ADAPTABLE is a webserver and database of antimicrobial peptides that uses sequence and property alignment to highlight their mode of action against the threat of resistance in medicine and agriculture.

HB43 (FAKLLAKLAKKLL) is a synthetic peptide active against cell lines derived from breast, colon, melanoma, lung, prostate, and cervical cancers. Despite its remarkable spectrum of activity, the mechanism of action at the molecular level has never been investigated, preventing further optimization of its selectivity. The alternation of charged and hydrophobic residues suggests amphipathicity, but the formation of alpha-helical structure seems discouraged by its short length and the large number of positively charged residues. Using different biophysical and in silico approaches we show that HB43 is completely unstructured in solution but assumes alpha-helical conformation in the presence of DPC micelles and liposomes exposing phosphatidylserine (PS) used as mimics of cancer cell membranes. Membrane permeabilization assays demonstrate that the interaction leads to the preferential destabilization of PS-containing vesicles with respect to PC-containing ones, here used as noncancerous cell mimics. ssNMR reveals that HB43 is able to fluidify the internal structure of cancer-cell mimicking liposomes while MD simulations show its internalization in such bilayers. This is achieved by the formation of specific interactions between the lysine side chains and the carboxylate group of phosphatidylserine and/or the phosphate oxygen atoms of targeted phospholipids, which could catalyze the formation of the alpha helix required for internalization. With the aim of better understanding the peptide biocompatibility and the additional antibacterial activity, the interaction with noncancerous cell mimicking liposomes exposing phosphatidylcholine (PC) and bacterial mimicking bilayers exposing phosphatidylglycerol (PG) is also described.

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Esophageal cancer is an aggressive lethal malignancy causing thousands of deaths every year. While current treatments have poor outcomes, cecropinXJ (CXJ) is one of the very few peptides with demonstrated in vivo activity. The great interest in CXJ stems from its low toxicity and additional activity against most ESKAPE bacteria and fungi. Here, we present the first study of its mechanism of action based on molecular dynamics (MD) simulations and sequence-property alignment. Although unstructured in solution, predictions highlight the presence of two helices separated by a flexible hinge containing P24 and stabilized by the interaction of W2 with target biomembranes: an amphipathic helix-I and a poorly structured helix-II. Both MD and sequence-property alignment point to the important role of helix I in both the activity and the interaction with biomembranes. MD reveals that CXJ interacts mainly with phosphatidylserine (PS) but also with phosphatidylethanolamine (PE) headgroups, both found in the outer leaflet of cancer cells, while salt bridges with phosphate moieties are prevalent in bacterial biomimetic membranes composed of PE, phosphatidylglycerol (PG) and cardiolipin (CL). The antibacterial activity of CXJ might also explain its interaction with mitochondria, whose phospholipid composition recalls that of bacteria and its capability to induce apoptosis in cancer cells.

K11 is a synthetic peptide originating from the introduction of a lysine residue in position 11 within the sequence of a rationally designed antibacterial scaffold. Despite its remarkable antibacterial properties towards many ESKAPE bacteria and its optimal therapeutic index (320), a detailed description of its mechanism of action is missing. As most antimicrobial peptides act by destabilizing the membranes of the target organisms, we investigated the interaction of K11 with biomimetic membranes of various phospholipid compositions by liquid and solid-state NMR. Our data show that K11 can selectively destabilize bacterial biomimetic membranes and torque the surface of their bilayers. The same is observed for membranes containing other negatively charged phospholipids which might suggest additional biological activities. Molecular dynamic simulations reveal that K11 can penetrate the membrane in four steps: after binding to phosphate groups by means of the lysine residue at the N-terminus (anchoring), three couples of lysine residues act subsequently to exert a torque in the membrane (twisting) which allows the insertion of aromatic side chains at both termini (insertion) eventually leading to the flip of the amphipathic helix inside the bilayer core (helix flip and internalization).

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Despite the remarkable similarity in amino acid composition, many anticancer peptides (ACPs) display significant differences in terms of activity. This strongly suggests that particular relative dispositions of amino acids (motifs) play a role in the interaction with their biological target, which is often the cell membrane. To better verify this hypothesis, we intentionally modify HB43, an ACP active against a wide variety of cancers. Sequence alignment of related ACPs by ADAPTABLE web server highlighted the conserved motifs that could be at the origin of the activity. In this study, we show that changing the order of amino acids in such motifs results in a significant loss of activity against colon and breast cancer cell lines. On the contrary, amino acid substitution in key motifs may reinforce or weaken the activity, even when the alteration does not perturb the amphipathicity of the helix formed by HB43 on liposomes mimicking their surface. NMR and MD simulations with different membrane models (micelles, bicelles, and vesicles) indicate that the activity reflects the insertion capability in cancer-mimicking serine-exposing membranes, supported by the insertion of N-terminal phenylalanine in the FAK motif and the anchoring to the carboxylate of phosphatidylserine by means of arginine side chains.