Phenotypic screening of 1,953 FDA-approved drugs reveals 26 hits with potential for repurposing for Peyronie’s disease

Abstract

Drug repurposing has been shown to bring safe medications to new patient populations, as recently evidenced by the COVID-19 pandemic. We investigated whether we could use phenotypic screening to repurpose drugs for the treatment of Peyronie’s disease (PD). PD is a fibrotic disease characterised by continued myofibroblast presence and activity leading to formation of a plaque in the penile tunica albuginea (TA) that can cause pain during erection, erectile dysfunction, and penile deformity. PD affects 3–9% of men with treatment options limited to surgery or injection of collagenase which can only be utilised at late stages after the plaque is formed. Currently there are no approved medications that can be offered to patients presenting with early disease before the formation of the plaque. Drug repurposing may therefore be the ideal strategy to identify medical treatments to address this unmet medical need in early PD. We used primary human fibroblasts from PD patients in a phenotypic screening assay that measures TGF-β1-induced myofibroblast transformation which is the main cellular phenotype that drives the pathology in early PD. A library of FDA-approved 1,953 drugs was screened in duplicate wells at a single concentration (10 μM) in presence of TGF-β1. The myofibroblast marker α-SMA was quantified after 72h incubation. A positive control of SB-505124 (TGF-β1 receptor antagonist) was included on each plate. Hits were defined as showing >80% inhibition, whilst retaining >80% cell viability. 26 hits (1.3%) were identified which were divided into the following main groups: anti-cancer drugs, anti-inflammation, neurology, endocrinology, and imaging agents. Five of the top-ten drugs that increase myofibroblast-transformation appear to act on VEGFR. This is the first phenotypic screening of FDA-approved drugs for PD and our results suggest that it is a viable method to predict drugs with potential for repurposing to treat early PD.

TGF-β1 induces formation of TSG-6-enriched extracellular vesicles in fibroblasts which can prevent myofibroblast transformation by modulating Erk1/2 phosphorylation

“…In terms of fibroblast heterogeneity, this would have potential to a significant contribution to the fibrosis field, as we previously reported that exposure to the same FDA-approved drugs can lead to different levels of myofibroblast transformation or prevention thereof in various tissues, i.e. drugs that show significant inhibition in one tissue type show no inhibition in the other 87 , 88 . In addition, in the current study, we only tested one time point of adding the myofibroblast vesicles (co-incubated with TGF-β1).…”

“…Human primary fibroblasts were isolated from TA samples from patients undergoing corrective surgery for PD as previously described [88][89][90][91] . Tissue samples were transferred to the labs from the hospital in ice cold DMEM F-12 with 10% foetal calf serum (FCS) and 1% penicillin/streptomycin in less than 3 h. Once the tissue arrived in the lab, it was dissected and corpus cavernosum was removed.…”

“…Cells were grown to confluency and expanded in T75 flasks (Thermo Fisher Scientific). The fibroblasts were characterised as previously described and cell identity was confirmed by expression of vimentin in the absence of expression of desmin [88][89][90] .…”

“…This concentration was previously determined to induce full myofibroblast transformation after 72 h [89][90][91] . Myofibroblasts could be distinguished visually (phase contrast microscopy) and by quantifying expression of the myofibroblast marker α-SMA using immunocytochemistry and in cell ELISA, as previously described [88][89][90][91] .…”

“…To quantify the expression of the myofibroblast marker alpha-smooth muscle actin, as well as the phosphorylation status of Smad2/3 and Erk1/2, we used the In-Cell ELISA (ICE) technique, as previously described and validated [88][89][90] . Depending on experimental design, different cell numbers were seeded onto 96-well optical flatbottom black microplates (Nunc, Rochester, New York).…”