BDBM50172756 N-benzyl-N-methylprop-2-yn-1-amine Eutron PARGYLINE US9603833, Pargyline CHEMBL673 Eutonyl Benzyl-methyl-prop-2-ynyl-amine
- Yang, HL; Cai, P; Liu, QH; Yang, XL; Li, F; Wang, J; Wu, JJ; Wang, XB; Kong, LY Design, synthesis and evaluation of coumarin-pargyline hybrids as novel dual inhibitors of monoamine oxidases and amyloid-? aggregation for the treatment of Alzheimer's disease. Eur J Med Chem 138: 715-728 (2017)
- ChEMBL_2105008 Inhibition of human recombinant MAO-A using pargyline as incubated for 20 mins measuring increase in emission signal at 310 nm multimode plate reader assay
- Receptor Binding Assay Isotope ligand 3H-8-OH-DPAT (67.0 Ci/mmol) was purchased from PerkinElmer Company; 5-HT was purchased from RBI Company; GF/C glass fiber filter paper was purchased from Whatman Company; Tris was imported and divided into aliquots; PPO, POPOP were purchased from Shanghai No. 1 Reagent Factory; lipid-soluble scintillation solution. Beckman LS-6500 Multi-function Liquid Scintillation Counter was used.Procedures(1) The prepared membrane was first applied with appropriate amount of buffer (0.05 M Tris-HCl buffer, containing 0.1% ascorbic acid, 10 um pargyline and 4 mM CaCl2), and homogenizer was used for evenly dispersing. 15 tubes were mixed into a 100 ml container, and appropriate amount of homogenized liquid was added to give 50 ml of membrane suspension, which was reserved for future use.(2) 100 uL of membrane preparation and 100 uL of buffer (0.05 M Tris-HCl buffer, containing 0.1% ascorbic acid, 10 um pargyline and 4 mM CaCl2) were added into each reaction tube.
- Radioligand Binding Assays The composition of the assay buffers was as follows: for 5-HT1AR: 50 mM Tris¿HCl, 0.1 mM EDTA, 4 mM MgCl2, 10 lM pargyline, and 0.1% ascorbate; for 5-HT6R: 50 mM Tris¿HCl, 0.5 mM EDTA, and 4 mM MgCl2; and for 5-HT7bR: 50 mM Tris¿HCl, 4 mM MgCl2, 10 lM pargyline, and 0.1% ascorbate. All assays were incubated in a total volume of 200 µL in 96-well microtiter plates for 1 h at 37 °C, except for 5-HT1AR which were incubated at room temperature for 1 h. The process of equilibration is terminated by rapid filtration through Unifilter plates with a 96-well cell harvester, and radioactivity retained on the filters was quantified on a Microbeta plate reader. For displacement studies, the assay samples contained as radioligands: 1.5 nM [3H]8-OH-DPAT (187 Ci/mM) for 5-HT1AR; 2 nM[3H]LSD (85.2 Ci/mM for 5-HT6R or 0.6 nM [3H]5-CT (39.2 Ci/mM) for 5-HT7R. Non-specific binding was defined with 10 µM of 5-HT in 5-HT1AR and 5-HT7R binding experiments, whereas 10 µM methiothepin was used in 5-HT6R assa
- Inhibition Assay A stock solution was prepared using a human MAO-B enzyme (purchased from Aldrich) and a Amplex Red monoamine oxidase assay kit according to a preparation manual. The kit includes a 5x reaction buffer, an Amplex red reagent (1 mg), HRP, DMSO, H2O2, p-tyramine (substrate of MAO-A, B), benzylamine (substrate of MAO-B), clorgiline (inhibitor of MAO-A), and pargyline (inhibitor of MAO-B). Among these reagents in the kit, benzylamine was used as a substrate for MAO-B, and pargyline was used as an MAO-B inhibitor. A solution as overall substrates was prepared as follows. 200 ul of a solution of 1 mg of Amplex red sufficiently dissolved in 200 ul of DMSO, 100 ul of a mixed solution of HRP and 1 ml of a 1x buffer, 200 ul of a solution of benzylamine dissolved in 1.2 ml of dH2O were added to 9.5 ml of a 1x buffer to reach a total volume of 10 mL, which is sufficient for 100 wells. 0.5 ul of a mixture of MAO-B inhibitor pargyline and 1 ml of dH2O was put into each well. First, the activity of MAO-B was determined using 10 uM of the synthesized compound. 