Comparative analysis of internalisation, haemolytic, cytotoxic and antibacterial effect of membrane-active cationic peptides: aspects of experimental setup
- 357 Downloads
Cationic peptides proved fundamental importance as pharmaceutical agents and/or drug carrier moieties functioning in cellular processes. The comparison of the in vitro activity of these peptides is an experimental challenge and a combination of different methods, such as cytotoxicity, internalisation rate, haemolytic and antibacterial effect, is necessary. At the same time, several issues need to be addressed as the assay conditions have a great influence on the measured biological effects and the experimental setup needs to be optimised. Therefore, critical comparison of results from different assays using representative examples of cell penetrating and antimicrobial peptides was performed and optimal test conditions were suggested. Our main goal was to identify carrier peptides for drug delivery systems of antimicrobial drug candidates. Based on the results of internalisation, haemolytic, cytotoxic and antibacterial activity assays, a classification of cationic peptides is advocated. We found eight promising carrier peptides with good penetration ability of which Penetratin, Tat, Buforin and Dhvar4 peptides showed low adverse haemolytic effect. Penetratin, Transportan, Dhvar4 and the hybrid CM15 peptide had the most potent antibacterial activity on Streptococcus pneumoniae (MIC lower than 1.2 μM) and Transportan was effective against Mycobacterium tuberculosis as well. The most selective peptide was the Penetratin, where the effective antimicrobial concentration on pneumococcus was more than 250 times lower than the HC50 value. Therefore, these peptides and their analogues will be further investigated as drug delivery systems for antimicrobial agents.
KeywordsCationic peptides Cell penetrating peptide Antimicrobial peptide Antibacterial drug carrier Haemolysis Tuberculosis
This work was supported by the Hungarian Research Fund (115431 and 104275) and by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (bo_87_15). The authors thank Dr. Hedvig Medzihradszky-Schweiger for the amino acid analysis and Mr. Sándor Dávid for the antimycobacterial testing.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This study was funded by the Hungarian Research Fund (115431 and 104275) and by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (bo_87_15).
The authors confirm that this work is new and original and not under consideration elsewhere. Our institute, the MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences, and all authors have agreed to the submission of this manuscript.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
- Baranyai Z, Krátký M, Vinšová J, Szabó N, Senoner Z, Horváti K, Stolaříková J, Dávid S, Bősze S (2015) Combating highly resistant emerging pathogen Mycobacterium abscessus and Mycobacterium tuberculosis with novel salicylanilide esters and carbamates. Eur J Med Chem 101:692–704. doi:10.1016/j.ejmech.2015.07.001 CrossRefPubMedGoogle Scholar
- Dathe M, Schümann M, Wieprecht T, Winkler A, Beyermann M, Krause E, Matsuzaki K, Murase O, Bienert M. (1996) Peptide helicity and membrane surface charge modulate the balance of electrostatic and hydrophobic interactions with lipid bilayers and biological membranes. Biochemistry 35:12612–12622. doi:10.1021/bi960835f CrossRefPubMedGoogle Scholar
- Davanço MG, Aguiar AC, Dos Santos LA, Padilha EC, Campos ML, de Andrade CR, da Fonseca LM, Dos Santos JL, Chin CM, Krettli AU, Peccinini RG (2014) Evaluation of antimalarial activity and toxicity of a new primaquine prodrug. PLoS One. doi:10.1371/journal.pone.0105217 PubMedPubMedCentralGoogle Scholar
- Horváti K, Bacsa B, Szabó N, Fodor K, Balka G, Rusvai M, Kiss É, Mező G, Grolmusz V, Vértessy B, Hudecz F, Bősze S (2015) Antimycobacterial activity of peptide conjugate of pyridopyrimidine derivative against Mycobacterium tuberculosis in a series of in vitro and in vivo models. Tuberculosis 95:S207–S211. doi:10.1016/j.tube.2015.02.026 CrossRefPubMedGoogle Scholar
- Magzoub M, Eriksson LE, Graslund A (2002) Conformational states of the cell-penetrating peptide penetratin when interacting with phospholipid vesicles: effects of surface charge and peptide concentration. Biochim Biophys Acta 1563:53–63. doi:10.1016/S0005-2736(02)00373-5 CrossRefPubMedGoogle Scholar
- Saberwal G, Nagaraj R (1994) Cell-lytic and antibacterial peptides that act by perturbing the barrier function of membranes: facets of their conformational features, structure-function correlations and membrane-perturbing abilities. Biochim Biophys Acta 1197:109–131. doi:10.1016/0304-4157(94)90002-7 CrossRefPubMedGoogle Scholar
- Stenger S, Hanson DA, Teitelbaum R, Dewan P, Niazi KR, Froelich CJ, Ganz T, Thoma-Uszynski S, Melián A, Bogdan C, Porcelli SA, Bloom BR, Krensky AM, Modlin RL (1998) An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 282:121–125. doi:10.1126/science.282.5386.121 CrossRefPubMedGoogle Scholar
- Sula L (1963) Who co-operative studies on a simple culture technique for the isolation of mycobacteria. 1. Preparation, lyophilization and reconstitution of a simple semi-synthetic concentrated liquid medium; culture technique; growth pattern of different mycobacteria. Bull World Health Organ 29:589–606 (PMCID: PMC2555071)PubMedPubMedCentralGoogle Scholar
- Sula L, Sundaresan TK (1963) Who co-operative studies on a simple culture technique for the isolation of mycobacteria. 2. Comparison of the efficacy of lyophilized liquid medium with that of Loewenstein–Jensen (L–J) medium. Bull World Health Organ 29:607–625 (PMCID: 2555071)PubMedPubMedCentralGoogle Scholar
- Toniolo C, Formaggio F, Woody RW (2012) Electronic circular dichroism of peptides. In: Berova N, Polavarapu PL, Nakanishi K, Woody RW (eds) Comprehensive chiroptical spectroscopy: applications in stereochemical analysis of synthetic compounds, natural products, and biomolecules, vol 2, pp 499–544. doi:10.1002/9781118120392.ch15