PROJECT SUMMARY/ABSTRACT An inherent challenge in drug discovery is that every target requires its own individualized strategy. Many targets are “undruggable” by small molecules, which, owing to their rapid onset and low cost, are the pharmacologic cornerstone of both clinical therapies and research tools alike. A generalizable solution to inhibit the function of any given protein with a small molecule is therefore a holy grail of biomedical research. A possible solution exists in a recently described small molecule which stalls the translation of a very limited number of protein targets. This class of molecules binds in the ribosome exit tunnel to allosterically alter the peptidyl transferase center and prevent elongation, doing so in a manner that is dependent on the sequence identity of nascent chain. Though this novel mechanism of action is exciting, the off-target effects of the current compounds precludes their use either as therapeutics or as a clean sequence-specific chemical probe. In this proposal, we will directly address whether these compounds can be developed into useful research tools. In Aim 1, we will probe the intrinsic limits of the specificity of this technology, doing so by developing an optimized chemical genetics pair between the known compounds and an evolved target peptide sequence. In Aim 2, we will evaluate whether the maximum efficacy of these compounds can be improved by developing covalent analogs that can target specific cysteine-containing nascent chains. In Aim 3, we will ask whether the structures of the compounds can be tuned to alter the sequence specificity of the nascent chains they target. We will do this via an innovative kinetic target-templated synthetic strategy, which will simultaneously probe the accessible chemical space of the binding pocket with the exit tunnel, while also producing nascent chain specific chemical probes. We anticipate that advances in any one of these Aims would allow this technology to be further developed into a powerful tool: the ability to stop, and then restart, the protein production of any target of interest.

PROJECT NARRATIVE Nearly all proteins in the cell are made by the ribosome, meaning that a method to inhibit ribosome function could, in theory, affect any protein of interest. Such a method would be a powerful way to rapidly test the validity of a protein target for any disease, while simultaneously forming the starting point for a therapy itself. In this project, we will evaluate an emerging technology – a small molecule which inhibits the translation of only specific proteins – to determine whether its specificity, efficacy, and generalizability could permit it to be this useful biomedical research tool.