A combined in vitro and in silico approach in the study of drug-induced mitochondrial dysfunction

Mitochondria are the cellular organelles that generate 95% of the energy needed for a cell to remain viable. Due to their vital role in energy metabolism and homeostasis, mitochondria are increasingly viewed as important off-targets in the study of drug-induced organ toxicities. Indeed, a significant body of evidence has shown that drugs from diverse chemical and therapeutic classes are responsible for mitochondrial dysfunction, which has led in some cases to ‘Black Box’ warnings and drug withdrawals by regulatory bodies. In this research, multiple in vitro assays using different model systems and mitochondrial endpoints were used to gain understanding on the mechanisms involved in drug-induced mitochondrial toxicity (DIMT), specifically those relevant to the operation of the mitochondrial electron transport chain (ETC). Furthermore, in silico methods, such as Quantitative Structure Activity Relationships (QSAR) and molecular docking were used to investigate the chemical features associated with inhibition of the ETC.

The first objective was to investigate the interactions between the alternative oxidase (AOX), a crucial respiratory protein present in the ETC of some pathogenic parasites, and inhibitors through in silico studies such as QSAR and molecular docking to highlight key residues and molecular properties required for inhibition. Given the relative structural simplicity of the AOX, this investigation offered an opportunity to develop a methodology that could be later applied to other more complex protein targets, such as the cytochrome bc1 complex.

The second objective was to study the in vitro effects of some well-known complex III and AOX inhibitors used as fungicides on both isolated mitochondria and whole cells. The aim was to gain a clearer understanding on the mechanisms of toxicity of this compound set, as well as to validate the techniques employed for future investigations on pharmaceutical drugs.

The third objective was to investigate the effects of a wide range of pharmaceutical drugs on various aspects of mitochondrial function using molecular docking and the in vitro techniques validated in previous chapters. This included the effects of drugs on oxygen consumption, mitochondrial membrane potential and reactive oxygen species using isolated mitochondria from rat liver, as well as cell viability and cell bioenergetics using intact and permeabilised HepG2 cells. The tested drugs covered a wide range of pharmacological classes including, but not limited to, antidiabetics, antihyperlipidemics, anti-inflammatories, antipsychotics and anticonvulsants. Many of the drugs tested showed an effect on various aspects of mitochondrial function, with the wealth of experimental results allowing an estimation of the mechanism of actions. Some of these mechanisms included direct inhibition or uncoupling of the ETC and, in some cases, a dual activity was observed depending on drug concentration and mitochondrial state.

Finally, QSAR analysis using stepwise regression with half maximal inhibitory concentrations (IC50 values) generated in previous chapters as the response variable, and hundreds of computed molecular descriptors of compounds as independent variables, resulted in three regression models with good prediction accuracy for inhibition of the cytochrome bc1 complex. These models highlighted several physicochemical and topological properties that could be responsible for the inhibitory effects of the compounds on the cytochrome bc1 complex.

In summary, the findings within this thesis have shed light on the underlying mechanisms of toxicity of several pharmaceutical drugs currently clinically used and have highlighted the importance of obtaining a complete understanding of DIMT through multiple assays and mitochondrial endpoints in order to improve the safety of pharmaceutical drugs.