lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): COC(=O)CN(CCCNC(=O)c1cc2nccc(Oc3ccc(NC(=O)Nc4cc(C)ccc4F)c(F)c3)c2s1)CC(=O)OC
InChIKey: SAJGQCIKURPXJS-UHFFFAOYSA-N
Target: Vascular endothelial growth factor receptor 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P17948
Assay: Kinase Assay: Kinase assays were performed in 96 well microtiter plates that were coated overnight with 30 μg of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.2-7.4. The plates were incubated with 1% BSA and then washed four times with PBS prior to starting the reaction. Reactions were carried out in 120 μL reaction volumes containing 3.6 μM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 0.5 ng of purified protein. Following a ten minute incubation at 25 °C., the reactions were washed four times with PBS containing 0.05% Tween-20. 100 μl of a monoclonal anti-phosphotyrosine antibody-peroxidase conjugate was diluted 1:10000 in PBS-Tween-20 and added to the wells for 30 minutes. Following four washes with PBS-Tween-20, 100 μl of 0-phenylenediamine Dihydrochloride in Phosphate-citrate buffer, containing urea hydrogen peroxide, was added to the wells for 7 minutes as a colorimetric substrate for the peroxidase. The reaction was terminated by the addition of 100 μl of 2.5N H2 SO4 to each well and read using a microplate ELISA reader set at 492 nm.
Measurement: IC50 = 10 nM (pChEMBL 8)
Descriptors: MW 655.68, ALogP 5.09, HBA 10, HBD 3, PSA 148.19, QED 0.13
Compound (SMILES): Cc1c(-c2ccc(C(F)(F)F)cc2)nnc(N2CCN(c3cnc(C(=O)N(c4ccccc4)C(C)C)cn3)[C@H](C)C2)c1C
InChIKey: QRUXAUDVXVLKOY-OAQYLSRUSA-N
Target: Protein smoothened (SINGLE PROTEIN), Mus musculus
UniProt: P56726
Assay: Inhibition of mouse recombinant Shh protein in mouse TMHh12 cells assessed as inhibition of 25 nM Smo agonist-induced hedgehog signalling response by Gli-luciferase reporter gene assay
Measurement: IC50 < 100 nM (pChEMBL 7)
Descriptors: MW 589.67, ALogP 6.34, HBA 7, HBD 0, PSA 78.35, QED 0.26
Compound (SMILES): O=C(Cc1ccncc1)NCC(O)CN1CCc2ccccc2C1
InChIKey: QXZIFFLQGVGPLZ-UHFFFAOYSA-N
Target: PRMT5/MEP50 complex (PROTEIN COMPLEX), Homo sapiens
UniProt: Q9BQA1
Assay: Inhibition of recombinant human fetal brain FLAG-tagged PRMT5/full length human testis 6His-tagged MEP50 expressed in baculovirus infected insect Sf9 cells using human histone H4 (1 to 15 residues) as substrate preincubated for 30 mins followed by [3H]-SAM addition
Measurement: IC50 = 10001 nM (pChEMBL 5)
Descriptors: MW 325.41, ALogP 1.16, HBA 4, HBD 2, PSA 65.46, QED 0.84
Compound (SMILES): Cc1nnc2c(NC3CCN(S(=O)(=O)CCCN(C)C)CC3)cc(N(C)Cc3ccccc3)nn12
InChIKey: PKELZFOCBUGMPL-UHFFFAOYSA-N
Target: Bromodomain-containing protein 4 (SINGLE PROTEIN), Homo sapiens
UniProt: O60885
Assay: AlphaScreen Assay: Compounds are diluted in serial dilution 1:5 in assay buffer from 10 mM stock in DMSO (100 μM start concentration) in white OptiPlate-384 (PerkinElmer). A mix consisting of 15nM GST-BRD4-BD1 protein (aa 44-168) or 150nM 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 μl of the mix is added to the compound dilutions. Subsequently, 6 μl of premixed AlphaLISA Glutathione Acceptor Beads and AlphaScreen Streptavidin Donor Beads from PerkinElmer (in assay buffer at a concentration of 10 μg/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.
