Abstract
SummaryTherapies halting the progression of fibrosis are ineffective and limited. Activated myofibroblasts are emerging as important targets in the progression of fibrotic diseases. Previously, we performed a high-throughput screen on lung fibroblasts and subsequently demonstrated that the inhibition of myofibroblast activation is able to prevent lung fibrosis in bleomycin-treated mice. High-throughput screens are an ideal method of repurposing drugs, yet they contain an intrinsic limitation, which is the size of the library itself. Here, we exploited the data from our “wet” screen and used “dry” machine learning analysis to virtually screen millions of compounds, identifying novel anti-fibrotic hits which target myofibroblast differentiation, many of which were structurally related to dopamine. We synthesized and validated several compounds ex vivo (“wet”) and confirmed that both dopamine and its derivative TS1 are powerful inhibitors of myofibroblast activation. We further used RNAi-mediated knock-down and demonstrated that both molecules act through the dopamine receptor 3 and exert their anti-fibrotic effect by inhibiting the canonical transforming growth factor β pathway. Furthermore, molecular modelling confirmed the capability of TS1 to bind both human and mouse dopamine receptor 3. The anti-fibrotic effect on human cells was confirmed using primary fibroblasts from idiopathic pulmonary fibrosis patients. Finally, TS1 prevented and reversed disease progression in a murine model of lung fibrosis. Both our interdisciplinary approach and our novel compound TS1 are promising tools for understanding and combating lung fibrosis.
Highlights
A major inducer of myofibroblast activation is transforming growth factor β (TGFβ) [4], which increases the secretion of extracellular matrix components, and the expression of the contractile protein alpha-smooth muscle actin, currently considered to be the most reliable marker of this cell type [5]
We comparatively evaluated the efficacy of TS1-6 to reduce the expression of both αSMA and collagen in primary lung fibroblasts stimulated with TGFβ
We applied a “dry” machine learning strategy to extrapolate our wet results onto a database of over 1 million drug-like compounds to eventually design and synthetize a few novel candidates, one of which (TS1) potently inhibited lung fibrosis in a “wet” context
Summary
We validated the [15,16,17,18] They have invariably failed, exhibiting severe predicted anti-fibrotic compounds in both primary cells and a side effects [6, 7, 19] and we did not consider them mouse model of lung fibrosis (“wet”). These results indicate that dopamine effectively reduces myofibroblast differentiation ex vivo through Drd3 It fails to significantly inhibit bleomycin-induced lung fibrosis in vivo, possibly because of residual collagen expression and/or its quick degradation [23]. We tested the activity of TS1 on human adult lung fibroblasts and confirmed that it significantly inhibits TGFβ-induced myofibroblast activation in vitro, similar to dopamine (Fig. 4G, H). TS1 reverses myofibroblast differentiation and fibrosis, both in preventive and curative approaches in mice, as well as in human IPF lung fibroblasts
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