A Novel High-Content Screening Assay Identified Belinostat as Protective in a FSGS—Like Zebrafish Model

Abstract

Background: Focal segmental glomerulosclerosis (FSGS) affects the complex three-dimensional morphology of podocytes, resulting in loss of filtration barrier function and the development of sclerotic lesions. Therapies to treat FSGS are limited, and podocyte-specific drugs are unavailable. To address the need for treatments to delay or stop FSGS progression, researchers are exploring the repurposing of drugs that have been approved by the US Food and Drug Administration (FDA) for other purposes. Methods: To identify drugs with potential to treat FSGS, we used a specific zebrafish screening strain to combine a high-content screening approach with an in vivo model. This strain expresses nitroreductase and the red fluorescent protein mCherry exclusively in podocytes (providing an indicator for podocyte depletion), as well as a circulating 78 kDa vitamin D–binding enhanced green fluorescent protein (eGFP) fusion protein (as a readout for proteinuria). To produce FSGS-like lesions in the zebrafish, we added 80 µM metronidazole into the fish water. We used a specific screening microscope in conjunction with advanced image analysis methods to screen a library of 138 drugs and compounds (including some FDA-approved drugs) for podocyte-protective effects. Promising candidates were validated to be suitable for translational studies. Results: After establishing this novel in vivo high-content screening assay, we identified seven drugs or compounds that were protective in our FSGS-like model. Validation experiments confirmed that the FDA-approved drug belinostat was protective against larval FSGS. Similar pan-histone deacetylase (HDAC) inhibitors also showed potential to reproduce this effect. Conclusions: Using an FSGS-like zebrafish model, we developed a novel in vivo high-content screening assay that identified belinostat and related pan-HDAC inhibitors as potential candidates for treating FSGS.

Nifurpirinol: A potent inducer of FSGS-like glomerular injury in transgenic zebrafish

“…Since pan-HDAC inhibitors such as Belinostat, Oxamflatin and Panobinostat were shown to impair edema formation in MTZ-treated larvae, we wanted to analyze whether these inhibitors also show beneficial effects on NFP-treated zebrafish larvae. 18…”

“…To address this, we developed a high-content drug screening in which transgenic zebrafish larvae were treated with Metronidazole (MTZ), a well-established prodrug that is converted into a toxin by the bacterial nitroreductase (NTR) selectively expressed in the podocytes in a transgenic zebrafish strain. 18 After the damage and loss of podocytes, the zebrafish larvae develop a FSGS-like injury. 27 Since MTZ, which is used at micromolar concentrations showed a high inter-individual variability in generation of FSGS, we wanted to test an alternative substrate for the bacterial nitroreductase.…”

“…Hereby, we identified potential drugs/small molecules that showed a beneficial effect on the development of FSGS in these zebrafish larvae. 18…”

“…Hereby, we identified potential drugs/small molecules that showed a beneficial effect on the development of FSGS in these zebrafish larvae. 18 The zebrafish is a well-known animal model to study renal function in vivo. [19][20][21][22] The simple kidney, the pronephros, is composed of one glomerulus combined with two tubules, and shows a high similarity in morphology and protein expression compared to glomeruli of mammals.…”

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis

“…Larvae were mounted in 96-well plates and imaged on an Acquifer Imaging Machine (Acquifer Imaging GmbH, Germany). Intravascular fluorescence intensities of the tail region were quantified with an automated FIJI script, segmenting blood vessels upon traversing blood cells in a short image sequence and measuring GFP fluorescence within the masks 49 .…”