The mechanopharmacology of Gsmtx4 and other cysteine knot peptides from spider venom is characterized by a hydrophobic patch stabilized with charged residues surrounded by lipid molecules. These amphipathic molecules modulate membrane proteins by binding to their hydrophobic surfaces and altering the lateral pressure in the lipid bilayer. The resulting changes in depth of the peptides allow them to function as “area reservoirs” that reduce the effective magnitude of stimulus needed for activation by the mechanosensitive channels.
Molecular dynamics simulations of WT Gsmtx4 show that the peptide primarily resides in a shallow mode in an equilibrium with deeper insertion as the free volume increases. Changing the lysine residues that surround the hydrophobic face of the peptide shifts this equilibrium by reducing its stability in the shallow mode and allowing it to penetrate deeper. This shift in depth alters the lateral pressure distribution in the lipid bilayer, modulating the size of the outer monolayer and inhibiting Piezo activity.
Analysis of the kinetics of peptide-lipid binding revealed that Gsmtx4 bound to POPC primarily by its hydrophobic surface. The net association (ka) and dissociation (kd) rate constants were largely unchanged by the K-E analogs, revealing independence from the net charge of the peptide.
Gsmtx4 significantly reduced the inward current evoked by mechanical strain in CaN/NFAT1+ chondrocytes and was nearly unaffected by the NFAT inhibitor VIVIT and the CaN inhibitor CsA. Additionally, gating changes with Gsmtx4 included decreased unitary conductance and an increase in open channel life time. These effects were independent of the polarity of the membrane, and were observed in both ACLT-induced OA rats and a human articular cartilage OA model. Intraarticular injections of Gsmtx4 also ameliorated OA in rat joints by preventing cartilage degradation as assessed by HE, Safranin-O/Fast Green, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining.