Rational Optimization of Small Molecules for Alzheimer's Disease

Rational Optimization of Small Molecules for Alzheimer’s Disease Premortem Diagnosis DISSERTATION Presented in Partial Fulfillment of Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Katryna Cisek Biophysics Graduate Program The Ohio State University 2012 Dissertation Committee: Jeffrey A. Kuret, PhD, Advisor Christopher Hadad, PhD Pui-Kai Tom Li, PhD Michael Tweedle, PhD Copyright by Katryna Cisek 2012 ABSTRACT Alzheimer’s disease is a debilitating, progressive neurodegenerative disorder that affects a large percentage of the elderly population. Currently, there is no definitive premortem diagnosis and no cure. Pathological brain tissue examination upon autopsy is used to identify the characteristic neurofibrillary tangles of tau protein and amyloid deposits that define this disease. These protein aggregates accumulate for many years before the onset of clinical symptoms; therefore, their in situ detection would be an invaluable tool for early premortem diagnosis. Because radiolabeled small molecules used for whole brain imaging, such as those used for PET imaging, have the advantage of tracking the spatiotemporal pattern of molecular targets, this approach has tremendous utility for neurodegenerative disorders. More specifically, the detection of tau-bearing neurofibrillary tangles is especially promising as the amount and spatiotemporal pattern of tau-aggregate deposition is the gold standard of postmortem disease assessment, as it correlates with loss of neurons. The main challenge in the development and optimization of a tau-selective imaging agent is the structure of these aggregates, which adopts a cross- ß-sheet of interdigitating monomers. Although multiple scaffold classes have been reported to bind cross-ß-sheet structure, their mechanism of binding and their ability to selectively bind different aggregates of varying protein composition are not well understood. There are no crystal or NMR structures that would reveal the atomic-level binding modes of this interaction. Most small molecule development studies focus on iterative structure-activity relationship modifications and testing of known scaffolds, such as the benzothiazole dye and commonly used tissue staining agent Thioflavin T. Even though quantitative structure activity relationship studies have been employed to investigate amyloid-binding ii compounds, these retrospective studies have not elucidated any novel compound molecular properties that could explain the mechanism of interaction. Moreover, these studies have not rationalized the binding activity or selectivity of cross-ß-sheet-binding ligands in the context of computational binding models. The project described herein is a ligand-based quantitative structure activity relationship approach to identify descriptors of binding affinity and selectivity for two series of over fifty closely related benzothiazole derivatives and indolinones reported to displace Thioflavin T fluorescent probe from synthetic aggregates composed of tau and ß-amyloid peptide. This is a novel computational approach in that it seeks to elucidate binding affinity, selectivity as well as binding site density in the context of existing experimental and computational data. I developed a two-step regression analysis for the identification of two-dimensional “global” descriptors of ligand potency and selectivity, in conjunction with a three-dimensional analysis for the identification of volumetric “local” regions for the optimization of R-group substituents of potent and selective ligands. The resulting models were statistically robust and predictive and provided clear guidelines for ligand optimization. For the indolinone series, the model was successful in predicting novel potent analogues, which upon synthesis and testing were shown to be very promising candidates for further preclinical studies. The dissertation comprises of six chapters; the 1st chapter introduces the key challenges of Alzheimer’s disease premortem diagnosis, namely the tau-based imaging strategy for early and differential diagnosis, and the current status of the field, including the limitations of current diagnostic agents, quantitative structure-activity relationships and computational models of compounds binding aggregates. The 2nd chapter is a feasibility study confirming that computational models are capable of identifying not only potency-driving features of ligands, but also descriptors that account for compound selectivity. Chapter 3 explains the mechanism of polarizability within a neural background that was identified in Chapter 2 as an important feature for affinity and selectivity, as well as its implication for binding site density. The following two chapters are quantitative structure-affinity relationship analyses of two