초록 ▼

1. $\alpha-helix$형 항생펩타이드인 IsCT, Piscidin, Pleulocidin, HP(2-20)의 삼차원 구조를 NMR로 연구하고 구조를 바탕으로 고기능성 항생펩타이드를 개발함.
2. 모델형 ${\alpha}$-helix구조 펩타이드(KLW)의 중앙부위의 hydrophobic helix face에 두 개의 Ala-peptoid를 치환시킨 $KLW-L9,13-\alpha\;(KWKKLLKKaLKLaKKLLK-NH_2$, a: Ala peptoid re

1. $\alpha-helix$형 항생펩타이드인 IsCT, Piscidin, Pleulocidin, HP(2-20)의 삼차원 구조를 NMR로 연구하고 구조를 바탕으로 고기능성 항생펩타이드를 개발함.
2. 모델형 ${\alpha}$-helix구조 펩타이드(KLW)의 중앙부위의 hydrophobic helix face에 두 개의 Ala-peptoid를 치환시킨 $KLW-L9,13-\alpha\;(KWKKLLKKaLKLaKKLLK-NH_2$, a: Ala peptoid residue)가 인체독성이 가장 없으며 높은 항균활성을 나타내었다.
3. $\beta-turn$구조 항균 펩타이인 tritrpticin의 5번째 및 9번째의 Pro을 Lys-peptoid residue로 치환한 $TPk\;(WRRFkWWWkFLRR-NH_2$, k: Lys peptoid residue)가 인체독성이 가장 없으며 높은 항균활성을 나타내었다.
4. Disulfide bnd를 갖는 갯지렁이에서 유래된 항균 펩타이드인 arenicine과 그 유도체의 구조를 연구 한 결과 V10, R11, G12번에서 turn이 이루어지고 antiparrallel $\beta-sheet$ structure를 가지고 있음을 밝힘.
5. Peptoid 합성, D-아미노산, alpha-helix, beta-turn, beta-sheet 모방형 펩타이드 합성에 필요한 각종 비자연계 아미노산을 비대칭 합성하였다.
5. CRAMP항생펩타이드의 대량생산을 위해 GST-CRAMP 발현시스템을 최적화하였다. 또한$^{15}N$ label된 CRAMP를 발현, 정제하여 구조 및 동력학 연구를 수행하였다.
6. 구조-활성간 상관관계 연구결과 숙주세포에 독성을 나타내지 않고 강한 항균활성을 나타내는 8종의 박테리아세포 특이성 고기능성 펩타이드를 개발 하였다 (KLW-L9-a, KLW-L9,13-a, Pis-PG, $(K^7,\;P^8,\;K^{11})-IsCT,\;IsCT-1,\;IsCT-2,\;IsCT-3$ 및 TPk). 박테리아 세포특이성을 가진 $KLW-L9,13-\alpha$ 및 TPk는 박테리아 세포내의 intracellular function의 inhibition에 의한 작용기작으로 항생활성을 알았다.

Abstract ▼

Many antimicrobial peptides have been isolated from a wide range of animal, plant and bacterial species. Antimicrobial peptides are known to play important roles in the host defense system and innate immunity of all-living species. These peptides have stimulated enormous interest as a new class of a

