Structure determination of Rv0569 from Mycobacterium tuberculosis and Lead compound development of HsPDF from Homo sapiens
결핵균 유래 Rv0569 단백질의 구조 규명 및 사람 유래 단백질 HsPDF의 선도물질 개발

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 제약학과, 2014. 8. 이봉진.-
dc.description.abstract구조 기반 약물 설계는 합리적인 약물 개발 방법의 하나로 목적 단백질의 구조를 밝히고 이후 결합자리 연구를 통해 선도물질을 도출한다. 결핵균 유래의 단백질 Rv0569 의 구조 연구를 통하여 구조기반 약물 설계의 초반 부분을 수행하였다. Rv0569 단백질의 클로닝, 대량발현, 단백질 정제의 과정을 거쳐 원편광이색성 (Circular Dichrosim) 실험을 통해 단백질의 2차구조를 예측하고 기본적인 물성을 알아 본 뒤 핵자기공명(Nuclear Magnetic Resonance) 실험을 통하여 단백질의 구조를 계산하였다. 계산된 구조와 구조 기반 기능 예측과 기존연구 분석을 통해 Rv0569 단백질이 결핵균의 잠복기 감염시 저산소 신호 전달에 관여하는 기능을 가짐을 제안하였다. 구조기반 약물 설계의 후반부분을 사람 유래 펩타이드 디포밀라제 단백질의 저해제 선도물질 도출연구를 통해 수행하였다. 43개의 박테리아 유래 저해제 라이브러리에서 결합 모의 실험(Docking Simulation)을 통해 10 종의 화합물을 선택하고 이의 항암능을 실험하였다. 항암능 실험결과에서 우수한 결과를 보인 4종의 화합물과 핵자기 공명법 실험을 통하여 사람유래의 펩타이드 디포밀라제 단백질과 결합 실험을 수행하였다. 4종 중 2종의 박테리아 유래의 저해제가 사람 유래의 단백질과 결합함을 확인하였고 이 2종의 화합물을 선도물질로 도출하였다.-
dc.description.abstractStructure-based drug design (SBDD) is one of the rational drug development method and can be divided largely into two parts. First, the structure of the target protein is revealed by using Nuclear Magnetic Resonance (NMR) or X-ray crystallography. Second, lead compounds are generated through binding site research. The structure of Rv0569 protein from Mycobacterium tuberculosis was calculated by NMR, achieving the early part of SBDD. After a serial process of cloning, massive expression, and purification of Rv0569, circular dichroism (CD) experiments was performed to predict the secondary structure and identify the basic physicochemical properties of the protein. The three-dimensional structure was calculated through NMR experiments. The bioinformatic research, structure calculation, and previous papers suggest that Rv0569 could be involved in hypoxia signal transduction. The latter part of SBDD was accomplished with peptide deformylase (HsPDF) derived from Homo sapiens. The lead compounds for HsPDF inhibitors drug were developed using library of bacterial peptide deformylase inhibitors. Forty-three species of the bacterial peptide deformylase inhibitors were selected by docking simulation. The species of top ten scores out of 43 were selected and their antitumor activities were tested. NMR binding experiments were conducted with selected four compounds showing the excellent antitumor activities to identify the HsPDF protein–ligand binding. Consequently, two species of bacteria-derived inhibitors were confirmed to bind human-derived protein, and were chosen as a lead.-
Abstract of the Dissertation i
Contents iii
List of Tables vi
List of Figures vii
List of Abbreviations xi

General Introduction
Structure-based drug design 1

Chapter 1. Structure Determination of Rv0569, Potent Hypoxic Signal Transduction Protein, from Mycobacterium tuberculosis

1.1 Abstract 4
1.2 Introduction 7
1.3 Experimental procedures 11
1.3.1 Protein preparation 11
1.3.2 Circular Dichroism measurement 13
1.3.3 NMR measurement 14
1.3.4 Structure calculation and validation 15
1.4 Results 16
1.4.1 Protein preparation 16
1.4.2 NMR spectrum analysis 18
1.4.3 Structure calculation and validation 23
1.5 Discussion 30
1.6 Conclusion 39

Chapter 2. Lead compound development of HsPDF from Homo sapiens

2.1 Abstract 40
2.2 Introduction 41
2.3 Experimental procedures 48
2.3.1 Virtual screening 48
2.3.2 MTT assay 50
2.3.3 Protein preparation 52
2.3.4 NMR experiment 55
2.3.5 Chemistry56
2.4 Results and Discussion 57
2.4.1 Initial screen 57
2.4.2 MTT assay 59
2.4.3 Various docking simulation 61
2.4.4 Structure of selected compounds 63
2.4.5 Molecular modeling 64
2.4.6 Codon optimization and protein purification 66
2.4.7 NMR binding experiment 70
2.5 Conclusion 73

Summary 74
Supplemental data 75
References 90
국문초록 103
감사의 글 105
dc.format.extent7529770 bytes-
dc.publisher서울대학교 대학원-
dc.subjectStructure–based drug design (SBDD)-
dc.subjectNuclear Magnetic Resonance (NMR)-
dc.subjecthypoxic signal transduction-
dc.subjectdocking simulation-
dc.subjectpeptide deformylase inhibitors-
dc.subject구조 기반 약물 설계-
dc.subject저산소 신호 전달-
dc.subject펩타이드 디포밀라제-
dc.subject결합 모의 실험-
dc.titleStructure determination of Rv0569 from Mycobacterium tuberculosis and Lead compound development of HsPDF from Homo sapiens-
dc.title.alternative결핵균 유래 Rv0569 단백질의 구조 규명 및 사람 유래 단백질 HsPDF의 선도물질 개발-
dc.citation.pagesxii, 108-
dc.contributor.affiliation약학대학 제약학과-
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College of Pharmacy (약학대학)Dept. of Manufacturing Pharmacy (제약학과)Theses (Ph.D. / Sc.D._제약학과)
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