Measurement: IC50 = 57 nM (pChEMBL 7.239999771118164)
Descriptors: MW 500.67, ALogP 2.23, HBA 9, HBD 1, PSA 98.97, QED 0.45
Compound (SMILES): CNC(=O)c1ccccc1Nc1nc(Nc2ccccc2OC)nc2c1CCN2
InChIKey: BZPYOXSDWSEONJ-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 < 200 nM (pChEMBL 6.698999881744385)
Descriptors: MW 390.45, ALogP 3.3, HBA 7, HBD 4, PSA 100.2, QED 0.51
Compound (SMILES): COc1cccc(OC)c1C1CCCCC(=O)N1Cc1cccc(-c2csc(C)n2)c1
InChIKey: JKTONSFRVQDZTK-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 = 3 nM (pChEMBL 8.520000457763672)
Descriptors: MW 436.58, ALogP 5.78, HBA 5, HBD 0, PSA 51.66, QED 0.49
Compound (SMILES): CC(C)(C)OC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3cccc(-c4cccc(Cl)c4)c3)n2)CC1
InChIKey: PJSGMXKNIHQEAU-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 = 1481 nM (pChEMBL 5.829999923706055)
Descriptors: MW 535.05, ALogP 5.69, HBA 7, HBD 2, PSA 126.27, QED 0.42
Compound (SMILES): COCCNc1cc(NC(=O)N2CCCc3cc(CO)c(C=O)nc32)ncc1C#N
InChIKey: SYRAVIOIIKVUFT-UHFFFAOYSA-N
Target: Fibroblast growth factor receptor 4 (SINGLE PROTEIN), Homo sapiens
UniProt: P22455
Assay: Displacement of reporter probe from human recombinant FGFR4 (391 to 802) expressed in Baculovirus infected Sf9 cells measured after 30 mins by proteros reporter displacement assay
Measurement: Kd = 4 nM (pChEMBL 8.399999618530273)
Descriptors: MW 410.43, ALogP 1.7, HBA 8, HBD 3, PSA 140.47, QED 0.46
Compound (SMILES): Cn1cnc2c(-c3cnc(OCCC4CCNCC4)c(C(F)(F)F)c3)nc(C#N)nc21
InChIKey: SJKIGKXSOQCXFK-UHFFFAOYSA-N
Target: Cathepsin S (SINGLE PROTEIN), Mus musculus
UniProt: O70370
Assay: Inhibition Assay: First, spleen cells collected from C57BL/6 mice were seeded in a 96-well plate so as to be a 1× 105 cells/well, then, 5-fold dilution of 10 mM DMSO solution of the test compound was serially performed with RPMI 1640 medium (including 10% fetal bovine serum (FCS), 5×10^−5 M 2-mercaptoethanol, 50 IU/mL penicillin and 50 μg/mL streptomycin) nine times such that the final concentration becomes 0.026 nM to 10 μM, and the resultant products were added thereto (the final DMSO concentration was 0.1%). At the same time, LPS (Sigma L4005) was added to the wells such that the final concentration becomes 2 μg/mL, and culture was performed at 37° C. for 48 hours in 5% CO2. After culturing, the cells were stained at 4° C. for 20 minutes with PE-labeled streptavidin (BD BIOSCIENCE 554061) and biotin-labeled YAe antibody (EBIOSCIENCE 13-5741-85) together with FITC-labeled anti-mouse B220 antibody (BD BIOSCIENCE 553088), and an expression level (fluorescence intensity of YAe-bioti
Measurement: IC50 = 0.81 nM (pChEMBL 9.09000015258789)
Descriptors: MW 431.42, ALogP 3.08, HBA 8, HBD 1, PSA 101.54, QED 0.66
Compound (SMILES): CC(C)COC(=O)N1CCC(CC#N)(n2cc(C(N)=O)c(Nc3ccccc3)n2)CC1
InChIKey: QOKBEMAXSKPNIQ-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 = 102 nM (pChEMBL 6.989999771118164)
Descriptors: MW 424.51, ALogP 3.22, HBA 7, HBD 2, PSA 126.27, QED 0.7
Compound (SMILES): CCCN(CCCC[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](Cc1ccccc1)NC(=O)c1ccc(C(C)(C)C)cc1)C(=O)N[C@@H](CO)C(=O)OC)C(C)C
InChIKey: ITTGNAWNEDCTNG-DUKNTCSXSA-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 = 32 nM (pChEMBL 7.5)
Descriptors: MW 823.09, ALogP 3.79, HBA 9, HBD 6, PSA 195.27, QED 0.07
Compound (SMILES): CC(C)(C)Nc1ncc2cccc(-c3cc4c([nH]3)CCNC4=O)c2n1
InChIKey: UKKVYNLBAIKQRV-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase pim-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9P1W9
Assay: Homogeneous Time Resolved Fluorescence Assay (HTRF): The assay for the determination of Pim activity is based on the formation of phosphorylated biotinylated-BAD peptide at the Serine 112 residue (S112) and employs HTRF® (homogeneous time resolved fluorescence) technology to detect the product in a 384-well plate format. The phosphorylation of biotinylated-BAD (S112) peptide by full length recombinant Pim-1, Pim-2, or Pim-3 protein was detected with streptavidin:Allophycocyanin (APC) conjugate and a europium (Eu) labeled antibody directed against phosphorylated-BAD (S112). Excitation of Eu by a high energy laser light (337 nm) leads to a transfer of energy to the APC molecule, and results in an emission at 665 nm. The fluorescence is directly proportional to the amount of phosphorylated BAD peptide present in the reaction. Compounds were prepared in DMSO by conducting 3-fold serial dilutions to give a 22-point dosing curve having a high dose of 1 μM. A reference compound was included on each assay plate [Costar 3658] in order to validate that plate; on one plate of every assay run, two additional reference compounds were included. The Reaction Buffer consisted of 45 mM Hepes, pH 7.0, 15 mM NaCl, and 1 mM MgCl. The quench/detection buffer consisted of 50 mM Tris, 100 mM NaCl, 0.05% BSA, 0.1% Tween and 3 mM EDTA. Biotinylated BAD peptide (Biopeptide), 10 mM ATP (Sigma), Labeled p-BAD (S112) mAb (Cell Signalling and Perkin Elmer) [with 0.05% BSA and 2 mM DTT added] streptavidin:Allophycocyanin [Perkin Elmer]. Final concentrations either Pim-1 enzyme [5 pM], or Pim-2 enzyme [0.5 pM], DMSO [1%], BLC BAD (S112) [0.5 μM], ATP [1.5 μM], streptavidin:Allophycocyanin [0.002 mg/mL] and biotinylated-BAD (S112) mAb [100 pM]. Initial incubations were carried out at RT (22° C.) for 30 min for both Pim-1 and for Pim-2. Pim enzyme is added to compound in buffer, and plates are incubated of 30 min. Biotinylated BAD and ATP are added and plates are incubated for 1 h. A mixture of labeled p-BAD (S112) mAb and quench/detection buffer are added and incubated for 2 h. Fluorescence was measured by an HTRF® Envision microplate reader.
Measurement: IC50 = 3.62 nM (pChEMBL 8.4399995803833)
Descriptors: MW 335.41, ALogP 3.12, HBA 4, HBD 3, PSA 82.7, QED 0.67
Compound (SMILES): CCOC(=O)CCCCOc1cc(C(c2ccc(Cl)cc2)c2ccc(Cl)cc2)cc2c(NC3CCN(S(=O)(=O)C(F)(F)F)CC3)ccnc12
InChIKey: FZTZIEYASIAHTL-UHFFFAOYSA-N
Target: Cannabinoid receptor 2 (SINGLE PROTEIN), Homo sapiens
UniProt: P34972
Assay: cAMP Assay: The following mixtures and buffer solutions were prepared: (a) Buffer 1: HBSS (Mediatech Cat#21-023-CV) with 5 mM HEPES (1 mM stock, Gibco BRL Cat#15630-056) and 0.1% BSA (7.5% stock, Invitrogen Cat#15260-037); (b) Buffer 2: 0.5 mM IBMX (200 mM stock in DMSO, Sigma 15879) in Buffer 1; (c) 1 uM cAMP Standard (50 uM stock, Perkin Elmer Cat# AD0262) diluted in Buffer 2 and serially diluted in Buffer 2, 12 doses at 1/2 dilutions resulting in a dose range of 1 uM to 0.5 nM; (d) d2 labelled cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 6 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); (e) anti-cAMP (CisBio HTRF Detection Kit Cat #62AM4PEB reconstituted with 5 ml dH2O) diluted 1/20 with lysis buffer (CisBio HTRF Detection Kit Cat #62AM4PEB); and (f) Forskolin (Sigma Cat# F6886, 10 mM in DMSO) diluted first in DMSO to 1 mM and then to 1.5 uM in Buffer 2.
Measurement: EC50 = 7100 nM (pChEMBL 5.150000095367432)
Descriptors: MW 738.66, ALogP 8.56, HBA 7, HBD 1, PSA 97.83, QED 0.08
Compound (SMILES): CS(=O)(=O)NC(=O)c1cccc2nc(/C=C/c3nc4c(N5CCOCC5)cc(Cl)nn4c3-c3cccnc3)ccc12
InChIKey: MLOPMAUQBHFKTF-CSKARUKUSA-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 = 58 nM (pChEMBL 7.239999771118164)
Descriptors: MW 590.07, ALogP 3.69, HBA 10, HBD 1, PSA 131.68, QED 0.31
Compound (SMILES): Cn1cc(-c2ccc3nc(NC(=O)c4ccc(Cl)cc4F)cn3n2)c(-c2ccc(F)cc2)n1
InChIKey: OGTJCBOFCMLEAH-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 = 6.3 nM (pChEMBL 8.199999809265137)
Descriptors: MW 464.86, ALogP 4.98, HBA 6, HBD 1, PSA 77.11, QED 0.41
Compound (SMILES): Cc1cc(=O)oc2cc(OCCCCn3cc(CCCCN4C[C@H](O)[C@@H](O)[C@H](O)[C@H]4CO)nn3)ccc12
InChIKey: VDPRGUYPFVCTHF-USXZWKKNSA-N
Target: Sucrase-isomaltase, intestinal (SINGLE PROTEIN), Rattus norvegicus
UniProt: P23739
Assay: Inhibition of rat small intestinal brush border membrane sucrase assessed as reduction in production of D-glucose using disaccharides as substrate incubated for 10 to 30 mins
Measurement: IC50 = 540 nM (pChEMBL 6.269999980926514)
Descriptors: MW 516.6, ALogP 0.63, HBA 11, HBD 4, PSA 154.31, QED 0.2
Compound (SMILES): CNC(=O)c1c(-n2ccc(C)cc2=O)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CC4)cc12
InChIKey: LBTNUWZANZKCJC-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhibition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 3.952 nM (pChEMBL 8.399999618530273)
Descriptors: MW 625.68, ALogP 5.03, HBA 8, HBD 1, PSA 119.44, QED 0.29
Compound (SMILES): N=C1NC(c2cccc(-c3cccnc3)c2)(C2CCCCC2)C(=O)N1C1CCOCC1
InChIKey: HEYINMYLERBOCR-UHFFFAOYSA-N
Target: Beta-secretase 1 (SINGLE PROTEIN), Homo sapiens
UniProt: P56817
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay : The recombinant human BACE1, Cathepsin D, and Cathepsin E enzymes were purchased from R&D Systems (catalog numbers are 931-AS, 1014AS and 1294AS, respectively). The enzymatic inhibition activity assays were determined using the fluorescence resonance energy transfer (FRET) assay. The assays were performed in a 384-well plate format. The recombinant human BACE-1 enzyme (R&D Systems, catalog#931-AS) was diluted to 20 ng/μL in assay buffer (100 mM sodium acetate pH 4.0), 10 point 1:3 serial dilutions of compound in DMSO were preincubated with the enzyme for 15 min at room temperature. The concentration of Cathepsin D was 20 ng/μL, and 1 ng/μL of Cathepsin E was used. CatD and CatE were activated by incubation in assay buffer (0.1 M NaOAc, 0.2 M NaCl, pH 3.5) at room temperature for 30 min. Subsequently, the rhBACE-1 substrate (R&D Systems, catalog# ES004), the Cathepsin D and Cathepsin E substrate (R&D Systems, Catalog # ES001) were added accordingly to final concentration
