# Validation of Small Molecule Enhancers of Mutant Glucocerebrosidase 1 Activity

> **NIH NIH R03** · JOHNS HOPKINS UNIVERSITY · 2024 · $163,750

## Abstract

PROJECT SUMMARY
The glucocerebrosidase 1 (GBA1) gene is the most common genetic risk factor for Parkinson's disease (PD)
and Dementia with Lewy Bodies (DLB). The L444P mutation is the most frequent occurrence and is known
to cause early onset and severe forms of PD and DLB. A deficiency in the functional enzyme, GBA1, occurs
in lysosomes due to misfolded GCase not properly transferring to the lysosomes and instead being retained
in the ER. The accumulation of misfolded mutant GCase leads to ER stress, which in turn causes GCase to
move to the cytoplasm and interact and stabilize soluble α-synuclein (α-syn) oligomers. This accelerates the
formation of pathological α-syn in PD pathology. The evidence linking GBA1 mutations to PD and DLB has
sparked interest in researching GCase as a target for therapy. Enzyme replacement therapy is the most
commonly used approach, but it has limitations in crossing the blood-brain barrier. Substrate reduction
therapy has also seen limited success in clinical trials. An alternative approach, using molecular chaperones
to aid the misfolded GCase and increase its translocation to lysosomes, has gained attention. This has led
to the screening of both pharmacological and small molecule chaperones for their ability to cross the blood-
brain barrier, making it a promising therapeutic strategy for PD and DLB. Discovered chaperones, both
inhibitory and non-inhibitory, through high-throughput screening (HTS) have demonstrated an improvement
in GCase activity, however, many have failed to be effective in clinical trials. This may be due to the limitations
of the model used for screening. The use of recombinant wild-type GCase and patient-derived fibroblasts
that do not accurately represent the mutant protein, as well as the tissue-specific expression of the protein to
reflect disease pathology, has limitations. HTS using patient-derived iPSCs can provide more accurate
results, but it is a labor-intensive and costly process to screen large libraries. To address these challenges,
we have devised an economical genetic model that employs low substrate concentration in SH-SY5Y cells
carrying the L444P mutation. This is supported by a platform that integrates fluorescence-based assay and
flow cytometry to assess GCase activity. In Aim 1, we will perform an initial screening of 11,280 small
molecule compounds to boost GCase activity using the SH-SY5Y GBA1L444P/L444P and GBA1L444P/+ cell lines.
Additionally, our aim is to select the top 5 hit compounds through secondary screening of the top 10. In Aim
2, we will assess the efficacy of these 5 hit compounds in alleviating the disease symptoms in human
dopaminergic (hDA) neurons with GBA1L444P/L444P mutation. We will prioritize assessing the efficacy of the top
five hit compounds in rescuing impaired differentiation of neuronal progenitor cells with the GBA1L444P/L444P
mutation into hDA neurons considering the time and budget constraints of the R03 award. This research will
lead to the...

## Key facts

- **NIH application ID:** 10988959
- **Project number:** 1R03NS135450-01A1
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Hanseok Ko
- **Activity code:** R03 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $163,750
- **Award type:** 1
- **Project period:** 2024-07-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10988959

## Citation

> US National Institutes of Health, RePORTER application 10988959, Validation of Small Molecule Enhancers of Mutant Glucocerebrosidase 1 Activity (1R03NS135450-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10988959. Licensed CC0.

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