96 wells were injected with positive and negative types, and the wile type. The positive type included only substrate and hydrogen peroxide, and the negative type included only substrate. For the wild type, corresponding wells were injected with the enzyme, substrate, and MAO-B inhibitor, but with no synthesized compound. Afterward, 2 ul of the synthesized compound (1 mM) was added into each well, and the human MAO-B enzyme was put only into the 1st row of wells. 0.5 ug of the human MAO-B was put into each well along with 100 ul of a 1x buffer. The human MAO-B enzyme was put into the 2nd row of the wells along with 0.5 ul of a pargyline, the MAO-B inhibitor. To reduce an experimental error for accuracy, the test was repeated three times for each compound. After 30 minutes, 100 ul of the substrate solution was added into each well in a darkroom. The test was performed in the darkroom due to light sensitivity of the Amplex reagent. Finally, a total volume of the reaction solution per well reached 200 ul. After about 2 to 3 hours, chromophoric degrees of the samples were measured. A variation in data values for the 1st and 2nd rows of the wells indicates the pure reaction activity of the MAO-B enzyme with the substrate. Using the samples with the synthesized compound the remaining activity of MAO-B after inhibited by the synthesized compound may be determined. This is because the activities of the other enzymes excluding the MAO-B enzyme may be excluded through this method. Compounds with high inhibitory activity at a concentration of 10 uM were screened from among the synthesized compounds at a compound concentration of 10 uM. Afterward, concentration-dependent IC50 values of these compounds may be obtained through an activity assay at different concentrations of 0.001 uM, 0.01 uM, 0.1 uM, 1 uM, and 10 uM.
- MAO Assay The reaction mixture (final volume 200 µL) contained 5 µg/mL MAO-A or MAO-B in a 50 mM sodium phosphate buffer pH 7.4, 1 mM p-tyramine (substrate for MAO-A) or 1 mM benzyl-amine (substrate for MAO-B), 1 U/mL HRP, and 200 µM AR was incubated, in the presence or absence of the inhibitors, at 37 °C for 45 min. The enzyme plus inhibitors were incubated at 37 °C for 20 min followed by the addition of the substrates, HRP and AR. The inhibitors were dissolved in 100% DMSO, and equivalent concentrations of DMSO alone (1-2%) were used as controls. Clorgyline (MAO-A inhibitor) or pargyline (MAO-B inhibitor) at 5 µM concentration was used as positive control for inhibition.
- Radioligand Binding Assay Radioligand binding assays for human 5-HT2A receptor was conducted using the 5-HT2 agonist [125I]DOI as radioligand. To define nonspecific binding, 10 μM DOI was used for all assays. For competitive binding studies, 0.5 nM [125I]DOI was used and compounds were assayed over a range of 0.01 nM to 10 μM. Assays were conducted in a total volume of 200 μl in 96-well Perkin Elmer GF/C filter plates in assay buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM MgCl2, and 10 μM pargyline). Assay incubations were performed for 60 mM at room temperature and were terminated by rapid filtration under vacuum pressure of the reaction mixture over Whatman GF/C glass fiber filters presoaked in 0.5% PEI using a Brandell cell harvester.
- Radioligand Binding Assays Radioligand binding assays for human 5-HT2A receptor was conducted using the 5-HT2 agonist [125]DOI as radioligand. To define nonspecific binding, 10 μM DOI was used for all assays. For competitive binding studies, 0.5 nM [125]DOI was used and compounds were assayed over a range of 0.01 nM to 10 μM. Assays were conducted in a total volume of 200 μl in 96-well Perkin Elmer GF/C filter plates in assay buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM MgCl2, and 10 μM pargyline). Assay incubations were performed for 60 min at room temperature and were terminated by rapid filtration under vacuum pressure of the reaction mixture over Whatman GF/C glass fiber filters presoaked in 0.5% PEI using a Brandell cell harvestor.
