Repositioning gram-positive antibiotics and FDA approved drugs against gram-negative bacteria using alanine mutation and lipidation of kink α-helical peptide as improved sensitizers = 그람 음성균에 대한 항균 활성을 나타내는 알라닌 치환과 지방화에 의한 꺾인 알파나선 펩타이드

다국어 초록 (Multilingual Abstract)

The crisis by increases of drug-resistant gram-negative bacteria is an important issue to overcome for public health. The major reason for this crisis is that there are relatively few drugs available against Gram-negative bacteria, since most of gram-positive antibiotics cannot pass through the formidable outer membrane in Gram-negative pathogens. Unfortunately, current new antibiotics are not available even in pipelines. As an alternative of developing new drugs, a few molecules are suggested as sensitizers of outer membrane to repositioning gram-positive antibiotics against gram-negative bacteria. We have synthesized a small library of the amphipathic helical and hinged peptides as sensitizers. And then we have mutated peptide for alanine, serine mutation and lipidation. The synthesized peptides help Gram-positive antibiotics, such as rifampicin, clarithromycin, and linezolid, to pass through membrane by rearranging the LPS layer of Gram-negative bacteria. Furthermore, our peptides potentiate E. coli than Polymixin B nonapeptide (PMBN), which is known as a hallmark of the potentiator peptide. Several biophysical methods, such as Laurdan generalized polarization (GP), were used for elucidating the mechanism of the potentiator peptide. The increases of fluidity changes in the membrane suggest that OctA 15-NL3AL7A stays at membrane, making LPS-rich nano- (or micro-) domains, demixing and facilitating antibiotics to get through outer membrane. Animal tests using various models, such as mouse thigh- and lung- infection models confirm the sensitization effect of OctA 15-NL3AL7A. Especially, peptide-raping liposome formulation further increases efficacy of the antibiotics. Taken together, the sensitizer peptide rearranges LPS-containing layer, facilitating the translocation of most of gram-positive antibiotics to eradicate gram-negative bacteria.

국문 초록 (Abstract)

약물에 내성이 있는 그람 음성균의 증가로 인한 위기는 공중 보건에서 극복해야 할 중요한 문제이다. 이러한 위기의 주된 이유는 그람 양성균 항생제 대부분이 그람 음성균의 외막을 통과할 수 없기 때문에 그람 음성균에 대항할 수 있는 약물이 상대적으로 매우 적기 때문이다. 불행하게도, 현재의 새로운 항생제는 파이프라인에서도 구할 수 없다. 신약 개발의 대안으로 그람 양성균 항생제를 그람 음성균에 대항하여 재배치하기 위한 외부 막의 감응제로 몇 개의 분자가 제안된다. 우리는 감응제로서 양면성 나선형 및 꺾은형 펩타이드 라이브러리를 합성했다. 그리고 알라닌, 세린 돌연변이, 지방화를 통해 펩타이드를 변형시켰다. 합성 펩타이드는 그람 음성균의 LPS층을 재정렬시켜 리팜피신, 클라리스로마이신, 리네졸리드 등 그람 양성균 항생제가 막을 통과하도록 돕는다. 더욱이, 우리의 펩타이드는 대표적으로 항균 펩타이드로 알려진 폴리믹신 B 노나펩타이드(PMBN)보다 대장균을 감응시킨다. 감응 펩타이드(Potentiator Peptide)의 메커니즘을 설명하기 위해 Laurdan generalized polarization (GP)과 같은 몇 가지 생물물리학적 방법이 사용되었다. 막의 유동성 변화 증가는 OctA 15-NL3AL7A가 막에 머무르면서 LPS가 풍부한 나노(또는 마이크로) 도메인을 만들고, 항생제가 외부 막을 통과하기 쉽게 한다는 것을 시사한다. 마우스 허벅지 및 폐 감염 모델과 같은 다양한 모델을 사용한 동물 실험은 OctA 15-NL3AL7A의 감응성 효과를 확인하였다. 특히 펩타이드 리포좀 제제는 항생제의 효능을 더욱 높여준다. 결론적으로 감응 펩타이드(sensitizer peptide)는 LPS 함유 층을 재배열하여 그람 음성 박테리아를 제거하기 위해 대부분의 그람 양성 항생제의 통과를 촉진한다.

목차 (Table of Contents)

• Introduction 1
• Experimental Section 7
• 1. Solid phase peptide synthesis 7
• 2. MIC assay 7
• 3. Hemolysis assay 8
• 4. WST-1 assay 8
• 5. Bacterial membrane leakage assay 9
• 6. Membrane fluidity change assay 9
• 7. Fluorescent dye accumulation assay 10
• 8. Neutropenic mouse thigh model 11
• 9. Neutropenic mouse pneumonia model 12
• Results and Discussion 13
• 1. Determine the Sensitive Effects of the Alanine Scanning Peptide Library 13
• 2. The effectiveness of Alanine and valine mutated 15-N peptide library 18
• 3. Choosing a peptide that has an increased sensitizing effect at low concentrations by lipidation 20
• 4. Choosing peptides that have improved the sensitizing effect by lidation of various fatty acids. 23
• 5. Determination of toxicity to host cells. (Hemolysis assay) 26
• 6. Determination of toxicity to host cells. (WST-1 assay) 28
• 7. Mechanism study of selected peptides on the outer membrane of gram-negative bacteria 30
• 8. Check the membrane breakability of the peptide (Enzyme assay) 32
• 9. Change in amount of compound entering bacteria in the presence of sensitizer. 34
• 10. Screening repositioning antibiotics by investigating MIC of FDA approved drugs in the presence of selected peptide against gram-negative bacteria 36
• 11. In vitro fold increase of antibiotic’s MIC in the presence of lipidated Dab peptides with various fatty acids against Gram-negative bacteria 41
• 12. Determination of toxicity to host cells. (Hemolysis assay) 45
• 13. Selection of peptides using Deconvolution Library to maximize Rifampicin sensitivity to four types of gram-negative bacteria 47
• 14. Re-synthesis of derived peptides and the sensitization effect of Rifampicin on four types of gram-negative bacteria 50
• 15. Sensitive effects of CMP1701 to CMP1709 peptides (MICassay) 53
• 16. Demixing Membrane Effect of Peptide (OctA 15-NL3AL7A) against Wild Type and
• arbapenem Resistant Entereobacteriaceae 56
• 17. Determination of toxicity to host cells. (Hemolysis & WST-1 assay) 58
• 18. Evaluation of Membrane Reorganization Effects and Effectiveness of Rifampicin/Peptide Combination Using Animal Experiments 60
• Conclusions 63
• References 66
• Abstract (in Korean) 67