lovingscience/chembl-v37-raw-activities huggingface dataset:
Compound (SMILES): Cc1c(-c2cccc(S(=O)(=O)N(C)C)c2)c2cc(O)ccc2n1C/C(F)=C/CN
InChIKey: VMZRQLIVPKQUDC-SXGWCWSVSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibit Human Recombinant Assay: MAO-B: The specificity of the compounds of this invention was tested by determining their ability to inhibit MAO-B activities in vitro. Recombinant human MAO-B (0.06 mg/mL; Sigma Aldrich) was used as source of MAO-B enzyme activities.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 417.51, ALogP 3.38, HBA 5, HBD 2, PSA 88.56, QED 0.64
Compound (SMILES): Cc1ccc(/C(=C(\CCCO)c2ccccc2)c2ccc(N3CCN(C)CC3)cc2)cc1
InChIKey: PZDWZAMMWRBRBP-ZIADKAODSA-N
Target: Steroid hormone receptor ERR2 (SINGLE PROTEIN), Homo sapiens
UniProt: O95718
Assay: Binding Assay: In an ERR alpha binding assay, GST-bound ERR alpha LBD was used, and all experiments other than that was the same as the ERR gamma binding assay.In an ERR beta binding assay, GST-bound ERR alpha LBD was used so that a final concentration was 10 nM and a fluorescein-conjugated coactivator PGC1a was 250 nM, and all experiments other than that was the same as the ERR gamma binding assay.In an ER alpha binding assay, a GST-bound ER alpha ligand-binding domain (LBD) was added to a 384 well plate to which the arylethene derivative of the present invention was added to a final concentration of 7.3 nM. Then, a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 250 nM and 5 nM, respectively, and beta-estradiol as an agonist was added to a final concentration of 4 nM. All subsequent experiments was the same as the ERR gamma binding assay.
Measurement: IC50 = 1017 nM (pChEMBL 5.989999771118164)
Descriptors: MW 426.6, ALogP 5.48, HBA 3, HBD 1, PSA 26.71, QED 0.51
Compound (SMILES): Cc1c(-c2cccc(S(=O)(=O)N(C)C)c2)c2cc(-c3nnn[nH]3)ccc2n1C/C(F)=C/CN
InChIKey: HFXSXVBYINJBIZ-MFOYZWKCSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibit Human Recombinant Assay: MAO-B: The specificity of the compounds of this invention was tested by determining their ability to inhibit MAO-B activities in vitro. Recombinant human MAO-B (0.06 mg/mL; Sigma Aldrich) was used as source of MAO-B enzyme activities.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 469.55, ALogP 2.86, HBA 7, HBD 2, PSA 122.79, QED 0.43
Compound (SMILES): NCc1ccc(S(=O)(=O)c2ccc(S(=O)(=O)CCN3CCCC3)cc2)s1
InChIKey: AWPLRLGRKNDWTQ-UHFFFAOYSA-N
Target: Protein-lysine 6-oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P28300
Assay: LOX Enzyme Activity Assay: The following reagents were prepared:Reagents500 mM CHES, pH 9 (with NaOH), filtered (MW=207.29)20.7 g in 200 ml in dH2O10% Pluronic F-127 (store at 4° C.)1 g in 10 ml in dH2O10 % BSA (store at 4° C.)−2.5 g in 25 ml dH2O1 M MgCl2.6H2O (MW=203)5.1 g in 25 ml dH2O5 M Urea (MW=60.06)60 g in 200 ml dH2O5 M NaCl (MW=58.44)14.6 g in 50 ml dH2O8.5 M Cadaverine Dihydrochloride (MW=175.10): Sigma, Cat No: C85611.49 g in 1 ml dH2O10 mM Na3VO4, pH 10 (with HCl), boil to activate (MW=183.91): Sigma, Cat No: S65080.1839 g in 10 ml dH2O20 mM Amplex Red (MW=257.25): Invitrogen, Cat No: A12222.5 mg in 1 ml DMSO (aliquot into 40 μl and store at −20° C.)1000 U/ml HRP in dH2O (store at 4° C.): Sigma, Cat No: P2088H2O2 (store at 4° C.): Sigma, Cat No: H1009DMSOLOX Assay Buffer: Volume (μl) Reagent 1 × plate Final Concn 500 mM CHES, pH 9 5000 100 mM 10% Pluronic F-127 125 0.05% 10% BSA 1250 0.5% 1M MgCl2 25  1 mM 5M Urea 5000 1M 5M NaCl 500 100 mM dH2O 13100 — Total Volume 25 000 —Inhibitors (Test Compounds):10 mM stock diluted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0.0003 mM in drug plate, resulting in a concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 uM in the assay.LOX enzyme:LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (˜4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls: 0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix: Volume Reagent 1 × plate 2 × plate Final Concn Assay Buffer 2 ml 3 ml — 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM 20 mM Amplex Red 10 μl 15 μl 100 μM 1000 U/ml HRP 10 μl 15 μl 5 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader: Excitation wavelength: 545 nm Emission wavelength: 585 nm HRP Counter Assay Protocol1. Dilute 5 μl H2O2 in 640 μl dH2O. Add 1 μl diluted H2O2 to 10 ml Assay Buffer. Vortex2. Add 40 μl H2O2+Assay Buffer into every well on the counter assay plate EXCEPT no H2O2 control wells (add 40 μl Assay Buffer only)3. Add 0.5 μl test compound serial dilutions, in duplicate4. Add 0.5 μl serial dilutions of positive control, Na3VO4, down column 11 (no duplication)5. Add following controls: 0.5 μl DMSO (100% activity control) 1 μl diluted H2O2 (blow out control)6. Incubate for 20 min at room temp on a plate shaker.
