ABSTRACT The Importance of Cathepsin B Research and Clinical Applications David M. Kosa Director: Amanda C. Sevcik, Ph.D. Cathepsin B is a cysteine protease of the papain family. It plays an important role in intracellular proteolysis, but it displays exopeptidase activity due to a unique structural element called the occluding loop. There is also a large amount of evidence that cathepsin B is involved in the development and progression of cancer, Alzheimer’s disease, and pathological conditions. Throughout this thesis cathepsin B is analyzed for its role in pathology, enzyme mechanisms, and protease activity. APPROVED BY DIRECTOR OF HONORS THESIS: _________________________________________________ Dr. Amanda Sevcik APPROVED BY THE HONORS PROGRAM: _______________________________________________ Dr. Elizabeth Corey, Director DATE: __________________________ THE IMPORTANCE OF CATHEPSIN B RESEARCH AND CLINICAL APPLICATIONS A Thesis Submitted to the Faculty of Baylor University In Partial Fulfillment of the Requirements for the Honors Program By David M. Kosa Waco, Texas May 2020 TABLE OF CONTENTS LIST OF FIGURES . iii LIST OF TABLES . iv DEDICATION . v CHAPTER ONE – Foundation of Cathepsin B Research . 1 CHAPTER TWO – Enzymology of Cathepsin B . 5 CHAPTER THREE – Inhibitors . 17 CHAPTER FOUR – Proposed Mechanisms in Medical Research . 23 CHAPTER FIVE – Concluding Words . 28 BIBLIOGRAPHY . 29 ii LIST OF FIGURES FIGURE 1 . 5 FIGURE 2 . 7 FIGURE 3 . 9 FIGURE 4 . 10 FIGURE 5 . 11 FIGURE 6 . 13 FIGURE 7 . 15 FIGURE 8 . 17 FIGURE 9 . 22 FIGURE 10 . 24 FIGURE 11 . 25 iii LIST OF TABLES TABLE 1 . 8 iv DEDICATION To my loving family, my dear friends, my Thesis Director Dr. Sevcik, and the dedicated professors of the Baylor science departments. v CHAPTER ONE Foundation of Cathepsin B Research Introduction to Cathepsin B The lysosomal cysteine protease, cathepsin B, plays a valuable role in proteolysis, the breakdown of proteins into smaller components for recycling and cellular housekeeping. Cathepsin B “is of significant importance to cancer therapy as it is involved in various pathologies and oncogenic processes in humans.”1 However, the specificity of the compound, and the complex role of cathepsin B in cancer treatment has hampered the advancement of clinical application.2 Although researchers have spent decades studying cathepsin B, little is actually known about the mechanisms of it in regards to activation, regulation, and inhibition. Despite the lack of information, scientists have pursued research of cathepsin B in hopes of producing notable work in the field of cancer and disease treatment. Historical Importance of Current Research Cathepsin B has been the focus of numerous researchers and experiments since the late 1900’s. The first known documented report on cathepsin B is in 1957 by Greenbaum and Fruton.3 Although they were only studying the purification and properties of the compound, their work began the cascade that would become crucial to the human understanding of proteases. Shortly afterwards, Greenbaum joined with Schoichet and Hirshkowitz to analyze the activation process of trypsinogen by cathepsin B.4 A few other scientists joined in the initial research on the protease in hopes of 1 understanding the methods of activation; however, cathepsin B was mostly unknown during the 1950’s. The experiments in the early 1900’s were focused on the effects of cathepsins as a whole with the occasional reference to a distinct “cathepsin C” in some articles with Fruton as a primary researcher. The 1952 article “On the proteolytic enzymes of animal tissues. Beef spleen cathepsin C” was the first article to mention a clearly distinguished compound known as cathepsin C.5 The second article on distinguished cathepsins was focused on the transamidation reactions that are catalyzed by cathepsin C.6 It was not until around the late 1970’s that cathepsin B became popularized across research groups and experiments. Part of this increase in popularity can be attributed to the Barrett assay of cathepsin B which was improved in 1976.7 The assay ultimately utilized the cleavage of the substrate benzyoyl-DL-arginine 2- napthylamide and the coupling of a diazonium salt with a napthylamine to generate a compound capable of being read at an absorbance of A520 nm. With new techniques being developed, the number of articles being written on cathepsin B rapidly began to increase until researchers began to realize that the inhibition of cathepsin B could be crucial in preventing cancerous growth because of its role as an extracellular membrane degrader. The first inhibitor of cathepsin B, haptoglobin, was found by Snellmann and Sylvén; it is natural inhibitor of cathepsin B activity where haptoglobin is a protein that is responsible for binding to free hemoglobin in circulation. However, it was not until the German scientists Appel, Huth, and Herrmann discovered the implications of cathepsin overexpression, that scientists began to realize that inhibition of cathepsin B could be a viable strategy for