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PIGS-CDG Cure Odyssey
We call them odysseys for good reason. The scientific method involves unexpected detours and cul-de-sacs. But persistence and patience in science pay: we finally have a clinically actionable hit.
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The results of the PIGS-CDG drug repurposing project that we are sharing in the spirit of open science below are novel preclinical research findings and therefore they do not constitute the practice of medicine. Please consult your physician or clinical care team if you’re considering off-label use of any approved drug. The same caution applies to nutraceuticals, supplements and “generally recognized as safe” compounds.
The PIGS-CDG cure odyssey has been the rockiest road we’ve had to traverse over the past year since launching our on-demand “drug repurposing as a service" powered by yeast avatars. In spite of hitting a wall over and over again, failure was not an option because there are no treatments right now for the handful of PIGS-CDG kiddos known to medicine, like pioneer Anna.
In the previous PIGS-CDG project update seven months ago, we thought we had an unimpeded line of sight on a screen of the Pharmakon library at the Small Molecule Discovery Center at UCSF. But then we had to pivot to a temperature-sensitive yeast avatar that was created by random mutagenesis versus engineering in specific patient variants. We executed the same pivot at multiple points along the length of the GPI anchor biosynthesis pathway: PIGA-CDG, PIGN-CDG and PIGW-CDG.
Recall that while the function of the PIGS proteins remains elusive, it is part of the multi-subunit GPI transamidase (GPI-TA) complex whose job is to attach proteins to the GPI anchor. In other words, PIGS is the last stop on the train, biosynthetically speaking. What happens when PIGS function is reduced or eliminated? Knowing CDGs like we do, at least one metabolic flux is out of balance.
Presumably unattached GPI anchors accumulate in the ER membrane, which might lead to a multi-car pileup upstream of PIGS. Perhaps a compound that partially inhibits the first or an early step in GPI anchor biosynthesis, i.e., the GPI-GnT complex that forms the natural bookend to the GPI-TA complex, might restore the flow of traffic?
We uncovered a similar “substrate reduction” rescue mechanism for SRD5A3-CDG. The SRD5A3 gene encodes an enzyme that converts polyprenol to dolichol. A top hit from a SRD5A3-CDG worm avatar screen is a statin drug that inhibits the enzyme HMG-CoA reductase, which functions at an early step in dolichol biosynthesis.
Another potential cellular coping mechanism would involve relieving the pressure caused by buildup and/or molecular crowding of unattached GPI anchors in the ER membrane. Perhaps an amphipathic compound that inserts into the ER membrane and leads to changes in the ER membrane’s biophysical properties might restore the balance of unattached GPI anchors to attached GPI anchors?
Our first successful Pharmakon screen using a heat-sensitive yeast avatar was for PGAP3-CDG. It resulted in a best-case scenario in terms of the clinical actionability of yeast “rescuer” hits: the discovery of several over-the-counter dietary supplements. A less optimal scenario is when rescuers from a yeast avatar screen are less clinically actionable. That happens for a host of complicating factors, including but not limited to narrow therapeutic index and lack of access to GMP/commercial supply. That happened with PIGA-CDG, where we’re now hoping common hits will be found between the yeast screen we completed in the Spring and an ongoing fly screen in Professor Clement Chow’s lab at University of Utah.
Thanks to the generosity of Professor Charlie Boone’s lab at the University of Toronto, we obtained a GPI17 (the yeast version of PIGS) temperature-sensitive mutant that is unable to grow at temperatures greater than 38˚C.
In the first seeding density experiment shown below, our yeast team at BADASS Labs was able to show that the temperature-sensitive PIGS yeast avatar behaves as expected when exposed to 38˚C heat shock.
We had been down this road, at this exact juncture, several times already only to run into a buzzsaw at the Z’ optimization step. Having tasted the bitterness of failure, the yeast team — Natalie, Brianna, James and Dr Mathura Thevandavakkam — along with project lead Dr Michelle Dookwah-Smith were determined to apply their collective learnings and quickly generate robust Z' optimization results that would give us high confidence to progress to the Pharmakon library screen. And that they did!
The baton was then passed to Jez at SMDC to run the 7-plate Pharmakon library screen. One week ago, we finally got the email attachment we’d been anticipating for months. Much to our simultaneous relief and delight, the data looked great. There are just over a dozen rescuers, and several dozen sensitizers, as shown here:
How does the PIGS Pharmakon screen compare to the other Pharmakon screens we’ve completed over the past year? As shown below, the ratio of rescuers to sensitizers varies widely. On one end there’s PIGA, which has a massive number of sensitizers relative to rescuers. On the other end there’s SURF1 (a mitochondrial disease), which has a large number of rescuers relative to sensitizers. PIGS falls somewhere in between.
A pairwise comparison between PIGS and PIGA is instructive because PIGA and PIGS are natural bookends of the GPI anchor assembly line. Remarkably, the strongest PIGA rescuer appears to be a weak PIGS sensitizer, and vice versa, the strongest PIGS rescuer is a strong PIGA sensitizer. Indeed, almost half of the PIGS rescuers are strong PIGA sensitizers, with at least one notable exception marked by the arrow, which happens to be the second strongest PIGS rescuer.
The identity of that compound is the nutritional supplement ascorbyl palmitate, or fat-soluble Vitamin C. We don’t know the mechanism by which ascorbyl palmitate rescues PIGS deficiency, but it doesn’t obviously appear to involve substrate reduction. Recall that in the PGAP3-CDG drug repurposing project, the yeast avatar screen identified three nutritional supplements.
We just had a call with Anna’s parents to discuss the results. The two options we’ll pursue in parallel are starting a 1-to-N observational study in consultation with a physician, and validating ascorbyl palmitate in a PIGS fly model in the Chow lab. Onward!