Nada Ilić

Computational design of highly selective antimicrobial peptides.

We have created a structure-selectivity database (AMPad) of frog-derived, helical antimicrobial peptides (AMPs), in which the selectivity was determined as a therapeutic index (TI), and then used the novel concept of sequence moments to study the lengthwise asymmetry of physicochemical peptide properties. We found that the cosine of the angle between two sequence moments obtained with different hydrophobicity scales, defined as the D-descriptor, identifies highly selective peptide antibiotics. We could then use this descriptor to predict TI changes after point mutations in known AMPs, and to aid the prediction of TI for de novo designed AMPs. In combination with an amino acid selectivity index, a motif regularity index and other statistical rules extracted from AMPad, the D-descriptor enabled construction of the AMP-Designer algorithm. A 23 residue, glycine-rich, peptide suggested by the algorithm was synthesized and the activity and selectivity tested. This peptide, adepantin 1, is less than 50% identical to any other AMP, has a potent antibacterial activity against the reference organism, E. coli, and has a significantly greater selectivity (TI > 200) than the best AMP present in the AMPad database (TI = 125).

Knowledge-based computational methods for identifying or designing novel, non-homologous antimicrobial peptides

We describe computational approaches for identifying promising lead candidates for the development of peptide antibiotics, in the context of quantitative structure–activity relationships (QSAR) studies for this type of molecule. A first approach deals with predicting the selectivity properties of generated antimicrobial peptide sequences in terms of measured therapeutic indices (TI) for known antimicrobial peptides (AMPs). Based on a training set of anuran AMPs, the concept of sequence moments was used to construct algorithms that could predict TIs for a second test set of natural AMPs and could also predict the effect of point mutations on TI values. This approach was then used to design peptide antibiotics (adepantins) not homologous to known natural or synthetic AMPs. In a second approach, many novel putative AMPs were identified from DNA sequences in EST databases, using the observation that, as a rule, specific subclasses of highly conserved signal peptides are associated exclusively with AMPs. Both anuran and teleost sequences were used to elucidate this observation and its implications. The predicted therapeutic indices of identified sequences could then be used to identify new types of selective putative AMPs for future experimental verification.

PGLa-H tandem-repeat peptides active against multidrug resistant clinical bacterial isolates.

Antimicrobial peptides (AMPs) are promising candidates for new antibiotic classes but often display an unacceptably high toxicity towards human cells. A naturally produced C-terminal fragment of PGLa, named PGLa-H, has been reported to have a very low haemolytic activity while maintaining a moderate antibacterial activity. A sequential tandem repeat of this fragment, diPGLa-H, was designed, as well as an analogue with a Val to Gly substitution at a key position. These peptides showed markedly improved in vitro bacteriostatic and bactericidal activity against both reference strains and multidrug resistant clinical isolates of Gram-negative and Gram-positive pathogens, with generally low toxicity for human cells as assessed by haemolysis, cell viability, and DNA damage assays. The glycine substitution analogue, kiadin, had a slightly better antibacterial activity and reduced haemolytic activity, which may correlate with an increased flexibility of its helical structure, as deduced using molecular dynamics simulations. These peptides may serve as useful lead compounds for developing anti-infective agents against resistant Gram-negative and Gram-positive species.

Antibacterial Activity Affected by the Conformational Flexibility in Glycine–Lysine Based α-Helical Antimicrobial Peptides

Antimicrobial peptides often show broad-spectrum activity due to a mechanism based on bacterial membrane disruption, which also reduces development of permanent resistance, a desirable characteristic in view of the escalating multidrug resistance problem. Host cell toxicity however requires design of artificial variants of natural AMPs to increase selectivity and reduce side effects. Kiadins were designed using rules obtained from natural peptides active against E. coli and a validated computational algorithm based on a training set of such peptides, followed by rational conformational alterations. In vitro activity, tested against ESKAPE strains (ATCC and clinical isolates), revealed a varied activity spectrum and cytotoxicity that only in part correlated with conformational flexibility. Peptides with a higher proportion of Gly were generally less potent and caused less bacterial membrane alteration, as observed by flow cytometry and AFM, which correlate to structural characteristics as observed by circular dichroism spectroscopy and predicted by molecular dynamics calculations.

Broad-spectrum resistance of Pseudomonas aeruginosa from shellfish: infrequent acquisition of novel resistance mechanisms

Pseudomonas aeruginosa is one the most common multidrug-resistant pathogens worldwide. It has been previously detected in marine shellfish, but its antibiotic resistance in such environment has not been explored. By combining PCR detection of acquired genes, and resistance-nodulation-cell division (RND) efflux studying, we investigated the multifactorial resistance traits of 108 P. aeruginosa isolates recovered from wild-growing Mediterranean mussels (Mytilus galloprovincialis) in Croatia. Eleven different resistance profiles were found, with the main mechanism being the overexpression of intrinsic efflux pump(s), particularly MexAB-OprM. Several acquired resistance determinants were detected, including the β-lactamase gene blaTEM-116, sulfamethoxazole resistance gene sul1, and the class 1 integron gene cassette carrying the streptomycin resistance gene aadA7. This study evidenced the multiple resistance in P. aeruginosa in shellfish from human-impacted marine environment, pointing to the underestimated role of the marine habitat for maintenance of multiresistant P. aeruginosa and, consequently, the potential risk for human and environmental health.

Designed peptide with a flexible central motif from ranatuerins adapts its conformation to bacterial membranes

The long-standing goal in the field of peptide antibiotics has been to design lead compounds that have a wide spectrum of excellent antibacterial activity but are nontoxic to human cells. Gram-negative and Gram-positive bacteria have very different membranes, which are additionally modified in some drug-resistant species, presenting a challenge for the design of a single membrane-active peptide able to adapt its conformation to various physical properties of membrane microenvironments. In this paper, we describe how a peptide sequence can be constructed starting from an adaptable dynamic turn tandem motif in a central location. The peptide, named flexampin, has been examined firstly by molecular dynamics simulations. It uses a flexible central motif and designed helix-forming cationic amphipathic arms to form a boomerang-like, L-shape, V-shape, and hairpin, super-secondary structures, whichever is the best in matching amphipathic and hydrophobic microenvironments it encounters. Secondly, activity measurements showed that flexampin is bactericidal at low micromolar concentrations against Gram-positive and Gram-negative strains including some multidrug resistant clinical isolates, while it is nontoxic for human circulating blood cells, does not cause DNA damage, and has good selectivity for bacterial cells in comparison to human cells. It is the first membrane-active peptide designed with the ability to self-adjust the orientation of its two cationic helical arms, 3D-hydrophobic moment, and dipole moment for obtaining a better grasp of anionic polar head groups at bacterial membrane surfaces.