Lapthorn_2021.pdf (10.09 MB)
Development of a high-throughput, phenotypic screening assay to identify novel medicines to prevent dermal scarring
thesisposted on 2023-08-30, 19:45 authored by Alice Lapthorn
Introduction: Dermal scarring affects 91% of burn patients every year and yet, there is currently no effective treatment for their prevention. Dermal scarring is often caused by the dysregulation in different stages of the wound healing process – particularly the sustained transformation of fibroblasts to alpha-smooth muscle actin (α-SMA) expressing myofibroblasts. The aim of this thesis was to identify novel drugs that can be re-purposed for the prevention of dermal scar formation, using a phenotypic, high-throughput screening (HTS) assay. Materials & Methods: Primary fibroblasts, derived from excised scar tissue, were exposed to transforming growth factor beta-1 (TGF-β1) to induce transformation to myofibroblasts. Using the In-Cell ELISA method, a HTS assay was optimised and validated before screening 1,954 approved drugs. Hits were defined as showing >80% inhibition of α-SMA expression, whilst maintaining >80% cell viability. Anti-myofibroblast activity of the candidate drugs was confirmed by construction of concentration response curves, before investigating their effects on cell viability, extracellular matrix (ECM) production and keratinocyte epithelial-mesenchymal transition (EMT). Results: A HTS assay measuring α-SMA expression was optimised and validated, yielding a Z-factor of 0.59 ± 0.03. The screening of 1,954 approved drugs identified 90 hits (4.6%) that successfully inhibited myofibroblast transformation. 10 hits were identified as having a desirable safety profile and could be used for topical application. The anti-myofibroblast activity of one drug class (hydroxypyridone anti-fungals) was confirmed – ciclopirox (IC50 = 16.7 ± 2.3 μM), ciclopirox ethanolamine (IC50 = 10.3 ± 0.8 μM) and piroctone olamine (IC50 = 1.4 ± 0.1 μM). Secondary assays showed that the hydroxypyridone anti-fungals could reduce ECM production and inhibit keratinocyte EMT, whilst maintaining cell viability. Conclusions: Using primary fibroblasts, a phenotypic HTS assay identified hydroxypyridone anti-fungals as being able to inhibit TGF-β1-induced myofibroblast transformation. These drugs exhibited further anti-fibrotic effect when measuring for ECM production and keratinocyte EMT. This is the first study to identify and investigate the anti-fibrotic effect of hydroxypyridone anti-fungals in dermal scarring, suggesting that these drugs could be re-purposed as medical therapy to prevent dermal scarring.
InstitutionAnglia Ruskin University
- Accepted version