Measurement: IC50 = 14700 nM (pChEMBL 4.829999923706055)
Descriptors: MW 418.54, ALogP 4.07, HBA 4, HBD 2, PSA 78.31, QED 0.78
Compound (SMILES): C=CC(=O)N1CCCC[C@@H](n2c(NC(=O)c3cccc(C(F)(F)F)c3)nc3cccc(C)c32)C1
InChIKey: DVTYPSOMACQPMD-LJQANCHMSA-N
Target: Epidermal growth factor receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P00533
Assay: Inhibition of EGFR L858R mutant phosphorylation in human patient derived H3255 cells incubated for 3 hrs by ELISA method
Measurement: IC50 = 14.84 nM (pChEMBL 7.829999923706055)
Descriptors: MW 470.5, ALogP 5.36, HBA 4, HBD 1, PSA 67.23, QED 0.52
Compound (SMILES): CC(=O)Nc1cc(Oc2ccc(NC(=O)Nc3cc(C(C)(C)C)nn3-c3ccc(C)cc3)c3ccccc23)ccn1
InChIKey: MPFNOCFGLHYSEQ-UHFFFAOYSA-N
Target: Mitogen-activated protein kinase 12 (SINGLE PROTEIN), Homo sapiens
UniProt: P53778
Assay: Fluorescence Resonance Energy Transfer (FRET) Assay: The enzyme inhibitory activities of compounds disclosed herein were determined by fluorescence resonance energy transfer (FRET) using synthetic peptides labelled with both donor and acceptor fluorophores (Z-LYTE, Invitrogen Ltd., Paisley, UK). Recombinant, phosphorylated p38 MAPKγ (MAPK12: Invitrogen) was diluted in HEPES buffer, mixed with the test compound at the desired final concentrations and incubated for 2 hr at RT. The FRET peptide (2 μM) and ATP (100 μM) were added to the enzyme/compound mixture and incubated for 1 hr. Development reagent (protease) was added for 1 hr prior to detection in a fluorescence microplate reader (Varioskan® Flash, ThermoFisher Scientific). The site-specific protease cleaves non-phosphorylated peptide only and eliminates the FRET signal. Phosphorylation levels of each reaction were calculated using the ratio of coumarin emission (donor) over fluorescein emission (acceptor) for which high ratios indicate high phosphorylation and low rat
Measurement: IC50 = 146 nM (pChEMBL 6.840000152587891)
Descriptors: MW 548.65, ALogP 7.42, HBA 6, HBD 3, PSA 110.17, QED 0.2
Compound (SMILES): Cc1ccc(-n2nccn2)c(C(=O)N2C[C@@H](C)OC[C@H]2Cc2cc(-n3ccnn3)ccc2F)c1
InChIKey: OSHBRXGINANDJA-YLJYHZDGSA-N
Target: Orexin receptor type 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43614
Assay: OX Inhibition Assay: 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 ug/mL G418, 100 U/mL penicillin, 100 ug/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.
Measurement: IC50 = 1110 nM (pChEMBL 5.960000038146973)
Descriptors: MW 461.5, ALogP 2.77, HBA 8, HBD 0, PSA 90.96, QED 0.45
Compound (SMILES): CN(C)C1(c2ccccc2)CCC(c2ccccc2)(N(C)CCCCO)CC1
InChIKey: UPOUETVJUDHXGZ-UHFFFAOYSA-N
Target: Mu-type opioid receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P35372
Assay: Binding Assay: The affinity to the human μ-opiate receptor was determined in a homogeneous preparation in microtiter plates. For this, dilution series of the respective compound to be tested were incubated for 90 minutes at room temperature with a receptor membrane preparation (15-40 mg of protein per 250 μl of incubation batch) of CHO-K 1 cells, which express the human μ-opiate receptor (RB-HOM receptor membrane preparation of NEN, Zaventem, Belgium), in the presence of 1 nmol/l of the radioactive ligand [3H'-naloxone (NET719, NEN, Zaventem, Belgium) and of 1 mg WGA-SPA beads (wheat germ agglutinin SPA beads from Amersham/Pharmacia, Freiburg, Germany) in a total volume of 250 μl. 50 mmol/l of tris-HCl supplemented by 0.05% by wt. of sodium azide and 0.06% by wt. of bovine serum albumin was used as incubation buffer. 25 μmol/l of naloxone were additionally added to determine the non-specific bond. After the ninety-minute incubation time had ended, the microtiter plates were centrifuged for 20 minutes at 1000 g and the radioactivity measured in a β-counter (Microbeta-Trilux, PerkinElmer Wallac, Freiburg, Germany).
