Mitochondrial fission, a dynamic process regulated by the interaction between dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1), plays a central role in maintaining cellular homeostasis. Aberrant activation of this pathway contributes to mitochondrial fragmentation and dysfunction, underlying several pathological states including cardiovascular and neurodegenerative diseases. Targeting this protein-protein interaction (PPI) through peptidomimetic design offers a promising therapeutic strategy but is often hindered by challenges in peptide stability, cell permeability, and bioavailability.
Here, we report the rational design, solid-phase synthesis, and optimization of CVP-350, a macrocyclic analog of the linear peptide P110, previously shown to inhibit the Drp1/Fis1 interaction. The macrocyclization strategy was guided by conformational modeling and structure-activity relationship (SAR) studies aimed at enhancing both the potency and metabolic stability of the inhibitor.
The synthetic route utilized Fmoc-based SPPS with orthogonal protection strategies to enable cyclization through a side chain-to-tail linkage, resulting in a stable and conformationally constrained macrocycle. This approach allowed rapid analog generation and purification, facilitating iterative SAR studies. A library of analogs was explored by varying amino acid residues to include basic, acidic, or natural side chains, along with systematic modifications to the N- and C-termini to assess their impact on peptide conformation and Drp1 binding affinity.
CVP-350 exhibited a six-fold increase in selective inhibition of the Drp1/Fis1 interaction compared to P110, as determined by in vitro binding assays. Notably, macrocyclization significantly improved proteolytic stability, with CVP-350 showing a prolonged half-life in serum and in protease-rich cellular environments. Circular dichroism (CD) spectroscopy and molecular dynamics simulations confirmed the stabilization of a bioactive conformation upon cyclization.
Binding kinetics measured by microscale thermophoresis revealed a dissociation constant (Kd) of 282 nM, with an IC50 of 480 nM in functional assays. Importantly, CVP-350 retained bioactivity in cellular models of mitochondrial stress and was further validated in a rodent model of ischemia/reperfusion injury, where it demonstrated enhanced myocardial protection.
This work illustrates how a rational peptide macrocyclization strategy, enabled by solid-phase synthesis and structure-guided design, can significantly improve the pharmacological properties of a peptide PPI inhibitor. CVP-350 represents a promising chemical probe and therapeutic candidate for diseases involving mitochondrial dysfunction, and highlights the value of macrocyclic peptidomimetics in modern drug discovery.