Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2015 Insights into the Development of Chemotherapeutics Targeting PFKFB Enzymes Robert Blake Crochet Louisiana State University and Agricultural and Mechanical College, [email protected] Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Recommended Citation Crochet, Robert Blake, "Insights into the Development of Chemotherapeutics Targeting PFKFB Enzymes" (2015). LSU Doctoral Dissertations. 311. https://digitalcommons.lsu.edu/gradschool_dissertations/311 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. INSIGHTS INTO THE DEVELOPMENT OF CHEMOTHERAPEUTICS TARGETING PFKFB ENZYMES A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Biological Sciences by Robert Blake Crochet B.S., Louisiana State University, 2009 December 2015 ACKNOWLEDGMENTS I would like to express my sincere appreciation and thanks to the many people who have guided, supported, and encouraged me throughout my graduate studies. I am deeply indebted to my advisor, Dr. Yong-Hwan Lee, for making this work possible. No words of thanks are enough for his mentorship, guidance, support and patience. I also thank the members of my committee, Dr. Grover Waldrop, Dr. Naohiro Kato, and Dr. Michal Brylinski for their invaluable support in improving my research project. Additional thanks are owed to my fellow lab members: Dr. Jeong-Do Kim, Dr. Michael Cavalier, and Dr. Minsuh Seo for the contributions they have made to my education. ii TABLE OF CONTENTS ACKNOWLEDGMENTS ...........................................................................................................ii LIST OF TABLES ......................................................................................................................v LIST OF FIGURES ......................................................................................................................vi ABSTRACT ..............................................................................................................................vii CHAPTER 1: REVIEW OF LITERATURE ……………………………....................................01 1.1 Glycolysis...................................................................................................................01 1.2 The Glycolytic Pathway Is Tightly Controlled .......................................................01 1.3 The Bifunctional Enzyme, PFKFB..........................................................................04 1.3.1 The Liver Isoform (PFKFB1) .............................................................09 1.3.2 The Heart Isoform (PFKFB2) .............................................................10 1.3.3 The Inducible Isoform (PFKFB3) .............................................................11 1.3.4 The Testis Isoform (PFKFB4) ............................................................12 1.4 Glycolytic Modification in Cancer Cells: The Warburg Effect ................................13 1.5 PFKFB and Cancer Metabolism ...............................................................................15 1.6 PFKFB as a Chemotherapeutic Target .....................................................................17 CHAPTER 2: REVIEW OF LITERATURE .............................................................................21 2.1 Drug Discovery and Development ..........................................................................21 2.2 Virtual Screening …..................................................................................................22 2.3 Docking-Based Approaches .....................................................................................25 2.3.1 Protein Flexibility .....................................................................................25 2.3.2 Search Algorithms .....................................................................................30 2.3.2.1 Ligand Orientation Algorithms ..................................................31 2.3.2.2 Ligand Sampling Algorithms .....................................................33 2.3.2.3 Scoring Functions ......................................................................36 2.4 Ligand-Based Approaches .......................................................................................39 2.4.1 Similarity Based Methods .........................................................................40 2.4.2 Pharmacophore Based Methods ................................................................41 2.4.3 QSAR Based Methods ...............................................................................43 CHAPTER 3: INVESTIGATING COMBINATORIAL APPROACHES IN VIRTUAL SCREENING ON HUMAN INDUCIBLE 6-PHOSPHOFRUCTO-2- KINASE/FRUCTOSE-2,6-BISPHOSPHATASE (PFKFB3): A CASE STUDY FOR SMALL MOLECULE KINASES. 3.1 Abstract ....................................................................................................................44 3.2 Introduction ..............................................................................................................45 3.3 Materials and Methods ……...................................................................................48 3.4 Results ......................................................................................................................50 3.5 Discussion .................................................................................................................54 iii CHAPTER 4: CRYSTAL STRUCTURE OF 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE- 2,6-BISPHOSPHATASE (PFKFB2) AND THE INHIBITORY INFLUENCE OF CITRATE ON SUBSTRATE BINDING. 4.1 Abstract ....................................................................................................................60 4.2 Introduction ..............................................................................................................61 4.3 Materials and Methods ………..................................................................................62 4.4 Results and Discussion ............................................................................................65 CHAPTER 5: CONCLUSIONS ..................................................................................................75 REFERENCES .......................................................................................................................78 VITA ...........................................................................................................................97 iv LIST OF TABLES Table 4.1. Statistics of reflection data and structure refinements ..............................................64 Table 4.2. Citrate inhibition properties on wild-type and mutant PFKFB2 .................................73 v LIST OF FIGURES Figure 1.1. Fructose-2,6-Bisphosphate, the most potent allosteric activator of glycolysis .........03 Figure 1.2. Structure of human PFKFB3 .....................................................................................05 Figure 1.3. P-loop NTPase ...........................................................................................................07 Figure 1.4. Substrate binding and mechanism .............................................................................08 Figure 1.5. Summary of Phosphorylation Sites and Enzyme Kinetics of PFKFB Isoforms .......13 Figure 1.6. Overview of the Metabolic Reprogramming of Tumor Cells ...................................16 Figure 1.7. Exploiting the Warburg Effect using Positron Emission Tomography .....................18 Figure 2.1. Standard Drug Development Pipeline ........................................................................21 Figure 2.2. Chronological overview of yearly publications for high-throughput and virtual screening methods .................................................................................................23 Figure 2.3. Approaches to Virtual Screening ...............................................................................25 Figure 2.4. Classification of methods for protein-ligand docking ...............................................26 Figure 3.1. Identification of potent PFKFB3 inhibitors via a single-dose primary screening assay ......................................................................................................51 Figure 3.2. Inhibition for the PFKFB3 2-kinase by NSC278631 ...............................................51 Figure 3.3. The selected actives from the throughput screening of the NCI Diversity Set II ....52 Figure 3.4. Pharmacophore map used in PFKFB3 virtual screening ..........................................53 Figure 3.5. Enrichment Comparison of Popular SBD Technologies on PFKFB3 ......................55 Figure 3.6. Performance comparison between sequential and non-sequential virtual screening protocols ...............................................................................................................56 Figure 4.1. Dimeric arrangement of heart form and a comparison of the structures of the human and bovine orthologues .........................................................................................67