Novel peptide inhibitors targeting the TGF-β receptor I/II complex modulate SMAD and ERK signaling in vitro

Transforming growth factor-β (TGF-β) contributes to fibrosis, immunosuppression, and tumor progression, but systemic TGF-β inhibition is limited by toxicity and lack of selectivity. This exploratory proof-of-concept study evaluated novel peptides designed to target the TGF-β receptor I/II complex and examined their effects on canonical and noncanonical TGF-β signaling in vitro. Peptides 2_5 and 2_6, selected by in silico docking of receptor-ligand interface-derived sequences, were tested in human embryonic kidney 293T (HEK293T) cells at 20 and 50 μM, with the TGF-β receptor I inhibitor SD-208 as a reference. Total and phosphorylated SMAD2, SMAD3, extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinase (JNK) were assessed by Western blotting and densitometry. Peptide 2_5 showed the strongest activity, reducing total ERK1/2 by 65% at 50 μM and phosphorylated ERK1/2 by 90.4% and 83.4% at 20 and 50 μM, respectively; SD-208 reduced total and phosphorylated ERK1/2 by 64.3% and 54.5%, respectively. Peptide 2_5 also reduced total SMAD2 by 81% at 50 μM, whereas its effect on SMAD2 phosphorylation was inconsistent. Peptide 2_6 showed minimal effects, JNK was largely unaffected, and SMAD3 results were inconclusive because of weak signals. These preliminary findings suggest that peptide 2_5 may differentially modulate TGF-β signaling, with a stronger effect on ERK1/2 than on SMAD2; however, receptor targeting and pathway selectivity require confirmation in fully replicated quantitative studies.