treating a variety of diseases and cancers. Appel et al.’s experiment involved the serum testing of 43 children with varying 2 alterations of health. Of the children with inflammatory diseases, specifically leukemia and pneumonia, most of them experienced an increase in a variety of protein compounds including cathepsin B and cathepsin C. Recent research with cathepsin B has provided a solid framework and guideline for future experimentation in the clinical field. Notably, the acceptance of cathepsin B being regarded as an effective biomarker for a plethora of cancers has pushed research advancement towards clinical experimentation.8–13 Research on cathepsin B involvement in cancer may be the most common, but there is almost an equivalent amount of evidence that it is involved in a wide variety of diseases and conditions. Given that cathepsin B is capable of crossing the blood-brain barrier and is involved in numerous pathological processes like inflammation, cell death, and the production of toxic peptides, it is highly influential in neurological diseases.14 Specifically in reference to neurological diseases, cathepsin B can be tied to epilepsy caused by apoptotic cell death, inhibitor treatments to prevent the decay of brain neurons and inflammation, and amyloid plaque production in Alzheimer’s disease.15–17 With such an extensive repertoire of clinical implications, cathpesin B is definitely a highly valued research target and the focus of numerous experimentations. Purpose of this Thesis Overall, the majority of the early research on cathepsin B was severely limited by the lack of information on the structure, composition, and specificity of the cysteine protease. However, this limitation was overcome as technological advances and interest grew. Eventually, the mechanistic models of cathepsin B began to advance as more information on the protein became available. Although the mechanistic models have 3 improved, the current understanding of cathepsin B is still limited. Because cathepsin B is highly involved in the clinical research of cancer and neurodegenerative diseases, there are increasingly more research groups and individuals wanting to learn about cathepsin B and the best methods to manipulate the mechanism of action in favor of inhibition. However, the complexity of cathepsin B has left significant gaps in the development of understanding cathepsin B. These gaps allow room for contradictory theories, and confusion in understanding the mechanisms of cathepsin B activity and regulation. The purpose of this thesis is to detail the comprehensive progress in the enzymology of cathepsin B while approaching theories and intricate mechanisms in order to show the potential of cathepsin B research and its clinical application. 4 CHAPTER TWO Enzymology of Cathepsin B Structure Because cathepsin B undergoes “considerable variation in its posttranslational modification,” it has been difficult to identify its numerous roles in “physiological and pathological processes.”18 The complete protein sequence was largely unknown until 1983 when Takio et al. published their work on rat liver enzymes.19 Today, it is known that the cathepsin B gene is located on chromosome 8, and it can be identified as the CTSB Gene. The location of the gene can be found below in Figure 1. Figure 1. Showing the Genomic Location of CTSB Gene in Chromosome 8. The red line located between p23 and p22 shows the location of the CTSB Gene. The exact location is marked as 8p23.1 20 In 1991, Musil et al. were finally able to decipher the crystal structure of human liver cathepsin B through Patterson search and heavy atom replacement methods. The Patterson function is equivalent to the Fourier transform of the intensities of the compound rather than the structure. The Patterson function was used in this approach because it is useful in solving the phase problem in X-ray crystallography. Shortly thereafter in 1996, Cygler et al. synthesized the inactive precursor of cathepsin B, procathepsin B. Both of these breakthroughs were pivotal in developing the fundamental 5 basis of cathepsin B specificity. Because of the X-ray data, Musil et al. were able to reconfirm the disulfide connectivities of bovine cathepsin B that were proven chemically correct by Bettie Evans and Elliott Shaw.21 Overall, Cathepsin B is a bilobal protein of approximately 30kDa with the active site and the substrate binding site located between two large lobes of the protein.22 The precursor, procathepsin B consists of 43~46 kDa. When the mature cathepsin B is formed, the procathepsin B is split apart into two chains linked together by a disulfide bridge. The heavy chain of cathepsin B is approximately 25 to 26 kDa, and the light chain is approximately 5 kDa.20 The enzyme is synthesized as a preproenzyme of 339 amino acids, and it has a signal peptide consisting of 17 amino acids. When matured and modified, human liver cathepsin B is reduced to a sequence length of 205 amino acids on the heavy chain and 47 amino acids on the light chain.23 Musil et al.