Please use this identifier to cite or link to this item: https://hdl.handle.net/10356/61757
Title: Designed antimicrobial peptides : structure-activity correlations and mode of action
Authors: Harini Mohanram
Keywords: DRNTU::Science::Biological sciences::Biophysics
Issue Date: 2013
Abstract: The discovery of antibiotics and its incredible usage in saving human lives has been considered as one of the major discoveries in medicine. However, fight against bacteria has not ended due to the emergence of resistant microbes. Super bugs or Multi- drug resistance (MDR) pathogens have become a common occurrence now. The situation is further complicated by the outburst of new diseases like H1N1, Dengue etc and its associated illnesses. The immune suppression of host system during such illness also invites other hospital infections and hence the treatment involves usage of multiple drugs. The mode of action of antibiotics is receptor mediated and hence there are high chances for the microbes to mutate such receptors and become resistance to that drug. Under such circumstances, the discovery of antimicrobial peptides incited a new path in development of future drugs, which the microbes would find it difficult to develop resistance. Lipopolysaccharides (LPS) are present in the outer layer of outer membrane of Gram negative bacteria. It serves dual role, as an important component in maintaining structural integrity of bacteria and also as membrane impermeability barrier to antibiotics and other hydrophobic substances. LPS is released in to the blood stream either during cell division or by the action of antibiotics. Such circulating LPS when accumulated in higher amounts were recognized to stimulate inflammatory pathway provoking septic shock syndrome. Sepsis is identified as one of the major mortality causing condition in hospital Intensive care units (ICU). Activity studies of antimicrobial peptides (AMPs) revealed that they could bind and neutralize the LPS before exerting its killing activity. Hence these AMPs from natural sources can be used as template for the synthesis of effective antiendotoxic antimicrobial peptides. In the current work, antimicrobial peptides were designed to characterize the structural features that govern the endotoxin neutralization and bacterial killing. First and second generation-boomerang peptides showed promising antimicrobial activity with LPS neutralization. Since N-terminal acylation and dimerization was found to increase antimicrobial activity. -boomerang peptides were acylated and dimerized through disulfide bridges in third generation. Isoleucine analogs (devoid of cysteine mediated disulphide link, YI13WF) and Cysteine analogs (YI13WFC) were further characterized for antiendotoxic antimicrobial activity. Both the analogs were also acylated with different length of acyl chains to define the appropriate hydrophobicity limit for the activity of the lipopeptides thus designed. Structure- activity correlation studies of the peptides explained that the cysteine analogs exhibited more efficient anti microbial activity than the isoleucine analogs. Further, the dimerized lipopeptides (acylated cysteine analogs) demonstrated broad spectrum of antibacterial activity with efficient LPS perturbation. Among the different chain lengths for acylation, C4 was found to be more appropriate as the increase in hydrophobicity complemented with antimicrobial antiendotoxic activity with reduced hemolytic activity. Atomic resolution structure of C4YI13WFC in aqueous state revealed that the peptide folds into characteristic boomerang shape with disulfide linkage between two cysteine residue bringing the residues W4 and F12 close together to form the aromatic fold. In the second project, the structured LPS binding motif (GG8WF) identified in second generation of -boomerang peptides was conjugated with the peptides that are found to aggregate on LPS layer, but active once trespasses the LPS layer. GG8WF peptide also found to fold into characteristic boomerang shape with aromatic fold between W2 and F7 upon interaction with LPS. GG8WF peptide was conjugated at C-terminus of Temporin A, Temporin B and synthetic K5L7 peptides that are found to aggregate on LPS layer. Three dimensional structure determination of the hybrid peptide revealed that the peptide folds into alpha helix upon membrane interaction and the positive charges in GG8 peptide initiates the binding of peptide with outer membrane. It was also found that the conjugation rendered alpha helix to boomerang motif also, but instead of the conserved aromatic lock between W2 and F7, cation-π interaction between W15 and R19 (in hybrid peptides) accounted for the remarkable activity of the hybrid peptides. When this R19 was mutated to alanine, it produced an inactive peptide. In the third project, cysteine deleted analogs of Protegrin-1 peptide, isolated from porcine leukocytes were characterized. The full length analog without cysteine, RR14 was found to be active followed by truncated analogs RR11 and LR10. The beta hairpin fold with π- π interactions between Tyr and Phe was found to be conserved, even after deletion of cysteine residue. SAR studies of these analogs revealed that the beta hairpin fold is important in exerting the antibacterial activity and that the first four residues RGGR provides the initial ionic interaction with outer membrane. When RGGR was deleted, the peptide showed impaired antibacterial activity. In the last project, antimicrobial peptides rich in arginine with appropriately placed tryptophan, leucine and isoleucine residues were designed. Increase in hydrophobic surface of the peptide confers salt resistance to antimicrobial peptides. Pharmacological development of antimicrobial peptides requires the peptide to be salt tolerant. These series of designed peptides were found to be salt tolerant and adopt alpha helix upon interaction with LPS outer membrane.
URI: https://hdl.handle.net/10356/61757
DOI: 10.32657/10356/61757
Fulltext Permission: open
Fulltext Availability: With Fulltext
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