Measurement: Ki = 415 nM (pChEMBL 6.380000114440918)
Descriptors: MW 380.58, ALogP 4.62, HBA 3, HBD 1, PSA 26.71, QED 0.68
Compound (SMILES): Cc1cccc(NC(=O)Nc2ccc(C#Cc3ccc4nccn4n3)cc2)c1
InChIKey: RUTCUMBDVHNVLK-UHFFFAOYSA-N
Target: Platelet-derived growth factor receptor beta (SINGLE PROTEIN), Homo sapiens
UniProt: P09619
Assay: PDGFRbeta Kinase Assay: Biochemical KDR kinase assays were performed in 96 well microtiter plates that were coated overnight with 75 ug/well of poly-Glu-Tyr (4:1) in 10 mM Phosphate Buffered Saline (PBS), pH 7.4. The coated plates were washed with 2 mls per well PBS+0.05% Tween-20 (PBS-T), blocked by incubation with PBS containing 1% BSA, then washed with 2 mls per well PBS-T prior to starting the reaction. Reactions were carried out in 100 uL reaction volumes containing 2.7 uM ATP in kinase buffer (50 mM Hepes buffer pH 7.4, 20 mM MgCl2, 0.1 mM MnCl2 and 0.2 mM Na3VO4). Test compounds were reconstituted in 100% DMSO and added to the reaction to give a final DMSO concentration of 5%. Reactions were initiated by the addition 20 ul per well of kinase buffer containing 200-300 ng purified cytoplasmic domain KDR protein (BPS Bioscience, San Diego, Calif.). Following a 15 minute incubation at 30° C., the reactions were washed 2 mls per well PBS-T.
Measurement: IC50 = 80 nM (pChEMBL 7.099999904632568)
Descriptors: MW 367.41, ALogP 4.08, HBA 4, HBD 2, PSA 71.32, QED 0.52
Compound (SMILES): O=C(N[C@H]1CCC[C@@H]1Nc1cnc(C(F)(F)F)cn1)c1cc(Cl)ccc1-n1cccn1
InChIKey: BVJAHTQQALEZIK-GJZGRUSLSA-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 450.85, ALogP 4.1, HBA 6, HBD 2, PSA 84.73, QED 0.61
Compound (SMILES): Cc1ccc2nc(NC3=NCC4(CN5CCC4CC5)O3)sc2c1
InChIKey: KOHDBHPGUHPVRI-UHFFFAOYSA-N
Target: 5-hydroxytryptamine receptor 3A (SINGLE PROTEIN), Homo sapiens
UniProt: P46098
Assay: Antagonist activity at human 5-HT3A expressed in HEK293 cells assessed as reduction in acetylcholine-induced activity preincubated for 30 mins followed by addition of acetylcholine measured for 2 mins by Fluo-4AM dye based FLIPR assay
Measurement: IC50 = 3800 nM (pChEMBL 5.420000076293945)
Descriptors: MW 328.44, ALogP 2.87, HBA 6, HBD 1, PSA 49.75, QED 0.87
Compound (SMILES): CCOc1cc(=O)n(C)cc1-c1cc(N(Cc2ccccc2Cl)S(=O)(=O)CC)ccc1Oc1ccc(F)cc1F
InChIKey: DMCHKXUBWIFAAV-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 = 13.5 nM (pChEMBL 7.869999885559082)
Descriptors: MW 589.06, ALogP 6.53, HBA 6, HBD 0, PSA 77.84, QED 0.21
Compound (SMILES): COc1cc([N+](=O)[O-])cnc1Nc1ccc(F)c([C@]2(C(F)F)COCC(N)=N2)c1
InChIKey: MALBXUIZEBZONF-KRWDZBQOSA-N
Target: Beta-secretase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9Y5Z0
Assay: BACE-2 Inhibition Assay: Recombinant BACE-2 (extracellular domain, expressed in baculovirus and purified using standard methods) at 0.1 to 1 nM concentrations is incubated with the test compound at various concentrations for 1 hour at room temperature in 100 mM acetate buffer, pH 4.5, containing 0.1% CHAPS. Activity was measured using a final concentration of 3 uM of the fluorescence-quenched substrate Q-C(HSO3)-Ile-Asp-Leu-Ala-Val-Leu-Asp-HN-CH2-CH2-Mca, where Q=2-nitro-5-amino benzoic acid and Mca=7-methoxy-4-coumarinyl acetic acid. Catalytic turnover was monitored in a Spectramax Gemini fluorescence plate reader (Molecular Devices) in black 96-well microplates using excitation/emission wavelength of 325 nm and 400 nm, respectively. Fluorescence increase was followed for 15 min, in 1 minute's intervals.
