Drug screens of NGLY1 deficiency in worm and fly models reveal catecholamine, NRF2 and anti-inflammatory-pathway activation as potential clinical approaches

Abstract

N-glycanase 1 (NGLY1) deficiency is an ultra-rare and complex monogenic glycosylation disorder that affects fewer than 40 patients globally. NGLY1 deficiency has been studied in model organisms such as yeast, worms, flies and mice. Proteasomal and mitochondrial homeostasis gene networks are controlled by the evolutionarily conserved transcriptional regulator NRF1, whose activity requires deglycosylation by NGLY1. Hypersensitivity to the proteasome inhibitor bortezomib is a common phenotype observed in whole-animal and cellular models of NGLY1 deficiency. Here, we describe unbiased phenotypic drug screens to identify FDA-approved drugs that are generally recognized as safe natural products, and novel chemical entities, that rescue growth and development of NGLY1-deficient worm and fly larvae treated with a toxic dose of bortezomib. We used image-based larval size and number assays for use in screens of a 2560-member drug-repurposing library and a 20,240-member lead-discovery library. A total of 91 validated hit compounds from primary invertebrate screens were tested in a human cell line in an NRF2 activity assay. NRF2 is a transcriptional regulator that regulates cellular redox homeostasis, and it can compensate for loss of NRF1. Plant-based polyphenols make up the largest class of hit compounds and NRF2 inducers. Catecholamines and catecholamine receptor activators make up the second largest class of hits. Steroidal and non-steroidal anti-inflammatory drugs make up the third largest class. Only one compound was active in all assays and species: the atypical antipsychotic and dopamine receptor agonist aripiprazole. Worm and fly models of NGLY1 deficiency validate therapeutic rationales for activation of NRF2 and anti-inflammatory pathways based on results in mice and human cell models, and suggest a novel therapeutic rationale for boosting catecholamine levels and/or signaling in the brain.

Keywords: Aripiprazole; Congenital disorder of deglycosylation; Disease model; N-glycanase 1 deficiency; NGLY1; Pngl; SKN-1.

Fig. 1.

Determining a half-maximal effective concentration (EC₅₀) for bortezomib in NGLY1-deficient worms and flies. (A) Wild-type N2 worms (top row) and png-1 homozygous mutant worms (bottom row) were grown in liquid media in the presence of increasing concentrations of bortezomib (left to right). (B) Wild-type and Pngl^+/− heterozygous mutant fly larvae were grown on solid media in the presence of either 5 µM or 10 µM bortezomib. Fly larvae were imaged and their sizes were plotted in arbitrary units (AU).

Fig. 2.

2560-compound drug-repurposing screens of png-1 homozygous mutant worms and Pngl heterozygous mutant flies. (A) 15 L1 png-1 mutant larvae were sorted into each well, and plates were incubated for 5 days at 20°C while shaking. Worm screen images of a representative positive control well (A01), a representative negative control well (C23), two presumptive suppressors (K12, K13), and two presumptive enhancers/toxic compounds (C18, N14). Worms were pseudo-colored blue during image processing and analysis. (B) Venn diagram of overlapping hits from three replicate screens. (C) Three fly first-instar Pngl heterozygous mutant larvae were sorted into each well, and plates were incubated for 3 days at room temperature. Z-score plot of three replicates of fly repurposing screen positive control (red circles) versus negative control (cyan circles) wells. (D) Z-score plot of three replicates of fly repurposing screen test compounds (red circles) versus negative (cyan circles) control wells.

Fig. 3.

Chemical structures of cross-validated drug repurposing hits. (A) Theaflavin monogallate; (B) pomiferin; (C) quercetin; (D) 3,4-didesmethyl-5-deshydroxy-3′-ethoxyscleroin; (E) aripiprazole; (F) phenylbutazone; (G) benserazide.

Fig. 4.

Aripiprazole worm and fly cross-validation data. (A) Fly Pngl^−/− homozygote larvae treated with 10 µM, 50 µM and 100 µM aripiprazole compared to untreated controls. (B) Worm png-1 homozygote larvae treated with 25 µM aripiprazole in the presence of 205 nM bortezomib (Bzb) or 27.5 µM carfilzomib (Czb).

Fig. 5.

20,240-compound novel lead-discovery screens of png-1 homozygous mutant worms and Pngl^+/− heterozygous mutant flies. (A) Worm screen Z-score plot of 20,240 test compounds in triplicate. Replicate 1 is shown as red circles. Replicate 2 is shown as red triangles. Replicate 3 is shown as red squares. The mean negative control Z-score is indicated by the red line. The mean positive control Z-score is indicated by the blue line. (B) Fly screen Z-score plot of 20,240 test compounds in triplicate. Replicate 1 is the left panel. Replicate 2 is the center panel. Replicate 3 is the right panel.