Macrocyclic peptides targeting human CD44 inhibit cell adhesion

CD44-hyaluronan interactions play a significant role in the progression of aggressive tumors. We generated non-native ligands for CD44 by identifying macrocyclic peptides that interact with the hyaluronan-binding domain of CD44. We investigated how the macrocyclic peptides L4-3 and D4-3 influence hyaluronan-engaged CD44 signaling in tumor cells, with a focus on glioma cells, as well as normal fibroblasts. Our findings indicated that inhibiting hyaluronan binding to CD44 in cultured U251MG glioma cells using the macrocyclic peptides L4-3 or D4-3 led to a reduction in cell adhesion and impacted various signaling pathways. The activation of hyaluronan-engaged CD44 during glioma progression - an aggressive and heterogeneous disease associated with multiple oncogenic alterations - cross-talk with epidermal growth factor receptor (EGFR) signaling. Specifically, the macrocyclic peptide L4-3 enhanced the negative feedback regulation of EGFR autophosphorylation at Tyr1068, an effect induced by phorbol 12-myristate 13-acetate (PMA). Furthermore, L4-3 inhibited EGF-mediated activation of the serine/threonine kinase AKT in glioma cells. These findings illuminate the relationship between the CD44-hyaluronan axis and EGFR signaling, suggesting a possible therapeutic strategy. Additionally, the macrocyclic peptide D4-3 suppressed hyaluronan-CD44-mediated adhesion and affected signaling related to cellular adhesion and migration in fibroblasts. However, it did not influence AKT and ERK1/2 signaling pathways. Thus, it is possible that the inhibitory effect of macrocyclic peptides L4-3 and D4-3 on hyaluronan-CD44 interaction have different effects in different cell types.