Research Article: Retro analog concept: comparative study on physico-chemical and biological properties of selected antimicrobial peptides

Date Published: July 29, 2017

Publisher: Springer Vienna

Author(s): Damian Neubauer, Maciej Jaśkiewicz, Dorian Migoń, Marta Bauer, Karol Sikora, Emilia Sikorska, Elżbieta Kamysz, Wojciech Kamysz.


Increasing drug resistance of common pathogens urgently needs discovery of new effective molecules. Antimicrobial peptides are believed to be one of the possible solutions of this problem. One of the approaches for improvement of biological properties is reversion of the sequence (retro analog concept). This research is based on investigation of antimicrobial activity against Gram-positive, Gram-negative bacteria, and fungi, hemolysis of erythrocytes, interpretation of the circular dichroism spectra, measurement of counter-ion content, and assessment of the peptide hydrophobicity and self-assembly using reversed-phase chromatography. The experiments were conducted using the following peptides: aurein 1.2, CAMEL, citropin 1.1, omiganan, pexiganan, temporin A, and their retro analogs. Among the compounds studied, only retro omiganan showed an enhanced antimicrobial and a slightly increased hemolytic activity as compared to parent molecule. Moreover, retro pexiganan exhibited high activity towards Klebsiella pneumoniae, whereas pexiganan was in general more or equally active against the rest of tested microorganisms. Furthermore, the determined activity was closely related to the peptide hydrophobicity. In general, the reduced hemolytic activity correlates with lower antimicrobial activity. The tendency to self-association and helicity fraction in SDS seems to be correlated. The normalized RP-HPLC—temperature profiles of citropin 1.1 and aurein 1.2, revealed an enhanced tendency to self-association than that of their retro analogs.

Partial Text

As a result of the widespread use of antibiotics, the appearance of infections caused by resistant organisms is on the rise. According to the reports of World Health Organization (WHO), the issue of antibiotic resistance is no longer the prediction for the future but it is the real problem nowadays (World Health Organization 2014). Since the treatment of common infections in the community and hospitals is being more difficult, patients remain infectious for a longer time and the healthcare costs rise. Acquisition of the non-susceptibility to antibiotics is also related to the circulation of genes in the environment and resistance determinants can be easily transferred to pathogenic microorganisms. The spread of these pathogens has been related to various epidemiological factors such as the lack of appropriate procedures in the healthcare facilities and the international transfer of patients coming from endemic areas (e.g., spread of carbapenem-resistant Klebsiella pneumoniae in Europe) (Girmenia et al. 2016). Increasing rate of multi-resistant bacteria forces the need of development of new antimicrobial substances that could be applicable in therapy.

All of the studied compounds exhibited antimicrobial activity against most of the tested strains; although, essentially different. Most of the tested peptide and retro peptide pairs exhibited a lower activity of original molecules, except r-omiganan. Additionally, omiganan differs from the remaining retro analogs by its higher hemolytic activity compared to the parent peptide. This study focuses partially on the interplay between peptide molecules. Thanks to chromatographic analysis over the wide temperature range it was possible to assess peptides’ tendency to self-association which was as a rule different between peptides in pairs. Being aware of the influence of self-association on retention time, it was expedient to apply the temperature gradient, this providing a reliable point of view on the peptides’ hydrophobicity. Moreover, the tendency to self-association and helicity fraction (fH) in SDS seems to be correlated. The normalized RP-HPLC—temperature profiles of citropin 1.1 and aurein 1.2, revealed an enhanced tendency to self-association than that of their retro analogs. Considering their mode of action, this tendency is likely to be involved in antimicrobial activity. It seems that it is possible to predict a substantial effect of sequence reversion on peptide hydrophobicity using side-chain hydrophobicity coefficients and thus to expect particular change in antimicrobial activity—enhance when hydrophobicity is increasing. However, this approach may fail if the difference is not high enough as it was found in this study (pexiganan and temporin A). Our results indicate that it is possible to reduce the number of those fails. If calculations based on hydrophobicity coefficients indicate that retro analog will be substantially more hydrophilic, there is a high risk that it will also be less antimicrobial active. Nevertheless, this approach does not allow to predict all changes in the biological activity, e.g., increase in activity against particular strain in antimicrobial assay (r-pexiganan and K. pneumoniae). Hypothetically, to increase the chance of obtaining more active compounds, retro analogs should be synthesized after initial calculations. However, this approach needs further studies to verify its correctness, range of applicability, and to determine guidelines to follow. Limited scope of this work does not allow us to draw more general statements but only to indicate which aspects should be taken into account. In conclusion, the retro analog concept applied to antimicrobial peptides presumably may contribute to development of more active peptides.




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