Many antimicrobial peptides have been isolated from a wide range of animal, plant and bacterial species. Antimicrobial peptides are known to play important roles in the host defense system and innate immunity of all-living species. These peptides have stimulated enormous interest as a new class of antibiotics with potential clinical value in the fight against multi drug resistant microorganisms. Their antimicrobial activities are related to their ability to adopt amphipathic structure including $\alpha-helices,\;\beta-sheet\;or\;\beta-turn.$ Their mode of antibiotic action appears to involve depolarization or permeabilization of the bacterial cell membrane, although the detail mechanisms are still not fully understood.
Proline is a potent ${\alpha}$-helix breaker, due to the lack of a hydrogen-bonding donor group and steric hindrance of hydrogen bonding to adjacent residues. Despite these unfavorable properties, proline residues have been found in the transmembrane helices of integral membrane proteins, and in a number of $\alpha-helical$ antimicrobial peptides that promote ion channel activity, including melittin, cecropin A, pardaxin, brevinins, gaegurin and buforin II. Peptoid residues, like proline, lack an amide nitrogen as a proton-donating group for hydrogen bonding to peptide backbone. Thus, the replacement of Pro residues with peptoid residues not only disrupts ${\alpha}$-helical structure but also induces a $\beta-turn$ in the tertiary structure of the peptide. To our knowledge, the effect of peptoid residues on the structure, biological function and mode of action of $\alpha-helical\;or\;\beta-turn$ antimicrobial peptides has not been previously examined.
To develop the cell-selective antimicrobial peptides having potent antimicrobial activity but no cytotoxicity against mammalian cells, we synthesized several antimicrobial peptides containing L-Pro, D-Pro or Peptoid residue. We developed a novel asymmetric synthetic method for the incorporation of unnatural amino acids including peptoids and D-amino acid. We characterized the biological activity of these peptides using human erythrocytes, other mammalian cell types and bacterial cells, and we examined their interaction with model phospholipid membranes by tryptophan fluorescence, lipid flip-flop, and membrane depolarization. The sites of action and the molecular mechanisms of antimicrobial activity were investigated by confocal laser scanning microscopy and DNA binding assays.
The tertiary structures of novel $\alpha-helical$ antimicrobial peptides, Piscidin 1, Pleurocidin, IsCT, HP(2-20), have been studied using NMR spectroscopy. These peptides have ${\alpha}$-helical structures and it has been found that the hinge structure introduced in the middle of the $\alpha-helices$ is important to provide the flexibilities in antibiotic peptides with bacterial cell selectivity. Based on the structure, we designed and synthesized several analogues of these peptides with Gly and/or Pro substitution. These results demonstrate that the flexibility of the position 8 of Pis-1 plays important role in enhancing antibiotic activity and membrane permeabilisation activity. Pis-1 PG has a bent structure at $Pro^8$ and exhibited low hemolytic activity but retained potent antibacterial activity, Pis-1 PG can be a potent candidate for new drug with high antibacterial activity without cytoxicity. In case of IsCT, $[K^7,\;P^8,\;K^{11}]$-IsCT, which has a bend in its middle region, exhibited the highest antibacterial activity without hemolytic activity, suggesting that its proline-induced bend is an important determinant of this selectivity.
Structure of $\beta-turn$ antimicrobial peptides, tritrpticin has been studied and it has amphiphilic $\beta-turn$ structure. Based on this structure, TPk with peptoid in Pro position has been designed and it is found that TPk penetrates into the bacterial cell membranes and shows high antimicrobial activities without hemolytic activities. Structure of antimicrobial peptides, Arenicin and it derivatives have been studied using NMR spectroscopy and Arenicin has an $\beta-sheet$ structure while Arenicin-1-S which has substitution of cyeteine with serine shows a-helical structure. Since Arenicin-1-S shows decrease of antimicrobial activity, f-sheet structure of arenicin may play critical roles in its antimicrobial activities.

목차 Contents

• 제 1 장 연구개발과제의 개요...18
• 제 2 장 국내외 기술개발 현황...20
• 제 3 장 연구개발수행 내용 및 결과...23
• 제 4 장 목표달성도 및 관련분야에의 기여도...73
• 제 5 장 연구개발결과의 활용계획...79
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보...80
• 제 7 장 참고문헌...82
• 제1세부과제: 고기능 항생펩타이드성 의약품 창출을 위한 설계, DNA재조합, 대량생산, 구조연구기술의 강호-Studies on design, structure, overexpression of peptide antibiotics...130
• 제2세부과제: Peptidornimetics기술을 이용한 모방형 펩타이드 항생제의 설계 및 활성연구-Study on design and antimicrobial cativity of model peptide antibiotics using peptidominetics technology...206
• 제3세부과제: Peptidornimetics기술을 이용한 모방형 펩타이드 항생제의 합성-Asymmetric syntheses of peptidomimetic antibiotics...295