A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor

Madrigal-Carrillo, Ezequiel-Alejandro; Díaz-Tufinio, Carlos-Alejandro; Santamaría-Suárez, Hugo-Aníbal; Arciniega, Marcelino; Torres-Larios, Alfredo

doi:10.1093/nar/gkz285

A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor

Journal Article · Thu Apr 18 00:00:00 EDT 2019 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkz285· OSTI ID:1507595

Madrigal-Carrillo, Ezequiel-Alejandro ^[1]; Díaz-Tufinio, Carlos-Alejandro ^[2]; Santamaría-Suárez, Hugo-Aníbal ^[1]; Arciniega, Marcelino ^[1]; ^[1]

Department of Biochemistry and Structural Biology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico
Department of Biochemistry and Structural Biology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico, Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, Mexico City, Mexico

Ribonucleoprotein (RNP) complexes and RNA-processing enzymes are attractive targets for antibiotic development owing to their central roles in microbial physiology. For many of these complexes, comprehensive strategies to identify inhibitors are either lacking or suffer from substantial technical limitations. Here, we describe an activity-binding-structure platform for bacterial ribonuclease P (RNase P), an essential RNP ribozyme involved in 5' tRNA processing. A novel, real-time fluorescence-based assay was used to monitor RNase P activity and rapidly identify inhibitors using a mini-helix and a pre-tRNA-like bipartite substrate. Using the mini-helix substrate, we screened a library comprising 2560 compounds. Initial hits were then validated using pre-tRNA and the pre-tRNA-like substrate, which ultimately verified four compounds as inhibitors. Biolayer interferometry-based binding assays and molecular dynamics simulations were then used to characterize the interactions between each validated inhibitor and the P protein, P RNA and pre-tRNA. X-ray crystallographic studies subsequently elucidated the structure of the P protein bound to the most promising hit, purpurin, and revealed how this inhibitor adversely affects tRNA 5' leader binding. This integrated platform affords improved structure-function studies of RNA processing enzymes and facilitates the discovery of novel regulators or inhibitors.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: Consejo Nacional de Ciencia y Tecnologıa (CONACyT); Fundacion Marcos Moshinsky, Mexico; Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor; USDOE Office of Science (SC); Universidad Nacional Autonoma de Mexico

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1507595

Alternate ID(s):: OSTI ID: 1543004

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 12 Vol. 47; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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