Proteomic profiling of adipose-derived mesenchymal stromal cell response to novel compounds targeting the Laminin Receptor – PEDF interaction

This study characterizes the proteomic responses of adipose-derived mesenchymal stromal cells (ASC) to novel compounds that target the interaction between pigment epithelium-derived factor (PEDF) and the 37 KDa Laminin Receptor (LR). Building on previous research, we introduced the second-generation analog, 02-09, for comparative analysis with C3, the original compound. C3 was identified through <i>in silico</i> screening and experimental validation, showing anti-inflammatory activity. Analog 02-09 was synthesized by modifying C3's aromatic ring, resulting in similar binding affinities to LR. The distinct molecular effects of C3 and 02-09, along with their potential as anti-inflammatory therapies from prior work, suggested their multi-functional utility. Proteomic profiling of ASC, under both undifferentiated and chondrogenic differentiation conditions, was performed using LC-MS/MS, with controls including the PEDF-derived peptide p18 and the cartilage-regenerative compound Kartogenin (Krt). Both C3 and 02-09 induced significant proteomic modulation compared to controls, with enrichment in pathways related to <i>integrin β1 signaling, extracellular matrix (ECM) organization</i>, and <i>TGF-β signaling</i>. C3 uniquely activated the <i>nuclear receptors meta-pathway,</i> whereas 02-09 was associated with <i>laminin interactions</i>. Protein-protein interaction and transcription factor enrichment analyses revealed distinct and overlapping regulatory networks for LR-targeting compounds. During chondrogenic differentiation, C3 primarily enhanced proteins linked to a chondrogenic phenotype with minimal fibrogenic activity, while 02-09 showed an influence on both chondrogenic and fibrogenic pathways. Comparative analysis of the proteomic response to C3 or 02-09 with a single-cell RNA-sequencing dataset confirmed that C3's modulated proteins primarily correlated with a chondrogenic differentiation phenotype, with minimal fibrogenic influence. The proteins modulated by 02-09 correlated with a dual profile (both chondrogenic and fibrogenic potential). Our results suggest that C3 has potential as a targeted chondrogenic agent, while 02-09 may serve as a multifaceted modulator of both cartilage repair and ECM protection. These findings indicate the utility of these compounds for regenerative medicine and the development of disease-modifying therapies for inflammatory and immune-related conditions.