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 411.34, ALogP 2.73, HBA 8, HBD 2, PSA 124.9, QED 0.55
Compound (SMILES): CNC(=O)c1c(-c2ccc(OC)nc2)oc2cc(N(C)S(C)(=O)=O)c(-c3ccc4c(n3)-c3cc5c(F)cccc5n3CO4)cc12
InChIKey: WXFDQSPVNCWBKS-UHFFFAOYSA-N
Target: Genome polyprotein (SINGLE PROTEIN), Hepatitis C virus genotype 1b (isolate Japanese) (HCV)
UniProt: P26662
Assay: Inhbition Assay: To initiate an assay, replicon cells were plated in the presence of a dilution series of test compound in the absence of G418. Typically, the assays were performed in a 96-well plate formate for manual operation, or a 384-well plate format for automated assay. Replicon cells and compound were incubated for 96 hours. At the end of the assay, cells were washed free of media and compound, and the cells were then lysed. RNA was quantified indirectly through detection of replicon-encoded NS3/4A protein levels, through an ELISA-based assay with an antibody specific for NS3/4A.
Measurement: IC50 = 8.4 nM (pChEMBL 8.079999923706055)
Descriptors: MW 627.65, ALogP 5.43, HBA 9, HBD 1, PSA 128.79, QED 0.26
Compound (SMILES): Cc1ccc(NC(=O)c2cccc(C(F)(F)F)c2)cc1-c1cc(N(C)C)nc(N2CCOCC2)c1
InChIKey: PABZMNZFBMSMPR-UHFFFAOYSA-N
Target: Serine/threonine-protein kinase B-raf (SINGLE PROTEIN), Homo sapiens
UniProt: P15056
Assay: In Vitro Enzyme Assay: Final assay conditions were 4 pM cRaf, 3 uM ATP, 10 nM biotin tagged MEK1 kinase dead protein substrate. Reactions were performed in Greiner384 well plates, catalog #784075. Reaction buffer was 50 mM Tris, pH 7.5, 50 mM NaCl, 10 mM MgCl2, 0.01% Tween-20, 0.05% BSA and 1 mM DTT. 5 ul of 2×cRaf plus MEK1 were dispensed into plates containing 0.25 ul of compound in 100% DMSO and incubated at room temperature for 30 minutes. Reactions were started by addition of 5 ul of 2×ATP. Reactions were run for two hours and terminated by addition of 10 ul of 2× antibody and Alphascreen bead mixture in Stop Buffer. Stop Buffer was 50 mM Tris, pH 7.5, 0.005% Tween-20, 25 mM EDTA.
Measurement: IC50 = 3.222 nM (pChEMBL 8.489999771118164)
Descriptors: MW 484.52, ALogP 5.23, HBA 5, HBD 1, PSA 57.7, QED 0.54
Compound (SMILES): C[C@H](Nc1nc(N)nc(N)c1C#N)c1nc2c(Cl)cccc2c(=O)n1-c1cccnc1
InChIKey: FCKCJTUBGNGWLU-JTQLQIEISA-N
Target: PI3-kinase p110-delta/p85-alpha (PROTEIN COMPLEX), Homo sapiens
UniProt: O00329
Assay: Inhibition of human full length PI3K p110delta/p85alpha using phosphatidylinositol 3,4,5-trisphosphate as substrate measured after 30 mins by TR-FRET assay
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 433.86, ALogP 2.43, HBA 10, HBD 3, PSA 161.42, QED 0.44
Compound (SMILES): C[C@]1(C#N)CS(=O)(=O)CC1n1cc(C(N)=O)c(Nc2ccc(F)cc2)n1
InChIKey: QPHALRREIYHCHW-VYIIXAMBSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Biochemical HTRF Assay: The ability of compounds to inhibit the activity of JAK1, JAK2, JAK3, and Tyk2 was measured using a recombinant purified GST-tagged catalytic domain for each enzyme (Invitrogen JAK1 #M4290, JAK2 #M4290, JAK3 #M4290, Tyk2 #M4290) in an HTRF format 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 μL of 1.11× enzyme and 1.11× substrate in 1× 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 ambient temperature to allow compound binding to equilibrate. After equilibration, 2 μL of 10×ATP in 1× assay buffer was added to initiate the kinase reaction and the plates were shaken and then incubated at ambient temperature for 120 minutes. At the end of the incubation, 20 μL of 2× 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. Plates were shaken and centrifuged and then incubated 60 minutes at ambient temperature and then read on a Perkin Elmer Envision (λex=337 nm, λem=665 and 615 nm, TRF delay time=20 μs). HTRF signal=10,000*665 nm reading/615 nm reading.
