Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability.
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.



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.



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.



Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore Section of Paediatrics, Department of Medicine, St Mary's Campus, Imperial College London, London W2 1PG, UK MRC Centre for Molecular Microbiology and Infection, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
Jasmeet Singh
JS
Khara
MRC Centre for Molecular Microbiology and Infection, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
Miles
M
Priestman
MRC Centre for Molecular Microbiology and Infection, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
Iria
I
Uhía
Section of Paediatrics, Department of Medicine, St Mary's Campus, Imperial College London, London W2 1PG, UK.
Melissa Shea
MS
Hamilton
MRC Centre for Molecular Microbiology and Infection, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
Nitya
N
Krishnan
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore.
Ying
Y
Wang
Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore.
Yi Yan
YY
Yang
Section of Paediatrics, Department of Medicine, St Mary's Campus, Imperial College London, London W2 1PG, UK.
Paul R
PR
Langford
Section of Paediatrics, Department of Medicine, St Mary's Campus, Imperial College London, London W2 1PG, UK.
Sandra M
SM
Newton
MRC Centre for Molecular Microbiology and Infection, Department of Medicine, Imperial College London, London SW7 2AZ, UK.
Brian D
BD
Robertson
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore phaeplr@nus.edu.sg.
Pui Lai Rachel
PL
Ee
D000596:Amino Acids / Q000494:pharmacology* / Q000633:toxicity; D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology* / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D008264:Macrophages / Q000382:microbiology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D051379:Mice; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D018613:Microscopy, Confocal; D009169:Mycobacterium tuberculosis / Q000187:drug effects*; D010455:Peptides / Q000494:pharmacology* / Q000633:toxicity; D000067996:RAW 264.7 Cells; D059008:Time-Lapse Imaging
D000596:Amino Acids; D000995:Antitubercular Agents; D008565:Membrane Proteins; D010455:Peptides