Date of Award

9-26-2024

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Natural products FR900359 and YM254890, selective inhibitors of the Gq/11 protein signaling pathway, are crucial for therapeutic development, particularly in treating uveal melanoma. This dissertation focuses on efforts to understand the mechanisms behind this selective inhibition. A versatile synthetic approach to these natural product analogs and a potential method for evaluating their biological activity on the microelectrode array are presented. Chapter 1 provides an overview of Gq/11 proteins, detailing their roles in cellular signaling. Mutations lead to constitutively active oncogenic Gq/11 proteins, contributing to uveal melanoma. The building-block synthetic concept is introduced in the YM/FR synthesis, enabling relatively scalable and versatile synthesis. The necessity of developing their analogs and new methods for rapid biological activity assessment is outlined. Chapter 2 describes the development of the hydroquinone/benzoquinone couple as a more stable mediator than the previously utilized iron-redox pair, providing more reproducible and reliable data. Methods for calibrating signals on the array, including varying the number of cycles for the placement reaction and pre-diluting peptide solutions, are also discussed. These methods, combined with the new redox mediators, were successfully applied in model studies with RGD/integrin and R6A/Gi1 interactions, improving the accuracy of binding information. Chapter 3 focuses on the synthesis of YM385781, an analog of natural product YM254890. Challenges and strategies of synthesizing this complex structure are discussed, such as oxazolone epimerization, beta-elimination, and diketopiperazine formation. The synthesized analog exhibits full efficacy in inhibiting wild-type and oncogenic Gq signaling pathways but with reduced potency compared to the natural product FR, consistent with previous studies. The next chapter focuses on the synthesis of FR analogs with potential potency improvement. Analogs with linkers designed for bio-labeling and array attachment are discussed. The building block synthesis was optimized using pseudo-proline strategies with improved synthetic efficiency. The synthesized FR analogs demonstrate full efficacy, but no improved potency compared to YM analogs was observed, suggesting further modifications are needed. The thesis concludes with a summary of the developed synthetic routes for YM and FR analogs, their biological evaluation, and the optimization of signaling studies on microelectrode arrays. Future directions include exploring enzymatic synthesis of (2S, 3R)-beta-hydroxyleucine, optimizing macrocyclization steps, reintroducing dehydroalanine residues, and further developing biological evaluation methods using microelectrode arrays. These efforts aim to enhance the potency and scalability of synthesized analogs, thereby improving our understanding of selective binding and advancing therapeutic development.

Language

English (en)

Chair and Committee

Kevin Moeller

Committee Members

Vladimir Birman, John-Stephen Taylor; Kendall Blumer; Timothy Wencewicz

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