Abstract
Phosphomannomutase 2 deficiency, or PMM2-CDG, is the most common congenital disorder of glycosylation affecting over 1,000 patients globally. PMM2 encodes an ancient metabolic enzyme that forms an obligate dimer that catalyzes an initial step in the conserved pathway leading to N-linked glycosylation of proteins in every cell of every animal throughout its life. All PMM2-CDG patients have at least some residual PMM2 enzymatic activity because complete loss of function is incompatible with life in humans as well as in yeast, flies, fish and mice. Here we describe the first nematode model of PMM2-CDG genetically engineered with the common F119L dimerization-defective patient allele, which has reduced PMM2 enzymatic activity, a constitutively activated ER stress response and an increased sensitivity to proteasome inhibition by bortezomib. We describe an unbiased phenotypic screen to identify FDA approved drugs and generally recognized as safe natural products that suppress bortezomib toxicity in PMM2 F119L homozygote mutant worms. Nine worm repurposing hits from this study and three yeast repurposing hits from a previous study were tested for their effect on PMM2 enzymatic activity in F119/R141H patient fibroblasts in a 96-well-plate assay. The yeast hit alpha-cyano-hydroxycinnamic acid (CHCA) and the worm hit pyrogallin rescue PMM2 enzymatic activity by 65.5% and 23% in PMM2 fibroblasts, respectively. CHCA shares the carboxylic acid-containing pharmacophore of aldose reductase inhibitors. We tested commercially available ARIs in worms and fibroblasts and we found that only epalrestat reproducibly rescues PMM2 enzymatic activity in both species. Epalrestat elevated PMM2 enzymatic activity in patient fibroblasts by 30% over baseline. Epalrestat reduced ER stress marker expression and increased PMM2 mRNA levels in PMM2 F119L homozygote mutant worms by a mechanism that appears to involve the transcriptional regulator NRF2. Epalrestat is the only safe, orally bioavailable and brain penetrant aldose reductase inhibitor approved for use in humans, and the first small molecule potentiator of PMM2 enzymatic activity.
