[보고서]천연물 라이브러리를 사용한 새로운 항결핵 후보 물질 도출 및 잠복 결핵 치료를 위한 약물 검색 모델 개발에 관한 연구

DELORME Vincent

[국가R&D연구보고서] 천연물 라이브러리를 사용한 새로운 항결핵 후보 물질 도출 및 잠복 결핵 치료를 위한 약물 검색 모델 개발에 관한 연구 원문보기

보고서 정보
주관연구기관	한국파스퇴르연구소
연구책임자	DELORME Vincent
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2017-05
과제시작연도	2016
주관부처	미래창조과학부 Ministry of Science, ICT and Future Planning
과제관리전문기관	한국연구재단 National Research Foundation of Korea
등록번호	TRKO201800004164
과제고유번호	1711037145
사업명	바이오.의료기술개발
DB 구축일자	2018-04-28
키워드	천연물.잠복결핵.결핵균.초고속 약효 탐색.약재내성결핵.Natural products.Latent tuberculosis.Mycobacterium tuberculosis.High-throughput screening.Drug-resistant tuberculosis.

초록 ▼

본 연구진은 해당 연구기간동안 대식세포내에서뿐만 아니라 액상 배지내에서의 결핵균 복제율에 대하여 11,088 방성균 추출물 라이브러리 스크리닝을 완료하였다. 이 스크리닝을 통해 선택된 활성 추출물은 공동 연구자인 식신약소재개발사업단의 이한기박사팀에 의하여 검증되었으나, 어떤 관련 균주에서도 유의성있는 결과를 찾기 어려웠다. 추가적으로 잠복결핵 및 비복제 결핵균과 세포외 유기체들을 사멸할 수 있는 화합물들을 발견할 수 있는 SS18b 결핵균주에 대한 분석방법이 최척화되어졌다. 향후 이 분석 방법을 사용하여 8,000 화합물들에 대한 파일럿 스크리닝을 시작할 것이다. 또한 자가 재생 뮤린 프라이머리 폐포 대식세포인 MPI(Max-Planck Institute) 세포주를 도입하여 이를 이용한 분석법을 개발하고 검증하였다. 마지막으로 박테리아 복제율의 감소가 관찰되는 지질 축적 대식세포(lipid-loaded macrophages) 모델을 도입하여 호스트와 병원체간의 상호작용을 연구할 수 있는 독특한 모델의 특성에 관한 연구가 현재 진행중에 있다.

(출처 : 보고서 요약서 3p)

Abstract ▼

Purpose & Contents
Tuberculosis (TB) is still a major health issue in the worldwide. Appearance and spread of extremely resistant strains from China and Russia represents a major threat for South Korea. Indeed, treating these infections requires expensive and lengthy treatments (6 months minimum for drug-sensitive TB, up to 2 years for drug-resistant TB), and together with ageing population, it could lead to huge financial repercussion for the country. Introduction of new drugs, less expensive and efficiently targeting resistant strains is then an important requirement for the control of TB. In addition, the discovery of new strategies to eliminate latent bacilli, persisting in the host for decades, is also critical. It could help i) to limit appearance of resistant strain, ii) to shorten the treatment duration and iii) to better control disease reactivation in elderly people. In this context, development of news assays and models allowing the study of such latent bacteria are highly desirable.

Results
We screened 11,088 raw extracts isolated from 1,112 actinomycetes strains isolated in Korea. This library, provided by our collaborator (CNPM), was assessed in two independent assays (replication in broth and in macrophages) and at two different dilutions (1/1,000 and 1/10,000 final dilution). A total of 27 candidate strains were surveyed by the CNPM using 16s rRNA sequencing, yielding only Streptomyces-related strains unfortunately of limited value for further investigation. In parallel, we developed spontaneous resistant mutants and probes related with a family of compounds displaying activity against drug-resistant isolates. Target identification will be performed by whole genome sequencing and pull-down assays. In addition, we generated a screening assay using the M. tuberculosis SS18b strain, based on alamar blue reduction. A fluorescent SS18b strain was also created, but attempts to develop an assay based on infected macrophages failed up to now. Finally, we developed a model of foamy (lipid-loaded) macrophages and different cell lines were explored, including the MPI cells, unique self-renewable murine alveolar macrophages.

Expected Contribution
We will continue to screen more natural extracts using external sources of funding, and aim at selecting pre-purified fractions without cellular toxicity to increase the relevance of the library. The robustness of the screening assay using SS18b strain will be assessed and compounds of interest used as tools to promote the identification of new mechanism of action and targets. Finally, the foamy macrophage model will be applied to better understand the host-pathogen interactions, as they are established during infection.

(출처 : SUMMARY 5p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
요약문 ... 4
SUMMARY ... 5
CONTENTS ... 6
목차 ... 7
제1장. 연구개발과제의 개요 ... 8
1. 연구개발 목적 ... 8
2. 연구개발의 필요성 ... 8
3. 연구개발 범위 ... 8
제2장. 국내외 기술 개발 현황 ... 9
제3장. 연구 수행 내용 및 성과 ... 13
제4장. 목표 달성도 및 관련 분야 기여도 ... 27
1. 연구개발의 최종목표 ... 27
제5장. 연구개발성과의 활용계획 ... 28
제6장. 연구 과정에서 수집한 해외 과학기술 정보 ... 29
제7장. 연구개발성과의 보안등급 ... 32
제8장. 국가과학기술종합정보시스템에 등록한 연구시설·장비 현황 ... 33
제9장. 연구개발과제 수행에 따른 연구실 등의 안전 조치 이행 실적 ... 36
제10장. 연구개발과제의 대표적 연구 실적 ... 37
제11장. 기타 사항 ... 37
제12장. 참고 문헌 ... 38
끝페이지 ... 39