- Scintillation Proximity Assay [125I]-(±)DOI binding experiments are carried out in SPA 96-well format. Membranes used in this assay are prepared from AV-12 cells stably expressing recombinant 5-HT2C receptor (human). The incubation is initiated by the addition of a mixture of WGA PVT SPA beads (0.5 mg/well, Perkin Elmer (MA, USA), RPNQ0001) and 2.5 μg membranes to assay buffer (50 mM Tris-HCl, 10 mM MgCl2, 0.5 mM EDTA, 10 μM pargyline, 0.1% ascorbic acid, pH7.4) containing 0.2 nM [[125I]-(±)DOI and varying concentrations of the test compound (10 point concentration response curves). Non-specific binding is determined in the presence of 20 μM 1-(1-Naphthyl) piperazine. Samples are incubated for 4 hr. at room temperature (22° C.) and then read in a Microbeta Trilux.
- Radioligand Binding Assay Radioligand binding assays for human 5-HT2A receptor was conducted using the 5-HT2 agonist [125I]DOI as radioligand. To define nonspecific binding, 10 μM DOI was used for all assays. For competitive binding studies, 0.5 nM [125I]DOI was used and compounds were assayed over a range of 0.01 nM to 10 μM. Assays were conducted in a total volume of 200 μl in 96-well Perkin Elmer GF/C filter plates in assay buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM MgCl2, and 10 μM pargyline). Assay incubations were performed for 60 min at room temperature and were terminated by rapid filtration under vacuum pressure of the reaction mixture over Whatman GF/C glass fiber filters presoaked in 0.5% PEI using a Brandell cell harvestor. Filters were then washing several times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4). Plates were then dried at room temperature and counted in a Wallac microBeta scintillation counter.
- [125I]DOI Radioligand Binding Assay Radioligand binding assays for human 5-HT2A receptor was conducted using the 5-HT2 agonist [125I]DOI as radioligand. To define nonspecific binding, 10 μM DOI was used for all assays. For competitive binding studies, 0.5 nM [125I]DOI was used and compounds were assayed over a range of 0.01 nM to 10 μM. Assays were conducted in a total volume of 200 μl in 96-well Perkin Elmer GF/C filter plates in assay buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM MgCl2, and 10 μM pargyline). Assay incubations were performed for 60 min at room temperature and were terminated by rapid filtration under vacuum pressure of the reaction mixture over Whatman GF/C glass fiber filters presoaked in 0.5% PEI using a Brandell cell harvestor. Filters were then washing several times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4). Plates were then dried at room temperature and counted in a Wallac microBeta scintillation counter.
- [125I]DOI Radioligand Binding Assay Radioligand binding assays for human 5-HT2A serotonin receptor were conducted using the 5-HT2 agonist [125I]DOI as radioligand. To define nonspecific binding, 10 μM DOI was used for all assays. For competitive binding studies, 0.5 nM [125I]DOI was used and compounds were assayed over a range of 0.01 nM to 10 μM. Assays were conducted in a total volume of 200 μl in 96-well Perkin Elmer GF/C filter plates in assay buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM MgCl2 and 10 μM pargyline). Assay incubations were performed for 60 min at room temperature and were terminated by rapid filtration under reduced pressure of the reaction mixture over Whatman GF/C glass fiber filters presoaked in 0.5% PEI using a Brandell cell harvester. Filters were then washed several times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4). Plates were then dried at room temperature and counted in a Wallac MicroBeta scintillation counter.
- [125I]DOI Radioligand Binding Assay Radioligand binding assays for human 5-HT2A serotonin receptor were conducted using the 5-HT2 agonist [125I]DOI as radioligand. To define nonspecific binding, 10 μM DOI was used for all assays. For competitive binding studies, 0.5 nM [125I]DOI was used and compounds were assayed over a range of 0.01 nM to 10 μM. Assays were conducted in a total volume of 200 μl in 96-well Perkin Elmer GF/C filter plates in assay buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM MgCl2 and 10 μM pargyline). Assay incubations were performed for 60 min at room temperature and were terminated by rapid filtration under reduced pressure of the reaction mixture over Whatman GF/C glass fiber filters presoaked in 0.5% PEI using a Brandell cell harvester. Filters were then washed several times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4). Plates were then dried at room temperature and counted in a Wallac MicroBeta scintillation counter.
