Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability
File(s)
Accepted version
Author(s)
Type
Journal Article
Abstract
Objectives The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities.
Methods Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy.
Results Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all D-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified L-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm.
Conclusions Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.
Methods Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy.
Results Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all D-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified L-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm.
Conclusions Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.
Date Issued
2016-04-26
Date Acceptance
2016-03-01
Citation
Journal of Antimicrobial Chemotherapy, 2016, 71 (8), pp.2181-2191
ISSN
1460-2091
Publisher
Oxford University Press (OUP)
Start Page
2181
End Page
2191
Journal / Book Title
Journal of Antimicrobial Chemotherapy
Volume
71
Issue
8
Copyright Statement
2017-04-26
This is a pre-copyedited, author-produced PDF of an article accepted for publication in Journal of Antimicrobial Chemotherapy following peer review. The version of record Jasmeet Singh Khara, Miles Priestman, Iria Uhía, Melissa Shea Hamilton, Nitya Krishnan, Ying Wang, Yi Yan Yang, Paul R. Langford, Sandra M. Newton, Brian D. Robertson, and Pui Lai Rachel Ee
Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability
J. Antimicrob. Chemother. (2016) 71 (8): 2181-2191 first published online April 26, 2016 is available online at: https://dx.doi.org/10.1093/jac/dkw107
Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability
J. Antimicrob. Chemother. (2016) 71 (8): 2181-2191 first published online April 26, 2016 is available online at: https://dx.doi.org/10.1093/jac/dkw107
Sponsor
British Society for Antimicrobial Chemotherapy
National Medical Res. Council
Grant Number
GA2011-01P
RL2012-001
Subjects
Microbiology
1115 Pharmacology And Pharmaceutical Sciences
0605 Microbiology
1108 Medical Microbiology
Publication Status
Published