Measurement: IC50 = 6 nM (pChEMBL 8.220000267028809)
Descriptors: MW 377.4, ALogP 1.36, HBA 7, HBD 2, PSA 130.87, QED 0.83
Compound (SMILES): CNCCCn1c(C(C)(C)C)cc(S(=O)(=O)Nc2cccc(C(F)(F)F)c2)c1C
InChIKey: CRVULPKLJRCXRJ-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 4 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9HBA0
Assay: Inhibition Assay: Test Example 1: (1) The TRPV4 inhibitory activity was measured using CHO-K1 cells stably expressing human TRPV4 (hTRPV4/CHO cells). (2) In the day before the experimental day, frozen cell were thawed and washed with the culture medium (MEM-α, 10% FBS, 2 mmol/L GlutaMax, 50 unit Penicillin, 50 μg/mL Streptomycin). Then cells were suspended in the culture medium. (3) The 384-well plates which hTRPV4/CHO cells were seeded at densities of 4000 cells/well in the culture medium and cultured in a CO2 incubator in the presence of 5% CO2 at 37° C. for overnight was used as assay plates. (4) The assay plate was washed with assay buffer (Hanks, 20 mmol/L HEPES, 2.5 mmol/L probenecid, pH7.4), and the buffer were remained at 20 μL/well. (5) 10 μl of dye loading buffer (9 μmol/L Fluo 4-AM, 0.09% Pluronic F-127/assay buffer) was added to each well, then the assay plate was incubated in a CO2 incubator in the presence of 5% CO2 at 37° C. for one hour. (final 3 μmol/L Fluo4-AM). (6) The assay plate was washed with the assay buffer, and the buffers were remained at 20 μL/well. Then the assay plate was incubated for 10 min at room temperature. (7) Diluted compound solution was dispensed to each well in the compound plate, and mixed with built-in Pipette and Mixer of the Fluorescence analysis system FLIPR TETRA (Molecular devices). (8) After incubation for 5 min, 20 μL of 4α-PDD solution were applied to each well of assay plate and mixed with the FLIPR TETRA. (final 1 Î¼mol/L 4α-PDD). (9) The fluorescent intensity was measured with FLIPR TETRA system for 10 min from the point of time addition the compound solution, at Ex 470-495 nm, Em 515-575 nm wavelength.
Measurement: IC50 = 9200 nM (pChEMBL 5.039999961853027)
Descriptors: MW 431.52, ALogP 4.52, HBA 4, HBD 2, PSA 63.13, QED 0.63
Compound (SMILES): CCCCCCC(=S)NCCCCC(NC(=O)Oc1ccccc1)C(=O)Nc1ccccc1
InChIKey: JCABVKCNCPVTLU-UHFFFAOYSA-N
Target: NAD-dependent protein deacetylase sirtuin-2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8IXJ6
Assay: Inhibition Assay: Different concentrations of TA, TB, TH, or TM were pre-incubated with 1 μM of the sirtuin and 1 mM NAD in 20 mM Tris-HCl buffer (pH 8.0) with 1 mM dithiothreitol (DTT) at 37° C. for 30 minutes. Then 0.05 mM acyl peptides (H3K9Ac for Sirt1, Sirt2 and Sirt3; H3K9Su for Sirt5; H3K9Myr for Sirt6 and Sirt7) were added to initiate the reactions. The total reaction volume iwas 60 mL. Then reactions were incubated at 37° C. for a certain amount of time (2 minutes for Sirt1, 10 minutes for Sirt2 and Sirt5, 30 minutes for Sirt3, and 1 hour for Sirt6 and Sirt7). The reactions were stopped by adding 60 mL of an aqueous solution containing 200 mM HCl and 320 mM acetic acid. After centrifugation to remove precipitated proteins, the supernatant was analyzed by HPLC with a reverse phase C18 column (250×4.6 mm, 90 A, 10 mm, GraceVydac, Southborough, Mass.), with a linear gradient of 0% to 20% B for 10 minutes (1 mL/min).
Measurement: IC50 = 120 nM (pChEMBL 6.920000076293945)
Descriptors: MW 469.65, ALogP 5.84, HBA 4, HBD 3, PSA 79.46, QED 0.24
Compound (SMILES): FC(F)(F)C(F)(F)CNC[C@H]1CC[C@H](c2nnn3cnc4[nH]ccc4c23)CC1
InChIKey: OKNRNFDKCVZCQF-XYPYZODXSA-N
Target: Tyrosine-protein kinase JAK2 (SINGLE PROTEIN), Homo sapiens
UniProt: O60674
Assay: Enzyme Assay: JAK1, JAK2, JAK3 and Tyk2 were purchased from Carna Biosciences, Inc. As the substrate, LANCE Ultra ULight-JAK1 Peptide (manufactured by PerkinElmer Co., Ltd. (PE)) was used. Dilute solutions of compounds and enzymes in assay buffer (50 mM HEPES pH7.5, 1 mM EGTA, 1 mM MgCl2, 2 mM DTT, 0.01% Tween20) were dispensed into wells of a 384-well black plate. After 5 minutes of preincubation, dilute solutions of the substrate and ATP (adenosine triphosphate) were added at a final concentration of 100 μM, and the plate was incubated at room temperature for 2 hours. After addition of a termination reagent containing EDTA (ethylenediamine tetraacetic acid) at a final concentration of 10 mM, LANCE Eu-W1024 Anti-phosphotyrosine (PT66) (manufactured by PE) was added, and after 1 hour of incubation, the fluorescences were measured with ARVO-HTS.