Measurement: IC50 = 550 nM (pChEMBL 6.260000228881836)
Descriptors: MW 414.57, ALogP 1.91, HBA 7, HBD 1, PSA 97.54, QED 0.74
Compound (SMILES): NCc1ccc(S(=O)(=O)c2ccc(-c3ccccc3)cc2)s1
InChIKey: OVQLNOGPBKMMBO-UHFFFAOYSA-N
Target: Protein-lysine 6-oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P28300
Assay: LOX Enzyme Activity Assay: The following reagents were prepared:Reagents500 mM CHES, pH 9 (with NaOH), filtered (MW=207.29)20.7 g in 200 ml in dH2O10% Pluronic F-127 (store at 4° C.)1 g in 10 ml in dH2O10 % BSA (store at 4° C.)−2.5 g in 25 ml dH2O1 M MgCl2.6H2O (MW=203)5.1 g in 25 ml dH2O5 M Urea (MW=60.06)60 g in 200 ml dH2O5 M NaCl (MW=58.44)14.6 g in 50 ml dH2O8.5 M Cadaverine Dihydrochloride (MW=175.10): Sigma, Cat No: C85611.49 g in 1 ml dH2O10 mM Na3VO4, pH 10 (with HCl), boil to activate (MW=183.91): Sigma, Cat No: S65080.1839 g in 10 ml dH2O20 mM Amplex Red (MW=257.25): Invitrogen, Cat No: A12222.5 mg in 1 ml DMSO (aliquot into 40 μl and store at −20° C.)1000 U/ml HRP in dH2O (store at 4° C.): Sigma, Cat No: P2088H2O2 (store at 4° C.): Sigma, Cat No: H1009DMSOLOX Assay Buffer: Volume (μl) Reagent 1 × plate Final Concn 500 mM CHES, pH 9 5000 100 mM 10% Pluronic F-127 125 0.05% 10% BSA 1250 0.5% 1M MgCl2 25  1 mM 5M Urea 5000 1M 5M NaCl 500 100 mM dH2O 13100 — Total Volume 25 000 —Inhibitors (Test Compounds):10 mM stock diluted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0.0003 mM in drug plate, resulting in a concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 uM in the assay.LOX enzyme:LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (˜4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls: 0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix: Volume Reagent 1 × plate 2 × plate Final Concn Assay Buffer 2 ml 3 ml — 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM 20 mM Amplex Red 10 μl 15 μl 100 μM 1000 U/ml HRP 10 μl 15 μl 5 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader: Excitation wavelength: 545 nm Emission wavelength: 585 nm HRP Counter Assay Protocol1. Dilute 5 μl H2O2 in 640 μl dH2O. Add 1 μl diluted H2O2 to 10 ml Assay Buffer. Vortex2. Add 40 μl H2O2+Assay Buffer into every well on the counter assay plate EXCEPT no H2O2 control wells (add 40 μl Assay Buffer only)3. Add 0.5 μl test compound serial dilutions, in duplicate4. Add 0.5 μl serial dilutions of positive control, Na3VO4, down column 11 (no duplication)5. Add following controls: 0.5 μl DMSO (100% activity control) 1 μl diluted H2O2 (blow out control)6. Incubate for 20 min at room temp on a plate shaker.
Measurement: IC50 = 1800 nM (pChEMBL 5.75)
Descriptors: MW 329.45, ALogP 3.71, HBA 4, HBD 1, PSA 60.16, QED 0.79
Compound (SMILES): NCc1ccc(S(=O)(=O)c2ccc(S(=O)(=O)CCN3CCCNCC3)cc2)s1
InChIKey: JVNMLAGLZHGLNG-UHFFFAOYSA-N
Target: Arachidonate 12-lipoxygenase (SINGLE PROTEIN), Sus scrofa
UniProt: P16469
Assay: LOX Enzyme Activity Assay: LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls:0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix:Volume Reagent 1 x plate 2 x plate Final ConcnAssay Buffer  2 ml    3 ml 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM20 mM Amplex Red 10 μl   15 μl 100 μM1000 U/ml HRP 10 μl   15 μl   35 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader:Excitation wavelength: 545 nmEmission wavelength: 585 nmHRP Counter Assay Protocol
Measurement: IC50 = 450 nM (pChEMBL 6.349999904632568)
Descriptors: MW 443.62, ALogP 1.11, HBA 8, HBD 2, PSA 109.57, QED 0.66
Compound (SMILES): CCOc1ccccc1-c1cc(S(C)(=O)=O)cc(S(=O)(=O)c2ccc(CN)s2)c1
InChIKey: CUIBMEWRAXNXOA-UHFFFAOYSA-N
Target: Arachidonate 12-lipoxygenase (SINGLE PROTEIN), Sus scrofa
UniProt: P16469
Assay: LOX Enzyme Activity Assay: LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls:0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix:Volume Reagent 1 x plate 2 x plate Final ConcnAssay Buffer  2 ml    3 ml 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM20 mM Amplex Red 10 μl   15 μl 100 μM1000 U/ml HRP 10 μl   15 μl   35 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader:Excitation wavelength: 545 nmEmission wavelength: 585 nmHRP Counter Assay Protocol
Measurement: IC50 = 7800 nM (pChEMBL 5.110000133514404)
Descriptors: MW 451.59, ALogP 3.51, HBA 7, HBD 1, PSA 103.53, QED 0.59
Compound (SMILES): Cc1ccc2c(N)c(C(=O)NCCc3ccc(N4CC[C@H]5NCC[C@H]54)cc3)sc2n1
InChIKey: IDEISMLLPVGWLL-RTBURBONSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 28 (SINGLE PROTEIN), Homo sapiens
UniProt: Q96RU2
Assay: Biochemical Assay: USP28: The assay was performed in a final volume of 20 uL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HCl, pH 8.0 solution; Corning 46-031-CM)), 2 mM CaCl2 (1M Calcium Chloride solution; Sigma #21114) 2 mM BME (2-Mercaptoethanol; Sigma 63689-25ML -F), 0.01% Prionex (0.22 uM filtered, Sigma #G-0411), and 0.01% Triton X-100. Stock compound solutions were stored at â20° C. as 10 mM in DMSO. Up to 1 month prior to the assay, 2 mM test compounds were pre-dispensed into assay plates (Black, low volume; Corning #3820) and frozen at â20° C. Prestamped assay plates were allowed to come to room temperature on the day of the assay. For the screen, 100 nL of 2 mM was pre-dispensed for a final screening concentration of 10 uM (DMSO(fc)=0.5%). Enzyme (USP28, construct USP28 (USP28-5(1-1077)-TEV-6*His; LifeSensors) concentration and incubation times were optimized for the maximal signal-to -background while maintaining initial velocity conditions at a fixed substrate concentration. The final concentration of the enzyme in the assay was 400 pM. Final substrate (Ub-Rh110; Ubiquitin -Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub-Rh110] Km. 10 uL of 2x enzyme was added to assay plates (pre-stamped with compound) either simultaneously with 2xUb-Rh110 or preincubated with USP28 40 minutes prior to the addition of 10 uL of 2xUb -Rh110 to compound plates. Plates were incubated stacked for 90 minutes at room temperature before fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 421.57, ALogP 3.1, HBA 6, HBD 3, PSA 83.28, QED 0.59
Compound (SMILES): Cc1c(-c2cccc(S(=O)(=O)N(C)C)c2)c2cccc(C(=O)N(C)C)c2n1C/C(F)=C/CN
InChIKey: SNHOXOFEINYVMA-PDGQHHTCSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Inhibit Human Recombinant Assay: MAO-B: The specificity of the compounds of this invention was tested by determining their ability to inhibit MAO-B activities in vitro. Recombinant human MAO-B (0.06 mg/mL; Sigma Aldrich) was used as source of MAO-B enzyme activities.
