Drug Repurposing Screening for Rare and Neglected Diseases

National Center For Advancing Translational Sciences

Investigators

Linked publications, trials & patents

Abstract

Drug Development Strategies for Haploinsufficiency. Haploinsufficiency is a genetic condition within a group of rare genetic diseases where a dominant loss-of-function mutation results in only one functional copy of a gene, leading to insufficient protein levels to maintain normal function. This unique feature makes haploinsufficiency diseases promising candidates for drug development by targeting the remaining normal allele to increase gene expression, enhance protein levels, or decrease protein degradation. Drug development strategies for haploinsufficiency diseases include gene therapies (such as CRISPR and AAV-based methods), nucleotide-based therapeutics (like antisense oligonucleotides and RNA interference), and small-molecule drugs (including activators, enhancers, and chaperones). These approaches aim to restore functional protein levels and alleviate disease symptoms, as described in our mini-review (Evans EF. et al. 2024. Drug Discovery Today. 29:104201. PMID: 39384033). We have applied these approaches in drug repurposing screens and drug development for several haploinsufficiency diseases, such as Alagille Syndrome and CTNNB1 syndrome, and will continue to utilize them in future haploinsufficiency disease projects. Methods for Detection of Cholesterol and Sphingosine. Niemann Pick Disease Type C (NPC), a rare genetic disease, shows characteristically accumulations of cholesterol and sphingosine. We have collaboratively developed an integrated platform for high-throughput extraction and mass spectrometry-based quantification of cholesterol and sphingosine from NPC cell samples. The semiautomated protocol using a liquid handling system to extract lipids from cells cultured in 384-well plates, enhancing reproducibility and throughput. By integrating this extraction method with RapidFire mass spectrometry (RF-MS), rapid and accurate quantification of cholesterol and sphingosine are enabled that allows efficient screening of small molecules such as drug repurposing screen for therapeutic potential. The platform demonstrated excellent linearity, reproducibility, and recovery rates, offering a robust tool for drug discovery in lipid storage diseases (Lin YH et al. 2025. Anal Chem. doi: 10.1021/acs.analchem.4c06628. PMID: 40554661). Oxidative Stress Phenotype in NGLY1 Deficiency Disease. NGLY1 deficiency is a rare genetic disease characterized by symptoms such as global developmental delay, hypolacrima/alacrima, movement disorders, and hypotonia, arising from mutations in the NGLY1 gene which impair the de-glycosylation enzyme critical for clearing misfolded glycoproteins. To facilitate drug development, we studied the cellular dysfunctions and disease phenotypic changes associated with NGLY1 deficiency using patient-derived iPSC-derived neural stem cells (NSCs) carrying the commonly occurring nonsense mutation c.1201A > T (p.R401X) and their CRISPR-corrected isogenic controls. The study reveals significant upregulation of ER stress markers, particularly components of the unfolded protein response (UPR) such as sXBP1 and the PERK-EIFα-ATF4 pathway. Additionally, there is a marked increase in autophagic flux and significant signs of oxidative stress, including elevated lipid peroxidation levels. These findings suggest that while autophagy may be a compensatory mechanism to mitigate protein misfolding and oxidative stress, highlighting potential therapeutic targets for this rare disease (Shyr ZA. Et al. 2025. Exp Cell Res 448, 114540. PMID: 40189184).