Enhancing the Mitochondrial Uptake of Phosphonium Cations by Carboxylic Acid Incorporation.

Laura Pala,Hans M Senn,Hiran A Prag,Tracy A Prime,Thomas P Bright,Richard C Hartley,Claire Wilson,Stuart T Caldwell,Michael P Murphy,Stefan Warrington

doi:10.3389/fchem.2020.00783

Abstract

There is considerable interest in developing drugs and probes targeted to mitochondria in order to understand and treat the many pathologies associated with mitochondrial dysfunction. The large membrane potential, negative inside, across the mitochondrial inner membrane enables delivery of molecules conjugated to lipophilic phosphonium cations to the organelle. Due to their combination of charge and hydrophobicity, quaternary triarylphosphonium cations rapidly cross biological membranes without the requirement for a carrier. Their extent of uptake is determined by the magnitude of the mitochondrial membrane potential, as described by the Nernst equation. To further enhance this uptake here we explored whether incorporation of a carboxylic acid into a quaternary triarylphosphonium cation would enhance its mitochondrial uptake in response to both the membrane potential and the mitochondrial pH gradient (alkaline inside). Accumulation of arylpropionic acid derivatives depended on both the membrane potential and the pH gradient. However, acetic or benzoic derivatives did not accumulate, due to their lowered pKa. Surprisingly, despite not being taken up by mitochondria, the phenylacetic or phenylbenzoic derivatives were not retained within mitochondria when generated within the mitochondrial matrix by hydrolysis of their cognate esters. Computational studies, supported by crystallography, showed that these molecules passed through the hydrophobic core of mitochondrial inner membrane as a neutral dimer. This finding extends our understanding of the mechanisms of membrane permeation of lipophilic cations and suggests future strategies to enhance drug and probe delivery to mitochondria.

Highlights

Mitochondrial dysfunction contributes to a wide range of pathologies, they are an important therapeutic target (Nunnari and Suomalainen, 2012; Gorman et al, 2016; Murphy and Hartley, 2018)
Uptake of the internal standards (IS) confirmed that the test compound did not significantly disrupt mitochondrial membrane potential and showed that the mitochondria continued to respond as expected to different additives known to affect the membrane potential
A hydrophobicity threshold is required for mitochondrial uptake of alkylTPP molecules (Ross et al, 2006; Finichiu et al, 2015; Hu et al, 2017), but when hydrophobicity was increased by replacing the methyl (3) with a hexyl group (4) there was still no mitochondrial uptake compared to IS (Figure 2D)

Summary

Introduction

Mitochondrial dysfunction contributes to a wide range of pathologies, they are an important therapeutic target (Nunnari and Suomalainen, 2012; Gorman et al, 2016; Murphy and Hartley, 2018). The delivery of probe molecules to the organelle in vivo is essential in understanding how mitochondrial dysfunction arises (Yousif et al, 2009; Smith et al, 2012; Logan et al, 2014; Jean et al, 2016). There are a number of strategies to deliver molecules to Enhancing Mitochondrial Uptake of Cations mitochondria in vivo, with conjugation to the alkyltriphenylphosphonium (TPP) cation being the most widespread (Smith et al, 2003, 2012; Yousif et al, 2009). An important aspect of the TPP targeting system is that its positive charge and the large mitochondrial membrane potential lead to these molecules accumulating ∼1,000-fold within energized mitochondria, as described by the Nernst equation (Ross et al, 2006; Smith et al, 2011). Even so, improving mitochondria-targeting head groups to enhance mitochondrial accumulation would lead to improved therapies and probes

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Results

Conclusion