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Small Molecule Modulators Reveal Mechanisms Regulating Cell Death

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Small Molecule Modulators Reveal Mechanisms Regulating Cell Death Investigating neurodegenerative diseases with small molecule modulators Reka Rebecca Letso Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2011 ©2011 Reka Rebecca Letso All Rights Reserved Abstract Investigating neurodegenerative diseases with small molecule modulators Reka Rebecca Letso Elucidation of the mechanisms underlying cell death in neurodegenerative diseases has proven difficult, due to the complex and interconnected architecture of the nervous system as well as the often pleiotropic nature of these diseases. Cell culture models of neurodegenerative diseases, although seldom recapitulating all aspects of the disease phenotype, enable investigation of specific aspects of these disease states. Small molecule screening in these cell culture models is a powerful method for identifying novel small molecule modulators of these disease phenotypes. Mechanistic studies of these modulators can reveal vital insights into the cellular pathways altered in these disease states, identifying new mechanisms leading to cellular dysfunction, as well as novel therapeutic targets to combat these destructive diseases. Small molecule modulators of protein activity have proven invaluable in the study of protein function and regulation. While inhibitors of protein activity are relatively common, small molecules that can increase protein abundance are quite rare. Small molecule protein upregulators with targeted activities would be of great value in the study of the mechanisms underlying many loss of function diseases. We developed a high- throughput screening approach to identify small molecule upregulators of the Survival of Motor Neuron protein (SMN), whose decreased levels cause the neurodegenerative disease Spinal Muscular Atrophy (SMA). We screened 69,189 compounds for SMN upregulators and performed mechanistic studies on the most active compound, a bromobenzophenone analog designated cuspin-1. Mechanistic studies of cuspin-1 revealed that increasing Ras signaling upregulates SMN protein abundance via translation, an effect which may be associated with the translational regulator mammalian target of rapamycin (mTOR). These findings suggest that controlled modulation of the Ras signaling pathway may benefit patients with SMA. Small molecule modulators of a disease phenotype, such as cell death, have the potential to reveal novel mechanisms regulating disease processes. This was exemplified by a screen for small molecule inhibitors of cell death caused by a pathogenic, misofolded mutant huntingtin protein in a cell culture model of Huntington’s Disease (HD). These cell death inhibitors were found to target protein disulfide isomerase (PDI), an oxidoreductase known to be important in endoplasmic reticulum quality control of protein folding. However, our studies utilizing the small molecule PDI inhibitors determined that the cell death observed in this system was due to a pro-apoptotic function of PDI involving proteins of the mitochondrial outer membrane. We have begun studies aimed at identifying the binding mode of these novel small molecule inhibitors of PDI, in efforts to develop more potent and efficacious analogs for testing in animal models of HD. These studies have helped defined a novel mechanism linking protein misfolding to cell death, and may prove to be relevant to a broader range of protein misfolding diseases. Table of contents List of Figures and Tables.……………………………………………………….….….....v Acknowledgements………………………………………………………………………vii Chapter 1: Spinal Muscular Atrophy.……………………………………….………….…1 I. Treating inherited neurodegenerative diseases………...………………….1 a. Gain of function neurodegenerative diseases…………………………2 b. Loss of function neurodegenerative diseases…………………………5 II. Spinal Muscular Atrophy Disease………………………………………...7 a. SMA disease pathogenesis……………………………………………8 b. SMA disease phenotypes……………………………………………...9 c. Genetics of SMA……………………………………………………..10 d. Animal models of SMA……………………………………………...15 III. Survival of Motor Neuron protein: Structure and Function……………...23 a. SMN expression and localization……………………………………23 b. SMN domain structure and functions………………………………..25 c. SMN interacting partners………………………………………….…28 i. The SMN Complex…………………………………………..29 ii. SMN interacts with hnRNP Q/R and β-actin mRNA………..30 iii. SMN and profilin…………………………………………….32 iv. The possible role of actin dynamics in Spinal Muscular Atrophy………………………………………………………33 d. Neuronal function(s) of SMN………………………………………..34 IV. Current state of small molecule therapeutics for SMA………………….46 i V. References……………………………………………………………….65 Chapter 2: Small molecule upregulator reveals regulation of SMN protein levels by Ras……………………………………………………………………………………….79 I. High-throughput screen for small molecule upregulators of SMN protein levels……………………………………………………………………..79 II. Screen development and optimization……………………….