표/그림 (18)

표 Number of MDR-TB cases estimated to occur among notified pulmonary TB cases in 2015.
표 Second-line regimen for MDR/RR-TB, with their most common adverse effects and other weaknesses.
표 Main competitors in the TB field and development stage of their respectve molecules.
표 The global clinical TB drug pipeline
표 Scheme presenting the process followed by the CNPM to prepare the ECUM library.
표 Properties of extracts displaying >50% inhibition threshold at Both 1000× and 10,000× dilutions in the extracellular assay (QUM).
표 Properties of extracts displaying >70% inhibition of infected surface at Both 1000× and 10,000× dilutions in the intracellular assay (QIM).
표 A. Scheme for the design of a generic probe. B. Protocol for pull-down assay using biotin as a tag. C. Scheme of the alkyne-azide 1,3-dipolar cycloaddition, catalyzed by CuI, giving access to 1,2,3-triazoles in good yield.
표 List of genetic variations identified in TTCA-01 resistant isolates. SNP, single nucleotide polymorphism; INS, insertion.
표 Scheme of two synthesized probes, with the linker and tag molecule attached A. on the left side or B. on the right side of the active molecule. C.
표 A. Structure of the two mycobactin synthesis gene clusters of M. tuberculosis. Genes of the cluster are indicated as gray arrows, while adjacent genes are in white. The iron-box operators (Fe-box) are indicated with hatches. B. Heat-map of the ln fold change (FC) of each gene of the mycobactin clusters (in bold), as well as the adjacent genes and the transcriptional regulator IdeR, showing the evolution of the expression with time and compounds concentration.
표 A. Representative picture of the SS18b-GFP strain visualized by fluorescence microscopy (Ex: 488 nm). Scale bar: 100 μm. B. Growth of the SS18b-GFP strain in presence (blue curve) or in absence (red curve) of streptomycin (STR), after an initial 7 days growth with STR (black curve).
표 A. Scheme illustrating the principle of the assay using the 18b strain. STR: streptomycin. B. Reproducibility of the screening assay, explored using a library of 8,356 bioactive/FDA approved molecules.
표 Detailed results of the SS18b assay for A. first-line anti-TB drugs, B. some second-line anti-TB drugs, C. the fluoroquinolone class and D. the aminoglycoside class. E. Legend for the reading of the box-plot presented in panel A-D.
표 A. General scheme of a mature granuloma, with a necrotic core in its center, circled by a ribbon of immune cells, including lymphocytes in the outer part and foamy macrophages in the inner side. B. Electron microscopy view of a foamy macrophage infected with M. avium. Note the accumulation of lipidic inclusions in the bacterial cytoplasm (*). LB, lipid bodies. Scale-bar: 1 μm. From Caire-Brandli et al. 2014 Infection Immunity.
표 A. Experiment timeline for the generation of foamy macrophages using THP-1 cells. B. Foam cells infected with H37Rv-GFP (MOI 0.05, green) were fixed at day 4 with formalin. Nuclei were stained with DAPI (blue) and lipid bodies with LipidTox (red). Scale-bar, 25 μm. C. Representative Brightfield pictures obtained day 6 post-infection. Bacteria were detected by fluorescence (green). Scale-bar, 50 μm. D. Quantification of the bacterial replication measured by fluorescence over a 7 days experiment.
표 A. Representative pictures of naive and VLDL (140 μg TAG/mL) treated THP-1 cells, MPI cells or BMDM (blue). Cells were stained with LipidTOX neutral lipid dye (red). Scale bar: 50 μm. B. Comparison of the ratio of LipidTOX stained cells in the presence and absence of VLDL, for different cell types (n=9). C. Proliferation of MPI cells in presence of various amount of VLDL after 4 days incubation. Significance compared to the non-treated (NT) sample is indicated for each concentration (n=9). Statistical significance was determined by Prism software using one-way ANOVA.
표 A. Representative pictures of DMSO and isoniazid treated MPI cells (blue) 5 days after batch infection (100 rpm, 2h) with H37Rv-GFP (green) at a MOI of 5. Scale bar: 50 μm. Insert from isoniazid treated cells shows magnified view of persistent bacterial infection in intact cells. Scale bar: 15 μm. B. Dose-response of isoniazid against H37Rv-GFP replicating in MPI cells (MOI 10). The ratio of infected cells is shown with black, closed circles; the number of cell is shown with gray, open squares. Fitting were performed by Prism software, using a sigmoidal dose-response model, and used to determine the concentration of isoniazid required to inhibit 50% of the bacterial colonization process (IC50). C. Comparison of isoniazid IC50 values (expressed in log scale) for different cell types.

과제명(ProjectTitle) :	-
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과제기간(DetailSeriesProject) :	-
총연구비 (DetailSeriesProject) :	-
키워드(keyword) :	-
과제수행기간(LeadAgency) :	-
연구목표(Goal) :	-
연구내용(Abstract) :	-
기대효과(Effect) :	-

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[국가R&D연구보고서] 천연물 라이브러리를 사용한 새로운 항결핵 후보 물질 도출 및 잠복 결핵 치료를 위한 약물 검색 모델 개발에 관한 연구 원문보기