Measurement: IC50 > 3000 nM (pChEMBL 5.5229997634887695)
Descriptors: MW 472.59, ALogP 3.38, HBA 5, HBD 1, PSA 88.64, QED 0.57
Compound (SMILES): Cn1cc(-c2cc(C(=O)O)cc(S(=O)(=O)c3cnc(CN)s3)c2)cn1
InChIKey: VGGFCLBUJBAJKZ-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Monoamine Oxidase B (MAO-B) Selectivity Assay: MAO-Glo Assay Kit: Promega, Cat No. V1402, MAO-B Enzyme: Sigma, Cat No. M7441.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 378.44, ALogP 1.53, HBA 8, HBD 2, PSA 128.17, QED 0.69
Compound (SMILES): [N-]=[N+]=NCCCS(=O)(=O)c1ccc(S(=O)(=O)c2ccc(CN)s2)cc1
InChIKey: SXJSNDMALCJGTG-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Monoamine Oxidase B (MAO-B) Selectivity Assay: MAO-Glo Assay Kit: Promega, Cat No. V1402, MAO-B Enzyme: Sigma, Cat No. M7441.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 400.51, ALogP 2.51, HBA 7, HBD 1, PSA 143.06, QED 0.31
Compound (SMILES): NCc1ccc(S(=O)(=O)c2ccc(S(=O)(=O)CCCO)cc2)s1
InChIKey: XFLLCXYGTGWHGM-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Monoamine Oxidase B (MAO-B) Selectivity Assay: MAO-Glo Assay Kit: Promega, Cat No. V1402, MAO-B Enzyme: Sigma, Cat No. M7441.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 375.49, ALogP 1.2, HBA 7, HBD 2, PSA 114.53, QED 0.75
Compound (SMILES): CC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(=N)N)C(=O)NCCCCC1CCN(CC(=O)N2c3ccccc3NC(=O)c3ccccc32)CC1
InChIKey: GLRXSDVYMJTKAU-CONSDPRKSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Displacement of [3H]-NMS from human muscarinic M4 receptor expressed in CHO cell membranes assessed as inhibition constant by radioligand competition analysis
Measurement: Ki = 63.1 nM (pChEMBL 7.199999809265137)
Descriptors: MW 732.93, ALogP 2.3, HBA 8, HBD 8, PSA 227.87, QED 0.06
Compound (SMILES): CC(C)N1CCN(c2ccc(/C(=C(/CCCO)c3ccccc3)c3ccc(NS(C)(=O)=O)cc3)cc2)CC1
InChIKey: LLLDMSCJOLSJFI-KTMFPKCZSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Binding Assay: In an ERR alpha binding assay, GST-bound ERR alpha LBD was used, and all experiments other than that was the same as the ERR gamma binding assay.In an ERR beta binding assay, GST-bound ERR alpha LBD was used so that a final concentration was 10 nM and a fluorescein-conjugated coactivator PGC1a was 250 nM, and all experiments other than that was the same as the ERR gamma binding assay.In an ER alpha binding assay, a GST-bound ER alpha ligand-binding domain (LBD) was added to a 384 well plate to which the arylethene derivative of the present invention was added to a final concentration of 7.3 nM. Then, a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 250 nM and 5 nM, respectively, and beta-estradiol as an agonist was added to a final concentration of 4 nM. All subsequent experiments was the same as the ERR gamma binding assay.
Measurement: IC50 = 413 nM (pChEMBL 6.380000114440918)
Descriptors: MW 533.74, ALogP 5.32, HBA 5, HBD 2, PSA 72.88, QED 0.35
Compound (SMILES): Cc1cccc(-c2cc(S(=O)(=O)c3ccc(CN)s3)ccc2S(C)(=O)=O)c1
InChIKey: IXHLNIHDDTYEDF-UHFFFAOYSA-N
Target: Arachidonate 12-lipoxygenase (SINGLE PROTEIN), Sus scrofa
UniProt: P16469
Assay: LOX Enzyme Activity Assay: LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls:0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix:Volume Reagent 1 x plate 2 x plate Final ConcnAssay Buffer  2 ml    3 ml 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM20 mM Amplex Red 10 μl   15 μl 100 μM1000 U/ml HRP 10 μl   15 μl   35 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader:Excitation wavelength: 545 nmEmission wavelength: 585 nmHRP Counter Assay Protocol
Measurement: IC50 = 1400 nM (pChEMBL 5.849999904632568)
Descriptors: MW 421.57, ALogP 3.42, HBA 6, HBD 1, PSA 94.3, QED 0.68
Compound (SMILES): CS(=O)(=O)c1cccc(S(=O)(=O)c2ccc(CN)s2)n1
InChIKey: IAYFKSQISJTPOA-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Monoamine Oxidase B (MAO-B) Selectivity Assay: MAO-Glo Assay Kit: Promega, Cat No. V1402, MAO-B Enzyme: Sigma, Cat No. M7441.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 332.43, ALogP 0.84, HBA 7, HBD 1, PSA 107.19, QED 0.89
Compound (SMILES): CC(C)(C)S(=O)(=O)c1cc2c(Nc3ccc4scnc4c3)ncnc2cc1OCCOP(=O)(O)O
InChIKey: MJLYDVMFNHZMLV-UHFFFAOYSA-N
Target: Receptor-interacting serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: O43353
Assay: Inhibition of RIPK2-mediated NF-kappaB signalling in MDP-treated human THP-1 cells preincubated for 30 mins followed by MDP addition measured after 24 hrs by luminescence based assay
Measurement: IC50 = 38 nM (pChEMBL 7.420000076293945)
Descriptors: MW 538.54, ALogP 4.04, HBA 10, HBD 3, PSA 160.83, QED 0.22
Compound (SMILES): CNC(=O)c1nnc(NC(=O)C2CC2)cc1Nc1cccc(-c2ncn(C)n2)c1OC
InChIKey: BZZKEPGENYLQSC-UHFFFAOYSA-N
Target: Non-receptor tyrosine-protein kinase TYK2 (SINGLE PROTEIN), Homo sapiens
UniProt: P29597
