New Design of an Activity Assay Suitable for High-Throughput Screening of Substrates and Inhibitors of the Mitochondrial Amidoxime Reducing Component (mARC)

Abstract

The mitochondrial amidoxime-reducing component (mARC) is one of the simplest molybdenum-containing enzymes. mARC is among a few known reducing enzymes playing an important role in drug metabolism in mammals. Here, an assay based on the fluorescence of NADH is reported for the rapid detection of substrates and potential inhibitors of mARC. So far unknown inhibitors might be useful for the development of drugs assigned to nonalcoholic fatty liver disease (NAFLD) and similar diseases. Kinetics of reactions catalyzed by mARC can be recorded with high sensitivity and precision. On a microtiter plate scale, the assay presented could be applied for highthroughput screening of substance libraries and detection of novel mARC substrate candidates. For instance, molnupiravir was also identified as a new substrate by this assay. For better comparison for such substances, the inhibitor or substrate-to-BAO ratio was introduced. After normalization of enzyme activities to the standard benzamidoxime, substrates can reproducibly be classified.

Reduction of Hydrogen Peroxide by Human Mitochondrial Amidoxime Reducing Component Enzymes

“…Since HEK-293T cells express onl low levels of mARC2, the MTARC1 knockout results in cells practically devoid of m activity (mARC2 expression levels do not increase to compensate the MTARC1 kno . The knockout was shown to be effective on the protein level by Western blot an Using our recently established fluorescence-based high-throughput assay [17], we were able to measure very similar conversion rates for the mARC-catalyzed reduction of hydrogen peroxide.…”

“…Alternatively, the enzyme activity was assayed by monitoring NADH consumption through a recently established fluorometric protocol [17]. Briefly, time-dependent change in NADH fluorescence (λ ex = 340 nm; λ em = 365 nm) was monitored with a TECAN Infinite 200 M Pro plate reader.…”