Chinese Traditional Medicines are top hits for AFG3L2, a progressive neurodegenerative disease
We discovered a class of natural products called saponins in a yeast-powered drug repurposing screen for AFG3L2. Saponins are also rescuers of FARS2, another ultra-rare mitochondrial disease.
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The results of the AFG3L2 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.
Genetic lightning can strike any family, anytime, anywhere. Sometimes it reveals parental superpowers. When I say superpower I mean a skill or expertise or personality trait that served the parents particularly well in their professional lives, and now this alpha has found a purpose like no other job or mission before. Once unleashed, kiddo-inspired superpowers spark into existence 1-to-N odysseys that begin with a single affected child and then spread across patient communities by word of mouth and social media like a chain reaction of hope.
Like every family we work with at Perlara, Rita and Daniel are compelled to lead even if that means going from zero to one. They launched Miracles for Mighty Milo, a cure-focused research foundation, on behalf of their son Milo, who suffers from Spastic Ataxia Type 5 (SPAX5). SPAX5 is caused by inheriting two pathogenic variants in the evolutionarily conserved gene AFG3L2.
The AFG3L2 gene encodes a protease, or a protein that chews up other proteins. Specifically, it’s a mitochondrially localized protease that is responsible for recycling unfinished or damaged proteins that reside in the mitochondrial membrane. For that reason, autosomal recessive AFG3L2 can be considered an ultra-rare mitochondrial disease. As we’ll show in the results of our yeast-powered drug repurposing screen for AFG3L2, the mitochondrial connection is quite salient.
Over the summer, we performed a 8,400-compound TargetMol screen at the HTSF in Berkeley, California. Here are the summary results. Per usual for our mitochondrial yeast avatar workflow, the positive control (magenta circles) is a wildtype yeast strain grown in 2% lactate media, and the negative control (cyan circles) is the mutant AFG3L2 knockout yeast strain grown in 2% lactate media.
The orange circles represent the mutant AFG3L2 knockout yeast strain grown in 2% lactate and treated with one of the 8,400 test compounds.
We observed exceptionally tight distributions of the positive and negative control well values, i.e., the magenta and cyan circles are all clustered around the mean (black bars) with very little spread. 26 compounds are discernible rescuers. Although none of the rescuers achieved wildtype growth levels, that’s not unusual in our experience across over many yeast drug repurposing projects. There are at least a dozen sensitizers depending on where we draw the cutoff.
Here’s the AFG3L2 TargetMol Z score summary figure with compound identities listed. The dark blue shaded rectangle contains the strongest rescuers. The light blue shaded rectangle contains the moderate to weak rescuers. Sensitizers are highlighted in the red shaded rectangle.
The enlarged orange circles and bolded names correspond to the sprawling family of Chinese Traditional Medicines called steroidal saponins. Intriguingly, steroidal saponins, quaternary amine containing lipids, and sphingolipid analogs are also rescuers of FARS2, another ultra-rare mitochondrial disease.
Just as we’ve seen convergence of rescue mechanisms in yeast between different congenital disorders of glycosylation (CDGs), the same pattern of convergence is emerging for mitochondrial diseases.
Three out of the top six AFG3L2 rescuers are saponins. Eight out of the 26 AFG3L2 rescuers are saponins. With the exception of FARS2, we have not previously seen such a strong enrichment of saponins as rescuers. The chemical structures of six out of the eight saponins are displayed below. Note that unlike in the case of FARS2, there appears to be a strict requirement for a disaccharide or trisaccharide attachment. Many of these Chinese Traditional Medicines (CTM) are available as over-the-counter extracts and in a variety of formulations. Batch-to-batch effects and consistency of manufacturing process are issues for therapeutic natural products like CTM.
Next we compared the AFG3L2 and FARS2 TargetMol Z score datasets so we could evaluate the degree of convergence. It’s immediately apparent that the upper right quadrant is populated with shared rescuers. However, the lower left quadrant is empty, meaning there are no shared sensitizers.
Here’s the same Z score plot with hit compound IDs added. The quaternary amine lipid octenidine is the top common rescuer. RP-54745, an immunomodulating preclinical tool compound, is another common rescuer. Diclazuril and to a lesser extent muscone are examples of FARS2-specific rescuers.
There are only a handful FARS2 sensitizers, while there are at least a dozen AFG3L2 sensitizers highlighted by the red shaded rectangle. Let’s zoom in closer on the AFG3L2 sensitizers. We set a cutoff of Z scores below -0.35, the dashed line in the figure below. The top 12 AFG3L2 sensitizers are listed below by name.
Remarkably, almost all AFG3L2 sensitizers are also SURF1 sensitizers! For example, the iron containing porphyrins shown here.
Two AFG3L2-specific sensitizers are particularly noteworthy because of their mechanism of action.
Genipen is a protein cross-linker, a chemical agent that randomly staples together proteins that happen to be close by each other. When AFG3L2 is missing, mitochondria have diminished capacity to deal with protein stress, also known as proteotoxicity. Treating AFG3L2-deficient yeast with genipen is adding fuel to the fire of mitochondrial protein misfolding and respiratory complex mis-assembly.
MKT077 is a HSP70 inhibitor. HSP70s are a family of heat shock proteins whose expression is increased during heat stress or heavy metal stress. Just as a protein cross-linker would make proteotoxicity matters worse, inhibiting HSP70 would reduce cellular capacity to deal with mitochondrial protein stress.
Finally, a third group of chemically and pharmacologically related AFG3L2 sensitizers are polyphenols — specifically phenylpropanoids — which for reasons that we don’t yet understand are rescuers in CDG yeast drug repurposing screens.
Piceatannol and oxyresveratol are both stilbenoids. As we’ve seen in previous screens, there’s a hidden pharmacophore, in this case a stilbenoid scaffold that is accented in purple in the structure of hypericin, the second most potent AFG3L2 sensitizer. Hypericin is actually a criss-crossed double stilbenoid in a locked conformation.
Over the summer, Rita and Daniel tried out a few CTM in a series of n-of-1 observational studies with Milo. There was no playbook to recommend dosing levels or dosing frequency. Flickers of efficacy were observed, but then seizures led them to pause CTM. Seizures are part of the natural history of SPAX5.
What are the saponins, quaternary amines and sphingolipid analogs doing to rescue AFG3L2 deficiency? Is it the same mechanism at work underlying rescue of FARS2 deficiency too? Our hypothesis is that changing the ratio of positively charged lipids to negatively charged lipids leads to increased cellular capacity to handle proteotoxic stress. Perhaps the saponins themselves are acting directly on mitochondrial membrane proteins as miniature heat shock proteins?
The mitochondrial convergence we’re seeing is striking because the monogenic driver genes affect different aspects of mitochondrial physiology. FARS2 is involved in mitochondrial protein production, AFG3L2 is involved in mitochondrial protein degradation, and SURF1 is involved in Complex IV assembly. It’s fascinating to speculate about shared rescue mechanisms that would impact a wide range of mitochondrial diseases.
However, more funding is required for hit validation studies in Milo’s fibroblasts. Consider donating to Miracles for Mighty Milo to build on these exciting preliminary results which may have implications beyond kiddos like Milo and AFG3L2-related mitochondrial disease like SPAX5.
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