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Our internal WCMR Science Seminar Series is a great way for members of the WCMR team and colleagues to hear about the breadth of exciting research taking place across the Centre. Recently, we heard from PhD student Gareth Ettridge about the development of a new assay to test potential medicines that could help those affected by mitochondrial disease. Read on to find out more.

Complex I Assay Development Summary

Therapeutic development is a slow and methodical process that ensures medicines are both safe and effective. This starts from the very beginning of medicine development, which can be separated into different stages: finding a hopeful molecule (lead identification), making it as effective as possible (lead optimisation), testing the safety of the molecule (pre-clinical assessment) and patient trials (ie, does the molecule actually help patients?). This entire process can take up to fifteen years to complete and only when a molecule passes all of these stages can it be called a medicine.

Mitochondrial Complex I (CI) deficiency is the most common biochemical problem in people affected by mitochondrial disease. In order to ensure the time to develop a potential medicine is most effectively spent, this project has targeted CI deficiency to try and help as many mitochondrial patients as possible in the future. The ongoing work in the WCMR is still in the very early stages of the medicine development path but has identified a potentially hopeful molecule that has been tested on cells in the lab. Our data show that this compound can improve how cells with mitochondrial disease grow, although this rate is still very slow and the experiments take a long time to complete (almost three weeks for each one). On top of this, the amount of molecule required to improve the growth rate of the cells would not be safe to give to a person. Therefore, a lot of work is needed to turn this molecule into a medicine.

In order to speed up the development of the hopeful molecule, i.e. to find versions of it that work as effectively as possible, we need a faster way of testing molecules. My work in progress talk outlined recent efforts in developing a new testing method, which takes just over one week to complete and can collect more information from the cells. This new immunofluorescent technique (colour labelling different proteins so a microscope can see them) allows us to measure the number of cells in a plate (just like the assay used previously to determine the growth rate of cells) but can also measure the amount of mitochondria and the levels of two key proteins found in CI.

We have successfully shown that we can see CI biochemical problems in CI deficient cells (image below), and that we can see the deficiency gradually improving when we give the cells more and more of our hopeful molecule. This technique also has the potential of testing many thousands of molecules with relative ease – compared to a growth rate experiment – which could allow us to find more hopeful molecules in the future. There is also the potential of expanding the number of mitochondrial proteins tested. At the moment, we are looking at two CI proteins, but we could look at one from CI, and one from another – such as one from mitochondrial complex IV (CIV), another common biochemical problem found in people affected by mitochondrial disease. In this manner, after much validation and making sure it is accurate, it is possible that this method could help identify biochemical issues faster than at present. However, the main goal remains to speed up the assessment of molecules that could become medicines to help mitochondrial disease patients with mitochondrial complex deficiencies.

The immunofluorescent microscopy images above show different mitochondrial proteins in cells from a healthy person (top) and a mitochondrial disease patient (bottom). The colours show DNA (blue), a mitochondrial label (green) and two proteins found in Mitochondrial Complex I (orange and red). The healthy cells show normal levels of orange and red, which line up with the green mitochondrial label (colour merge image). The cells from a mitochondrial disease patient show very low levels of orange and red – indicating a deficiency in Mitochondrial Complex I.

 

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