TR-FRET is measured by excitation of the terbium-donor with 340 nm light and subsequent (delay time 100 us) measurement of terbium and fluorescein emission at 495 nm and 520 nm, respectively, over a time window of 1000 us. The measurement is repeated 10 times for fluorescein and 10 times for terbium emission with a 2000 us time window between repeats. TR-FRET measurements are performed on a Biomek Synergy plate. The TR-FRET signal is calculated as the emission-ratio at 520 nm over 495 nm.
Measurement: IC50 = 549 nM (pChEMBL 6.260000228881836)
Descriptors: MW 290.37, ALogP 3.47, HBA 3, HBD 1, PSA 55.71, QED 0.8
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)c4cc(F)nc(F)c4)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: XVGWIGDRSYKRDS-UHFFFAOYSA-N
Target: Casein kinase I isoform delta (SINGLE PROTEIN), Homo sapiens
UniProt: P48730
Assay: Kinase Assay: The kinase assay was performed in V-bottom 384-well plates. The final assay volume was 30 ul prepared from 15 ul additions of enzyme, substrates (fluoresceinated peptide FL-AHA-KRRRAL-PSER-VASLPGL-OH and ATP) and test compounds in assay buffer (20 mM HEPES pH 7.4, 30 mM MgCl2, 0.015% Brij35 and 4 mM DTT). The reaction was incubated at room temperature for 22 hours and terminated by adding 45 ul of 35 mM EDTA to each sample. The reaction mixture was analyzed on the Caliper LABCHIPÂ®3000 (Caliper, Hopkinton, Mass.) by electrophoretic separation of the unphosphorylated substrate and phosphorylated product Inhibition data were calculated by comparison of the no enzyme control reactions for 100% inhibition and vehicle-only reactions for 0% inhibition. The final concentration of reagents in the assay were 200 pM CK1ε or CK1δ, 50 uM ATP, 1.5 uM FL-AHA-KRRRAL-PSER-VASLPGL-OH, and 1.6% DMSO. Dose response curves were generated to determine the concentration required to inhibit 50% of the kinase activity (IC50). Compounds were dissolved at 10 mM in dimethylsulfoxide (DMSO) and evaluated at eleven concentrations. IC50 values were derived by non-linear regression analysis.
Measurement: IC50 = 1.1 nM (pChEMBL 8.960000038146973)
Descriptors: MW 449.4, ALogP 3.86, HBA 7, HBD 1, PSA 90, QED 0.42
Compound (SMILES): NCCC1CCN(c2nc(N3CCOCC3)nc(-n3c(C(F)F)nc4ccccc43)n2)CC1
InChIKey: VFXKRHROIHCRIA-UHFFFAOYSA-N
Target: Phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit beta isoform (SINGLE PROTEIN), Homo sapiens
UniProt: P42338
Assay: Inhibition of PI3Kbeta (unknown origin) expressed in Escherichia coli-infected fall armyworm sf21 cells co-expressing p85 by kinase-glo luminescence assay
Measurement: IC50 = 190 nM (pChEMBL 6.71999979019165)
Descriptors: MW 458.52, ALogP 2.55, HBA 9, HBD 1, PSA 98.22, QED 0.6
Compound (SMILES): O=C1N[C@H](c2cncc(C#C[C@H]3C[C@H](O)C3)c2)[C@@H](c2cccc(F)c2)O1
InChIKey: IZDJAZZKINJIGM-HAFFEQERSA-N
Target: Metabotropic glutamate receptor 5 (SINGLE PROTEIN), Homo sapiens
UniProt: P41594
Assay: Positive allosteric modulation activity at human mGluR5A expressed in HEK293(ZF) cells co-expressing rat glutamate-aspartate transporter assessed as increase in L-glutamate-induced Ca2+ flux preincubated for 60 mins followed by L-glutamate addition measured for 100 sec by calcium-4 dye based FLIPR assay
Measurement: EC50 > 1600 nM (pChEMBL 5.796000003814697)
Descriptors: MW 352.37, ALogP 2.87, HBA 4, HBD 2, PSA 71.45, QED 0.82
Compound (SMILES): CCN(CC)CCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CO)C(=O)OC
InChIKey: WHANHEKCAHJBNW-KCAPAQDDSA-N
Target: Chromobox protein homolog 7 (SINGLE PROTEIN), Homo sapiens
UniProt: O95931
Assay: Antagonist activity at recombinant human C-terminal His-tagged CBX7 chromodomain (8 to 62 residues) expressed in Escherichia coli Rosetta BL21(DE3)pLysS using biotin-labeled H3K9me3 measured after 30 mins by AlphaScreen assay
Measurement: IC50 = 1700 nM (pChEMBL 5.769999980926514)
Descriptors: MW 791.99, ALogP 2.12, HBA 9, HBD 7, PSA 211.06, QED 0.05