Image2_In Vivo Dopamine Neuron Imaging-Based Small Molecule Screen Identifies Novel Neuroprotective Compounds and Targets.TIF (4.67 MB)
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Image2_In Vivo Dopamine Neuron Imaging-Based Small Molecule Screen Identifies Novel Neuroprotective Compounds and Targets.TIF

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posted on 18.03.2022, 04:32 by Gha-hyun J. Kim, Han Mo, Harrison Liu, Meri Okorie, Steven Chen, Jiashun Zheng, Hao Li, Michelle Arkin, Bo Huang, Su Guo

Parkinson’s disease (PD) is the second most common neurodegenerative disorder with prominent dopamine (DA) neuron degeneration. PD affects millions of people worldwide, but currently available therapies are limited to temporary relief of symptoms. As an effort to discover disease-modifying therapeutics, we have conducted a screen of 1,403 bioactive small molecule compounds using an in vivo whole organism screening assay in transgenic larval zebrafish. The transgenic model expresses the bacterial enzyme nitroreductase (NTR) driven by the tyrosine hydroxylase (th) promotor. NTR converts the commonly used antibiotic pro-drug metronidazole (MTZ) to the toxic nitroso radical form to induce DA neuronal loss. 57 compounds were identified with a brain health score (BHS) that was significantly improved compared to the MTZ treatment alone after FDR adjustment (padj<0.05). Independently, we curated the high throughput screening (HTS) data by annotating each compound with pharmaceutical classification, known mechanism of action, indication, IC50, and target. Using the Reactome database, we performed pathway analysis, which uncovered previously unknown pathways in addition to validating previously known pathways associated with PD. Non-topology-based pathway analysis of the screening data further identified apoptosis, estrogen hormone, dipeptidyl-peptidase 4, and opioid receptor Mu1 to be potentially significant pathways and targets involved in neuroprotection. A total of 12 compounds were examined with a secondary assay that imaged DA neurons before and after compound treatment. The z’-factor of this secondary assay was determined to be 0.58, suggesting it is an excellent assay for screening. Etodolac, nepafenac, aloperine, protionamide, and olmesartan showed significant neuroprotection and was also validated by blinded manual DA neuronal counting. To determine whether these compounds are broadly relevant for neuroprotection, we tested them on a conduritol-b-epoxide (CBE)-induced Gaucher disease (GD) model, in which the activity of glucocerebrosidase (GBA), a commonly known genetic risk factor for PD, was inhibited. Aloperine, olmesartan, and nepafenac showed significant protection of DA neurons in this assay. Together, this work, which combines high content whole organism in vivo imaging-based screen and bioinformatic pathway analysis of the screening dataset, delineates a previously uncharted approach for identifying hit-to-lead candidates and for implicating previously unknown pathways and targets involved in DA neuron protection.