Surgery (St Vincent's) - Theses
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ItemNovel mitochondrial Drp1 inhibitors for cardioprotection( 2023)Mitochondria are dynamic organelles, constantly undergoing fusion and fission in a balanced manner to maintain cellular health. In the setting of myocardial ischaemia-reperfusion injury, mitochondrial morphology shifts towards excessive fission, which is associated with cardiomyocyte death and heart dysfunction. Inhibiting the mitochondrial fission protein dynamin-related protein 1 (Drp1) has been shown to reduce excessive mitochondrial fission and attenuate the pathological consequences of myocardial ischaemia-reperfusion injury. However, the most widely used inhibitor, Mdivi-1, is an unreliable inhibitor of Drp1 because of its off-target effects and inconsistent cytoprotection in different cell types, including mammalian cells. Mdivi-1 was originally developed to inhibit the GTPase enzymatic activity of Dnm1, a yeast homologue of human Drp1 protein, which has less than 50% similarity compared to human Drp1. These lines of evidence indicate that Mdivi-1 may not be a specific inhibitor of human Drp1. The overall aim of this thesis is to identify potential inhibitors of Drp1 that directly bind to, and inhibit the GTPase activity of human Drp1, and impart protection against in vitro and in vivo models of acute myocardial ischaemia-reperfusion injury. In Chapter 3, I investigated the interaction between Mdivi-1, yeast Dnm1 and human Drp1 using molecular modelling. Molecular docking analysis predicted that Mdivi-1 is docked more consistently in an open binding site conformation of both species with greater number of molecular interactions between the compound and yeast Dnm1 compared to human Drp1. Biological analysis of Mdivi-1 to human Drp1 was inconclusive due to differing results in direct binding assays, GTPase activity assay and mitochondrial morphology assays in Drp1 wildtype and knockout mouse embryonic fibroblasts. These results are likely confounded by the formation of Mdivi-1 aggregates at concentrations above 18.5 uM. These findings suggest that studies employing Mdivi-1 as an inhibitor of Drp1 warrant cautious interpretation as its effect may not be entirely Drp1-specific. In Chapter 4, further study was then conducted to identify a novel potential inhibitor of human Drp1. The drug discovery campaign for this project had already begun prior to my PhD study and three hit compounds, DRP1i1, DRP1i2 and DRP1i3 were previously identified. The three hit compounds represent three compound classes with distinct scaffolds, namely the diazabicyclic scaffold, tryptophan-like scaffold and the diazaspirocyclic scaffold. Direct binding assays, GTPase activity assays and mitochondrial morphology assays using Drp1 wildtype and knockout mouse embryonic fibroblasts indicate that DRP1i1, DRP1i2 and DRP3 directly bind to human Drp1, can inhibit its GTPase activity and supress Drp1-mediated mitochondrial fission. The most potent hit compound, DRP1i1 (KD value 3.23 uM), was selected for further investigation in in vitro and in vivo models of acute ischaemia-reperfusion injury in Chapter 5. In Chapter 5, DRP1i1 reduced cell death of HL1 cells and human cardiomyocytes derived from induced pluripotent stem cells subjected to hydrogen peroxide-induced oxidative stress and simulated ischaemia-reperfusion injury. In general, this protection was accompanied by reduced mitochondrial fragmentation, decreased mitochondrial superoxide production and improved mitochondrial membrane potential. The protective effect of DRP1i1 was also demonstrated in an in vivo mouse model of acute myocardial ischaemia-reperfusion injury, where I observed a reduction of infarct size accompanied by reduced phosphorylation of Drp1 at Ser616 and reduced circularity of myocardial interfibrillar mitochondria. Collectively, these results suggest that direct inhibition of the Drp1 protein with DRP1i1 possess a cytoprotective effect in in vitro and in vivo models of myocardial ischaemia-reperfusion injury. Due to the moderate affinity of the three hit compounds (within micromolar range; 3.23 uM for DRP1i1, 352 uM for DRP1i2 and 215 uM for DRP1i3), our lab had previously searched for structural analogues of each compound class in a two-dimensional analogue search based on the Tanimoto similarity index of 0.8. A total of 26 structural analogues of DRP1i1, 7 of DRP1i2 and 30 of DRP1i3 were identified. 10 additional analogues of DRP1i2 were also designed by our collaborator, giving us a total of 17 structural analogues for DRP1i2. In Chapter 6, I assessed these analogues for direct binding to human Drp1 and conducted molecular docking studies against human Drp1 to elucidate their structure activity relationship. Molecular docking analysis showed that DRP1i2 and its active analogues displayed the most consistently docked binding mode to the open conformation of human Drp1, whereas analogues of DRP1i1 and DRP1i3 did not show a clear consistency in binding mode. Regardless, hydrogen bond interactions between active compounds and amino acids Lys38 and Ser39 could be important for compound activity in all compound class and the effect of stereochemistry on binding affinity to human Drp1 protein was clearly demonstrated. Among all compound classes, only structural analogues of DRP1i2 and DRP1i3 that could potentially be more potent than their parent compounds. Collectively, the information on the structure activity relationship of these structural analogues will provide the essential fundamental knowledge to design better and more potent inhibitors of human Drp1 in future studies.