The Development of Phenotypic Screening Methods for Drug Discovery in Mitochondrial Dysfunction

Abstract

This project both develops and employs high throughput phenotypic screening in the mitochondrial dysfunction research space. Initially, mitochondrial proliferation was investigated as it has emerged as a potential therapeutic pathway of interest. Collaborative efforts with the small biotechnology company, Nanna Therapeutics, have resulted in the screening of millions of bacterial natural product extracts for mitochondrial proliferation. The current project develops a multi-stage assay pipeline for the further screening of the hits identified in the work conducted by Nanna Therapeutics. A total of 10 hit extracts were identified, each capable of inducing increases in multiple parameters associated with mitochondrial biogenesis, in human cell lines. Four of the hits were selected for further investigation, as a result of maximal mitochondrial density increases being achieved with the minimum dose - in comparison with the remaining hit population. Mutations affecting the assembly of mitochondrial complexes were also investigated in the project. Mitochondrial Complex I (CI) deficiency is the leading biological hallmark of mitochondrial disease. As such, a high throughput screen has been optimised for the identification of novel therapeutic agents for the rescue of CI deficiency. As a result of the screen optimisation process, the current project also demonstrates the activity of lead compound for CI deficiency rescue, MOA2. The developed CI immunofluorescent staining protocol was also applied to Mitochondrial Complex IV (CIV) and Mitochondrial Complex II. Successful optimisation of a quadruple immunofluorescent technique targeting CI, CII and CIV followed, which was used to further show novel effects of MOA2. This project has given rise to multiple outputs. This thesis details the development of a high throughput assay pipeline for the discovery of small molecules capable of inducing mitochondrial proliferation, identifying ten hits. It has also demonstrated the effects of mitochondrial biogenesis on mitophagy. Finally, the current project has developed a novel in vitro method for the assessment of mitochondrial complex deficiencies in cells using immunofluorescent staining, with potential applications to the mitochondrial disease diagnostic space with further validation.