Assay: TYK2 Kinase Domain Enzymatic Activity Assay: A reaction buffer containing the following components: an enzyme buffer (1×), 5 mM MgCl, 1 mM DTT, 10 nM SEB (Cisbio, Cat. No. 61SEBALB), 0.625 mM EGTA and 0.01% Brij35 from the kit; a human recombinant TYK2 kinase (JH1) domain protein (Carna Biosciences, Cat. No. 08-147) diluted to a solution of 0.25 ng/μL with the reaction buffer; a substrate reaction solution containing 11.25 μM ATP and a biotinylated tyrosine kinase substrate diluted to 0.5 μM with the reaction buffer; a detection solution containing 0.1 ng/μL Eulabeled cage antibody (Cisbio, Cat. No. 61T66KLB) and 25 nM streptavidin-labeled XL665 (Cisbio, Cat. No. 610SAXLB) in the reaction buffer. The test compound is dissolved to 1 mM in DMSO, followed by a serial 4-fold dilution with DMSO to a minimum concentration of 61 nM. Each concentration is further diluted 40-fold with the reaction buffer. To a 384-well assay plate (Corning, Cat. No. 3674) are added 4 μL of compound solution and 2 μL of TYK2 kinase solution. The mixture is incubated at room temperature for 15 minutes, and then added with 4 μL of the substrate reaction solution. After further incubation at room temperature for 40 minutes, the reaction mixture is added with an equal volume of 10 μL detection solution and allowed to stand at room temperature for 30 minutes. An Envision plate reader (Perkin Elmer) is then used to measure the progress of the reaction at 620 nm and 665 nm. The ratio of absorbances at 665 nm and 620 nm is positively correlated with the degree of substrate phosphorylation, therefore the activity of TYK2 kinase is detected.
Measurement: IC50 = 2100 nM (pChEMBL 5.679999828338623)
Descriptors: MW 422.45, ALogP 1.73, HBA 9, HBD 3, PSA 135.95, QED 0.52
Compound (SMILES): NCc1ncc(S(=O)(=O)c2cc(C(=O)O)cc(-c3ccccc3)c2)s1
InChIKey: CCLJTQVYKROXIX-UHFFFAOYSA-N
Target: Protein-lysine 6-oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P28300
Assay: LOX Activity in Cysts Assay: Cell Culture and TransfectionAll cell lines used in this study was purchased from American Type Culture Collection (ATCC). Mycoplasma contamination was routinely monitored by PCR. Cells used were not found to be Mycoplasma positive. MDCK cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin Streptomycin solution (Pen Strep). For GFP constructs transfection in MDCK cells, lipofectamine 3000 was used according to manufactures protocols. Cells were either selected with G418 (Life Technologies) at 5 mg/ml. All cell culture reagents were purchased from Life Technologies.To produce MDCK cysts, cells were cultured on Matrigel (Corning) with 2% Matrigel supplemented in DMEM with 10% FBS. Cysts were allowed to form for 10 days before subsequent studies.Cloning of LOX Expression ConstructsMouse LOX cDNA was purchased from OriGene. Full length LOX cDNA was then PCR cloned into pEGFP-N1 (Clonetech), or biosensor vector proGFP2-N1 (Hanson, 2004) using the following primers, GAGAGAGCTAGCATGCGTTTCGCCTGGG (SEQ. ID NO. 1) (forward primer) and TCTCTCCTCGAGATACGGTGAAATTGTGCAGCC (SEQ. ID NO. 2) (reverse primer). For the insertion into pEGFP-N1 or proGFP2-N1, Nhel and Xhol restriction sites were added to forward and reverse primers accordingly. Mutant LOX constructs were made using QuickChange II site-directed mutagenesis kit (Agilent Technologies) following manufacture's protocol using LOX-GFP as template. To generate, roGFP2 versions of LOX mutant constructs, LOX mutant cDNA was transferred from pEGFP-N1 to proGFP2-N1 using Nhel and Xhol.Confocal Imaging and Imaging AnalysisAll photomicrographs were taken with a Leica TCS SP8 X confocal system. For LOX biosensor imaging, live MDCK cysts were used. The oxidised biosensor was excited using a 405 nm laser, while the reduced biosensor was excited with a 488 nm laser. Emission of the biosensor was recorded at 500 nm˜530 nm range using sequential scans. Ratio images were generated following a published protocol {Kardash, 2011 #376}. Note, while the published protocol generates YFP/CFP ratio images, we used it to generate Oxidised/Reduced (roGFP2 ratio) ratio images. The roGFP2 ratio at the basal surface of MDCK cysts was used to indicate LOX inhibition. LOX inhibitors were added 30min prior to imaging at 20 uM.
Measurement: IC50 = 700 nM (pChEMBL 6.159999847412109)
Descriptors: MW 374.44, ALogP 2.8, HBA 6, HBD 2, PSA 110.35, QED 0.71
Compound (SMILES): CCNCCS(=O)(=O)c1ccc(S(=O)(=O)c2ccc(CN)s2)cc1
InChIKey: YAPCQJOMLHRJLJ-UHFFFAOYSA-N
Target: Arachidonate 12-lipoxygenase (SINGLE PROTEIN), Sus scrofa
UniProt: P16469
Assay: LOX Enzyme Activity Assay: LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls:0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix:Volume Reagent 1 x plate 2 x plate Final ConcnAssay Buffer  2 ml    3 ml 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM20 mM Amplex Red 10 μl   15 μl 100 μM1000 U/ml HRP 10 μl   15 μl   35 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader:Excitation wavelength: 545 nmEmission wavelength: 585 nmHRP Counter Assay Protocol
Measurement: IC50 = 400 nM (pChEMBL 6.400000095367432)
Descriptors: MW 388.54, ALogP 1.42, HBA 7, HBD 2, PSA 106.33, QED 0.66
Compound (SMILES): CCN1CCN(c2ccc(/C(=C(/CCCO)c3ccccc3)c3ccc(C)cc3)cc2)CC1
InChIKey: YPWHCQNHYZKJHW-FLWNBWAVSA-N
Target: Estrogen-related receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P62508
Assay: Binding Assay: In an ERR alpha binding assay, GST-bound ERR alpha LBD was used, and all experiments other than that was the same as the ERR gamma binding assay.In an ERR beta binding assay, GST-bound ERR alpha LBD was used so that a final concentration was 10 nM and a fluorescein-conjugated coactivator PGC1a was 250 nM, and all experiments other than that was the same as the ERR gamma binding assay.In an ER alpha binding assay, a GST-bound ER alpha ligand-binding domain (LBD) was added to a 384 well plate to which the arylethene derivative of the present invention was added to a final concentration of 7.3 nM. Then, a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 250 nM and 5 nM, respectively, and beta-estradiol as an agonist was added to a final concentration of 4 nM. All subsequent experiments was the same as the ERR gamma binding assay.
