Ester Prodrugs of Malonate with Enhanced Intracellular Delivery Protect Against Cardiac Ischemia-Reperfusion Injury In Vivo

Abstract

Purpose Mitochondrial reactive oxygen species (ROS) production upon reperfusion of ischemic tissue initiates the ischemia/ reperfusion (I/R) injury associated with heart attack. During ischemia, succinate accumulates and its oxidation upon reperfusion by succinate dehydrogenase (SDH) drives ROS production. Inhibition of succinate accumulation and/or oxidation by dimethyl malonate (DMM), a cell permeable prodrug of the SDH inhibitor malonate, can decrease I/R injury. However, DMM is hydrolysed slowly, requiring administration to the heart prior to ischemia, precluding its administration to patients at the point of reperfusion, for example at the same time as unblocking a coronary artery following a heart attack. To accelerate malonate delivery, here we developed more rapidly hydrolysable malonate esters. Methods We synthesised a series of malonate esters and assessed their uptake and hydrolysis by isolated mitochondria, C2C12 cells and in mice in vivo. In addition, we assessed protection against cardiac I/R injury by the esters using an in vivo mouse model of acute myocardial infarction. Results We found that the diacetoxymethyl malonate diester (MAM) most rapidly delivered large amounts of malonate to cells in vivo. Furthermore, MAM could inhibit mitochondrial ROS production from succinate oxidation and was protective against I/R injury in vivo when added at reperfusion. Conclusions The rapidly hydrolysed malonate prodrug MAM can protect against cardiac I/R injury in a clinically relevant mouse model.

Acid enhancement of ROS generation by complex-I reverse electron transport is balanced by acid inhibition of complex-II: Relevance for tissue reperfusion injury

“…The role of succinate oxidation by Cx-II as a driver of pathological ROS generation has been exploited for its potential as a drug target in IR injury [ 25 , 26 ]. We and others have demonstrated that Cx-II inhibition at the start of reperfusion is protective in animal and perfused organ models of IR injury [ 15 , [27] , [28] , [29] , [30] ]. In addition, it is known that prolongation of ischemic tissue acidosis into the reperfusion period can improve recovery from IR injury [ 31 , 32 ].…”

Selective Delivery of Dicarboxylates to Mitochondria by Conjugation to a Lipophilic Cation via a Cleavable Linker

“…LC-MS/MS measurement of malonate was performed using an LCMS-8060 mass spectrometer (Shimadzu, UK) with a Nexera UHPLC system (Shimadzu, UK), as described previously. 31 Samples were stored in a refrigerated autosampler (4 °C) until injection of 5 μL into a 15 μL flowthrough needle. A SeQuant ZIC-HILIC column (3.5 μm, 100 Å, 150 × 2.1 mm, 30 °C column temperature; Merck Millipore, UK) with a ZIC-HILIC guard column (200 Å, 1 × 5 mm) was used for liquid chromatography.…”

“… 31 , 32 This production of ROS is dependent on succinate availability to SDH, which can be prevented by the delivery of malonate, enabling this delivery to be assessed. 31 , 33 We found that this mitochondria-targeted strategy could deliver malonate to mitochondria, provided that the linker between TPP and malonate was hydrophobic enough to counteract the polarity of the free carboxylic acid and thus enable membrane permeation. Here, we show that a TPP–undecyl monoester of malonic acid (TPP 11 –malonate) accumulated in mitochondria in response to the proton motive force, releasing malonate within mitochondria in cells and in vivo .…”

NLR in eXile: Emerging roles of NLRX1 in immunity and human disease

“…Ischaemia–reperfusion injury, a leading cause of disabling disease and death worldwide, 100,101 is intrinsically linked to mitochondrial biology, with succinate build‐up and reverse‐electron transport‐mediated ROS production responsible for cellular damage 102,103 . Considering this mitochondrial link, it is therefore no surprise NLRX1 has been implicated 104–106 …”