Leveraging Natural Product-derived Ribosome Inhibitors to Study Translation Dynamics

Abstract

Protein synthesis is the most energy-consuming process in cells and therefore is tightly regulated, often in cell type- specific manners. These regulatory mechanisms are defective in numerous diseased states. Moreover, recent discovery of cis-acting messenger RNA (mRNA) elements and ribosome collisions indicates that translation events are interdependent, and that the cellular translation machinery can recognize and respond to multiple translation events on a single transcript. If and how such regulatory events may control protein synthesis in physiological and diseased states remains to be fully examined and depends on new methods to study translation genome-wide and in high special and temporal resolution. Ribosome profiling (also known as Ribo-seq) has enabled transcriptome-wide mapping of actively translating ribosomes, shedding light on novel open reading frames, transcript-specific translation dynamics and quality control. However, it critically relies on ribonuclease digestion of mRNA outside of ribosome occupancy, physically destroying the original mRNA structure including the poly(A) tail and endogenous post-transcriptional mRNA modifications. It is also impossible to unravel whether multiple footprints arise from a single mRNA molecule or multiple mRNAs. Additionally, low-input ribosome profiling often requires careful titration of ribonuclease owing to the necessity of minimizing the non-coding RNA contaminants. In this thesis, I describe our effort toward a novel technology called Ribo-MaP, which maps translating ribosomes without ribonuclease digestion. For this purpose, we have developed iii chemical probes that facilitate modification of mRNA in a ribosome occupancy-dependent manner. To design such probes, I studied three natural product ribosome inhibitors with the intention of repurposing them into heterobifunctional ligands, which simultaneously inhibit elongating ribosomes and enable modification of mRNA residues in vicinity. In chapter 2, I discuss our total synthesis and structure-activity relationship (SAR) study of one of the most ubiquitously used ribosome inhibitors, cycloheximide (CHX). By designing a novel total synthesis route, we discovered previously unappreciated SAR of the cyclohexanone moiety of CHX and found C13- modified analogs with unexpected potency. In chapter 3, I describe our second-generation route to the C13-modified CHX analogs with improved efficiency and scalability. Through this improved route, we discovered highly accessible C13-benzamide CHX analog, and we demonstrated through our structural and biochemical characterization that the analog was more efficient at stalling the actively translating ribosomes in ribosome profiling application. Despite these attempts, modifying CHX with the goal of engineering an mRNA-targeting probe was unsuccessful. Therefore, in chapter 4, I shifted my focus to two more ribosome inhibitors, amicoumacin A and emetine, which inhibit protein synthesis by stabilizing mRNA-ribosome interaction in the E site. Through the SAR studies and cryo-EM collaboration with the Shao Lab at Harvard Medical School, we discovered that emetine can be extensively modified while maintaining translation inhibition activity. Lastly, I describe our recent progress in engineering emetine analogs as mRNA- modifying agents in two strategies. In the first strategy, a FKBP ligand was attached to emetine to recruit an RNA-editing enzyme to enzymatically modify the mRNA. In the second strategy, a reactive functional group was attached to emetine to chemically crosslink the mRNA near the emetine binding site. Preliminary results for both strategies suggest successful mRNA modification in ribosome occupancy-dependent manner. Optimization of the probes and further validation of ribosome mapping detection is in progress.