High throughput drug discovery towards novel therapeutics for snakebite dermonecrosis
File(s)
Author(s)
Tuersley, Joseph
Type
Thesis
Abstract
Snakebite envenomation is a neglected tropical disease that kills over 100,000 people per year and injures more than 400,000. Brazil suffers more than 26,000 snakebites per year. It is estimated that 90% of all snakebite accidents in the Amazon region are caused by Bothrops species pit vipers. Envenomations are associated with acute, complex, and severe pathological manifestations, including blistering, oedema, dermonecrosis, myonecrosis, and haemorrhage, resulting in sequelae that includes tissue wasting, and permanent disability. Whilst antivenoms remain the gold-standard therapy, their paucity in resolving the local tissue effects urgently warrants the development of tissue-centric therapies. I explore the development of a small molecule cytoprotective approach that can protect cells in damaged tissues from necrosis driven by B. atrox venom. In collaboration with AstraZeneca, we validated a cell-based high throughput screening viability assay against B. atrox venom in keratinocytes. Screening yielded sixteen prioritised hits, of which eight subsequently revalidated at Imperial. In line with our rationale, biochemical activity assays confirmed that largely, identified hits did not inhibit toxin activity directly. Next, I developed a platform of medium throughput phenotypic assays focussing on the distinct pathological hallmarks of envenomation in keratinocytes and endothelial cells. I demonstrate that B. atrox venom induces morphological disruption, via perturbation of cell-matrix contacts, cell-cell adherens junctions, and cytoskeletal F-actin. I also report the induction of nuclear shrinkage, ROS overload, and mitochondrial dysfunction, but notably the absence of autophagy in response to B. atrox venom. Finally, I reveal that most revalidated hits rescue venom phenotypes across the distinct assays, highlighting the utility of identified hits in broadly rescuing the detrimental effects of toxin exposure in vitro. Identified hits represent novel pharmacophores as starting points with scope for development into drug-like scaffolds. Clinically successful small molecules would represent a first-in-class therapy, with utility as an adjunct to antivenom therapy in necrotoxic envenomings to ameliorate dermonecrosis.
Version
Open Access
Date Issued
2023-01-19
Date Awarded
01/06/2023
Advisor
Braga, Vania
Tate, Edward
Sponsor
Biotechnology and Biological Sciences Research Council (Great Britain)
Publisher Department
National Heart & Lung Institute
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)