Abstract Body: Background: Cardiac fibrosis is a central pathological process that leads to worse outcomes in various cardiomyopathies. Recent evidence shows that CDCP1 (CUB domain-containing protein 1) modulates myocardial recovery by attenuating human cardiac fibroblast (HCF) proliferation. This study investigates the hypothesis that selective targeting of the CDCP1cleavage domain can mitigate fibroblast proliferation, thereby offering a novel therapeutic approach to reducing cardiac fibrosis.

Methods: A high-throughput in silico screening was performed using the Enamine compound library, focusing on structural regions that include the cleavage and adjacent sites of CDCP1. A total of 147 candidate compounds were prioritized based on docking scores and binding stability profiles. Selected compounds were subsequently evaluated in vitro using HCF cultures seeded at 3,000 cells/well in 96-well plates. Each compound was administered at 10 μM concentration, and cell proliferation and viability were assessed by time-lapse imaging in the Incucyte® system at 24 and 96 hours. All experiments were done in triplicate with appropriate DMSO vehicle controls.

Results: Compound C3 significantly reduced HCF proliferation (Fig B) to 27.3±2.4% relative to DMSO controls (99.9±0.5%, P<0.001) at 96 hours. Detailed structural modeling indicated that C3 binds to the CDCP1 cleavage site (Fig C) with a favorable Glide XP docking score of -7.66 kcal/mol. The interaction is stabilized by hydrogen bonds with Lys365 and Cys374 (Fig D), and supplementary hydrophobic interactions between aromatic rings and aliphatic groups, which contribute to a strong inhibitory effect.

Conclusion: Targeting the CDCP1 cleavage domain with compound C3 effectively diminishes HCF proliferation in vitro. Therefore, these results substantiate the therapeutic potential of CDCP1 inhibition in cardiac fibrosis. Ongoing in vivo studies will explore the translational relevance of these findings.