- [125I]DOI Radioligand Binding Assay. Radioligand binding assays for human 5-HT2A serotonin receptor was conducted using the 5-HT2 agonist [125I]DOI as radioligand. To define nonspecific binding, 10 μM DOI was used for all assays. For competitive binding studies, 0.5 nM [125I]DOI was used and compounds were assayed over a range of 0.01 nM to 10 μM. Assays were conducted in a total volume of 200 μl in 96-well Perkin Elmer GF/C filter plates in assay buffer (50 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 5 mM MgCl2, and 10 μM pargyline). Assay incubations were performed for 60 min at room temperature and were terminated by rapid filtration under vacuum pressure of the reaction mixture over Whatman GF/C glass fiber filters presoaked in 0.5% PEI using a Brandell cell harvester. Filters were then washed several times with ice-cold wash buffer (50 mM Tris-HCl, pH 7.4). Plates were then dried at room temperature and counted in a Wallac MicroBeta scintillation counter.
- Biological Assay The binding affinity of the compound of this invention to human 5-HT6 receptor expressed in CHO cell was evaluated by radioligand binding assay as follows.32 μg membrane proteins of CHO cell expressing human 5-HT6 receptor, 2 nM of radioactive marker [3H]LSD, the compound of the present invention having different test concentrations and a buffer solution were mixed uniformly, and the resulting mixture was incubated at 37° C. for 120 mins, in which the buffer solution was comprised of 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.5 mM EDTA, 10 μM pargyline and 20 mg/L protease inhibitor.100 μM of 5-HT was added to the mixture to eliminate nonspecific binding sites. After incubation, the resulting mixture was filtered with glassfabricfilter in vacuo, and the glassfabricfilter should be preimpregnated with 0.3% PEI before filtering and washed with 50 mM of Tris-HCl several times after filtering. After the filter was dried, the radioactivity of the scintillation mixture was determined by liquid scintillation counting with a scintillometer. The reference standard was 5-HT, and IC50 values were calculated from plots of competitive inhibition curves formed based on several inhibition ratios and the corresponding compound concentrations.
- Radioligand Binding Assay 32 μg membrane proteins of CHO cell expressing human 5-HT6 receptor, 2 nM of radioactive marker [3H]LSD, a compound of the present invention having different test concentrations, 100 μM 5-HT (5-HT was used to eliminate nonspecific binding sites) and a buffer solution were mixed uniformly. Then the resulting mixture was incubated at 37° C. for 120 min, in which the buffer solution comprised 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2, 0.5 mM EDTA, 10 μM pargyline and 20 mg/L protease inhibitor.After incubation, the resulting mixture was filtered by a fiberglass filter (GF/B, Packard) in vacuo, and the filter membrane of the fiberglass filter was preimpregnated with 0.3% PEI before the filtering and washed with 50 mM of Tris-HCl for several times after the filtering. After the filter membrane was dried, and the radioactivity of filter membrane was determined by liquid scintillation counting by using a scintillometer (Topcount, Packard). The reference standard was 5-HT, and competitive inhibition curves were plotted based on several inhibition ratios and the corresponding compound concentrations. IC50 values were calculated by non-linear regression analysis using Hill equation curves, and the Ki values were calculated from IC50 by using the ChengPrusoff equation.
- 5-HT3 Receptor Assay Membranes from HEK293 cells which express recombinant human 5-HT3 receptor (RB-HS3, Receptor Biology, Inc., MD, USA) are diluted in accordance with the manufacturer's instructions in incubation buffer (50 mM tris base, pH 7.4, 5 mM MgCl2, 0.5 mM EDTA, 0.1% (w/v) ascorbic acid, 10 μM pargyline and incubated in a volume of 200 μl (amount of membrane protein 3 μg) in the presence of 0.5 nM of the selective 5-HT3 receptor radio ligand [3H]-GR65630 (NET 1011, Du Pont) and various concentrations of the test substance at 21° C. for 60 min. The nonspecific binding is determined by incubation in the presence of 100 μM 5-HT (5-hydroxytryptamine). The incubation is stopped by filtering through type A/E glass fiber filters (Gelman Sciences) or GF/B filters (Whatman) which have previously been placed in 0.3% (v/v) polyethyleneimine (PEI) for at least 1 h. The filters are washed three times with 3 ml of washing buffer (50 mM Tris-HCl, pH 7.4; 4° C.), and the bound radioactivity is determined by scintillation measurement. All the assays are carried out in triplicate. The dissociation constant Ki of the test substance is determined from the IC50 of the compounds (concentration of the test substance at which 50% of the ligand bound to the receptor is displaced), the dissociation constant KD and the concentration L of [3H]GR65630 (Ki=IC50/(1+L/KD)).