Measurement: IC50 = 50 nM (pChEMBL 7.300000190734863)
Descriptors: MW 440.63, ALogP 5.87, HBA 3, HBD 1, PSA 26.71, QED 0.44
Compound (SMILES): NCc1ccc(S(=O)(=O)c2ccc(S(=O)(=O)CCO)cc2)s1
InChIKey: KJRFSOOHGATYEC-UHFFFAOYSA-N
Target: Arachidonate 12-lipoxygenase (SINGLE PROTEIN), Sus scrofa
UniProt: P16469
Assay: LOX Enzyme Activity Assay: LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls:0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix:Volume Reagent 1 x plate 2 x plate Final ConcnAssay Buffer  2 ml    3 ml 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM20 mM Amplex Red 10 μl   15 μl 100 μM1000 U/ml HRP 10 μl   15 μl   35 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader:Excitation wavelength: 545 nmEmission wavelength: 585 nmHRP Counter Assay Protocol
Measurement: IC50 = 300 nM (pChEMBL 6.519999980926514)
Descriptors: MW 361.47, ALogP 0.81, HBA 7, HBD 2, PSA 114.53, QED 0.79
Compound (SMILES): CN(C)C(=O)c1cc(-c2ccccc2)cc(S(=O)(=O)c2cnc(CN)s2)c1
InChIKey: DLDHQUYJALROQX-UHFFFAOYSA-N
Target: D-amino-acid oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P14920
Assay: Diamine Oxidase (DAO) Selectivity Assay: ReagentsPromega ROS-Glo H2O2 Assay Kit, 50 ml. Cat No. G8821Inhibitors (Test Compounds):10 mM stock diluted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0.0003 mM in drug plate, resulting in a concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 uM in the assay.Positive Control (Aminoguanidine):1 mM stock diluted to 1 to 0.0001 and 0.00003 mM in drug plate, resulting in a concentration of 3 to 0.001 and 0.0003 uM in the assay.DAO:DAO batch is obtained from Sigma Enzyme is reconstituted in sodium phosphate buffer at 10 mg/mLAssay Plates: 96 well white polystyrene, flat bottom plates: Fisher, Cat No. DPS-134-050AProcedure:1. Dilute 400 ul DAO enzyme in 4 mL Assay Buffer (enough for one plate). Add 40 ul DAO enzyme to all wells (excluding G+H 12, assay buffer only)3. Add 0.5 μl test compound serial dilutions, in duplicate4. Add 0.5 μl serial dilutions of Aminoguanidine down column 11 and to E+F 125. Add 0.5 μl DMSO (100% activity control) to C+D 126. Cover plate and incubate for 20 minutes at room temp on a plate shaker7. Prepare Start Mix: Volume Reagent 1 × plate 2 × plate Final ConcnAssay Buffer 2 ml 3 ml —8.5M Cadaverine 23 μl 34.5 μl 97.8 mMH2O2 Substrate (1:80) 25 μl 37.5 μl 125 μM8. Add 10 μl Start Mix to all wells9. Incubate for 60 min at room temp on a plate shaker10. Prepare Detection Reagent: Volume Reagent 1 × plate 2 × plate Luciferin 5 ml 10 ml D-cysteine (1:100) 50 μl 100 μl Signal Enhancer (1:100) 50 μl 100 μl11. Add 50 μl Detection Regent to each well EXCEPT No Luciferin controls (A+B 12, add 50 μl assay buffer)12. Incubate for 20 min at room temp on a plate shaker, protected from light13. Measure luminescence using a plate reader (integration 500 ms)Values for the blank (no DAO controls) are subtracted from all samples. DMSO controls are set as 100% activity and samples are calculated as a % of the DMSO control. Data is plotted using Graphpad Prism software, and a non-linear regression line is calculated using a variable slope sigmoidal dose response equation.:Y=Bottom+(Top-Bottom)/(1+10{circumflex over ( )}((LogIC50−X)*HillSlope)).Where X is the logarithm of concentration and Y is the response. The IC50 is the concentration of the drug that produces a percentage control fluorescence value midway between the saturation and zero effect plateaus. Two independent assays are usually performed, and the mean IC50 is reported.
Measurement: IC50 > 100000 nM (pChEMBL 4)
Descriptors: MW 401.51, ALogP 2.8, HBA 6, HBD 1, PSA 93.36, QED 0.71
Compound (SMILES): CC(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)NCCCCC1CCN(CC(=O)N2c3ccccc3NC(=O)c3ccccc32)CC1
InChIKey: XKYMIJTXUGXDLR-IKYRTNQJSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Displacement of [3H]-NMS from human muscarinic M4 receptor expressed in CHO cell membranes assessed as inhibition constant by radioligand competition analysis
Measurement: Ki = 39.81 nM (pChEMBL 7.400000095367432)
Descriptors: MW 869.04, ALogP 5.08, HBA 8, HBD 7, PSA 205.07, QED 0.06
Compound (SMILES): CC(C)N1CCN(c2ccc(/C(=C(/CCCO)c3ccccc3)c3ccc(O)cc3)cc2)CC1
InChIKey: RUUIHSHMHGOGMZ-QVIHXGFCSA-N
Target: Estrogen-related receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P62508
Assay: Binding Assay: ERRγ: The arylethene derivative of the present invention was sequentially added to a 384 well plate from a concentration of 10 μM to a final concentration of two-fold dilution. Then, a GST-bound ERR gamma ligand-binding domain (LBD) was added to a final concentration of 5 nM, and a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 500 nM and 5 nM, respectively. After all reagents were added, a reaction was carried out with gentle shaking at 20° C. for 1 hour, and after the reaction, a binding activity was measured by a TR-FRET method. That is, excitation at 340 nm was performed, each emission value at 495 nm and 520 nm was measured, the result assay was a value measured at 490 nm/a value measured at 520 nm, and an analysis program was Prism 6.
