Monoamine Oxidase Inhibitor Screening Service

Monoamine Oxidase Activity Assay Control Tests: Km for MAO A and MAO B and IC50 Determination for Clorgyline and Pargyline

Before determining the IC50's for both Monoamine Oxidase A (MAO A) and Monoamine Oxidase B (MAO B) inhibitors, we performed some initial experiments to establish appropriate assay conditions. We assessed conditions for human MAO A and MAO B. First we titrated the MAO's using 1 mM p-tyramine as the substrate. From these titrations we decided to use 0.13 U/reaction for the MAO A and 0.25 U/reaction for the MAO B. We next titrated DMSO to determine the highest concentration that would be tolerated by each MAO. The Km of p-tyramine for each MAO was then measured in the presence of the highest tolerable DMSO concentration. Finally, the IC50 for a known MAO A inhibitor, clorgyline, and MAO B inhibitor, pargyline, was measured for each MAO. For the MAO A, we determined the maximum DMSO concentration for the test compounds to be 10 v% (final reaction concentration of 0.5 v%). We determined the p-tyramine Km to be 55.6 ± 3.7 mM for MAO A and 24.1 ± 4.8 mM for MAO B. Using 55 mM p-tyramine, we observed an IC50 = 11 nM for clorgyline for the MAO A. Using 24 mM p-tyramine, we observed an IC50 = 404 nM for pargyline for the MAO B.

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MAO A and MAO B Titrations
In order to determine an appropriate amount of MAO to use for the IC50 determinations, we titrated each MAO. The MAO A and MAO B were titrated from 1 U/reaction. Both titrations were performed using 1 mM final [p-tyramine] as the substrate. The MAO's titration was performed in a black 96-well plate.

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From these results we opted to use 0.13 U/rxn for the MAO A and 0.25 U/rxn for the MAO B. At these concentrations the reaction would remain linear for at least 20 mins.

DMSO Titration
In order to determine tolerance for DMSO of the MAO, we ran DMSO titrations for MAO A from 0-10 v%. For these titrations we used 0.13U per reaction for the MAO A. The reaction was initiated by adding 1 mM final [p-tyramine] and was allowed to proceed for 20 mins. We then computed the ΔF (F20min - F0min), which is proportional to the rate of reaction, for each DMSO concentration. The ΔF was then plotted versus DMSO concentration. From these result we discovered that DMSO actually inhibited the activity of MAO. For the MAO A, we decided to move forward with <10 v% DMSO to used to dilute the inhibitors (final <0.5 v/v% per reaction)

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Km of MAO A and MAO B
In order to determine the Km of p-tyramine for MAO A and B, we titrated p-tyramine from 0 - 100 mM with either 0.13 U/rxn MAO A or 0.25 U/rxn MAO B and computed the ΔF (F20min - F0min), which is proportional to the rate of reaction, for each p-tyramine concentration. The ΔF's were then plotted versus p-tyramine and the Km was computed using Prism 4 (GraphPad Software Inc.). The MAO A and MAO B Km determination was performed in the presence of 0.5 v% DMSO (final reaction concentration). We found the Km = 55.6 mM for the MAO A and the Km = 24 mM for the MAO B.

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Clorgyline and Pargyline IC50 Determination
The IC50 determination required two steps: 1) 15 min pre-incubation of 45 µL MAO (0.13 U/rxn MAO A or 0.25 U/rxm MAO B) with 5 µL clorgyline in 10% DMSO for MAO A and 5 µL pargyline in 10% DMSO for MAO B, and 2) monoamine oxidase reaction with 0.53 mM final [p-tyramine] for MAO A and 0.23 mM for MAO B for 20 min at 25°C. We titrated clorgyline from 0-0.025 mM and pargyline inhibitor from 0-10 µM and computed the ΔF for each inhibitor concentration. The ΔF's were then plotted versus the inhibitor concentration and the IC50 was computed using Prism 4 (GraphPad Software Inc.). We observed an IC50 = 11 nM for MAO A and an IC50 = 404 nM for MAO B.

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