Introduction. Fibrosis is characterized by excessive extracellular matrix (ECM)
deposition leading to organ dysfunction. Connective Tissue Growth Factor (CTGF)
is a key mediator of fibrosis, promoting ECM production and fibroblast activation.
Its inhibition can attenuate fibrotic progression. Structurally, CTGF comprises four
conserved domains organized into N-terminal (domains 1-2) and C-terminal
(domains 3-4) regions, connected by a protease-sensitive hinge. The C-terminal
region has been associated with profibrotic activity. We previously identified a
peptide capable of inhibiting CTGF, however, its mechanism remains unclear. This
study aimed to define the CTGF-peptide interaction to guide the rational design of
improved CTGF-targeting binders.
Methods. Structural models of CTGF alone and in complex with the peptide were
generated using AlphaFold2 and AlphaFold-Multimer, followed by 1.2 μs molecular
dynamics simulations. Binding contributions were assessed by alanine scanning
using Rosetta Flex ddG. Experimental validation included ELISA alanine scanning
and co-immunoprecipitation assays with full-length and truncated CTGF. Functional
effects were assessed in fibroblasts via fibronectin deposition and stress fibers
formation. Statistical analysis was performed using two-way ANOVA.
Results. Computational models suggested dual peptide interaction with both
CTGF regions, promoting conformational compaction and reduced proteolytic
accessibility. Experimental data revealed predominant binding to the C-terminal
region and identified key peptide residues critical for binding and functional activity.
Conclusion. These findings define a CTGF-peptide interaction interface and
support the rational design of targeted antifibrotic binders.
Acknowledgements. This work is supported by FONDECYT-N°1230054,
Proyecto CCTE Ciencia y Vida Basal-FB210008, FONDEF-ID25I10016, and
ANID/BECA DOCTORADO NACIONAL-21241714.

Co-authors: Enrique Brandan, Sebastián Bazaes, Raul Araya-Secchi, Felipe García-Olave, Tiaren Ruiz