Identification of chemical and pharmacological chaperones to treat Zellweger Spectrum patients with the common allele, PEX1-Gly483Asp. N. E. Braverman1, S. J. Steinberg2, S. Heibler2, G. E. Maclean1 1) Human Genetics, McGill University, Montreal, Quebec, Canada; 2) Neurogenetics, Kennedy Krieger Institute, Baltimore, Maryland, USA.

   Zellweger spectrum disorder (ZSD) results from recessive defects in any one of 13 PEX proteins, which are required for peroxisome biogenesis. ZSD causes progressive multisystem dysfunction. One mutation in PEX1, a AAA ATPase required for peroxisome matrix enzyme import, Gly843Asp (G843D), represents up to 40% of all ZSD alleles. Thus, identifying treatments for this allele will benefit many patients. We recently showed that PEX1-G843D behaves as a misfolded protein whose function can be improved by chemical and pharmacologic chaperones. In a small molecule screen, we reported recovery of peroxisome matrix enzyme import by flavonoid compounds. Flavonoids are known to bind ATPases, and thus could bind specific regions of PEX1-G843D, such as the ATP binding domain, to improve conformation. In order to develop a lead, we evaluated 70 flavonoids using our cell based phenotype assay. We used a PEX1-G843D/null patient fibroblast cell line, expressing a GFP-PTS1 reporter. Functional recovery of peroxisome matrix enzyme import was determined visually by scoring the number of cells with peroxisomal vs. cytosolic GFP fluorescence. We used a second, independent assay to confirm these results. SAR was determined for the compounds, and diosmetin was found to be the most effective, reaching 75-100% recovery of the GFP reporter into the peroxisome at 10 M. Interestingly, our group of effective drugs included 5 related flavones and 4 of their corresponding flavonols. Structure comparisons showed that methoxylation or hydroxylation at the 4 position and hydroxylation at the 5 and 7 positions increased efficacy, flavones were more effective than the flavonols, and flavanones, isoflavones, and chalcones were ineffective. Additional comparisons showed that a set of modifications were uniquely best for our specific target, possibly allowing the drug to bind PEX1 reversibly. Finally, combination therapy with a chemical chaperone, betaine, allowed us to achieve equivalent recovery of import using 5 M diosmetin. In summary, we have identified a target for pharmacological chaperone therapy that captures a large group of children with a rare genetic disease. We have also identified a group of potential pharmacological chaperones that could be uniquely optimized once their mechanism of action is validated. As our assay measures downstream recovery of peroxisome matrix enzyme import, it has the potential to identify additional compounds with a variety of mechanisms of action.

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