Validation of small molecule enhancers of… (R03NS135450) – Project Grant to The Johns Hopkins University

Abstract: 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 identification of innovative drug candidates for treating GBA1-related PD/DLB and sporadic PD/DLB, and IT will also offer a new platform for drug screening for patients with this condition.

Validation of small molecule enhancers of mutant glucocerebrosidase 1 activity

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