ABSTRACT
The mitochondrial amidoxime reducing component (mARC) is a human molybdoenzyme known to catalyze the reduction of various N-oxygenated substrates. The physiological function of mARC enzymes, however, remains unknown. In this study, we examine the reduction of hydrogen peroxide (H2O2) by the human mARC1 and mARC2 enzymes. Furthermore, we demonstrate an increased sensitivity toward H2O2 for HEK-293T cells with an MTARC1 knockout, which implies a role of mARC enzymes in the cellular response to oxidative stress. H2O2 is a reactive oxygen species (ROS) formed in all living cells involved in many physiological processes. Furthermore, H2O2 constitutes the first mARC substrate without a nitrogen-oxygen bond, implying that mARC enzymes may have a substrate spectrum going beyond the previously examined N-oxygenated compounds.
Subject(s)
Hydrogen Peroxide , Oximes , Humans , Oximes/pharmacology , Mitochondria , CatalysisSubject(s)
Pharmaceutical Preparations , Proteomics , Animals , Disease Models, Animal , Humans , Models, Animal , Species SpecificityABSTRACT
Under different pathological conditions, aberrant induction of neuronal nitric oxide synthase (nNOS) generates overproduction of NO that can cause irreversible cell damage. The aim of this study was to develop an amidoxime prodrug of a potent nNOS inhibitor, the benzhydryl acetamidine. We synthesized the benzhydryl acetamidoxime, which was evaluated inâ vitro to ascertain the potential NOS inhibitory activity, as well as conducting bioconversion into the parent acetamidine. The prodrug was also profiled for inâ vitro physicochemical properties, by determining the lipophilicity, passive permeation through the human gastrointestinal tract and across the blood-brain barrier by PAMPA, and chemical, enzymatic, and plasma stability. The obtained data demonstrate that the amidoxime prodrug shows an improved pharmacokinetic profile with respect to the acetamidine nNOS inhibitor, thus suggesting that it could be a promising lead compound to treat all those pathological conditions in which nNOS activity is dysregulated.
Subject(s)
Amidines/pharmacology , Benzhydryl Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Nitric Oxide Synthase Type I/antagonists & inhibitors , Prodrugs/pharmacology , Amidines/chemical synthesis , Amidines/chemistry , Benzhydryl Compounds/chemical synthesis , Benzhydryl Compounds/chemistry , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Molecular Structure , Nitric Oxide Synthase Type I/metabolism , Prodrugs/chemical synthesis , Prodrugs/chemistry , Recombinant Proteins/metabolismABSTRACT
For the development of new drugs, the investigation of their metabolism is of central importance. In the past, the focus was mostly on the consideration of established enzymes leading to oxidations such as cytochrome P450. However, reductive metabolism by the mARC enzyme system can play an important role in particular for nitrogen containing functional groups. A rapid test was established to give developers of new drugs in the preclinical stage the opportunity to test the metabolism by mARC. To demonstrate the relevance and validity of the new test system, known and potential substrates were applied to this new assay. All known substrates could be detected by the system. Furthermore, several new substrates were found including long-established drugs such as hydroxyurea and new compounds in development such as epacdadostat.
