Membrane Association Modes of Natural Anticancer Peptides: Mechanistic Details on Helicity, Orientation, and Surface Coverage.

Anticancer peptides (ACPs) could potentially offer many advantages over other cancer therapies. ACPs often target cell membranes, where their surface mechanism is coupled to a conformational change into helical structures. However, details on their binding are still unclear, which would be crucial to reach progress in connecting structural aspects to ACP action and to therapeutic developments. Here we investigated natural helical ACPs, Lasioglossin LL-III, Macropin 1, Temporin-La, FK-16, and LL-37, on model liposomes, and also on extracellular vesicles (EVs), with an outer leaflet composition similar to cancer cells. The combined simulations and experiments identified three distinct binding modes to the membranes. Firstly, a highly helical structure, lying mainly on the membrane surface; secondly, a similar, yet only partially helical structure with disordered regions; and thirdly, a helical monomeric form with a non-inserted perpendicular orientation relative to the membrane surface. The latter allows large swings of the helix while the N-terminal is anchored to the headgroup region. These results indicate that subtle differences in sequence and charge can result in altered binding modes. The first two modes could be part of the well-known carpet model mechanism, whereas the newly identified third mode could be an intermediate state, existing prior to membrane insertion.

Peptide-based chemical models for lytic polysaccharide monooxygenases.

Copper(II) complexes of HPH-NH2 (L1) and HPHPY-NH2 (L2) peptides have been studied as small molecular models of lytic polysaccharide monooxygenases by pH-potentiometry and UV-vis, CD and EPR spectroscopy. The coordination properties of these ligands are fundamentally different from those of other non-protected N-terminal HXH-sequences concerning the metal binding ability of amide nitrogens. The proline units prevent the formation of fused chelates with the participation of amide nitrogens; therefore, instead of ATCUN-type {NH2,2N-,Nim} coordination, dimer complexes (Cu2HxL2, where x = -1, -2, and -3 for L1 and 1, 0, and -1 for L2) are formed in equimolar systems above pH 5. Using H2O2 as the oxidant and PNPG as the activated substrate, these dimer complexes were proved to be relevant functional models of LPMOs, even at neutral pH. Although the tyrosine residue in L2 participates in the coordination at pH 7-9.6, it does not seem to play a role in the oxidation process. In the presence of H2O2, the dimer complexes partially dissociate to form mononuclear hydroperoxo complexes, which are stable for 1-2 hours in equimolar concentrations of H2O2. On the other hand, with excess H2O2 both their formation and their decomposition are faster. The decay of (hydro)peroxo complexes, after longer reaction times, results in the evolution of dioxygen bubbles and the formation of Cu(I) (probably through catalytic disproportionation). However, in the presence of PNPG, the formation of dioxygen bubbles was not observed. Therefore, we assumed that the formed Cu(I) complexes bind H2O2 and enter into a similar catalytic cycle as suggested recently for native LPMOs.

New short cationic antibacterial peptides. Synthesis, biological activity and mechanism of action.

We report a theoretical and experimental study on a new series of small-sized antibacterial peptides. Synthesis and bioassays for these peptides are reported here. In addition, we evaluated different physicochemical parameters that modulate antimicrobial activity (charge, secondary structure, amphipathicity, hydrophobicity and polarity). We also performed molecular dynamic simulations to assess the interaction between these peptides and their molecular target (the membrane). Biophysical characterization of the peptides was carried out with different techniques, such as circular dichroism (CD), linear dichroism (LD), infrared spectroscopy (IR), dynamic light scattering (DLS), fluorescence spectroscopy and TEM studies using model systems (liposomes) for mammalian and bacterial membranes. The results of this study allow us to draw important conclusions on three different aspects. Theoretical and experimental results indicate that small-sized peptides have a particular mechanism of action that is different to that of large peptides. These results provide additional support for a previously proposed four-step mechanism of action. The possible pharmacophoric requirement for these small-sized peptides is discussed. Furthermore, our results indicate that a net +4 charge is the adequate for 9 amino acid long peptides to produce antibacterial activity. The information reported here is very important for designing new antibacterial peptides with these structural characteristics.