i ii Characterization of Allosteric Modulators of Glutamate Transporters: Selectivity and Potency Screening Implications for Drug Development and for Understanding Allosteric Modulation of Glutamate Transport APEKSHA KHATIWADA AUGUST 2018 A Dissertation Presented to Faculty of Drexel University College of Medicine In partial fulfilment of the Requirement for the Degree of Masters of Science in Drug Discovery and Development iii ACKNOWLEDGMENTS I would like to acknowledge everyone who believed in me and has been a constant motivator throughout my journey. Firstly, I would like to extend my sincere gratitude to my mentor, Dr. Andreia Mortensen for her guidance, support and encouragement towards my work. She has been so nice to me and taught all the laboratory techniques, helped me improved my presentation and critical thinking skills. I feel so fortunate to have this wonderful opportunity to do my master’s thesis under her supervision. This project would not have been possible without Dr. Joseph Salvino. All the synthetic analogs used for the project has been synthesized in medicinal chemistry lab under his supervision. I would also like to thank Dr. Wagner F. Santos (University of São Paulo, Brazil) for kindly providing us additional compound for my study. I would like express my heartfelt thanks to members of my thesis committee: Dr. Ole Mortensen, Dr. Joanne Mathiasen, and Dr. Joseph Salvino for their time and support. Every suggestions and feedbacks from them has been a tremendous tool towards making my work as best as possible. I would also like to thank Dr. Paul McGonigle and Dr. Joanne Mathiasen for making the Drug Discovery and Development program so interesting and for all the valuable feedbacks, interactions and words of encouragement. I would also like to thank Dr. Ole Mortensen for his insights and knowledge that really helped me understand the neuropharmacology and for stimulating the discussions during presentations that helped me build my confidence. iv I would also like to thank previous lab members Ryan Wretz, Romulo Falcucci and Srawastie Sarker for their contributions toward the project. I would also like to thank Rafaela Scalco, an exchange student from Brazil who trained me with all the basic skills for my project. I would like to thank my current lab member Jennifer Green for her support and contribution towards my work. I would also like to thank Stacia Lewandowski, Caitlyn Rice and Shaili Agarwal and Xiaonan Liu for their useful inputs and suggestions towards my work. They have been amazing support to me. I would also like to thank the Meucci lab for providing some of the animals for my study, the Hu lab for providing the dissection hood and the front office for being so kind and cooperative all the time. There is nothing greater than family’s love and support and I feel extremely blessed to have amazing family and super supportive husband Pratic. They have always always believed in me and encouraged me to do better. I would never be able to achieve what I have without their support. I owe them a huge thanks. v Contents List of Figures and Tables ...................................................................................................................... vii Abstract ...................................................................................................................................................... ix 1. INTRODUCTION ..................................................................................................................................... 1 1.1. GLUTAMATE AND GLUTAMATERGIC SYNAPSE ................................................................... 1 1.2. GLUTAMATE RECEPTORS ............................................................................................................. 3 1.3. GLUTAMATE TRANSPORTERS: LOCALIZATION AND MOLECULAR FUNCTIONING ........... 5 1.4. GLUTAMATE EXCITOTOXICITY ................................................................................................. 10 1.5. PREVIOUS STRATEGIES TO TREAT THE HARMFUL EFFECTS OF GLUTAMATE EXCITOTOXICITY AND THEIR LIMITATIONS ................................................................................. 12 1.6. TARGETING GLUTAMATE TRANSPORTERS: AN EMERGENT STRATEGY........................... 14 1.7. ALLOSTERIC MODULATION ....................................................................................................... 16 1.8. IDENTIFICATION OF PAMs OF EAAT2: PREVIOUS STUDIES ................................................ 17 1.9. PROGRESSION OF COMPOUNDS: FIRST TO THIRD GENERATION ...................................... 19 2. AIMS ....................................................................................................................................................... 21 3. MATERIALS AND METHODS .......................................................................................................... 22 3.1. MATERIALS ................................................................................................................................... 22 3.2. EQUIPMENT .................................................................................................................................. 24 3.3 Synthesis of compounds and workflow chart ......................................................................... 24 3.4. Experiments with COS-7 cells .................................................................................................... 27 3.4.1. Transfection of COS-7 cells ................................................................................................. 27 3.4.2. Glutamate uptake assays in COS-7 cells ........................................................................... 27 3.5. Experiments with primary cultures of glia ............................................................................. 29 3.5.1 Glia dissection and plating .................................................................................................. 29 3.5.2 GLUTAMATE UPTAKE ASSAYS IN GLIA .............................................................................. 31 3.6 DOSE RESPONSE ASSAYS IN OTHER TYPES OF NEUROTRANSMITTER TRANSPORTERS 33 3.6.1 DOSE RESPONSE ASSAYS IN GABA (GAT-1) and GLYCINE (Glyt1, Glyt3) TRANSPORTERS ............................................................................................................................... 34 3.6.2 DOSE RESPONSE ASSAYS IN MONOAMINE TRANSPORTERS (hDAT, hNET and hSERT) ............................................................................................................................................... 35 3.7. IN VITRO NEUROPROTECTION STUDIES .................................................................................. 35 3.8. DATA ANALYSIS ............................................................................................................................ 36 vi 4. RESULTS ................................................................................................................................................ 38 4.1. DOSE RESPONSE ASSAYS IN COS-7 CELLS ................................................................................ 38 4.1.1. Validation of previous results ............................................................................................ 38 4.1.2 Non-selective stimulators of glutamate uptake .............................................................. 39 4.1.3. Stimulators of glutamate transport: selective to EAAT1 and EAAT2 ......................... 42 4.1.4. Stimulator of glutamate transport: selective to EAAT2 only ....................................... 45 4.1.5. Inactive compounds ............................................................................................................. 46 4.1.6. Inhibitor compounds ........................................................................................................... 47 4.2. DOSE RESPONSE ASSAYS IN GLIA .............................................................................................. 52 4.3. KINETICS STUDIES ....................................................................................................................... 56 4.3.1 KINETICS STUDIES IN COS-7 CELLS .................................................................................... 56 4.3.2 KINETICS STUDY IN GLIA ..................................................................................................... 60 4.4 DOSE RESPONSE ASSAYS IN OTHER TYPES OF NEUROTRANSMITTER TRANSPORTERS 64 4.4.1 DOSE RESPONSE IN GABA AND GLYCINE TRANSPORTERS ............................................ 64 4.4.2 DOSE RESPONSE IN MONOAMINE TRANSPORTERS ........................................................ 65 4.4. IN VITRO NEUROPROTECTION STUDIES .................................................................................. 67 4.4.1 NEUROPROTECTION STUDIED IN EXCITOTOXIC GLUTAMATE INSULTS .................... 67 4.4.2 NEUROPROTECTION STUDIED AFTER OXYGEN/GLUCOSE INSULTS ........................... 69 5. CONCLUSIONS ................................................................................................................................... 72 6. DISCUSSION AND FUTURE DIRECTIONS ...................................................................................... 81 7. REFERENCES ..................................................................................................................................... 88 vii List of Figures and Tables Figure 1. Glutamatergic synapse showing release of glutamate from