Dissertation Discovery of anti-tuberculosis drug candidates targeting Mycobacterium tuberculosis ClpC1 through cell-free in-vitro assay Graduate School, Myongji University “ Department of Interdisciplinary Program of Bio-modulation ” Minh Duc Nguyen Thesis Advisor Joo Won Suh June, 2019 1 Discovery of anti-tuberculosis drug candidates targeting Mycobacterium tuberculosis ClpC1 through cell-free in-vitro assay Submitted in partial fulfillment of the requirements for the “ Ph. degree in Biological Science ” June, 2019 “Graduate School. Myongji University Department of Interdisciplinary Program of Bio-modulation ” Minh Duc Nguyen 2 Discovery of anti-tuberculosis drug candidates targeting Mycobacterium tuberculosis ClpC1 through cell-free in-vitro assay Graduate School, Myongji University “ Department of Interdisciplinary Programs of Bio-modulation Minh Duc Nguyen We at this moment recommend that the dissertation by the above candidate for the Ph. degree in Biological science be accepted.
” Chair, Evaluation Committee “ Name Signature Member, Evaluation Committee Name Signature Member, Evaluation Committee Name Signature Member, Evaluation Committee Name Signature Member, Evaluation Committee Name Signature ” June, 2019 3 Acknowledgments I would like to express my sincere gratitude to Professor Joo Won Suh and Professor Lee Hanki, my research supervisors, for their patient guidance, enthusiastic encouragement, and useful critiques of this research work. I would also like to thank Dr. YongU Kim, for his advice and assistance in keeping my progress on schedule. My thanks are also extended to Mr.
Johnson Eldin for his help in doing data analysis, to Ms. Jinhua Cheng for her support in my experiments. I would also like to extend my thanks to the technicians of the laboratory of the center for Nutraceutical and Pharmaceutical Materials for their help in offering me the resources in running the program. Finally, I wish to thank my family for their support and encouragement throughout my study.
4 “Table of Contents ” “List of Tables ” iv “List of Figures ” 0v Abstract vii Introduction 01 Tuberculosis 01 MDR-, XDR-TB and new anti-tuberculosis drugs development 5 ATP-dependent protease 10 Clp protease family 14 Clp ATPase and ClpP – the proteolytic component 15 Mycobacterium tuberculosis and its protein degradation system 16 Cell-free protein synthesis system from Escherichia coli cells 18 Molecular Docking 20 N-terminal of ClpC1 protein 22 The aim of this dissertation 24 Chapter 1 - Development of biochemical assay using ClpC1 for screening compounds targeting ClpC1 26 1.1 Materials and Methods “ ” 26 1.1 Bacterial strains and Medium cultures “ ” 26 1.2 Cloning and Purification DNA 27 1.3 Expression and Purification recombinant ClpC1 protein 28 1.4 SDS-PAGE for protein electrophoresis 29 1 1.5 ATPase activity assay 30 BIOMOL® GREEN reagent 30 ADPTM Glo reagent 31 1.6 The anti-tuberculosis drugs 32 1.1 Cloning, Expression and Purification of ClpC1 33 1.2 ClpC1 Displays Basal ATPase activity 37 1.3 Comparison of Luminescence ADP Production Assay and Fluorescence Free- phosphate Released Assay with Ecumicin and Rufomycin I 40 1.4 Testing the ATPase activity of ClpC1 with 1-, 2- line anti-TB drugs 45 Chapter 2 - Development of biochemical assay using ClpC1, ClpP1 and ClpP2 for screening anti-tuberculosis lead compounds 48 2.1 Materials and Methods 48 2.1 Expression and Purification ClpP1 and ClpP2 protein 48 2.2 Measurement concentration protein with Bradford method 50 2.3 Exchanged buffer with PD-10 Desalting Column 51 2.4 Proteolytic activity of ClpC1/ P1/ P2 complex 52 FITC – Casein 52 SsrA – eGFP 53 2.1 Cloning, Expression and Purification of ClpP1 and ClpP2 54 2.2 Comparison of FITC-Casein degradation and SsrA-eGFP degradation with Ecumicin and Rufomycin I 57 2 2.3 Testing the Proteolysis activity of ClpC1/ P1/ P2 complex with 1-, 2- line anti-TB drugs 62 Chapter 3 - Screening anti-tuberculosis lead compounds through biochemical and biophysical assay developed 65 3.1 Materials and Method 65 3.1 Prestwick Chemical Library 65 3.2 Isolation and Purification procedure of Ecumicin analogues 66 3.3 Protein and lead compounds structure 68 3.1 The ATPase activity and Proteolysis activity with Ecumicin analogues 71 3.2 Screening anti-tuberculosis lead compounds from the Prestwick Chemical Library 77 3.3 Docking to ClpC1 84 Conclusion 97 References 101 Korean Abstract 109 3 List of Tables Table 1. First- and second-line anti-tuberculosis drugs based on the World Health Organization classification 7 Table 2. ATPase activity of ClpC1 with Ecumicin and Rufomycin I 43 Table 3. The list of 10 anti-tuberculosis drugs with MIC unit and Molecular Weight 46 Table 4.
