Repurposing the aldose reductase inhibitor and diabetic neuropathy drug epalrestat for the congenital disorder of glycosylation PMM2-CDG

Abstract

Phosphomannomutase 2 deficiency, or PMM2-CDG, is the most common congenital disorder of glycosylation and affects over 1000 patients globally. There are no approved drugs that treat the symptoms or root cause of PMM2-CDG. To identify clinically actionable compounds that boost human PMM2 enzyme function, we performed a multispecies drug repurposing screen using a novel worm model of PMM2-CDG, followed by PMM2 enzyme functional studies in PMM2-CDG patient fibroblasts. Drug repurposing candidates from this study, and drug repurposing candidates from a previously published study using yeast models of PMM2-CDG, were tested for their effect on human PMM2 enzyme activity in PMM2-CDG fibroblasts. Of the 20 repurposing candidates discovered in the worm-based phenotypic screen, 12 were plant-based polyphenols. Insights from structure-activity relationships revealed epalrestat, the only antidiabetic aldose reductase inhibitor approved for use in humans, as a first-in-class PMM2 enzyme activator. Epalrestat increased PMM2 enzymatic activity in four PMM2-CDG patient fibroblast lines with genotypes R141H/F119L, R141H/E139K, R141H/N216I and R141H/F183S. PMM2 enzyme activity gains ranged from 30% to 400% over baseline, depending on genotype. Pharmacological inhibition of aldose reductase by epalrestat may shunt glucose from the polyol pathway to glucose-1,6-bisphosphate, which is an endogenous stabilizer and coactivator of PMM2 homodimerization. Epalrestat is a safe, oral and brain penetrant drug that was approved 27 years ago in Japan to treat diabetic neuropathy in geriatric populations. We demonstrate that epalrestat is the first small molecule activator of PMM2 enzyme activity with the potential to treat peripheral neuropathy and correct the underlying enzyme deficiency in a majority of pediatric and adult PMM2-CDG patients.

Keywords: Aldose reductase inhibitor; Congenital disorder of glycosylation; Drug repurposing; Epalrestat; PMM2-CDG; Phosphomannomutase 2 deficiency.

Fig. 1.

Plot of PMM2 enzymatic activity in whole-animal lysates of pmm-2^F126L mutant worms. PMM2 activity is measured by production of NADPH concentration (mmol/L) over time. The blue line indicates PMM2 activity in wild-type N2 worms. The orange line indicates PMM2 activity in COP1626 worms (pmm-2^F125L homozygote mutant). The gray line indicates PMM2 activity in GR2246 worms, a png-1 deletion mutant, which serves as a negative control. The same amount of protein was used in lysates from each strain.

Fig. 2.

pmm-2 F125L homozygote mutant worms are more than ten times more sensitive to bortezomib than wild-type worms. (A) Side-by-side comparison of representative wells of clear-bottom 384-well plates containing wild-type worms (left) and pmm-2 F125L/F125L worms (right) treated with 13.6 µM bortezomib, the highest dose tested. (B) Z-score box and whisker plot comparing pmm-2 F125L/F125L worms (gray boxes) and wild-type worms (black boxes) treated with the following range of concentrations of bortezomib: 1 nM, 14 nM, 23 nM, 45 nM, 91 nM, 230 nM, 450 nM, 1.6 µM, 2.3 µM, 6.8 µM, 13.6 µM. Z-score labels on the y-axis are 0, −4, −8 and −12. Individual black dots represent statistical outliers.

Fig. 3.

Summary of drug repurposing screen of pmm-2 F125L/F125L mutant worms. (A) Three replicates of the Microsource Spectrum library screen (replicate 1, gray circles; replicate 2, gray squares; replicate 3, gray triangles). Black circles represent the 20 hit compounds with Z-scores greater than five in all three replicates. Each column represents one of the eight library plates. The lower black line indicates Z-score of 0. The upper black line indicates Z-score of 5. (B) Image of a representative positive control well into which L1 DMSO-treated pmm-2 F125L/F125L mutant worms were dispensed and incubated at 20°C for 5 days. 12 adults are visible along with their progeny. (C) Representative negative control well into which L1 11 µM bortezomib-treated pmm-2 F125L/F125L mutant worms were dispensed and incubated at 20°C for 5 days. 16 animals are visible, ranging from L1-L4 larval stages. (D) Representative suppressor (hit) well into which L1 pmm-2 F125L/F125L mutant worms were dispensed and incubated at 20°C for 5 days. 12 adults are visible along with their progeny. (E) Representative enhancer/toxic well into which L1 11 µM-bortezomib-treated pmm-2 F125L/F125L mutant worms were dispensed and incubated at 20°C for 5 days. No living larvae are visible.

