Applications of molecular modeling techniques in the design of xanthine based adenosine receptor antagonists and the development of the protein function annotation method SALSA by Joslynn S. Lee B.S. in Chemistry-Biochemistry and Cellular Molecular Biology, Fort Lewis College A dissertation submitted to: The Faculty of the College of Science of Northeastern University In partial fulfillment of the requirements for the degree of Doctor of Philosophy February 21, 2014 Dissertation directed by Mary Jo Ondrechen Professor of Chemistry and Chemical Biology Graham B. Jones Professor of Chemistry and Chemical Biology © 2014 Joslynn S. Lee ALL RIGHTS RESERVED Dedication I dedicate this dissertation to my grandparents, Susie & William Pino and Tom & Alice Lee, who inspired me to get a higher education since they were not given the opportunity to go to college. On the Navajo reservation, shinálí asdzáá (my maternal grandmother) would take me to herd goats and pick wild tea (Thelesperma sp). She would use this plant would help with stomach aches and digestion. This was my first memorable experience that peaked my interest in science. Their stories and beliefs passed down to me, have shaped the person I am today. Ahéhee’ and Dawaeh (thank you in Navajo and Kerese) Hózhóogo naasháa doo (In beauty I walk) Shitsijí' hózhóogo naasháa doo (With beauty before me I walk) Shikéédéé hózhóogo naasháa doo (With beauty behind me I walk) Shideigi hózhóogo naasháa doo (With beauty above me I walk) T'áá altso shinaagóó hózhóogo naasháa doo (With beauty around me I walk) Hózhó náhásdlíí' (It has become beauty again) Da wa `eh, he numeh, Joslynn Lee, shawiiti hanu stah `che, Naya eh, Na sge ya, ma mie, stru gu na ma, eh za, uum`atsi, du dra ne, ha ya, s`au du me`tra, no ya zi, eh sw ue no 'ta. (Thank-you, from me, Joslynn Lee of the Parrot Clan of my people. To our Mother Earth and Father Creator, I'm thankful for the blessings and help bestowed upon me to complete my education.) Acknowledgements I want to thank my advisor, mentor and friend Professor Mary Jo Ondrechen for allowing me to join her lab to conduct research. Mary has always shown unconditional support and patience during my time at NU. I want to thank my co-advisor, mentor and friend, Professor Graham Jones for building a collaboration for molecular modeling and research for the A2AAR project (Part I) and support during my time at NU. I want to extend my gratitude, feedback and support to my thesis committee members Professor Carla Mattos and Professor Gene Cooperman. Their guidance has been invaluable. I cannot give enough thanks to previous ORG members: Dr. Srinivas Somarowthru, Dr. Jaeju Ko, Dr. Leo Murga, Dr. Heather Brodkin, Dr. Pengcheng Yin and Dr. Zhouxi Wang, for their help, patience and discussions for my research. I thank the current ORG members Dr. Ramya Parasuram, Caitlyn Mills, Lisa Ngu for their time discussion my research and for their support! My research would not be a great story without the collaborations for the A2AAR and SALSA projects. Rhiannon Thomas-Tran is an amazing individual who helped get the A2AAR project going and also Vincent Chevalier for the scale-up of molecules for in vitro studies. The in vitro studies were performed in the lab of Professor Michail Sitkovsky by Dr. Stephen Hatfield and Dr. Kaisa Selesniemi. The SALSA-DT program could not have been written or optimized without the help from Rohan Garg (CCIS), Liang Tian (Math), Jiajun Cao (CCIS), Professor Gene Cooperman (CCIS) and Professor Alexandru Suciu (Math). The wonderful staff in the Chemistry and Chemical Biology department, Jean Harris, Cara Shockley, Richard Pumphrey and Andrew Bean, they were happy and helpful individuals. I want to thank my undergraduate professors who made an impact on my research career. From FLC, Dr. Leslie Sommerville, Dr. Ron Estler and Dr. Rob Milofsky, were the first to encourage me to pursue research in chemistry. Les for his unconditional support and continued mentoring. Dr. Harry Higgs from Dartmouth University who let me conduct summer research in his lab and give me a preview of graduate school level work, pairing me up with Dr. Susie Nicholson- Dykstra. She helped me build my confidence as a budding scientist! Before applying to grad school, Dr. Christopher Brummel from Vertex Pharmaceuticals, assigned me challenging projects to tackle as a research associate but most importantly encouraged me to leave my position to pursue graduate level studies full-time. Dr. Daniel Jay, from Tufts Med School, from the Academy for Future Faculty (AFF) provided his support, along with the amazing cohort of doctoral students, who all inspire me to pursue my goal of becoming a professor. My family has been pivotal in my pursuit of a graduate degree, becoming the first