title: Designing α-helical peptides with enhanced synergism and selectivity against Mycobacterium smegmatis: Discerning the role of hydrophobicity and helicity.
abstract: Recently, we reported on a series of short amphipathic α-helical peptides, comprising the backbone sequence (LLKK)2, with the ability to kill susceptible and drug-resistant Mycobacterium tuberculosis. In this study, the effect of key physicochemical parameters including hydrophobicity and helicity of α-helical peptides on anti-mycobacterial activity and synergism with rifampicin was investigated. The most hydrophobic analogue, W(LLKK)2W, displayed low selectivity against mycobacteria while peptides with intermediate hydrophobicity were shown to be equally active, yet significantly less toxic. Furthermore, proline substitution impeded the formation of stable amphipathic structures, rendering P(LLKK)2P as one of the least active analogues. Terminal capping with isoleucine was found to promote α-helical folding and the resultant peptide demonstrated the highest selectivity and minimal cytotoxicity against mammalian macrophages. Flow cytometric analysis revealed that enhancements in hydrophobicity and α-helicity increased the rate and extent of peptide-mediated membrane permeabilization. This finding corroborated the hypothesis that synergism between the peptides and rifampicin was likely mediated via peptide-induced pore formation. The rapid, concentration-dependent membrane depolarization, leakage of intracellular ATP and calcein release from PE/PG LUVs supported the membrane-lytic mechanism of action of the peptides. Together, these findings suggest that hydrophobicity and α-helicity significantly impact anti-mycobacterial activity and optimization of both parameters is necessary to develop synthetic analogues with superior selectivity indices and enhanced synergistic potential with conventional antibiotics.
  
  
  CONCLUSIONS
  There is an urgent clinical need for the discovery of new antimicrobials, effective not just for drug susceptible, but also rapidly emerging drug-resistant TB. Recently, we reported on a series of short amphipathic α-helical peptides, comprising the backbone sequence (LLKK)2, with the ability to kill susceptible and drug-resistant M. tuberculosis. In this study, we evaluated a series of synthetic α-helical (LLKK)2 peptides over a range of hydrophobicities for their activity against mycobacteria and provide the first report on the modulating effect of hydrophobicity and α-helicity on the antimicrobial mechanisms of synthetic AMPs and their synergism with first-line antibiotics. These findings demonstrate the applicability of strategies employed here for the rational design of AMPs with the aim of improving cell selectivity and synergistic interactions when co-administered with first-line antibiotics in the fight against drug-resistant tuberculosis.
authors.affiliation: Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Jasmeet Singh
authors.initials: JS
authors.lastname: Khara
authors.affiliation: Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Fang Kang
authors.initials: FK
authors.lastname: Lim
authors.affiliation: Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore.
authors.forename: Xi-Yu
authors.initials: XY
authors.lastname: Ke
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore.
authors.forename: Zhi Xiang
authors.initials: ZX
authors.lastname: Voo
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, Singapore 138669, Singapore.
authors.forename: Yi Yan
authors.initials: YY
authors.lastname: Yang
authors.affiliation: Singapore Eye Research Institute, 11 Third Hospital Avenue, Singapore 168751, Singapore; Duke-NUS Medical School, SRP Neuroscience and Behavioral Disorders, Singapore 169857, Singapore.
authors.forename: Rajamani
authors.initials: R
authors.lastname: Lakshminarayanan
authors.affiliation: Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543, Singapore. Electronic address: phaeplr@nus.edu.sg.
authors.forename: Pui Lai Rachel
authors.initials: PLR
authors.lastname: Ee
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000494:pharmacology; D002460:Cell Line; D002942:Circular Dichroism; D006461:Hemolysis / Q000187:drug effects; D057927:Hydrophobic and Hydrophilic Interactions; D008264:Macrophages / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D008855:Microscopy, Electron, Scanning; D020102:Mycobacterium smegmatis / Q000187:drug effects*; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology; D011487:Protein Conformation; D051381:Rats
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides
keywords: Amphipathic α-helix; Antimicrobial peptides; Hydrophobicity; Mycobacterium tuberculosis; Synergy