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

Abstract

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.

Authors:
 [1];  [2];  [1];  [1]; ORCiD logo [1]
  1. Department of Biochemistry and Structural Biology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico
  2. 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
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC); Fundacion Marcos Moshinsky, Mexico; Consejo Nacional de Ciencia y Tecnologıa (CONACyT); Universidad Nacional Autonoma de Mexico; Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor
OSTI Identifier:
1507595
Alternate Identifier(s):
OSTI ID: 1543004
Grant/Contract Number:  
AC02-06CH11357; CB-82308; CB- 252316; CB-253323; PDCPN2014-47543; IN202416; IA202917
Resource Type:
Published Article
Journal Name:
Nucleic Acids Research
Additional Journal Information:
Journal Name: Nucleic Acids Research Journal Volume: 47 Journal Issue: 12; Journal ID: ISSN 0305-1048
Publisher:
Oxford University Press
Country of Publication:
United Kingdom
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Madrigal-Carrillo, Ezequiel-Alejandro, Díaz-Tufinio, Carlos-Alejandro, Santamaría-Suárez, Hugo-Aníbal, Arciniega, Marcelino, and Torres-Larios, Alfredo. A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor. United Kingdom: N. p., 2019. Web. doi:10.1093/nar/gkz285.
Madrigal-Carrillo, Ezequiel-Alejandro, Díaz-Tufinio, Carlos-Alejandro, Santamaría-Suárez, Hugo-Aníbal, Arciniega, Marcelino, & Torres-Larios, Alfredo. A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor. United Kingdom. doi:10.1093/nar/gkz285.
Madrigal-Carrillo, Ezequiel-Alejandro, Díaz-Tufinio, Carlos-Alejandro, Santamaría-Suárez, Hugo-Aníbal, Arciniega, Marcelino, and Torres-Larios, Alfredo. Thu . "A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor". United Kingdom. doi:10.1093/nar/gkz285.
@article{osti_1507595,
title = {A screening platform to monitor RNA processing and protein-RNA interactions in ribonuclease P uncovers a small molecule inhibitor},
author = {Madrigal-Carrillo, Ezequiel-Alejandro and Díaz-Tufinio, Carlos-Alejandro and Santamaría-Suárez, Hugo-Aníbal and Arciniega, Marcelino and Torres-Larios, Alfredo},
abstractNote = {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.},
doi = {10.1093/nar/gkz285},
journal = {Nucleic Acids Research},
number = 12,
volume = 47,
place = {United Kingdom},
year = {2019},
month = {4}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1093/nar/gkz285

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