Measurement: IC50 = 60 nM (pChEMBL 7.21999979019165)
Descriptors: MW 456.63, ALogP 5.65, HBA 4, HBD 2, PSA 46.94, QED 0.43
Compound (SMILES): Cc1cnc(Cc2nc3cnc4ccc(Cl)cc4c3n2[C@@H]2CCO[C@H](C)C2)cn1
InChIKey: YYSUHJMDOKKANR-RHSMWYFYSA-N
Target: Leucine-rich repeat serine/threonine-protein kinase 2 (SINGLE PROTEIN), Homo sapiens
UniProt: Q5S007
Assay: LRRK2 Assay: LRRK2 kinase activity was measured using Lantha Screen technology from Invitrogen. GST-tagged truncated LRRK2 from Invitrogen (Cat #PV4874) was incubated with a fluorescein-labeled peptide substrate based upon ezrin/radixin/moesin (ERM), also known as LRRKtide (Invitrogen cat #PR8976A), in the presence of a dose response of compound. Upon completion, the assay was stopped and detected with a terbium labeled anti-phospho-ERM antibody (Invitrogen, cat #PR8975A). The assay was carried out under the following protocol: The compound dose response was prepared by diluting compound to a top concentration of 0.3 mM in 100% DMSO and serial diluted by half-log in DMSO to give an 11 point curve, 100× final assay concentration. Using Echo acoustic dispensing, 60 nL of compound was transferred to a low volume Corning 384-well assay plate. 3 μL of a working solution of substrate (200 nM LRRKtide, 2 mM ATP) prepared in assay buffer (50 mM HEPES, pH 7.5, 3 mM MgCl2, with 2 mM DTT and 0.01% Brij35 added fresh) was added to the 60 nL compound assay plate. The kinase reaction was started with 3 μL of a working solution of LRRK2 enzyme at a concentration of 4 μg/m L. The final reaction concentrations were 100 nM LRRKtide, 1 mM ATP, 2 μg/mL LRRK2 enzyme and a compound dose response with a top dose of 3 μM. The reaction was allowed to progress at room temperature for 30 minutes and then stopped with the addition of 6 μL of detection buffer (20 mM Tris pH 7.6, 0.01% NP-40, 6 mM EDTA with 2 nM terbium labeled antiphospho-ERM). After an incubation of 1 hour at room temperature, the plate was read on an Envision with an excitation wavelength of 340 nm and a reading emission at both 520 nm and 495 nm. The ratio of the 520 nm and 495 nm emission was used to analyze the data. Inhibition of mutant G2019S LRRK2 (Invitrogen cat #PV4881) was measured in the exact same method. All final concentrations of substrate ATP and enzyme were the same.
Measurement: IC50 = 2.16 nM (pChEMBL 8.670000076293945)
Descriptors: MW 407.91, ALogP 4.67, HBA 6, HBD 0, PSA 65.72, QED 0.49
Compound (SMILES): Cc1cc(C)c(CNC(=O)c2cc(Cl)c3c(c2C)O[C@@](C)([C@H]2CC[C@H](N(C)C)CC2)O3)c(=O)[nH]1
InChIKey: SSDRNUPMYCFXGM-UYPAYLBCSA-N
Target: Histone-lysine N-methyltransferase EZH1 (SINGLE PROTEIN), Homo sapiens
UniProt: Q92800
Assay: Inhibition of wildtype EZH1 (unknown origin)
Measurement: IC50 = 23 nM (pChEMBL 7.639999866485596)
Descriptors: MW 488.03, ALogP 4.49, HBA 5, HBD 2, PSA 83.66, QED 0.65
Compound (SMILES): CC(C)(C)CNc1nccc(N2CCc3nc[nH]c3C2)n1
InChIKey: GYJOUMCDNZCUAE-UHFFFAOYSA-N
Target: Histamine H4 receptor (SINGLE PROTEIN), Mus musculus
UniProt: Q91ZY2
Assay: Inhibition of UR-DEBa242 binding to mouse recombinant NLuc/GPCR-fused H4R expressed in HEK293T cells measured after 30 mins by furimazine substrate based BRET assay
Measurement: Ki = 1.23 nM (pChEMBL 8.90999984741211)
Descriptors: MW 286.38, ALogP 2.22, HBA 5, HBD 2, PSA 69.73, QED 0.91
Compound (SMILES): NCc1ccc(S(=O)(=O)c2ccc(C(=O)NCCO)cc2)s1
InChIKey: AUZDMUSWBKKNHM-UHFFFAOYSA-N
Target: Protein-lysine 6-oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P28300
Assay: LOX Enzyme Activity Assay: The following reagents were prepared:Reagents500 mM CHES, pH 9 (with NaOH), filtered (MW=207.29)20.7 g in 200 ml in dH2O10% Pluronic F-127 (store at 4° C.)1 g in 10 ml in dH2O10 % BSA (store at 4° C.)−2.5 g in 25 ml dH2O1 M MgCl2.6H2O (MW=203)5.1 g in 25 ml dH2O5 M Urea (MW=60.06)60 g in 200 ml dH2O5 M NaCl (MW=58.44)14.6 g in 50 ml dH2O8.5 M Cadaverine Dihydrochloride (MW=175.10): Sigma, Cat No: C85611.49 g in 1 ml dH2O10 mM Na3VO4, pH 10 (with HCl), boil to activate (MW=183.91): Sigma, Cat No: S65080.1839 g in 10 ml dH2O20 mM Amplex Red (MW=257.25): Invitrogen, Cat No: A12222.5 mg in 1 ml DMSO (aliquot into 40 μl and store at −20° C.)1000 U/ml HRP in dH2O (store at 4° C.): Sigma, Cat No: P2088H2O2 (store at 4° C.): Sigma, Cat No: H1009DMSOLOX Assay Buffer: Volume (μl) Reagent 1 × plate Final Concn 500 mM CHES, pH 9 5000 100 mM 10% Pluronic F-127 125 0.05% 10% BSA 1250 0.5% 1M MgCl2 25  1 mM 5M Urea 5000 1M 5M NaCl 500 100 mM dH2O 13100 — Total Volume 25 000 —Inhibitors (Test Compounds):10 mM stock diluted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0.0003 mM in drug plate, resulting in a concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 uM in the assay.LOX enzyme:LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (˜4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls: 0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix: Volume Reagent 1 × plate 2 × plate Final Concn Assay Buffer 2 ml 3 ml — 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM 20 mM Amplex Red 10 μl 15 μl 100 μM 1000 U/ml HRP 10 μl 15 μl 5 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader: Excitation wavelength: 545 nm Emission wavelength: 585 nm HRP Counter Assay Protocol1. Dilute 5 μl H2O2 in 640 μl dH2O. Add 1 μl diluted H2O2 to 10 ml Assay Buffer. Vortex2. Add 40 μl H2O2+Assay Buffer into every well on the counter assay plate EXCEPT no H2O2 control wells (add 40 μl Assay Buffer only)3. Add 0.5 μl test compound serial dilutions, in duplicate4. Add 0.5 μl serial dilutions of positive control, Na3VO4, down column 11 (no duplication)5. Add following controls: 0.5 μl DMSO (100% activity control) 1 μl diluted H2O2 (blow out control)6. Incubate for 20 min at room temp on a plate shaker.
Measurement: IC50 = 2700 nM (pChEMBL 5.570000171661377)
Descriptors: MW 340.43, ALogP 0.76, HBA 6, HBD 3, PSA 109.49, QED 0.72
Compound (SMILES): CN1CCN(c2ccc(/C(=C(/CCCO)c3ccccc3)c3ccc(Br)cc3)cc2)CC1
InChIKey: RVPDDVIQFCWUAC-DQSJHHFOSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Binding Assay: In an ERR alpha binding assay, GST-bound ERR alpha LBD was used, and all experiments other than that was the same as the ERR gamma binding assay.In an ERR beta binding assay, GST-bound ERR alpha LBD was used so that a final concentration was 10 nM and a fluorescein-conjugated coactivator PGC1a was 250 nM, and all experiments other than that was the same as the ERR gamma binding assay.In an ER alpha binding assay, a GST-bound ER alpha ligand-binding domain (LBD) was added to a 384 well plate to which the arylethene derivative of the present invention was added to a final concentration of 7.3 nM. Then, a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 250 nM and 5 nM, respectively, and beta-estradiol as an agonist was added to a final concentration of 4 nM. All subsequent experiments was the same as the ERR gamma binding assay.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 491.47, ALogP 5.93, HBA 3, HBD 1, PSA 26.71, QED 0.42
Compound (SMILES): CC(C)N1CCN(c2ccc(/C(=C(/CCCO)c3ccccc3)c3ccc(Br)cc3)cc2)CC1
InChIKey: SEPBORHBTVHWRY-FLWNBWAVSA-N
Target: Estrogen-related receptor gamma (SINGLE PROTEIN), Homo sapiens
UniProt: P62508
Assay: Binding Assay: ERRγ: The arylethene derivative of the present invention was sequentially added to a 384 well plate from a concentration of 10 μM to a final concentration of two-fold dilution. Then, a GST-bound ERR gamma ligand-binding domain (LBD) was added to a final concentration of 5 nM, and a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 500 nM and 5 nM, respectively. After all reagents were added, a reaction was carried out with gentle shaking at 20° C. for 1 hour, and after the reaction, a binding activity was measured by a TR-FRET method. That is, excitation at 340 nm was performed, each emission value at 495 nm and 520 nm was measured, the result assay was a value measured at 490 nm/a value measured at 520 nm, and an analysis program was Prism 6.
Measurement: IC50 = 32 nM (pChEMBL 7.5)
Descriptors: MW 519.53, ALogP 6.71, HBA 3, HBD 1, PSA 26.71, QED 0.34
Compound (SMILES): CS(=O)(=O)c1ccc(S(=O)(=O)c2cnc(CN)s2)cc1
InChIKey: MRLYBPQXYAIYJO-UHFFFAOYSA-N
Target: Protein-lysine 6-oxidase (SINGLE PROTEIN), Homo sapiens
UniProt: P28300
Assay: LOX Enzyme Activity Assay: The following reagents were prepared:Reagents500 mM CHES, pH 9 (with NaOH), filtered (MW=207.29)20.7 g in 200 ml in dH2O10% Pluronic F-127 (store at 4° C.)1 g in 10 ml in dH2O10 % BSA (store at 4° C.)−2.5 g in 25 ml dH2O1 M MgCl2.6H2O (MW=203)5.1 g in 25 ml dH2O5 M Urea (MW=60.06)60 g in 200 ml dH2O5 M NaCl (MW=58.44)14.6 g in 50 ml dH2O8.5 M Cadaverine Dihydrochloride (MW=175.10): Sigma, Cat No: C85611.49 g in 1 ml dH2O10 mM Na3VO4, pH 10 (with HCl), boil to activate (MW=183.91): Sigma, Cat No: S65080.1839 g in 10 ml dH2O20 mM Amplex Red (MW=257.25): Invitrogen, Cat No: A12222.5 mg in 1 ml DMSO (aliquot into 40 μl and store at −20° C.)1000 U/ml HRP in dH2O (store at 4° C.): Sigma, Cat No: P2088H2O2 (store at 4° C.): Sigma, Cat No: H1009DMSOLOX Assay Buffer: Volume (μl) Reagent 1 × plate Final Concn 500 mM CHES, pH 9 5000 100 mM 10% Pluronic F-127 125 0.05% 10% BSA 1250 0.5% 1M MgCl2 25  1 mM 5M Urea 5000 1M 5M NaCl 500 100 mM dH2O 13100 — Total Volume 25 000 —Inhibitors (Test Compounds):10 mM stock diluted to 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.003, 0.001, and 0.0003 mM in drug plate, resulting in a concentration of 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0.003 uM in the assay.LOX enzyme:LOX enzyme was obtained from pig skin by the method of Shackleton et al 1990.Assay Plates:Black, flat bottom 96 well platesProcedure:1. Dilute LOX enzyme in Assay Buffer (˜4 ml for one plate, 8 ml for two plates) (dilution dependent on batch activity)2. Add 0.5 μl test compound serial dilutions, in duplicate3. Add 0.5 μl serial dilutions of positive control, BAPN, down column 11 (no duplication)4. Add following controls: 0.5 μl DMSO (100% activity control)5. Cover plate and incubate for 20 min at room temperature on a plate shaker6. Prepare Start Mix: Volume Reagent 1 × plate 2 × plate Final Concn Assay Buffer 2 ml 3 ml — 8.5M Cadaverine 23 μl 34.5 μl 97.8 mM 20 mM Amplex Red 10 μl 15 μl 100 μM 1000 U/ml HRP 10 μl 15 μl 5 U/ml7. Add 10 μl Start Mix to No HRP control wells8. Add HRP to the Start Mix. Add 10 ul to all wells EXCEPT No HRP control wells9. Incubate for 45 min at room temp on a plate shaker, protected from light10. Measure fluorescence using a plate reader: Excitation wavelength: 545 nm Emission wavelength: 585 nm HRP Counter Assay Protocol1. Dilute 5 μl H2O2 in 640 μl dH2O. Add 1 μl diluted H2O2 to 10 ml Assay Buffer. Vortex2. Add 40 μl H2O2+Assay Buffer into every well on the counter assay plate EXCEPT no H2O2 control wells (add 40 μl Assay Buffer only)3. Add 0.5 μl test compound serial dilutions, in duplicate4. Add 0.5 μl serial dilutions of positive control, Na3VO4, down column 11 (no duplication)5. Add following controls: 0.5 μl DMSO (100% activity control) 1 μl diluted H2O2 (blow out control)6. Incubate for 20 min at room temp on a plate shaker.
