Structural characterization and antimicrobial activity of innovative compounds: a novel strategy for in silico peptides design

Infectious diseases account for approximately 13.3 million deaths worldwide each year. With the increasing emergence of multidrugresistant microorganisms, the demand for new and more effective antimicrobial agents has never been more urgent and several strategies have been so far used to develop novel antimicrobial peptide agents. Recently it has been reported that different antimicrobial peptides exhibit an immediate cytotoxic effect against cancer cells, selectively causing a mitochondrial membrane damage and subsequent triggering apoptosis [1]. Therefore, it can be supposed that mitochondrial targeting peptides could also display antimicrobial activity by binding and disrupting the plasma membrane of bacterial cells. In this context, two peptides named rNterC and rTM2C including 13 and 15 amino acids, respectively, were projected starting from protein sequences which are known to bind or to be targeted to the outer mitochondrial membrane. By site-directed mutation modeling using wild-type sequences as scaffolds, the peptides were modified to possibly increase antimicrobial potency taking into account several important aspects specifically concerning the appropriate balance among the total net charge, amphipathicity and hydrophobicity [2]. Usually, a large proportion of antimicrobial peptides are unstructured in aqueous solution but they adopt amphipathic a-helices when bound to a phospholipid bilayer. However, in different solvents (such as TFE) these peptides can change conformation if they have a tendency to fold. Aim of the study was to investigate the structural properties of the two peptides and to evaluate their antimicrobial activity against Listeria monocytogenes (NCTC 11994-HPA--London) using different concentrations (10, 50, 100 lg/mL); for the assays we suspended a lenticula of L. monocytogenes (4.3 9 103-2.1 9 104 UFC/lenticule disc) and the growth of the microorganism was observed in two different culture media: blood agar (Biolife-Italia) and ALOA medium (Biolife-Italia). Results revealed that the highest concentration produced the best antimicrobial activity for both peptides analyzed. Specifically, at 100 lg/mL concentration, rTM2C efficiently inhibited the growth of L. monocytogenes by 98.5 or 99.0 %, on blood agar or ALOA medium, respectively, while at the same concentration rNterC inhibited the growth of L. monocytogenes, by 94.7 or 95.5 %, on the two culture media, respectively. Finally, circular dichroism (CD) analyses performed in different environment and temperature conditions revealed that rTM2C assumed a a-helix structure in TFE 50 % which was stable until 50 C and a combined a-helix/b-turn structure in 3 and 10 mM SDS which remained stable until 90 C. In contrast the rNterC peptide showed CD spectra typical of a random-coil structure in all experimental conditions investigated. Moreover, preliminary studies by NMR spectroscopy suggested a large number of inter-residue NH-NH contacts in rTM2C and an ahelix structure at the C-terminal region even if the spectra were acquired in a strong denaturing solvent such as DMSO.