Smith Pilot Project Summary

The rise of antimicrobial resistance presents a new challenge to public health, and this crisis is further exacerbated by the dearth of new antibiotic agents. Targeting new interactions in microbial resistance mechanisms is a potentially fruitful avenue to both probe key regulatory processes, but also to provide leads toward new antibiotic therapeutics. Pyridoxal 5’-phosphate (PLP)-binding protein YggS has been demonstrated to support the trans-translation pathway through binding to a distal site in transfer-messenger RNA (tmRNA). Inhibition of this RNA-binding by YggS knockout significantly increased the sensitivity of Escherichia coli (E. coli) to aminoglycoside antibiotics. A central goal of this work is the development of a high-throughput screening platform to mine for small molecule inhibitors of this interaction. To this end, we propose a microscale thermophoresis inhibitor screen assay as an ideal bioanalytical technique to study the interaction with speed and in the near native state. A key outcome of the proposed study is a set of molecular features that for generalized inhibition across the family of PLP-binding protein interactions with RNA. This work will lay the foundation for rapid inhibitor creation in the future for therapeutically relevant interactions.

A second aim of this proposal is to develop catalytic technologies for the photoreactive, spatial labeling of tmRNA using YggS as a chromophore vehicle. By leveraging the native covalent linkage of PLP to YggS, reactive oxygen generating chromophores can be synthetically appended to the cofactor. Light-driven modification of tmRNA upon binding to YggS will offer both a reporter for YggS-like proteins and their interactions with tmRNAs that can be applied to other bacterial genera, but also will illustrate the specific YggS-binding region on tmRNA. This approach will allow for comparative interactome profiling across different pathogenic bacterial strains which is valuable for the future generation of novel antimicrobial agents and potentiators.