Structural Insights Into Inhibitory Mechanisms of Cathepsin K
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STRUCTURAL INSIGHTS INTO INHIBITORY MECHANISMS OF CATHEPSIN K by Simon Sau Yin Law B.Sc., The University of British Columbia, 2013 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (BIOCHEMISTRY AND MOLECULAR BIOLOGY) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2018 © Simon Sau Yin Law, 2018 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Structural Insights into Inhibitory Mechanisms of Cathepsin K submitted by Simon Sau Yin Law in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry and Molecular Biology Examining Committee: Dieter Bromme, Oral Biological and Medical Sciences Supervisor Chris Overall, Oral Biological and Medical Sciences Supervisory Committee Member Christian Kastrup, Biochemistry and Molecular Biology Supervisory Committee Member Katherine Ryan, Chemistry University Examiner Filip Van Petegem, Biochemistry and Molecular Biology University Examiner ii Abstract Cathepsin K (CatK) is a lysosomal cysteine protease highly expressed in osteoclasts and is responsible for the degradation of bone. Implication of CatK in various musculoskeletal disorders including osteoporosis highlights CatK as an important target for drug development. This thesis aims to identify and characterize different classes of CatK inhibitors, which target the active site and collagenolytically relevant ectosteric sites using X-ray crystallography combined with mutational and kinetic studies. In chapter 2, odanacatib, a specific CatK inhibitor recently abandoned in clinical trials due to adverse side effects, was investigated for its selectivity for mouse CatK over the human counterpart. Structural, mutagenic, and kinetic studies identified two structural features in the mouse enzyme which determines odanacatib’s selectivity. Replacement of these features from the human counterpart was able to restore inhibitory activity in the mouse enzyme. In chapters 3 and 4, composite docking and high throughput fluorescence polarization (FP) assay methods were developed to identify novel ectosteric inhibitors of CatK by targeting the protein oligomerization site required for collagen degradation. Screening of the NCI Database (280,000 compounds) using three distinct molecular modeling methods identified nine active compounds. The best compound had an IC50 value around 300 nM in cell-based resorption assays. Over 5,000 compounds were also screened using a developed FP assay and nine collagenase inhibitors were identified. Three of these compounds were active in subsequent cell- based assays. In chapter 5, NSC-13345, the structure of a putative allosteric inhibitor of CatK described in literature was determined in complex with the enzyme using X-ray crystallography. Previous characterization was performed using an inactive variant and the allosteric site affected by the iii presence of an extra loop. Structural determination with the fully processed enzyme identified three binding sites. One site located above the active site may explain its substrate selective inhibition. In addition, the crystal structure of T-06, a potent collagenase inhibitor of CatK, in complex with CatK was determined and provided insight into its ectosteric inhibition. These findings suggest that NSC-13345 and T-06 function as a substrate selective ectosteric inhibitors for CatK. iv Lay Summary Osteoporosis affects 50% of women aged 50 years or older and medical costs are estimated to be 17-20 billion dollars in the US and over two billion dollars in Canada, annually. Cathepsin K (CatK) is an enzyme highly expressed in the cells responsible for bone degradation. Inhibiting this enzyme has been shown to be effective at alleviating symptoms associated with osteoporosis. Various CatK inhibitors have been tested in clinical trials but they have all failed due to side effects with unknown causes. Therefore, it is important to study the origins of these side effects and to investigate other potential therapeutics. This thesis covers the investigation of the potency of odanacatib, a highly promising drug for treating osteoporosis abandoned after Phase III clinical trials due to complicating side effects. It also covers the development of novel drug development methods to identify compounds specifically targeting the bone remodeling activities of the enzyme. v Preface Dr. Dieter Brömme was the principal investigator of the research projects. I was responsible for design, performance and analysis of the research as well as all the manuscript preparation for the work described in this thesis with details and exceptions outlined below. Chapter 2 A version of chapter 2 has been published in the Biochemical Journal. (Law, S., Andrault, P., Aguda, A., Nguyen, N., Kruglyak, N., Brayer, G., Brömme, D., Identification of mouse cathepsin K structural elements that regulate the potency of odanacatib. Biochemical Journal, Vol. 474, No. 5, pp. 851-864, Feb., 2017.) I was responsible for the research design and performance of the experiments and the manuscript preparation. Dr. Pierre-Marie Andrault assisted with the characterization of the kinetic properties of the enzyme as well as general revision of the manuscript prior to submission and publication. Dr. Adeleke Aguda aided with the structural determination of the crystal structure of the inhibitor- free human wild-type CatK and Dr. Gary Brayer provided insight for the other CatK structures. Chapter 3 A version of chapter 3 has been published in PLOS One. (Law, S., Panwar, P., Li, J., Aguda, A., Jamroz, A., Guido, R., Brömme, D., A composite docking approach for the identification and characterization of ectosteric inhibitors of cathepsin K, PLoS One, Vol. 12, No. 10, pp. e0186869, Oct., 2017.) I was responsible for the research design and performing of the experiments and the manuscript preparation. Co-op student Jody Li assisted under my supervision in the evaluation of the compounds identified through composite docking for their collagenase properties. Dr. Preety Panwar provided her expertise and assisted in the cell-based vi osteoclast resorption assays. Dr. Rafael Guido provided guidance and resources for the development of the composite docking method. Chapter 4 Former Ph.D. graduate Xin Du generated the screening data for the compounds from the KD2 library in collaboration with the Centre for Drug Research and Development (CDRD). Dr. Preety Panwar assisted with the cell-based bone resorption assays and subsequent analysis. A manuscript containing a modified version of the chapter is in preparation. Chapter 5 Udit Dalwadi under my supervision generated and characterized the allosteric binding site mutant CatK as part of his undergraduate thesis. Dr. Adeleke Aguda provided his guidance and expertise in the structural determination of the CatK-NSC-13345 and CatK-T-06 complex. A manuscript prepared containing the data presented is under preparation. In addition to the projects outlined above, I also conducted additional projects and experiments which have resulted in contributions to four other published peer-reviewed research articles. A complete list of other co-authored publications can be found in the Appendices with a short description of my relative contributions to each manuscript. Manuscripts have been reproduced with permission from the respective copyright holders. vii Table of Contents Abstract ......................................................................................................................................... iii Lay Summary ................................................................................................................................ v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ................................................................................................................................. x List of Figures ............................................................................................................................... xi Abbreviations ............................................................................................................................. xiii Acknowledgements ..................................................................................................................... xv 1. Introduction ........................................................................................................................... 1 1.1. Structure and Function of Proteases ............................................................................ 1 1.2. Proteases in the Extracellular Matrix .......................................................................... 6 1.3. Cysteine Proteases .......................................................................................................... 9 1.3.1. Cathepsin K ........................................................................................................... 13 1.3.2. Cathepsin K and Bone Remodeling..................................................................... 16 1.4. Protease Inhibition ......................................................................................................