- Inhibition Assay LOX: The assay was developed using either 384 or 96 well format. Briefly, in a standard 384 well plate assay 25 μL of a dilution of any of the isoenzymes and orthologues in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were added into each well in the presence of 1 μM mofegiline and 0.5 mM pargyline (to inhibit SSAO and MAO-B and MAO-A, respectively). Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 11 data points, typically in the micromolar or nanomolar range after incubation with the enzyme for 30 min at 37° C. Twenty five μL of a reaction mixture containing twice the KM concentration of putrescine (Sigma Aldrich, e.g. 20 mM for LOX, or 10 mM for LOXL2 and LOXL3), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were then added to the corresponding wells. The above volumes were doubled in the case of 96 wells plate. The fluorescence (RFU) was read every 2.5 min for 30 min at a range of temperatures from 37° to 45° C., excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).
- Assay for Dopamine Reuptake Inhibition Uptake inhibition assay for the dopamine transporter was conducted in rat brain synaptosomes as described elsewhere with minor modifications (Rothman et al., Synapse 39, 32-41 (2001)). Freshly removed caudate was homogenized in 10% ice-cold sucrose with 12 strokes of a hand-held Potter-Elvehjem homogenizer followed by centrifugation at 1000×g for 10 min. The supernatants were saved on ice and used immediately. Transporter activity was assessed using 5 nM [3H]dopamine. The assay buffer was Krebs-phosphate buffer containing 154.4 mM NaCl, 2.9 mM KCl, 1.1 mM CaCl2, 0.83 mM MgCl2, 5 mM glucose, 1 mg/mL ascorbic acid, and 50 μM pargyline. The selectivity of the uptake assay for DAT was optimized by including 100 nM citalopram and 100 nM desipramine as blockers of SERT and NET in the sucrose solution and assay buffer. Uptake inhibition assays were conducted at 25° C. and were initiated by adding 100 μl of tissue to 900 μL assay buffer containing test drug and [3H]dopamine. Test drugs were diluted in assay buffer containing 1 mg/mL bovine serum albumin. Nonspecific uptake was measured by incubating in the presence of 10 μM indatraline. The reactions were stopped after 15 minutes by rapid vacuum filtration with a cell harvester (BRANDEL) over GF/B filters (Whatman) presoaked in wash buffer maintained at 25° C. (10 mM Tris-HCl, pH 7.4/150 mM NaCl). Filters were rinsed with 6 mL wash buffer and retained tritium was quantified by a MicroBeta liquid scintillation counter (PerkinElmer) after overnight extraction in 0.6 mL of liquid scintillation cocktail (Cytoscint, ICN). The data from three experiments were pooled and fit to a dose-response curve equation (using Kaleidagraph), to yield an Emax and EC50 value.
- Inhibition Assay LOX and LOXL2: Lysyl oxidase (LOX) is an extracellular copper dependent enzyme which oxidizes peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. This catalytic reaction can be irreversibly inhibited by β-aminopropionitrile (BAPN) that binds to the active site of LOX (Tang S. S., Trackman P. C. and Kagan H. M., Reaction of aortic lysyl oxidase with beta-aminopropionitrile. J Biol Chem 1983; 258: 4331-4338). There are five LOX family members; these are LOX, LOXL1, LOXL2, LOXL3 and LOXL4. LOX and LOXL family members can be acquired as recombinant active proteins from commercial sources, or extracted from animal tissues like bovine aorta, tendons, pig skin; or prepared from cell cultures. The inhibitory effects of the compounds of the present invention were tested against the given LOX-LOXL preparation using a high-throughput coupled colorimetric method (Holt A. and Palcic M., A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat. Protoc. 2006; 1: 2498-2505). The assay was developed using either 384 or 96 well format. Briefly, in a standard 384 well plate assay 25 μL of a dilution of any of the isoenzymes and orthologues in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were added into each well in the presence of 1 μM mofegiline and 0.5 mM pargyline (to inhibit SSAO and MAO-B and MAO-A, respectively). Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 11 data points, typically in the micromolar or nanomolar range after incubation with the enzyme for 30 min at 37° C. Twenty five μL of a reaction mixture containing twice the KM concentration of putrescine (Sigma Aldrich, e.g. 20 mM for LOX, or 10 mM for LOXL2 and LOXL3), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were then added to the corresponding wells. The above volumes were doubled in the case of 96 wells plate.
