Anti-mycobacterial activities of synthetic cationic a-helical peptides and their synergism with rifampicin Jasmeet S. Khara a , Ying Wang a , Xi-Yu Ke b , Shaoqiong Liu b , Sandra M. Newton c , Paul R. Langford c , Yi Yan Yang b, ** , Pui Lai Rachel Ee a, * a Department of Pharmacy, National University of Singapore,18 Science Drive 4, Singapore 117543, Singapore b Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore c Section of Paediatrics, Division of Medicine, St Mary’s Campus, Imperial College, London W2 1PG, United Kingdom article info Article history: Received 20 August 2013 Accepted 13 November 2013 Available online 4 December 2013 Keywords: Antimicrobial peptides a-Helical Drug resistance Synergy Mycobacterium tuberculosis abstract The rapid emergence of multi-drug resistant tuberculosis (TB) and the lack of effective therapies have prompted the development of compounds with novel mechanisms of action to tackle this growing public health concern. In this study, a series of synthetic cationic a-helical antimicrobial peptides (AMPs) modified with different hydrophobic amino acids was investigated for their anti-mycobacterial activity, both alone and in synergistic combinations with the frontline anti-tuberculosis drug rifampicin. The addition of thiol groups by incorporating cysteine residues in the AMPs did not improve anti- mycobacterial activity against drug-susceptible and drug-resistant Mycobacterium tuberculosis, while the enhancement of peptide hydrophobicity by adding methionine residues increased the efficacy of the primary peptide against all strains tested, including clinically isolated multidrug-resistant mycobacteria. The peptide with the optimal composition M(LLKK) 2 M was bactericidal, and eradicated mycobacteria via a membrane-lytic mechanism as demonstrated by confocal microscopic studies. Mycobacteria did not develop resistance after multiple exposures to sub-lethal doses of the peptide. In addition, the peptide displayed synergism with rifampicin against both Mycobacterium smegmatis and Mycobacterium bovis BCG and additivity against M. tuberculosis. Moreover, such combination therapy is effective in delaying the emergence of rifampicin resistance. The ability to potentiate anti-TB drug activity, kill drug-resistant bacteria and prevent drug resistance highlights the potential utility of the peptide in combating multidrug-resistant TB. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Tuberculosis (TB) is a preventable and curable infectious disease caused by Mycobacterium tuberculosis. However, it remains a major cause of mortality and morbidity worldwide as the effectiveness of standard treatment is often compromised by side effects, patients’ failure to complete the lengthy course of treatment and the development of drug resistance in the form of multi-drug resistant (MDR) and extensively drug resistant (XDR) TB [1]. The current available treatment for MDR-TB not only requires a longer period of time, but is also less efficacious, more expensive and more toxic than the standard treatment [1,2], presenting a double whammy for TB treatment and control. New anti-TB drug development has generally been slow, with bedaquiline being the first drug to be approved in 40 years by the US Food and Drug Administration (FDA) last year [3]. Inspired by nature, many antimicrobial peptides (AMPs) are currently under clinical development for treating various bacterial infections [4]. There is a huge diversity in sequences although their common cationic and amphiphilic nature is apparent [5]. With their overall positive charges, AMPs contact the bacterial cell sur- faces by associating with the acidic polymers and other negatively charged molecules, after which they insert themselves into the membrane and disrupt its physical integrity via membrane thin- ning, transient pore formation and/or disruption of the barrier function [6e8]. Some studies have also shown that they are capable of translocating across the membrane and acting on internal targets for bacterial kill [9]. In the TB context, even though the composition of the mycobacterial cell wall is inherently different with a higher proportion of lipids such as mycolic acids, natural AMPs, including cathelicidin LL-37 and defensin human neutrophil peptides (HNPs), * Corresponding author. Tel.: þ65 6516 2653; fax: þ65 6779 1554. ** Corresponding author. Tel.: þ65 6824 7106; fax: þ65 6478 9084. E-mail addresses: yyyang@ibn.a-star.edu.sg (Y.Y. Yang), phaeplr@nus.edu.sg (P.L.R. Ee). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biomaterials.2013.11.035 Biomaterials 35 (2014) 2032e2038