Ph.D. My sister, Rhiannon Lee, who provided her unconditional love and support during graduate school. To her wife, Eliza, for providing me with laugh and constant support, especially a place to stay while writing my dissertation. My parents, Thomas & Valeria Lee, for everything. They believed in me and instilled my cultural beliefs that have shaped the woman I am today. Their continuous support to further my education. My brother Clifton, his wife KC and their kids for being my cheerleading squad filled with unconditional love! My projects were financially supported by the National Science Foundation under grants MCB- 0843603/1158176 and CHE-1305655. Receiving the prestigious NSF graduate research fellowship allowed me to share my results internationally and necessary instrumentation in lab. Other funding sources include Dana Farber Institute and IGERT-NU. Abstract of Dissertation In the field of molecular modeling, theoretical and computational methods are used to study biological structures, dynamics and interactions. Using functional site predictors to identify the binding sites in 3D protein structures provides information that can be used in the fields of drug design and protein function annotation. This dissertation is divided in two parts, each describes the application of molecular modeling techniques in the development of antitumor immunotherapies to target the human A2A adenosine receptor and the development of an automated program, Structurally Aligned Local Sites of Activity (SALSA), to assign function based on local sites of alignment and chemical properties. Cancer cells undergo tumor hypoxia in the earlier stages of growth. Hypoxic tumors generally have poor prognosis and can become resistant to traditional radiation and chemotherapy treatments. In the tumor microenvironment, an overaccumulation of adenosine is produced through the hypoxia-adenosinergic tissue-protecting mechanism that activates A2A adenosine receptors (A2AAR) on the surface of surrounding cells, leading to the protection of cancerous tissues. Targeting the A2AAR with antagonists will disrupt A2AAR signaling, thus preventing the protection of tumor cells. Molecular modeling techniques were applied to the design and optimization of xanthine-based A2AAR antagonists. In silico docking studies, using the programs AutoDock 4.0 and Schrodinger’s GLIDE, yielded similar and favorable binding poses to identify a lead compound. The lead compound was converted to a PEG derivative tested in two in vitro functional binding assays to confirm efficacy. The lead compound performed better than a previously known antagonist. In 2000, the Protein Structure Initiative (PSI) was launched to make the 3D structures of proteins easily attainable from the knowledge of their corresponding DNA sequences. The PSI project generated a large set of 3D structures named structural genomics proteins. To date, there are 12,900 structural genomics (SG) protein structures reported in the Protein Data Bank. These SG proteins were annotated (assigned function) using sequence or structural similarity matching tools. Unfortunately, a number of these SG proteins are listed with unknown, putative and hypothetical function. The computational method, Structurally Aligned Local Sites of Activity (SALSA) is able to reliably assign function to these SG proteins. The strategy of SALSA is to define functional subclasses of characterized members from a protein superfamily. Functional site predictors identify active site residues to create unique signatures for each subclass. Incorporating multiple structure alignment tools, a local spatial pattern of the active site residues is established for each functional subclass and this can be used to sort a superfamily according to biochemical function. An automated version of SALSA, SALSA-DT, utilizes Delaunay triangulation to better match the local active sites of 3D protein structures for faster structural alignments within the superfamily. The application of the SALSA and SALSA-DT methods applied to the ribulose phosphate binding barrel (RPBB) superfamily, successfully sorts the superfamily and 27 SG proteins are evaluated. Additionally, SALSA revealed more information about the structural architecture of the active sites within the superfamily. Table of Contents Dedication …………………………………………………………………………… iii Acknowledgements ……………..………………………………………………………... iv Abstract of Dissertation …………………………………………………………………. vi Table of Contents ……..………………………………………………………..………. viii List of Figures …………………………………………………………………………… xv List of Tables …………………………………………………………………………… xxii List of Abbreviations …….………………………………………………………………… xxv Part I – Using molecular modeling techniques in designing small molecules to target the human A2A adenosine receptor