Employed for over half a century to study protein synthesis, cycloheximide (CHX, 1) is a small molecule natural product that reversibly inhibits translation elongation. More recently, CHX has been commonly used in ribosome profiling – a method for mapping ribosome positions on mRNA and translation genome-wide. Despite CHX’s extensive use, CHX treatment often results in incomplete translation inhibition due to its rapid reversibility, prompting the need for improved reagents. Described here is the total synthesis of CHX and analogues, allowing the establishment of structure–activity relationships responsible for translation inhibition. These studies aided the design of more potent compounds modified at the C13 position, one of which effectively, irreversibly blocks ribosomal elongation. Motivated by the beneficial properties of these inhibitors, we developed a simplified and rapid synthetic route to access novel C13-amide functionalized CHX derivatives that display significantly increased potencies toward protein synthesis inhibition and perform as superior reagents for ribosome profiling experiments. Cryogenic electron-microscopy revealed that benzamide 2 occupies the same site as CHX to compete with the 3' end of E-site tRNA. We demonstrate that 2 is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.

Natural product derivatives as powerful tools to study translation

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