Measurement: IC50 = 34 nM (pChEMBL 7.46999979019165)
Descriptors: MW 402.37, ALogP 3.67, HBA 5, HBD 2, PSA 70.9, QED 0.64
Compound (SMILES): CCN(CC)C(=O)c1cc(-c2cccs2)nc2c1c(C)nn2-c1ccccc1C
InChIKey: SFKDMBHAUPXODT-UHFFFAOYSA-N
Target: Dual specificity calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1B (SINGLE PROTEIN), Homo sapiens
UniProt: Q01064
Assay: In Vitro Enzyme Assay: In vitro enzyme assays were conducted via the Ba(OH)2 precipitation method of Wang et al. (32) using recombinant human PDE11A4, PDE10A1, PDE5A1, PDE6C, PDE8A (BPS Bioscience Inc.), PDE7A (BIOMOL International), and PDE4A10 enzymes (gift from HengmingKe), in the presence of 100 nM cGMP, 30 nM cAMP, 500 nM cGMP, 1.7 μM cGMP, 10 nM cAMP, 15 nM cAMP, and 625 nM cAMP, respectively. Inhibitor concentrations that reduce enzyme activity by 50% (IC50) are presented. The values are means of at least three independent experiments.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 404.54, ALogP 5.25, HBA 5, HBD 0, PSA 51.02, QED 0.46
Compound (SMILES): N#C/C(=C\C1CC1)C(=O)N1CCCC(n2nc(-c3ccc(Oc4ccccc4)cc3)c3c(N)ncnc32)C1
InChIKey: SKHARCJJALHOPW-RCCKNPSSSA-N
Target: Tyrosine-protein kinase BTK (SINGLE PROTEIN), Homo sapiens
UniProt: Q06187
Assay: Enzymatic Activity Assay: A Caliper-based kinase assay (Caliper Life Sciences, Hopkinton, Mass.) was used to measure inhibition of Btk kinase activity of a compound of Formula (IA), (I′) or (I). Serial dilutions of test compounds were incubated with human recombinant Btk (2 nM), ATP (40 μM) and a phosphoacceptor peptide substrate FAM-GEEPLYWSFPAKKK-NH2 (1 μM) at room temperature for 3 h. The reaction was then terminated with EDTA, final concentration 20 mM and the phosphorylated reaction product was quantified on a Caliper Desktop Profiler (Caliper LabChip 3000). Percent inhibition was calculated for each compound dilution and the concentration that produced 50% inhibition was calculated.
Measurement: IC50 = 1.7 nM (pChEMBL 8.770000457763672)
Descriptors: MW 505.58, ALogP 4.89, HBA 8, HBD 1, PSA 122.95, QED 0.29
Compound (SMILES): CCC(CC)(NC(=O)c1ccc(C2CC2)c(OCC2CCOCC2)n1)C(=O)O
InChIKey: HXOMGSVTAKWIMX-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 = 475 nM (pChEMBL 6.320000171661377)
Descriptors: MW 390.48, ALogP 3.14, HBA 5, HBD 2, PSA 97.75, QED 0.67
Compound (SMILES): CC(C)(O)c1cnc(C2=CCN(C(=O)Nc3ccc(C(F)(F)F)cc3)CC2)c(Cl)c1
InChIKey: MZJZMKVSTCPTID-UHFFFAOYSA-N
Target: Transient receptor potential cation channel subfamily V member 1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q8NER1
Assay: pH-Based Assay: Two days prior to performing this assay, cells are seeded on poly-D-lysine-coated 96-well clear-bottom black plates (commercially available from Becton-Dickinson) at 75,000 cells/well in growth media containing 5 μM PonA (commercially available from Invitrogen) to induce expression of TRPV1. On the day of the assay, the plates are washed with 0.2 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 1.6 mM CaCl2 and 20 mM HEPES, pH 7.4 ("wash buffer"), and loaded using 0.1 mL of wash buffer containing Fluo-4 (3 μM final concentration, commercially available from Molecular Probes). After 1 h, the cells are washed twice with 0.2 mL wash buffer and resuspended in 0.05 mL 1× Hank's Balanced Salt Solution (commercially available from Life Technologies) containing 3.5 mM CaCl2 and 10 mM Citrate, pH 7.4 ("assay buffer"). Plates are then transferred to a FLIPR for assay. The test compound is diluted in assay buffer, and 50 μL of the resultant solution is added to the cell plates and the solution is monitored for two minutes. The final concentration of the test compound is adjusted to range from about 50 picoM to about 3 μM. Agonist buffer (wash buffer titrated with 1N HCl to provide a solution having a pH of 5.5 when mixed 1:1 with assay buffer) (0.1 mL) is then added to each well, and the plates are incubated for 1 additional minute.
Measurement: IC50 = 81.1 nM (pChEMBL 7.090000152587891)
Descriptors: MW 439.87, ALogP 5.3, HBA 3, HBD 2, PSA 65.46, QED 0.68
Compound (SMILES): O=C(Nc1cnc(Oc2ccnc(NC(=O)C3CC3)c2)c(F)c1)Nc1ccc(Cl)c(C(F)(F)F)c1
InChIKey: SILQMMMQYPKYGU-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 = 9.1 nM (pChEMBL 8.039999961853027)
Descriptors: MW 509.85, ALogP 6.07, HBA 5, HBD 3, PSA 105.24, QED 0.35
Compound (SMILES): NC(=O)c1cccc([C@H]2C[C@H]3CC[C@@H](C2)N3CCN(CC23CC4CC(CC(C4)C2)C3)C(=O)CO)c1.O=C(O)C(F)(F)F
InChIKey: SPEPWEVEUGRBST-ZYJOMFFYSA-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 479.67, ALogP 3.53, HBA 4, HBD 2, PSA 86.87, QED 0.6