Drug multipurposing for PIGA-CDG using yeast
We began working with CDG CARE and several pioneer CDG communities last summer. PIGA-CDG is one of 170+ CDGs, most of which are ultra-rare. CDG stands for Congenital Disorder of Glycosylation.
The extraordinary degree of evolutionary conservation of glycosylation pathways across the animal kingdom combined with the extraordinary unmet medical need of CDG communities led last summer to the launch of several yeast-powered drug multipurposing projects, including one for PIGA-CDG. It took longer than expected to complete the assay development stage but we’re finally ready for the drug screening stage. Here’s a summary of the journey thus far.
Collaborators
On behalf of their son Romeo living with PIGA-CDG, Mariana and Paul were among dozens of parents of children living with a CDG who convened almost a year ago in San Diego at the 2022 Rare Disease Day symposium & CDG/NGLY1 Family conference.
Several months later, we had a contract and statement of work in place with CDG Care, which serves as a fiscal sponsor and collaboration platform for pioneer CDG families seeking to fund research.
The X-linked PIGA gene encodes a protein that forms a part of the multi-subunit glycosylphosphatidylinositol N-acetylglucosamine transferase (GPI-GnT) complex that carries out the first step in GPI anchor synthesis.
PIGA is the bookend to PIGS, which encodes a protein subunit of the GPI-TA complex that catalyzes the final step in GPI anchor biosynthesis. Perlara Cure Guide and CDG Program Director Dr Kristin Kantautas nicely summarized what is known about PIGA-CDG on CDG Hub here.
Romeo is heterozygous for a PIGA missense mutation at a position that is evolutionarily conserved in all animals: V49M. This case report describes Romeo’s diagnosis and clinical assessment.
V49M is computationally predicted to be damaging but the exact defect or defects caused by each mutation – protein misfolding, protein instability, protein mis-localization, catalytic site inactivation, among others – has not yet been elucidated.
The genome of baker’s yeast (Saccharomyces cerevisiae) contains one PIGA homolog called SPT14 (it also goes by the aliases GPI3 and CWH6). We attempted to create a Romeo V49M yeast avatar along with a catalytically dead E297D yeast avatar.
Unfortunately, the plasmids we used did not support adequate expression of the mutant SPT14 protein. As a backup plan, we had ordered a SPT14 DAmP mutant strain, which represents a generic hypomorphic allele.
Initial attempts last Fall to demonstrate a robust growth defect of the SPT14 DAmP strain under standard growth conditions were unsuccessful. So like in the PIGS-CDG project, we had to pivot to a sensitization paradigm to stress the SPT14 DAmP strain into slower growth. Preliminary results looked promising using hygromycin B as a stressor.
Frustratingly, when we switched from 96-well plates to 384-well plates we lost the sensitized growth phenotype. Normally we would only proceed to drug screening in 384-well plates, but we made an exception for this project.
However, when we continued to optimize in 96-well plate format, we weren’t able to reproduce results from one experiment to the next. The delta between the SPT14 DAmP strain and the wildtype strain was not large enough or too inconsistent to advance to the drug screening stage.
Temperature-sensitive mutant to the rescue!
We ordered a conditional SPT14 mutant from the Euroscarf collection. As expected, it grows just fine at 30˚C but has a severe growth defect at 26˚C, or room temperature. We have an assay window large enough to drive a Mack truck through.
Although the temperature-sensitive SPT14 mutant does not harbor Romeo’s pathogenic variant, we believe that the spt14 temperature-sensitive is a representative loss-of-function mutant. Z’ optimization experiments are currently underway.
The plan is to perform the Pharmakon screen at SMDC next week.