Drug Drop Test: How to Quickly Identify Potential Therapeutic Compounds for Mitochondrial Diseases Using Yeast Saccharomyces cerevisiae

Abstract

Mitochondrial diseases (MDs) refer to a group of clinically and genetically heterogeneous pathologies characterized by defective mitochondrial function and energy production. Unfortunately, there is no effective treatment for most MDs, and current therapeutic management is limited to relieving symptoms. The yeast Saccharomyces cerevisiae has been efficiently used as a model organism to study mitochondria-related disorders thanks to its easy manipulation and well-known mitochondrial biogenesis and metabolism. It has been successfully exploited both to validate alleged pathogenic variants identified in patients and to discover potential beneficial molecules for their treatment. The so-called "drug drop test", a phenotype-based high-throughput screening, especially if coupled with a drug repurposing approach, allows the identification of molecules with high translational potential in a cost-effective and time-saving manner. In addition to drug identification, S. cerevisiae can be used to point out the drug's target or pathway. To date, drug drop tests have been successfully carried out for a variety of disease models, leading to very promising results. The most relevant aspect is that studies on more complex model organisms confirmed the effectiveness of the drugs, strengthening the results obtained in yeast and demonstrating the usefulness of this screening as a novel approach to revealing new therapeutic molecules for MDs.

Keywords: Saccharomyces cerevisiae; drug drop test; drug repurposing; mitochondrial diseases; yeast model.

Figure 1

The drug drop test method. The mutant yeast strain is spread on square plates containing solid medium supplemented with a non-fermentable carbon source that allows basal minimal growth. Subsequently, small sterile filters are placed on the agar surface and spotted with molecules belonging to chemical libraries. In addition, negative and positive controls (i.e., the solvent in which molecules are dissolved and a wild-type spot, respectively) are deposed. The plate is then incubated for several days. After incubation, the positive compounds (active compounds) determine the appearance of a halo of enhanced growth around the corresponding filter. Created with BioRender.com, accessed on 29 May 2023.

Figure 2

From yeast to patients: the drugs’ path. A huge number of molecules (belonging to chemical libraries) could be tested in yeast. Active compounds could be further analyzed in other model organisms and/or cell models, and each step determines a reduction in the number of potential therapeutic compounds. In the end, the most promising molecules could be administered to patients to treat MDs. Two outcomes could be envisaged: personalized medicine, when a drug is active only on a specific mutation and therefore applicable to a single or few patients; alternatively, some drugs could present a broad spectrum of activity, being suitable for a higher number of patients presenting mutations in different genes. Created with BioRender.com on 22 June 2023.