Antimicrobial and Anti-inflammatory Activities and Mechanism of Action of Cathelicidin-derived Synthetic Antimicrobial Peptides

Abstract

ABSTRACT

Antimicrobial and Anti-inflammatory Activities and Mechanism of Action of Cathelicidin-derived Synthetic Antimicrobial Peptides

Binu Jacob Advisor: Prof. Song Yub Shin Department of Bio-Materials Graduate School of Chosun University

PART Ⅰ

KR-12 (residues 18-29 of LL-37) was known to be the smallest peptide of human cathelicidin LL-37 possessing antimicrobial activity. In order to optimize -helical short antimicrobial peptides having both antimicrobial and antiendotoxic activities without mammalian cell toxicity, we designed and synthesized a series of KR-12 analogs. Highest hydrophobic analogs KR-12-a5 and KR-12-a6 displayed greater inhibition of LPS-stimulated TNF-production and higher LPS-binding activity. We have observed that antimicrobial activity is independent of charge but LPS neutralization requires a balance of hydrophobicity and net positive charge. Among KR-12 analogs, KR-12-a2, KR-12-a3 and KR-12-a4 showed much higher cell specificity for bacteria over erythrocytes and retained antiendotoxic activity, relative to parental LL-37. KR-12-a5 displayed the strongest antiendotoxic activity, whereas almost similar cell specificity as compared to LL-37. Also, these KR-12 analogs (KR-12-a2, KR-12-a3, KR-12-a4 and KR-12-a5) exhibited potent antimicrobial activity (MIC: 4 M) against methicillin-resistant Staphylococcus aureus. Taken together, these KR-12 analogs have the potential for future development as novel class of antimicrobial and anti-inflammatory therapeutic agents.

PART Ⅱ

To develop short antimicrobial peptide (AMPs) with improved cell selectivity and reduced mammalian cell toxicity compared to sheep myeloid antimicrobial peptide-29 (SMAP-29) and elucidate the possible mechanisms responsible for their antimicrobial action, we synthesized an N-terminal 18-residues peptide amide (SMAP-18) from SMAP-29 and its Trp-substituted analog (SMAP-18-W). Due to their reduced hemolytic activity and retained antimicrobial activity, SMAP-18 and SMAP-18-W showed higher cell selectivity than SMAP-29. In addition, SMAP-18 and SMAP-18-W had no cytotoxicity against three different mammalian cells such as RAW 264.7, NIH-3T3 and HeLa cells even at 100 M. These results suggest that SMAP-18 and SMAP-18-W has potential for future development as novel therapeutic antimicrobial agent. Unlike SMAP-29, SMAP-18 and SMAP-18-W showed relatively weak ability to induce dye leakage from bacterial membrane-mimicking liposomes, N-phenyl-1-napthylamine (NPN) uptake and o-nitrophenyl--galactoside (ONPG) hydrolysis. Similar to SMAP-29, SMAP-18-W led to a significant membrane depolarization (>80%) against Staphylococcus aureus at 2  MIC. In contrast, SMAP-18 didn’t cause any membrane depolarization even at 4  MIC. In confocal laser scanning microscopy, we observed translocation of SMAP-18 across the membrane in a non-membrane disruptive manner. SMAP-29 and SMAP-18-W was unable to translocate the bacterial membrane. Collectively, we propose here that SMAP-29 and SMAP-18-W kill microorganisms by disrupting/perturbing the lipid bilayer and forming pore/ion channels on bacterial cell membranes, respectively. In contrast, SMAP-18 may kill bacteria via intracellular-targeting mechanism.

PART III

Sheep myeloid antimicrobial peptide-29 (SMAP-29) is a potent but equally cytotoxic antimicrobial peptide. Its highly hydrophobic C-terminal region (19-29 residues) containing three isoleucines is responsible for hemolytic and cytotoxic activities. To study the membrane interaction of this hydrophobic region, D-enantiomer (SMAP-29D), and diastereomer (SMAP-29DA) containing D-allo-isoleucines had prepared. To improve cell selectivity and LPS neutralization ability of SMAP-29, 2 isomers consist of L-form (1-18 residues) and D-form (19-28 residues) segments were also synthesized (SMAP-29HD and SMAP-29HDA). The non-hemolytic N-terminal region (SMAP-18, 1-18 residues) is having an intracellular target. We had prepared D-enantiomer (SMAP-18D) and diastereomer (SMAP-18DA) of SMAP-18 to probe the cytoplasmic chiral interaction and to study the membrane penetrating efficiency of enantiomeric peptides. Except SMAP-29HD and SMAP-29HDA, all peptides showed a typical -helical conformation in the presence of SDS or LPS. SMAP-29DA, SMAP-29HD and SMAP-29HDA had significantly lesser relative hydrophobicity, hemolytic activity, and several fold increase in therapeutic index than the corresponding L- and D-enantiomer of SMAP-29. SMAP-29 peptides but not SMAP-18 peptides showed a powerful inhibition of LPS-induced production of NO, TNF-α, IL-6 and MCP-1 in RAW264.7 cells. SMAP-29HD and SMAP-29HDA were less cytotoxic to RAW264.7 cells. SMAP-29 peptides showed instant membrane depolarization against Staphylococcus aureus, but SMAP-18 peptides did not induce membrane depolarization. SMAP-29L, SMAP-29D and SMAP-29DA readily released dye from bacterial membrane mimicking liposomes. SMAP-29HD and SMAP-29HDA took longer time to reach full release of dye from liposomes. SMAP-18D and SMAP-18DA showed 4- or 8-fold higher antimicrobial activity against five bacteria in broth dilution assay. In agar diffusion assay, SMAP-18L had smaller but distinct clear zone but SMAP-18D and SMAP-18DA had border but blurred clear zone. Both SMAP-18L and SMAP-18D peptides binds with bacterial plasmid DNA at 2M. SMAP-18L killed E. coli faster than SMAP-18D. Both SMAP-29 and SMAP-18 peptides showed good activity against 3 MRSA strains.