…………..81 III. Screening and hit validation………………………………………….…..83 IV. Structure activity relationship of cuspin-1…………………………….…86 V. Cuspin-1 reveals link between Ras signaling and SMN protein levels….88 VI. Mechanistic studies of Ras-mediated upregulation of SMN protein levels……………………………………………………………………..93 VII. The Raf-MEK-Erk pathway is not responsible for Ras-mediated upregulation of SMN…………………………………………………….96 VIII. Mammalian target of rapamycin modulates SMN protein levels…...….101 IX. The starvation response downregulates SMN protein levels…………...102 X. SMN downregulation is stimulated by serum deprivation………….….102 XI. Inhibition of mTOR by the small molecule rapamycin reduces SMN protein levels…………………………………………………………....104 XII. SMN downregulation is mediated by the proteasome………………….109 XIII. Discussion……………………………………………………………....114 XIV. Methods…………………………………………………………………117 a. Cell culture………………………………………………………….117 b. Small molecule libraries selected for screening…………………….118 ii c. Preparation of compound libraries for screening…………………...120 d. Primary screen……………………………………………………...120 e. Cytoblot fixation, permeabilization and antibody staining…………121 f. Cytoblot signal detection…………………………………………...121 g. Dose-response experiments…………………………………...……122 h. Robotic scripts and settings………………………………………...123 i. Analog synthesis……………………………………………………123 j. Western blot analysis………………………………………….……123 k. Retroviral preparation and transduction…………………………….125 l. RT-qPCR of SMN transcripts……………………………………….126 m. 35S-methionine/cysteine translation rate assay……………………...126 IX. References…………………………………………………………………..128 Chapter 3: High-throughput screening hit validation for Huntington’s Disease…….…133 I. Phenotypic small molecule screening in neurodegenerative diseases….133 II. Pathogenesis of Huntington’s Disease………………………………….133 III. Huntingtin protein……………………………………………………....134 IV. Protein misfolding and chaperones in neurodegenerative disease…...…135 V. Protein disulfide isomerase in Huntington’s Disease…………………..138 VI. Validation of PDI as a mitochondrially-mediated pro-apoptotic stimuli…………………………………………………………………..140 a. Rescue of mutant Htt-induced cell death by Bcl-2………………....140 b. Overexpression of PDI isoforms A1 and A3……………………….144 c. Mitochondrial outer membrane permeabilization (MOMP)………..146 iii d. Crystallization condition screening for the a domain of PDI isoforms A1 and A3…………………………………………………………..146 VII. Discussion………………………………………………………………151 VIII. Methods…………………………………………………………………152 a. Cell lines and reagents……………………………………………...152 b. Q103 PC12 plate-based viability assay……………………………..153 c. PDI overexpression……………………………………………...….153 d. Bcl-2 overexpression……………………………………………….154 e. Mitochondrial isolation…………………………………….……….154 f. MOMP assay……………………………………………..…………155 g. Expression and purification of PDI A1 and A3 a domains…………155 h. Crystallization condition screening…………………………………157 IX. References………………………………………………………...…….158 Chapter 4. Conclusions and Future Directions…………………………………………163 I. Spinal Muscular Atrophy……………………………………………….163 a. Summary………………………………………………………….....163 b. Significance……………………………………………………..…...164 c. Future Directions……………………………………………..……...164 II. Huntington’s Disease………...................................................................169 a. Summary………………………………………………………….....169 b. Significance…………………………………………………..……...170 c. Future Directions………………………………………………...…..170 III. References……………………………………………...……………….172 iv Figures and Tables Figure i. Low levels of SMN protein lead to SMA……………………………….…......13 Figure ii. SMN protein domain architecture…………………...........…………….…......26 Figure iii. SMN knockdown results in axonal pathfinding defects in zebrafish................44 Figure iv. The pathogenic events occurring in motor neurons in SMA.............................47 Figure 1. Schematic of primary screening workflow……………………………….…...82 Figures 2. Dose-response of the SMN-upregulating compounds 72 and 81 …..………..84 Figure 3. Cuspin-1 upregulates SMN protein levels in SMA patient fibroblast cells…...85 Figure 4. Structure-activity relationship of cuspin-1…………………………………….89 Figure 5. Increased Ras signaling upregulates SMN protein abundance……….……….91 Figure 6. Increased Ras signaling enhances SMN translation rate………………………94 Figure 7. SMN upregulation due to increased Ras signaling is not mediated by the Raf- MEK-Erk effector pathway………………………………………………………….…..97 Figure 8. Small molecule inhibition of downstream Ras effector kinases does not inhibit oncogenic Ras-mediated upregulation of SMN protein levels………………….……...100
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