different compound libraries, the benzothiazole-aryls and indolinones. The studies interpret features iii identified as the most significant drivers of binding affinity and selectivity and provide rational approaches for optimizing the scaffolds. Chapter 6 concludes with limitations of the computational studies and the significance of polarizability for aggregation inhibitors. iv ACKNOWLEDGEMENTS First and foremost I would like to thank my advisor, Dr. Jeff Kuret, who has been an excellent advisor, teacher and mentor. His leadership has allowed me to grow as a scientist, to develop my critical thinking and writing skills, and most importantly to fearlessly pursue challenging projects. I feel very lucky to have been a student in his lab. Big thanks to all of my lab colleagues. Dr. Nicolette Honson started the project and taught me many experimental methods; she and Jordan Jensen generated a lot of data in the lab and were instrumental to the success of the project and many publications that followed. Thank you to Kelsey Schafer for TEM imaging, as well as experimental work on inhibitors where I was able to contribute computational results. I would also like to thank current lab members Kristin Funk for confocal images, and Grace Cooper for continuing the project. Thank you to former lab members Edward Chang, Sohee Kim, Swati Naphade, Vandana Kumari and In Hee Park who were wonderful to work with. I would like to thank my committee; Dr. Christopher Hadad, for sharing his expertise with computational methods, Dr. Michael Tweedle and Dr. Pui-Kai Tom Li for their collaboration on the project. Dr. Michael Zhu and Dr. Dennis McKay have generously provided the use of their plate readers and Dr. Michael Darby for synthesis. Very special thanks to my family, especially my parents, Anna and Roman Cisek, whose unconditional support was paramount to my success in graduate school, as well as my brothers, Andrew and Lukas, sister-in-law Agnes, and other family and friends who have encouraged me from the very beginning. v VITA 2005……………………………………………Bachelor of Science in Computer Science University of Northern Iowa 2005……………………………………………………..……Bachelor of Arts in German University of Northern Iowa 2011……………………………………..………..………Master of Science in Biophysics Ohio State University 2005-Present…………………………………………………Graduate Research Associate Ohio State University PUBLICATIONS Jensen, J., Cisek, K., and Kuret, J. (2012) Benzothiazole-aryl compounds as potential tau- directed imaging agents [In Preparation]. Cisek, K., Jensen, J., Funk, K., Bhasin, D., Li, PK, Darby, M., and Kuret, J. (2012) Indolinone compounds as potential tau-directed imaging agents [In Preparation]. Cisek K, Kuret J. QSAR-guided development of tau-directed imaging agents. (2012) Int J Alzheimers Dis. [In Preparation]. Cisek K, Jensen JR, Honson NS, Schafer KN, Cooper GL and Kuret J. (2012) Ligand electronic properties modulate tau filament binding site density [submitted]. Cisek K, Kuret J. (2012) QSAR studies for prediction of cross-β sheet aggregate binding affinity and selectivity. Bioorg Med Chem. 20(4):1434-41. Kuret J, Jensen J, Cisek K. Benzothiazole and indolinone ligands for pre-mortem detection of aggregated tau proteins, provisional US patent filed July 27, 2011 [OSU Tech Licensing ID 2011-196]. vi Jensen JR, Cisek K, Funk KE, Naphade S, Schafer KN, Kuret J. (2011) Research towards tau imaging. J Alzheimers Dis. 26 Suppl 3:147-57. Jensen JR, Cisek K, Honson NS, Kuret J. (2011) Ligand polarizability contributes to tau fibril binding affinity.Bioorg Med Chem. 19(17):5147-54. Schafer KN, Murale DP, Kim K, Cisek K, Kuret J, Churchill DG. (2011) Structure- activity relationship of cyclic thiacarbocyanine tau aggregation inhibitors. Bioorg Med Chem Lett. 21(11):3273-6. Kim S, Jensen JR, Cisek K, Funk KE, Naphade S, Schafer K, Kuret J. (2010) Imaging as a strategy for premortem diagnosis and staging of tauopathies, Curr Alzheimer Res. 7(3):230-4. Fuh B, Sobo M, Cen L, Josiah D, Hutzen B, Cisek K, Bhasin D, Regan N, Lin L, Chan C, Caldas H, DeAngelis S, Li C, Li PK, Lin J. (2009) LLL-3 inhibits STAT3 activity, suppresses glioblastoma cell growth and prolongs survival in a mouse glioblastoma model, Br J Cancer. 100(1):106-12. Bhasin D, Cisek K, Pandharkar T, Regan N, Li C, Pandit B, Lin J, Li PK. (2008) Design, synthesis, and studies of small molecule STAT3 inhibitors, Bioorg Med Chem Lett. 18(1):391-5. FIELD OF STUDY Major Field: Biophysics Graduate Program vii TABLE OF CONTENTS Abstract……………………………………………………………………………………ii Acknowledgements………………………………....……………………………….…….v Vita…………………………………………………………………………………..……vi List of Tables…………………………………………………………………………….xii List of Figures……………………………………………………………………...…....xiii List of Abbreviations…………………………………………………………...…….…xiv

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