Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa

Abstract

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2,934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, i.e., potential pan-disease therapeutics. Nine of eleven leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.

NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation

“…To assess NTR 2.0/MTZ-induced ablation specificity more directly, selectivity of cell death was compared between neighboring rod (NTR 2.0-expressing) and cone (control) photoreceptor cells. A transgenic line co-expressing YFP and NTR 2.0 in rod cells, Tg(rho:GAP-YFP-2A-nfsB_Vv F70A/F108Y)jh405 (hereafter, rho:YFP-NTR2.0 ) 17 , was treated ±MTZ (400 μM) for 24 h (5 to 6 dpf). Retinas were fixed 24 h later (7 dpf), sectioned, and processed for immunohistochemistry with rod (Ab-1-rho, anti-rhodopsin, α-rho, aka 1D1) 30 and cone (zpr-1, anti-arrestin 3a, α-arr3a, aka Fret 43) 31 labeling antibodies.…”

“…Importantly, fusion proteins between NTR and fluorescent reporters retain MTZ-inducible cell-specific ablation activity 10 . We therefore adapted the NTR/MTZ ablation system to zebrafish 11 to expand studies of cellular regeneration 12 , reasoning that co-expression of NTR with reporters would enable visualization 13 , 14 and quantification 14 – 17 of MTZ-induced cell loss, and subsequent cell replacement, in vivo . In general, the NTR/MTZ system has proven useful for targeted cell ablation, and has had widespread uptake 12 .…”

Overcoming Therapy Resistance in Colon Cancer by Drug Repurposing

“…These approaches can help narrow down the list of candidate drugs that can be used for repurposing [146]. Nevertheless, experimental strategies relying on large-scale drug testing using in vitro and in vivo models remain to be an essential bridge to early phase clinical trials [145,147].…”

Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina

“…Mouse retinal slices from WT, myc-PLD1, and HA-PLD2 mice were prepared according to established protocols ( 28 ). Briefly, mice were anesthetized using isoflurane and decapitated.…”