A small library of anticancer, cell-permeating, stapled peptides based on potent dual-specific antagonist of p53-MDM2/MDMX interactions, PMI-N8A, was synthesized, characterized and screened for anticancer activity against human colorectal cancer cell line, HCT-116. Employed synthetic modifications included: S-alkylation-based stapling, point mutations increasing hydrophobicity in key residues as well as improvement of cell-permeability by introduction of polycationic sequence(s) that were woven into the sequence of parental peptide. Selected analogue, ArB14Co, was also tested in vivo and exhibited potent anticancer bioactivity at the low dose (3.0 mg/kg). Collectively, our findings suggest that application of stapling in combination with rational design of polycationic short analogues may be a suitable approach in the development of physiologically active p53-MDM2/MDMX peptide inhibitors.

Background

Many gram-positive bacteria produce pore-forming exotoxins that contain a highly conserved, 12-residue domain (ECTGLAWEWWRT) that binds cholesterol. This domain is usually flanked N-terminally by arginine and C-terminally by valine. We used this 14-residue sequence as a template to create a small library of peptides that bind cholesterol and other lipids.

Methodology/results

Several of these peptides manifested anti-inflammatory properties in a predictive in vitro monocyte chemotactic assay, and some also diminished the pro-inflammatory effects of low-density lipoprotein in apoE-deficient mice. The most potent analog, Oxpholipin-11D (OxP-11D), contained D-amino acids exclusively and was identical to the 14-residue design template except that diphenylalanine replaced cysteine-3. In surface plasmon resonance binding studies, OxP-11D bound oxidized (phospho)lipids and sterols in much the same manner as D-4F, a widely studied cardioprotective apoA-I-mimetic peptide with anti-inflammatory properties. In contrast to D-4F, which adopts a stable alpha-helical structure in solution, the OxP-11D structure was flexible and contained multiple turn-like features.

Conclusion

Given the substantial evidence that oxidized phospholipids are pro-inflammatory in vivo, OxP-11D and other Oxpholipins may have therapeutic potential.