Measurement: IC50 = 4800 nM (pChEMBL 5.320000171661377)
Descriptors: MW 332.43, ALogP 0.84, HBA 7, HBD 1, PSA 107.19, QED 0.89
Compound (SMILES): Cc1ccc(-c2cccc(S(=O)(=O)c3cnc(CN)s3)c2)cc1
InChIKey: ACWFOKNSHMVIEB-UHFFFAOYSA-N
Target: Amine oxidase [flavin-containing] B (SINGLE PROTEIN), Homo sapiens
UniProt: P27338
Assay: Monoamine Oxidase B (MAO-B) Selectivity Assay: MAO-Glo Assay Kit: Promega, Cat No. V1402, MAO-B Enzyme: Sigma, Cat No. M7441.
Measurement: IC50 = 17000 nM (pChEMBL 4.769999980926514)
Descriptors: MW 344.46, ALogP 3.41, HBA 5, HBD 1, PSA 73.05, QED 0.79
Compound (SMILES): CC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(=N)N)C(=O)NCCN1CCN(CCCCC2CCN(CC(=O)N3c4ccccc4NC(=O)c4ccccc43)CC2)CC1
InChIKey: ABRYVCKEQWCHSC-UEQQSRGXSA-N
Target: Muscarinic acetylcholine receptor M4 (SINGLE PROTEIN), Homo sapiens
UniProt: P08173
Assay: Displacement of [3H]-NMS from human muscarinic M4 receptor expressed in CHO cell membranes assessed as inhibition constant by radioligand competition analysis
Measurement: Ki = 1.995 nM (pChEMBL 8.699999809265137)
Descriptors: MW 916.19, ALogP 1.42, HBA 11, HBD 9, PSA 263.45, QED 0.04
Compound (SMILES): Cc1ccc(/C(=C(\CCCO)c2ccccc2)c2ccc(N3CCN(C)CC3)cc2)cc1
InChIKey: PZDWZAMMWRBRBP-ZIADKAODSA-N
Target: Estrogen receptor (SINGLE PROTEIN), Homo sapiens
UniProt: P03372
Assay: Binding Assay: In an ERR alpha binding assay, GST-bound ERR alpha LBD was used, and all experiments other than that was the same as the ERR gamma binding assay.In an ERR beta binding assay, GST-bound ERR alpha LBD was used so that a final concentration was 10 nM and a fluorescein-conjugated coactivator PGC1a was 250 nM, and all experiments other than that was the same as the ERR gamma binding assay.In an ER alpha binding assay, a GST-bound ER alpha ligand-binding domain (LBD) was added to a 384 well plate to which the arylethene derivative of the present invention was added to a final concentration of 7.3 nM. Then, a fluorescein-conjugated coactivator PGC1a and a Tb-a-GST antibody were added to 250 nM and 5 nM, respectively, and beta-estradiol as an agonist was added to a final concentration of 4 nM. All subsequent experiments was the same as the ERR gamma binding assay.
Measurement: IC50 > 10000 nM (pChEMBL 5)
Descriptors: MW 426.6, ALogP 5.48, HBA 3, HBD 1, PSA 26.71, QED 0.51
Compound (SMILES): Cc1ccc2c(N)c(C(=O)NCCc3ccc(N4CC[C@H]5NCC[C@H]54)cc3)sc2n1
InChIKey: IDEISMLLPVGWLL-RTBURBONSA-N
Target: Ubiquitin carboxyl-terminal hydrolase 25 (SINGLE PROTEIN), Homo sapiens
UniProt: Q9UHP3
Assay: Biochemical Assay: USP25: The assay was performed in a final volume of 9 uL in assay buffer containing 20 mM Tris-HCl (pH 8.0, (1M Tris-HC1, pH 8.0 solution; Corning 46-031-CM)), 3 mM BME (2-Mercaptoethanol; Sigma 63689-25ML-F), 0.03% BGG (0.22 uM filtered, Sigma, G7516-25G), and 0.01% Triton X-100 (Sigma, T9284-10L). Nanoliter quantities of 10-point, 3-fold serial dilution in DMSO was pre-dispensed into 1536 assay plates (Corning, #3724BC) for a final test concentration of 25 uM to 1.3 nM, top to lowest dose, respectively. Enzyme USP25, construct USP25-His6, (Boston Biochem E-546). Concentration and incubation times were optimized for the maximal signal-to-background while maintaining initial velocity conditions at a fixed substrate concentration. The final concentration of the enzyme in the assay was 75 pM. Final substrate (Ub -Rh110; Ubiquitin-Rhodamine 110, R&D Systems #U-555) concentration was 25 nM with [Ub -Rh110]Â  Km. 3 uL of 2x enzyme was added to assay plates (pre-stamped with compound) preincubated with USP25 for 30 minutes and then 3 uL of 2x Ub-Rh110 was added to assay plates. Plates were incubated for 45 minutes at room temperature before addition of 3 uL of stop solution (final concentration of 10 mM citric acid (Sigma, 251275-500G)). Fluorescence was read on the Envision (Excitation at 485 nm and Emission at 535 nm; Perkin Elmer) or on the PheraSTAR (Excitation at 485 nm and Emission at 535 nm; BMG Labtech).
Measurement: IC50 = 1100 nM (pChEMBL 5.960000038146973)
Descriptors: MW 421.57, ALogP 3.1, HBA 6, HBD 3, PSA 83.28, QED 0.59