ATPase activity of ClpC1 with Ecumicin analogues 73 Table 5. List lead compounds of the Prestwick Chemical Library 80 List of Figures 4 Figure 1. Global tuberculosis (TB) death rate from 1992 to 2017 (WHO) 2 Figure 2. Estimated TB incidence rates, 2017 3 Figure 3.
Current global pipeline of new anti-tuberculosis drugs 9 Figure 4. Architecture and mechanism of ATP-dependent proteases 11 Figure 5. ClpP1P2 and ClpP1P1 structures 17 Figure 6. Structural Comparison of ClpA/C AAA + ATPase Domains 23 Figure 7.
Electrophoretic analysis of affinity chromatography using Ni-TED resin for purification of recombinant Mycobacterium tuberculosis ClpC1 35 Figure 8. LB Broth medium and SOB medium for expression recombinant ClpC1 protein 36 Figure 9. Basal ATPase activity of recombinant ClpC1 protein 38 Figure 10. The stability of recombinant ClpC1 protein in ATPase activity 39 Figure 11.
Structures of Ecumicin and Rufomycin I. ClpC1 ATPase activity in response to Ecumicin (ECU) and Rufomycin I (RUFI) treatment 44 Figure 13. The ClpC1 ATPase activity in response to drugs treatment 47 Figure 14. Electrophoretic analysis of affinity chromatography using Ni - TED resin for purification of recombinant Mycobacterium tuberculosis ClpP1 55 Figure 15.
Electrophoretic analysis of affinity chromatography using Ni-TED resin for purification of recombinant Mycobacterium tuberculosis ClpP2 56 Figure 16. The proteolytic activity of the ClpC1/ P1/ P2 complex in response to Ecumicin (ECU) and Rufomycin I (RUFI) treatment 60 Figure 17. The Proteolysis activity of ClpC1/ P1/ P2 complex with SsrA – eGFP as a substrate 61 5 Figure 18. The Proteolytic activity of the ClpC1/ P1/ P2 complex in response to drugs treatment 64 Figure 19.
Structures of Ecumicin and analogues 67 Figure 20. N-terminal domain of Mycobacterium tuberculosis ClpC1 3WDB 70 Figure 21. Basal ATPase activity of ClpC1 with Ecumicin and Ecumicin analogues 74 Figure 22. The degradation FITC – Casein of analogues Ecumicin by ClpC1/ P1/ P2 complex 76 Figure 23.
ClpC1 ATPase activity (A) and proteolytic activity of the ClpC1/ P1/ P2 complex (B) after treated with 19 hit compounds 82 Figure 24. Images of a Docking Result and Structure Evaluations of 19 hit compounds 86 6 Discovery of anti-tuberculosis drug candidates targeting Mycobacterium tuberculosis ClpC1 through cell-free in-vitro assay Minh Duc Nguyen Department of Environmental Engineering and Biotechnology Graduate School, Myongji University Advisor Joo-Won Suh Tuberculosis has been with humans for a long time, a disease that should have only belonged to the past but is still growing today. Despite all efforts of humans to prevent and control of tuberculosis, it still affects 8 - 9 million new TB cases and 2 million people died from the disease annually. The global TB rate continues to increase by 1 % per year with the widespread of drug-resistant TB.
Therefore, the development and research to find new anti-TB drugs are becoming an extremely urgent mission. Our studies will focus on research and development the high throughput anti-tuberculosis drugs screening system, especially for in- vitro active with specific target protein of Mycobacterium tuberculosis, ClpC1 protein and ClpCP complex, through Cell-free protein synthesis (CFPS) systems. To be able to screen lead anti-tuberculosis drugs, currently, researchers have to carry out directly on the cells of Mycobacterium tuberculosis. Thus, the requirement set out that all of the experiments must be performed in bio-security facilities level 3 or 4, to prevent infection from pathogens.
