Browsing by Subject "High-throughput screening"

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    Identification of compounds inhibiting a Leishmania RNA editing reaction.
    (2010-05) Liang, Shuang
    Several species of Leishmania are human pathogens that afflict more than 12 million people worldwide, and the current treatment options are limited. An RNA editing reaction that is both essential and specific to the parasites is an attractive target for new drug development. The editing reaction involves the post-transcriptional modification of specific mitochondrial mRNAs through the precise deletion or insertion of uridylates. Many aspects of the editing mechanism are still unclear, and the lack of specific inhibitors to probe the reaction has hindered the field. Although high-throughput screening of chemical libraries is a powerful strategy often used to identify inhibitors, the available in vitro editing assays do not have the necessary sensitivity and format for this approach to be feasible. A novel editing assay was developed in this thesis that overcame previous limitations as it can both detect edited product in the low femtomole range and is ideal for high-throughput format. The reporter for the assay consists of an RNA editing substrate linked to a streptavidin-binding domain that is initially held within an inactive conformation. An in vitro selection strategy optimized the linkage so that the streptavidin-binding domain is only activated by an editing-induced conformational change. The reporter RNA is labeled with a ruthenium complex, and an electrochemiluminescent signal results from the ruthenium label when the reporter is bound to the bottom of a streptavidin-coated microtiter plate where it can be stimulated by a carbon electrode. Chemical probing, mutagenesis and binding affinity measurements were used to characterize the reporter. This highly sensitive assay was optimized and validated for use in high-throughput screening, and a pilot screen of a 1280 compound library identified compounds that are the first specific inhibitors of the editing reaction. Some of the identified inhibitors will have value as probes of the editing reaction and have already provided insights into possible regulatory mechanisms. The identification of novel drugs through screens of large chemical libraries is now possible with the new assay.
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    Targeting Pathological Protein Interactions in Drug Discovery
    (2021-07) Young, Malaney
    The interaction of proteins with themselves or other molecules is essential to biological function. The dysregulation of normal protein structure, folding, and interactions forms the basis of many human diseases. Elucidating the protein interactions which underlie various disease states is crucial to understanding disease progression and identifying protein targets for therapeutic development. This dissertation focuses on targeting pathological protein interactions in cancer, non-alcoholic fatty liver disease (NAFLD), and neurodegenerative disorders. The tools developed in this dissertation are not only relevant to these specific disease states, but can be modified to pursue novel protein targets for drug discovery in other diseases as well. Chapter 1 of this dissertation provides an explanation of the scientific background and previous research that has motivated the current work. Chapter 2 presents the first high-throughput screening (HTS) platform that detects structural changes in death receptor 5 (DR5), a membrane protein which regulates apoptosis. In this chapter we have identified small-molecules which modulate DR5 signaling and sensitize TRAIL-resistant cancer cells to TRAIL-induced apoptosis. In chapter 3, the same HTS platform is used to discover novel inhibitors of DR5-mediated TRAIL-induced apoptosis for therapeutic intervention of NAFLD and Alzheimer’s Disease. Chapter 4 provides a summary of various projects which involved research on alpha-synuclein (α-syn), the primary protein involved in Parkinson’s Disease. In chapter 4.2, yeast display is implemented as a screening platform to identify inhibitors of α-syn uptake via targeting neurexin-1β. Chapter 4.3 provides an in-depth protocol on the purification of α-syn for laboratory studies. Chapter 4.4 summarizes an attempt to create a cell-free FRET-based biosensor to target the pathological aggregation of α-syn. Chapter 4.5 details a protocol for producing dopamine-modified α-syn oligomers for laboratory studies on dopamine-induced α-syn toxicity. Finally, chapter 4.6 identifies threonine 75 in α-syn as a critical amino acid for fibrillization. This work has overall contributed to the field of drug discovery by providing novel high-throughput screening tools to study DR5 and other protein-protein interactions, and has further elucidated important aspects of α-syn aggregation to be targeted in Parkinson’s Disease.