Targeting Mitochondria with Small Molecules: The Preparation of MitoB and MitoP as Exomarkers of Mitochondrial Hydrogen Peroxide

Abstract

Small molecules can be physicochemically targeted to mitochondria using the lipophilic alkyltriphenylphosphonium (TPP) group. Once in the mitochondria the TPP---conjugate can detect or influence processes within the mitochondrial matrix directly. Alternatively, the conjugate can behave as a prodrug, which is activated by release from the TPP group either using an internal or external instruction. Small molecules can be designed that can be used in any cell line, tissue or whole organism, allow temporal control, and be applied in a reversible dose---dependent fashion. An example is the detection and quantification of hydrogen peroxide in mitochondria of whole living organisms by MitoB. Hydrogen peroxide produced within the mitochondrial matrix is involved in signalling and implicated in the oxidative damage associated with aging and a wide range of age---associated conditions including cardiovascular disease, neurodegeneration and cancer. MitoB accumulates in mitochondria and is converted into the exomarker, MitoP, by hydrogen peroxide in the mitochondrial matrix. The hydrogen peroxide concentration is determined from the ratio of MitoP to MitoB after a period of incubation, and this ratio is determined by mass spectrometry using d15---MitoP and d15---MitoB as standard. 2Here we describe the synthesis of MitoB and MitoP and the deuterated standards necessary for this method of quantification.1. Introduction Mitochondria---targeted drugs and prodrugsSmall molecule drugs are vital to medicine (1). They can often be administered orally, and produce rapid dose---dependent effects. Similarly, small molecules are useful tools to the molecular biologist seeking to elucidate biological processes. A key advantage to small molecules is that in theory they can be used in any cell line, tissue, organ or organism. Their use does not require the manipulation of proteins and gene expression through mutation and RNA---dependent gene silencing, so they can be applied to native tissues and organisms. Furthermore, a small molecule that is useful for the study of a biological process can often be a lead compound for drug discovery, and vice versa.Mitochondria play a central role in metabolism, supplying most of the ATP used by cells, and also are key to signalling, homeostasis, and the events leading up to apoptosis and necrosis (2,3). Mitochondrial dysfunction contributes to almost every age---associated disease including cardiovascular diseases, neurodegeneration and cancer (2), and is implicated in the process of aging itself (4,5).Drugs can act on targets in the mitochondria without having an independent mechanism for their accumulation there. However, efficacy would be increased and side---effects decreased if the concentration of a drug is elevated near its site of action.For this reason, it is desirable to have a mechanism of targeting small molecules to the mitochondria, and in particular the mitochondrial matrix where much of 3 metabolism is sited. Fortunately, there are a variety of approaches for the delivery of molecular...

Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System

“…H 2 O 2 concentration is determined by assessing the ratio of MitoP to MitoB as measured by MS with d15-MitoP and d15-MitoB used as standards. 90,91 As such, this assay can be used in cultured cells and tissue. A limitation of this method is the need for sophisticated equipment not readily available in many laboratories.…”

Trypanocidal action of bisphosphonium salts through a mitochondrial target in bloodstream form Trypanosoma brucei

“…The triphenylphosphonium (TPP) moiety has been used extensively as a vehicle to deliver drugs to mitochondrial targets ( Cairns et al., 2015 , Cortes et al., 2015 , Smith et al., 2011 ). Among many applications, TPP has been used to deliver functional probes ( Cairns et al., 2015 ), antioxidants ( Kelso et al., 2001 , Teixeira et al., 2012 ), anti-cancer drugs ( Jara et al., 2014 ), and even liposomes ( Benein et al., 2015 ) to mitochondria. TPP-linked natural compounds botulin and betulinic acid showed in vitro and in vivo activity against Schistosoma mansoni ( Spivak et al., 2014 ).…”

“…The mitochondrial accumulation of TPP-coupled drugs is driven by the strong inside-negative potential across the inner membrane of the mitochondrion. TPP, being a lipophilic cation with a highly dispersed charge is believed to diffuse freely across the inner membrane and be highly concentrated in the mitochondrial matrix, to an extent determined by the mitochondrial membrane potential Ψm and the plasma membrane potential V m , as described by the Nernst equation ( Cairns et al., 2015 ). Consistent with the extensive TPP literature, Luque-Ortega et al.…”

Azidophosphonium salt-directed chemoselective synthesis of (E)/(Z)-cinnamyl-1H-triazoles and regiospecific access to bromomethylcoumarins from Morita–Baylis–Hillman adducts

“…Their synthetic utility was not only confined to catalysis, but they were also used as intermediates for the synthesis of 1H-indazoles [37], as promoters for stereoselective rearrangements [38], and as temporary protectors of O,P-acetals [39], which branded them as promising motifs. The above reports and the Lewis acid character of quaternary phosphonium salts (QPS) [40][41][42][43][44][45][46][47][48] qualifies them as reliable catalysts for the proposed methodology. The most elaborate process in the proposed methodology is the protection and elimination of the allylic hydroxy group.…”