Targeting DDR1 with novel synthesized antagonists delays cellular aging and enhances wound healing in mesenchymal stem cells

Abstract

Mesenchymal stem cells (MSCs) are central to regenerative medicine; however, their clinical utility is constrained by reduced proliferative capacity and replicative senescence during ex vivo expansion. Here, we report on the rational design and biological characterization of novel phthalimide-based inhibitors targeting discoidin domain receptor 1 (DDR1). Structure–activity relationship optimization efforts led to the identification of AC-4067 and AC-4061 as potent DDR1 inhibitors, with IC₅₀ values of 30.9 nM and 53.6 nM, respectively. AC-4067 demonstrated 18-fold selectivity for DDR1 over DDR2 and exhibited no detectable cytotoxicity in normal or cancer cell lines. In Wharton’s jelly–derived MSCs (WJ-MSCs), pharmacological DDR1 inhibition with AC-4067 enhanced proliferative capacity and accelerated scratch-wound closure without compromising expression of key pluripotency markers (OCT3/4, SOX2, NANOG). Importantly, sustained DDR1 inhibition significantly attenuated replicative senescence, as evidenced by reduced senescence-associated β-galactosidase activity and coordinated downregulation of the senescence regulators p16, p21, and p53. Mechanistically, DDR1 inhibition markedly reduced γH2AX protein levels, indicating suppression of persistent DNA damage signaling and alleviation of chronic DNA damage response activation in late-passage MSCs. This reduction in genomic stress markers was associated with preservation of cellular function and delayed onset of senescence. Collectively, these findings identify DDR1 as a critical regulator of MSC aging and genomic stability and establish AC-4067 as a promising pharmacological agent for preserving MSC functional integrity and enhancing their therapeutic potential in regenerative medicine.