- Inhibition Assay LOX and LOXL2: Lysyl oxidase (LOX) is an extracellular copper dependent enzyme which oxidizes peptidyl lysine and hydroxylysine residues in collagen and lysine residues in elastin to produce peptidyl alpha-aminoadipic-delta-semialdehydes. This catalytic reaction can be irreversibly inhibited by β-aminopropionitrile (BAPN) that binds to the active site of LOX (Tang S. S., Trackman P. C. and Kagan H. M., Reaction of aortic lysyl oxidase with beta-aminoproprionitrile. J Biol Chem 1983; 258: 4331-4338). There are five LOX family members; these are LOX, LOXL1, LOXL2, LOXL3 and LOXL4. LOX and LOXL family members can be acquired as recombinant active proteins from commercial sources, or extracted from animal tissues like bovine aorta, tendons, pig skin; or prepared from cell cultures. The inhibitory effects of the compounds of the present invention were tested against the given LOX-LOXL preparation using a high-throughput coupled colorimetric method (Holt A. and Palcic M., A peroxidase-coupled continuous absorbance plate-reader assay for flavin monoamine oxidases, copper-containing amine oxidases and related enzymes. Nat. Protoc. 2006; 1: 2498-2505). The assay was developed using either 384 or 96 well format. Briefly, in a standard 384 well plate assay 25 μL of a dilution of any of the isoenzymes and orthologues in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were added into each well in the presence of 1 μM mofegiline and 0.5 mM pargyline (to inhibit SSAO and MAO-B and MAO-A, respectively). Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 11 data points, typically in the micromolar or nanomolar range after incubation with the enzyme for 30 min at 37° C. Twenty five L of a reaction mixture containing twice the KM concentration of putrescine (Sigma Aldrich, e.g. 20 mM for LOX, or 10 mM for LOXL2 and LOXL3), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 1.2 M urea, 50 mM sodium borate buffer (pH 8.2) were then added to the corresponding wells. The above volumes were doubled in the case of 96 wells plate.
- Recombinant SSAO/VAP-1 Inhibtion Assay Briefly, HMEC cell expressing human SSAO/VAP-1 were grown in several 10 cm petri dishes, once the cells reached 100% confluency, cells were harvested and homogenates were prepared. Cells were washed twice with 5 mL of chilled HES buffer (20 mM HEPES, 1 mM EDTA, 250 mM sucrose, pH 7.4). HES buffer containing 1× protease inhibitor (Sigma Aldrich) and added and cells were incubated on ice for 3 min. Buffer was removed and cells were scraped and transferred to a centrifuge tube. Cell lysates were prepared by passing through 23 G needle for, 10 times and followed by 27 G needle for 10 times. Alternatively the cell lysates were prepared by using IKA Ultra-Turrax T 10 homogenizer for 3 min for every 10 mL of cell suspensions. Cells were then spun for 5 min at 300×g. The clear supernatant was transferred to new centrifuge tube and stored at −80 °C. until colorimetric assay was performed. Prior to the assay, 0.5 mM pargyline was added in order to inhibit any residue MAO activities. Briefly, 50 μL of cell lysate was incubated with test compounds for 30 min at 37 °C. Reaction mixtures were added and kinetic was read as described in detail in Example 5: Briefly, in a standard 96 well plate assay 50 μL of purified human SSAO/VAP-1 (0.25 μg/mL) in 0.1 M NaPO4 buffer (pH 7.4) was added into each well. Test compounds were dissolved in DMSO and tested in a Concentration Response Curve (CRC) with 4-9 data points, typically in the micromolar or nanomolar range after incubation with human SSAO/VAP-1 for 30 min at 37 °C. After 30 min incubation, 50 μL of the reaction mixture containing 600 μM benzylamine (Sigma Aldrich), 120 μM Amplex Red (Sigma Aldrich) and 1.5 U/mL horseradish peroxidase (Sigma Aldrich) prepared in 0.1 M NaPO4 buffer (pH 7.4) were added to the corresponding well. The fluorescence unit (RFU) was read every 2.5 min for 30 min at 37 °C. excitation 565 nm and emission 590 (Optima; BMG labtech). The slope of the kinetics for each well was calculated using MARS data analysis software (BMG labtech) and this value was used to deduce the IC50 value (Dotmatics).