Backbone N-Methylation Improves PPCA-Binding of a Stylissatin A Analogue via Conformational Modulation

Obesity is associated with dysregulated intracellular signaling that controls metabolic homeostasis and inflammatory responses. The marine-derived cyclic heptapeptide stylissatin A (SA) and its analog d-Tyr¹(tBu)-SA (1) localize to lysosomes and inhibit the interaction between the protective protein cathepsin A (PPCA) and neuraminidase 1 (Neu1), thereby promoting lipid droplet degradation and suppressing fat accumulation. To improve cellular uptake and therapeutic efficacy, we introduced N-methylation into the macrocyclic backbone of 1 and synthesized a new analog, d-Tyr¹(tBu)-N-Me-Ile⁴-SA (2), via solid-phase peptide synthesis followed by solution-phase macrocyclization. N-methylation at Ile⁴ induced a marked conformational change from cis-Pro³ to trans-Pro³ and increased conformational flexibility in solution. Upon binding to PPCA, this flexibility triggered a transition at Pro⁵ from trans to cis. The enhanced hydrophobicity further promoted favorable interactions within the binding pocket. Consequently, 2 exhibited improved anti-inflammatory and similar anti-adipogenic activities compared to 1. Furthermore, the biotin analog of 2 exhibited a higher binding affinity to activated PPCA with a K_D value 29-fold smaller than that of 1, demonstrating the functional advantage of backbone N-methylation. This PPCA-Neu1 interaction-inhibitory mechanism differs from that of glucagon-like peptide-1 (GLP-1) receptor agonists, highlighting a distinct intracellular therapeutic strategy for the development of next-generation anti-obesity agents.