Fig. 4.

Chemical structures of 20 drug repurposing candidates discovered in a worm pmm-2 bortezomib chemical modifier screen. Fully capitalized compounds are active in the human PMM2 enzyme activity assay performed in R141H/F119L PMM2-CDG fibroblasts as described in Materials and Methods. Underlined compounds are active in at least one of three yeast PMM2-CDG models described by Lao et al. (2019). The yellow box indicates compounds that are active in the Keap1-Nrf2 cellular reporter assay described in Materials and Methods. The purple box indicates compounds that are catecholamines. The grey box indicates structural singletons with nonselective antioxidant properties. (1) pyrogallin, (2) fiseti,. (3) purpurogallin-4-carboxylic acid, (4) rhamnetin, (5) quercetin tetramethyl ether, (6) gossypetin, (7) ellagic acid, (8) hieracin (tricetin), (9) baicalein, (10) koparin, (11) epicatechin monogallate, (12) theaflavin monogallate, (13) 3-methoxycatechol, (14) 2,3,4-trihydroxy-4-methoxybenzophenone, (15) 3,4-didesmethyl-5-deshydroxy-3-ethoxyschleroin, (16) levodopa, (17) ethylnorepinephrine, (18) dobutamine, (19) amidol, (20) edaravone.

Fig. 5.

Aldose reductase inhibitors CHCA and epalrestat increase PMM2 enzymatic activity in worms and patient fibroblasts. (A) Percentage of PMM2 enzymatic activity normalized to the pmm-2 F125L/F125L (F119L) homozygote mutant treated with DMSO vehicle. From left to right: pmm-2 F125L/F125L (F119L) homozygote mutant treated with DMSO (grey), 15 µM epalrestat for 24 h (P=0.0352) (yellow) or 15 µM CHCA for 24 h (P=0.4175) (orange). (B) Percentage of PMM2 enzymatic activity relative to wild type (100%). 50% represents the activity level of unaffected heterozygous carriers. From left to right: wild-type control fibroblasts treated with DMSO vehicle (blue), GM20942 R141H/F119L PMM2-CDG patient fibroblasts treated with DMSO vehicle (grey), 10 µM epalrestat (P=0.00825) (yellow) or 10 µM CHCA (P=0.0436) (orange). The same amount of protein was used in lysates from each strain. Error bars represent standard error. P-values were determined by unpaired t-test (T.TEST function in Excel).

Fig. 6.

Epalrestat boosts human PMM2 enzymatic activity in multiple PMM2-CDG patient fibroblasts. (A) Supplemented samples were treated with 10 µM epalrestat for 24 h. PMM2 enzymatic activity was measured in PMM2-CDG fibroblasts with the genotype R141H/E139K (blue circles), in PMM2-CDG fibroblasts with the genotype R141H/N216I (red squares) and in PMM2-CDG fibroblasts with the genotype R141H/F183S (green triangles). (B) Bar plot of mean PMM2 enzymatic activity with or without epalrestat treatment (P=0.0321). Each black circle indicates the mean of replicates for each fibroblast. Error bars in the bar graphs indicate standard error of means. P-value was determined by t-test (T.TEST function in Excel).

Fig. 7.

Epalrestat treatment does not increase PMM2 protein abundance. (A) Immunoblots showing PMM2 protein levels in epalrestat-treated PMM2-CDG patient fibroblasts and control fibroblasts. Actin was used a loading control. (B) Quantification of PMM2 protein abundance based on band intensity. Black circles represent 10 µM epalrestat-treated PMM2-CDG fibroblasts. Black squares represent untreated control fibroblasts. (C) PMM2 protein abundance fold change before and after 10 µM epalrestat treatment for 24 h. Black circles represent epalrestat-treated PMM2-CDG fibroblasts. Black squares represent untreated control fibroblasts. (D) Plot of PMM2 enzymatic activity as a function of PMM2 protein abundance. Black circles represent three PMM2-CDG patient fibroblast lines and three control fibroblasts.