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DEVELOPMENT OF METHODS TO SCREEN AND EVALUATE SMALL MOLECULE BINDERS OF THE PROTEASOME

thesis
posted on 27.04.2021, 20:18 by Marianne Elizabeth MareshMarianne Elizabeth Maresh

The proteasome is responsible for the majority of protein degradation in the cell. Dysregulation of proteasome activity is implicated in a variety of diseases such as cancer, neurodegeneration, and aging. As a result, it has emerged as therapeutic target and significant research to elucidate the impacts of modulating its activity with small molecules is ongoing. While a variety of small molecule binders have been identified and shown to be effective in treating diseases such as cancer, there is a lack of standardized assays to identify new binders and evaluate their impacts in biologically relevant settings. In this work, we present a suite of assays that can be used to screen proteasome subunits for new binders and determine the impacts of small molecules in yeast with reduced proteasome capacity. We also explore stimulation of the immunoproteasome (iCP), a proteasome isoform that is expressed during inflammatory conditions.

We first developed a method to screen one-bead-one-compound (OBOC) libraries in which hits could be prioritized. Our method involves labeling a target protein with a near-infrared range fluorophore and incubating it with OBOC library beads in a well plate. The plate can then be imaged for fluorescence and the signal can be quantified. This provides a basis to rank hits. We used this screening method to identify TMM-6, a peptoid that covalently binds the oncoprotein gankyrin. Since this method does not require the function of a protein to be known, it is ideal for screening noncatalytic proteasome subunits.

We then optimized an assay to monitor the chronological lifespan (CLS) of yeast in response to dosage with small molecules that stimulate the proteasome. Through alterations of growth media, we successfully reduced the time required to perform this assay from over a month to just one week. Similarly, the assay was scaled down to a 96-well plate format to make it more amendable to high-throughput applications. We used this assay to evaluate the impacts of the proteasome stimulator ursolic acid (UA) on the CLS of yeast with reduced proteasome capacity. We found that dosage with UA resulted in slight lifespan extension.

The iCP generates peptides that are compatible with major-histocompatibility complexes type I (MHC-I) and several viruses are known to deploy proteins that interact with the iCP to inhibit its activity. We hypothesize that iCP inhibition could be rescued with a small molecule stimulator and in turn increase the pool of MHC-I compatible peptides, allowing virally infected cells to be recognized by the immune system. Here, we report UA and structurally similar compounds as potent iCP stimulators. Stimulation was demonstrated in both a biochemical assay and in cells induced to express the iCP.

This work resulted in several new assays that can be used to quickly identify quality binders to proteasome subunits and evaluate their impacts in aged yeast. We anticipate usage of these assays will result in the discovery of new proteasome binders that impact its activity. Particularly, we are interested in determining the impacts of targeting noncatalytic proteasome subunits, as their therapeutic potential remains largely underexplored. We also identify new iCP stimulators and plan to further explore the impacts of iCP stimulation on MHC-I expression.

History

Degree Type

Doctor of Philosophy

Department

Medicinal Chemistry and Molecular Pharmacology

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Darci Trader

Additional Committee Member 2

Vincent Jo Davisson

Additional Committee Member 3

Michael Wendt

Additional Committee Member 4

Qing Deng