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

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.

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

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.

Yeast Models of Phosphomannomutase 2 Deficiency, a Congenital Disorder of Glycosylation.

Phosphomannomutase 2 Deficiency (PMM2-CDG) is the most common monogenic congenital disorder of glycosylation (CDG) affecting at least 800 patients globally. PMM2 orthologs are present in model organisms, including the budding yeast Saccharomyces cerevisiae gene SEC53 Here we describe conserved genotype-phenotype relationships across yeast and human patients between five PMM2 loss-of-function missense mutations and their orthologous SEC53 mutations. These alleles range in severity from folding defective (hypomorph) to dimerization defective (severe hypomorph) to catalytic dead (null). We included the first and second most common missense mutations - R141H, F119L respectively- and the most common compound heterozygote genotype - PMM2R141H/F119L - observed in PMM2-CDG patients. Each mutation described is expressed in haploid as well as homozygous and heterozygous diploid yeast cells at varying protein expression levels as either SEC53 protein variants or PMM2 protein variants. We developed a 384-well-plate, growth-based assay for use in a screen of the 2,560-compound Microsource Spectrum library of approved drugs, experimental drugs, tool compounds and natural products. We identified three compounds that suppress growth defects of SEC53 variants, F126L and V238M, based on the biochemical defect of the allele, protein abundance or ploidy. The rare PMM2 E139K protein variant is fully functional in yeast cells, suggesting that its pathogenicity in humans is due to the underlying DNA mutation that results in skipping of exon 5 and a nonfunctional truncated protein. Together, these results demonstrate that yeast models can be used to characterize known and novel PMM2 patient alleles in quantitative growth and enzymatic activity assays, and used as patient avatars for PMM2-CDG drug screens yielding compounds that could be rapidly cross-validated in zebrafish, rodent and human organoid models.

A three dimensional micropatterned tumor model for breast cancer cell migration studies.

Breast cancer cell invasion is a highly orchestrated process driven by a myriad of complex microenvironmental stimuli, making it difficult to isolate and assess the effects of biochemical or biophysical cues (i.e. tumor architecture, matrix stiffness) on disease progression. In this regard, physiologically relevant tumor models are becoming instrumental to perform studies of cancer cell invasion within well-controlled conditions. Herein, we explored the use of photocrosslinkable hydrogels and a novel, two-step photolithography technique to microengineer a 3D breast tumor model. The microfabrication process enabled precise localization of cell-encapsulated circular constructs adjacent to a low stiffness matrix. To validate the model, breast cancer cell lines (MDA-MB-231, MCF7) and non-tumorigenic mammary epithelial cells (MCF10A) were embedded separately within the tumor model, all of which maintained high viability throughout the experiments. MDA-MB-231 cells exhibited extensive migratory behavior and invaded the surrounding matrix, whereas MCF7 or MCF10A cells formed clusters that stayed confined within the circular tumor regions. Additionally, real-time cell tracking indicated that the speed and persistence of MDA-MB-231 cells were substantially higher within the surrounding matrix compared to the circular constructs. Z-stack imaging of F-actin/α-tubulin cytoskeletal organization revealed unique 3D protrusions in MDA-MB-231 cells and an abundance of 3D clusters formed by MCF7 and MCF10A cells. Our results indicate that gelatin methacrylate (GelMA) hydrogel, integrated with the two-step photolithography technique, has great promise in the development of 3D tumor models with well-defined architecture and tunable stiffness.