P16 Atopic eczema: genetic risk to therapeutic target validation

Abstract Introduction and aims Skin barrier impairment is a central pathophysiological mechanism in prevalent diseases including eczema, for which null mutations in FLG, the gene encoding the skin barrier protein filaggrin are the strongest genetic risk factor. However, filaggrin has not yet been targeted therapeutically and there are no treatments which specifically target skin barrier formation or function. Our work defined EMSY as a transcriptional regulator controlling epidermal structure, skin barrier function and filaggrin expression. Methods We developed and validated a high-content phenotypic assay in human primary keratinocytes and screened 50 000 compounds for their ability to reduce nuclear EMSY expression. Overall, 130 putative hit compounds were validated by dose–response analysis (8-point; 10–0.03 µmol L−1) of nuclear EMSY expression (n = 3), FLG quantitative reverse-transcriptase polymerase chain reaction (n = 5) and cytotoxicity (lactate dehydrogenase release) (n = 5). The most promising compound, termed X (to protect intellectual property), was evaluated by mRNA sequencing (n = 6) and in epidermal skin organoids [histology and trans epidermal water loss (TWEL) analysis, TEWL] (n = 6). The keratinocyte-specific mechanism of action of X was determined by chemical capture affinity proteomics and confirmed by Western blotting (n = 3). Results Treatment of primary human keratinocytes with X induced a dose-dependent increase in FLG mRNA (5 µmol L−1 3.49 ± 0.75 fold) in the absence of cytotoxicity and transcriptome analysis demonstrates promotion of multiple aspects of skin barrier formation. Skin organoid models treated with X show quantitative improvement in biophysical measures of epidermal structure and function (TEWL, 5 µmol L−1 0.85 ± 0.03 fold). X has an established molecular target; however, published data (Human Protein Atlas, GTEx) and our own analyses indicate an alternative mechanism. Our chemical capture affinity proteomic analysis has identified novel protein binding partners of compound X in keratinocytes and analysis of the relevant pathway indicates it is an inhibitor. Conclusions We have established novel experimental methods in primary keratinocytes and identified compound X as a candidate therapeutic for the enhancement of skin barrier formation and function.