These difficulties posed new challenges for the development of new drugs against Mycobacterium tuberculosis. Based on this reason, the result from our studies demonstrated that could perform the screening of anti-TB drug candidates in bio-security facilities level 1 laboratory with the using Cell-free protein synthesis (CFPS) systems based on Escherichia coli cell extract. 7 ClpC1 is a common stress protein which is also involved in the heat-shock protein HSP100 family. Beside, ClpC1 is also an integral part of the Mycobacterium tuberculosis genome.
We have focused on the ATP hydrolysis activity of ClpC1 and prevented protein aggregation of ClpC1/ P1/ P2 protein complex to create a specific research direction for the high-throughput anti-tuberculosis drugs screening system. Thus, ClpC1 protein was overexpressed, purified and functionally characterized (93. The steady-state growth of recombinant ClpC1 protein in Luria-Bertani (LB) Broth High Salt medium is maintained and stabilize after extraction. In Mycobacterium tuberculosis, ClpC1 is an ATP-dependent molecular chaperone.
Thus, the determination ATPase activity of ClpC1 was carried out by measurement the released phosphate with BIOMOL® GREEN reagent or ADP generated from the reaction in ADP GloTM kinases assay. This activity was not changed significantly and remained the original activity in 10 days after extraction from Escherichia coli Cell-free protein synthesis systems. Several potential compounds such as Ecumicin and Rufomycin I were chosen becoming control compounds with expected ATP hydrolysis activities (Vmax = 4983.008 of Ecumicin and Rufomycin I, respectively). Clp proteases are involved in several cellular processes such as degradation of misfolded proteins, regulation of short-lived proteins and housekeeping removal of dysfunctional proteins.
To gain proteolytic activity, the ClpP multimer associates with one or two hexameric rings of Clp ATPases, forming the ClpP‐containing proteolytic complex (designated the ClpP protease). Therefore, in parallel with studies on ClpC1, experiments on over-expression, purified and functional characteristics of ClpP1 and ClpP2 were carried out simultaneously, 21. 8 The proteolysis experiments of the ClpC1/ P1/ P2 complex was carried out based on the degradation of β - Casein or measurement SsrA - eGFP initial degradation. We showed not only the ability of degradation protein substrate of the ClpCP complex but also the significant differences under the effects on protease activity of Ecumicin and Rufomycin I treatment similar to published studies.
Under the effect of Ecumicin and Rufomycin I, ClpC1's ATPase activities were abnormally stimulated up to 2 - 3 times (Hill coefficient = 1.008 of Ecumicin and Rufomycin I, respectively) and proteolytic activities changed markedly. The biochemical assay showed that lead compounds stimulate ClpC1’s ATPase activity by two different mechanisms while inhibiting the proteolysis activity of ClpC1/ P1/ P2 complex. Finally, we have used this high throughput screening system with three analogues of Ecumicin and ten anti-TB drugs. Also, a Chemical library with more than 1,000 compounds was accepted by the FDA, which has also been screened.
Notably, we found 19 potential compounds with a positive effect on ClpC1's ATPase activity and degradation protein substrate of the ClpCP complex. We also analyzed the docking between ClpC1 N-terminal and potential compounds in 3D structure. The results indicated that the presence of four amino acids (ASN26; TYR27, VAL119, and ARG83) have appeared in Hydrogen bonds and Van der Waal bonds which are weak and popular bonds in the intracellular. Based on these results, we can build 2D or 3D pharmacopeia models for Pharmacophore-based virtual screening with high- performance computing in the future.
Pharmacophore approaches are successful subfields of computer-aided drug design (CADD), which have become one of the essential tools in hit identification, lead optimization, and rational design of novel anti-TB drugs. 9 Introduction Tuberculosis Tuberculosis is known to be one of the most dangerous diseases in human history. Tuberculosis is most common in the lungs but can also affect the central nervous system (meningitis), lymphatic system, circulatory system, genitourinary system, bones, and joints 61. Currently, TB is the most common of bacterial infection, affecting 2 billion people or 1/3 of the population, with 9 million new cases annually, causing 1.5 million deaths (estimated 2016), most of them in developing countries.
Most (90 %) cases of TB infection are asymptomatic. 10 % of those in their lifetime will develop symptoms of tuberculosis, and if left untreated, it will kill 50 % of the victims. In 2016, about 4,100 people died every day, compared with 3,300 cases in a few years ago, making it the world's deadliest infectious disease.
