Yeast-powered drug repurposing for AARS2

Project Inspiration

Aubrie is living with AARS2 deficiency, one of 1,500+ inherited metabolic diseases that currently have no approved treatments. The first step toward a cure is drug repurposing or finding new uses for old medicines, including supplements and nutraceuticals.Aubrie is an AARS2 pioneer, but she’s not alone. This fully onchain project will create a path to scaling up ultra-fast and ultra-cheap yeast-powered drug repurposing screens to 20 related ARS diseases. The umbrella—group and parent-led CureARS Foundation is ready to spread the word about the results of this project to their micro-communities and organize rapid 1-to-N clinical testing in kiddos.

Project Background

The goal of this project is to identify an AARS2 patient-specific drug repurposing clinical candidate using a rapid turnaround, phenotypic screening approach that starts with the development and phenotypic characterization of genetically personalized yeast strains – “patient avatars” – and culminates in a drug repurposing screen, a method originally developed for the congenital orders of glycosylation PMM2-CDG (Lao et al., 2019) and NGLY1-CDG (Iyer et al., 2019).

Aminoacyl-tRNA synthetases (ARSs) are conserved enzymes involved in protein synthesis. They link amino acids to their corresponding tRNAs, ensuring translation fidelity and overall cellular function. Each proteinogenic amino acid is matched with its specific ARS, as reflected in their names (e.g., AARS for alanine-tRNA synthetase). Since translation occurs in both the cytosol and mitochondria, mammalian (and yeast) cells possess distinct cytosolic and mitochondrial ARSs for most tRNAs. Mutations in ARS genes have been implicated in various diseases, including neuropathies, encephalopathies, and other rare genetic disorders. These diseases, often early-onset and multisystemic, lack effective treatments, highlighting the urgent need for innovative therapeutic strategies.

Yeast strains expressing disease-causing hypomorphic variants exhibit growth defects directly proportional to the residual protein activity. Slow growth can be rescued in a pathway-agnostic high-throughput chemical suppressor screen. Perlara has previously implemented drug repurposing screens for FARS2 (mitochondrial phenylalanine tRNA synthetase) and NARS1 (cytoplasmic asparagine tRNA synthetase), identifying several potential clinical candidates.

Under this proposal, an ALA1-deficient yeast patient avatar representative of AARS1 and AARS2 dysfunction in humans will be used to discover drug repurposing candidates for AARS2 deficiency. This work builds upon Perlara’s proven track record of using yeast as a rapid turnaround platform to uncover therapeutic opportunities for rare genetic diseases.

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Project Details

The AARS gene encodes the Alanyl tRNA synthetase, with AARS1 functioning as the cytoplasmic enzyme and AARS2 as the mitochondrial enzyme. AARS1 deficiency is associated with Leukoencephalopathy, Charcot-Marie-Tooth disease, axonal, type 2N, and/or Developmental and epileptic encephalopathy, while AARS2 deficiency is linked to fatal infantile hypertrophic mitochondrial cardiomyopathy and Progressive Leukoencephalopathy With Ovarian Failure.

The genome of baker’s yeast (Saccharomyces cerevisiae) contains a single AARS1 and AARS2 ortholog, ALA1, which is highly conserved in function with the human AARS1 and AARS2.  Human AARS proteins can functionally complement ALA1 loss in yeast, making yeast an ideal model for studying AARS deficiencies. ALA1 yeast models have been previously used to elucidate the pathogenicity of mitochondrial AARS2 mutations (Figuccia, 2023), validate disease-related AARS2 variants (Antonellis et al., 2017), and study the effects of defective editing on translation fidelity (Zhang et al., 2021). Investigations into cytosolic AARS1 relocalization under stress (Debard et al., 2017) and the evolutionary origins of AARS genes (Chang et al., 2012) provide additional insights into its diverse functions. Yeast has also been instrumental in linking AARS mutations to neurological disorders like Charcot-Marie-Tooth disease (McLaughlin et al., 2012) and identifying therapeutic targets for broader ARS deficiencies (Rajendran et al., 2018).

The temperature-sensitive (ts) ALA1 yeast model can be used as a yeast patient avatar to investigate AARS1 and AARS2 deficiency by modulating culture conditions. Under normal conditions (standard YPD media), ALA1 localizes to the cytoplasm, mimicking AARS1 function. Under aerobic conditions (YP + 2% lactate), ALA1 localizes to mitochondria, mimicking AARS2 function. The ALA1-test yeast patient avatar provides a rapid turnaround and cost-effective disease model for AARS deficiency drug discovery.

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About Perlara

Perlara PBC is Curetopia’s yeast-screening laboratory partner and the first public benefit biotech company dedicated to discovering and developing therapies for rare genetic diseases in partnership with families and foundations.

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Key Milestones

• Using a luminescent-based growth assay, the growth rate of ALA1 temperature-sensitive yeast (ALA1-ts) in media conditions that recapitulate AARS1 (cytoplasmic) or AARS2 (mitochondrial) deficiency will be assessed relative to positive and negative controls.
• A 384-well plate high-throughput growth-based assay will be optimized using the AARS2 model*, and the yeast strain will be deployed in a drug repurposing screen (~8,400 compounds) to identify candidates that rescue AARS2 deficiency.
• A report summarizing the repurposing candidates identified from the screen and Perlara’s recommendations for top candidates to prioritize in validation studies will be provided.

_{* Although unlikely, If the AARS2 model does not exhibit a significant growth defect amenable to high-throughput screening, a GARS1 yeast avatar will be used to discover repurposing candidates for GARS1 deficiency. GARS1 encodes glycyl-tRNA synthetase, and GARS1 deficiency is associated with Charcot-Marie-Tooth disease type 2D, distal hereditary motor neuropathy type 5A, and infantile-onset spinal muscular atrophy.}