Date of Award

Fall 1-1-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Pyle, Anna

Abstract

Retinoic acid-inducible gene I (RIG-I) is a human pattern recognition receptor (PRR) that plays a key role in the innate immune response against RNA viruses, including influenza viruses, coronaviruses, and flaviviruses. As a ubiquitously expressed protein in nearly all nucleated cells, RIG-I resides primarily in the cytoplasm, where it detects foreign RNA. Although RIG-I can bind to a variety of RNA molecules, it is selectively activated by viral RNAs that have specific structural features—such as blunt-ended double-stranded regions and 5′-triphosphates. Beyond its antiviral role, RIG-I has emerged as a promising target in cancer immunotherapy, largely due to its ability to modulate immune responses by inducing interferons and promoting immunogenic cell death. The efforts on the discovery of RIG-I agonists with the detailed understanding of RIG-I's activation mechanism has been crucial in exploring its therapeutic potential. While RNA-based RIG-I agonists show great promise in recent antiviral and anti-tumor preclinical exploration, they face limitations related to stability, manufacturing, and delivery. Consequently, there is significant interest in identifying small-molecule RIG-I modulators. The small molecule therapeutics are advantageous due to their favorable pharmacokinetics, cost-effectiveness, ease of administration, and better stability compared to RNA-based drugs. However, the discovery of small-molecule RIG-I agonists has lagged behind, with only a few identified through phenotypic assays that indirectly measure downstream activation events, such as IRF3 signaling. Despite their unclear mechanisms of action, some of these molecules have shown efficacy, including as adjuvants in influenza vaccination. My thesis work has undertaken this challenge and focused on the discovery of RIG-I small molecule agonists. To address the lack of direct and target-specific RIG-I agonists, I started out by developing the first high-throughput target-based biochemical assay designed to detect small molecules that directly activate RIG-I (Chapter 2). The assay reports on the essential early step of RIG-I activation: CARD ejection. Using biorthogonal click chemistry and sortase-mediated labeling, a fluorophore and a quencher were successfully conjugated to the CARD and Hel2i domains, respectively. This design enabled precise and sensitive detection of conformational changes associated with RIG-I activation. Through this innovative design, I successfully optimized the assay conditions to enable a high-quality high-throughput (HTP) screening platform (Chapter 3). Leveraging this platform, I carried out a large-scale screening campaign and identified several small-molecule RIG-I agonists with in vitro activity. To further refine and expand upon these initial hits, I conducted structure–activity relationship (SAR) studies, which led to the development of a series of RIG-I agonists featuring a novel and specific chemotype (Chapter 4). These compounds were further characterized for their biochemical properties, including potency, binding affinity, and mechanism of action. Subsequently, I explored whether these in vitro agonists could activate RIG-I in a cellular context (Chapter 5). Using various cellular systems, and employing luciferase reporter assays and RT-qPCR to detect RIG-I activation, I identified agonists capable of activating RIG-I in cells and inducing a robust interferon response. Overall, my thesis work has advanced the field by first establishing a high-throughput screening assay that enables precise RIG-I targeting. Additionally, the labeling schemes developed during assay optimization serve as a model for labeling large, multidomain proteins. The novel chemotype I identified for RIG-I agonists holds promise for future development as antiviral and immunotherapeutic agents with broad clinical potential.

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