skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

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]
+ Show Author Affiliations
  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.
Copy to clipboard
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.
Copy to clipboard
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.
Copy to clipboard
@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}
}
Copy to clipboard
Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
DOI: 10.1093/nar/gkz285
Copyright Statement
Other availability
Search WorldCat Search WorldCat to find libraries that may hold this journal
Citation Metrics:
Cited by: 1 work
Citation information provided by
Web of Science
Save / Share:
Export Metadata
Save to My Library
You must Sign In or Create an Account in order to save documents to your library.

Works referenced in this record:

Ribonucleases P/MRP and the Expanding Ribonucleoprotein World
journal, May 2012

  • Hernandez-Cid, Aaron; Aguirre-Sampieri, Sergio; Diaz-Vilchis, Adelaida
  • IUBMB Life, Vol. 64, Issue 6
  • DOI: 10.1002/iub.1052

Inhibition of Bacterial RNase P RNA by Phenothiazine Derivatives
journal, September 2016

  • Wu, Shiying; Mao, Guanzhong; Kirsebom, Leif
  • Biomolecules, Vol. 6, Issue 3
  • DOI: 10.3390/biom6030038

AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility
journal, December 2009

  • Morris, Garrett M.; Huey, Ruth; Lindstrom, William
  • Journal of Computational Chemistry, Vol. 30, Issue 16
  • DOI: 10.1002/jcc.21256

Small Molecule Inhibitors of Staphylococcus aureus RnpA Alter Cellular mRNA Turnover, Exhibit Antimicrobial Activity, and Attenuate Pathogenesis
journal, February 2011

A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P
journal, September 2014

  • Liu, Xin; Chen, Yu; Fierke, Carol A.
  • Nucleic Acids Research, Vol. 42, Issue 20
  • DOI: 10.1093/nar/gku850

The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning
journal, August 2012

  • Reiter, Nicholas J.; Osterman, Amy K.; Mondragón, Alfonso
  • Nucleic Acids Research, Vol. 40, Issue 20
  • DOI: 10.1093/nar/gks744

Evidence for Induced Fit in Bacterial RNase P RNA-mediated Cleavage
journal, October 2007

  • Brännvall, Mathias; Kikovska, Ema; Wu, Shiying
  • Journal of Molecular Biology, Vol. 372, Issue 5
  • DOI: 10.1016/j.jmb.2007.07.030

Flavonol Activation Defines an Unanticipated Ligand-Binding Site in the Kinase-RNase Domain of IRE1
journal, April 2010

RNase P: At last, the key finds its lock
journal, July 2011

Trials, Travails and Triumphs: An Account of RNA Catalysis in RNase P
journal, April 2010

  • McClain, William H.; Lai, Lien B.; Gopalan, Venkat
  • Journal of Molecular Biology, Vol. 397, Issue 3
  • DOI: 10.1016/j.jmb.2010.01.038

The Diversity of Ribonuclease P: Protein and RNA Catalysts with Analogous Biological Functions
journal, May 2016

Antibiotics and RNase P
journal, May 2016

Expression, purification, crystallization and preliminary diffraction analysis of RNase P protein from Thermotoga maritima
journal, June 2002

  • Krivenko, Angelika A.; Kazantsev, Alexei V.; Adamidi, Catherine
  • Acta Crystallographica Section D Biological Crystallography, Vol. 58, Issue 7
  • DOI: 10.1107/S0907444902007965

Comparison of simple potential functions for simulating liquid water
journal, July 1983

  • Jorgensen, William L.; Chandrasekhar, Jayaraman; Madura, Jeffry D.
  • The Journal of Chemical Physics, Vol. 79, Issue 2
  • DOI: 10.1063/1.445869

The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme
journal, December 1983

Structure of a bacterial ribonuclease P holoenzyme in complex with tRNA
journal, November 2010

  • Reiter, Nicholas J.; Osterman, Amy; Torres-Larios, Alfredo
  • Nature, Vol. 468, Issue 7325
  • DOI: 10.1038/nature09516

PAINS in the Assay: Chemical Mechanisms of Assay Interference and Promiscuous Enzymatic Inhibition Observed during a Sulfhydryl-Scavenging HTS
journal, February 2015

  • Dahlin, Jayme L.; Nissink, J. Willem M.; Strasser, Jessica M.
  • Journal of Medicinal Chemistry, Vol. 58, Issue 5
  • DOI: 10.1021/jm5019093

RNA Conformation and Folding Studied with Fluorescence Resonance Energy Transfer
journal, March 2001

GROMACS 4.5: a high-throughput and highly parallel open source molecular simulation toolkit
journal, February 2013

Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet
journal, June 2008

  • Abdiche, Yasmina; Malashock, Dan; Pinkerton, Alanna
  • Analytical Biochemistry, Vol. 377, Issue 2
  • DOI: 10.1016/j.ab.2008.03.035

Of proteins and RNA: The RNase P/MRP family
journal, July 2010

Evaluation of Bacterial RNase P RNA as a Drug Target
journal, September 2003

  • Willkomm, Dagmar K.; Gruegelsiepe, Heike; Goudinakis, Olga
  • ChemBioChem, Vol. 4, Issue 10
  • DOI: 10.1002/cbic.200300674

Multiple magnesium ions in the ribonuclease P reaction mechanism
journal, May 1993

Ribonuclease P
journal, October 2011

  • Altman, Sidney
  • Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 366, Issue 1580
  • DOI: 10.1098/rstb.2011.0142

ACPYPE - AnteChamber PYthon Parser interfacE
journal, January 2012

XDS
journal, January 2010

  • Kabsch, Wolfgang
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2
  • DOI: 10.1107/S0907444909047337

Small molecule inhibitors of HIV RT Ribonuclease H
journal, January 2010

  • Grandi, Martin Di; Olson, Matthew; Prashad, Amar S.
  • Bioorganic & Medicinal Chemistry Letters, Vol. 20, Issue 1
  • DOI: 10.1016/j.bmcl.2009.10.043

Novel inhibitors of Staphylococcus aureus RnpA that synergize with mupirocin
journal, April 2018

  • Lounsbury, Nicole; Eidem, Tess; Colquhoun, Jennifer
  • Bioorganic & Medicinal Chemistry Letters, Vol. 28, Issue 6
  • DOI: 10.1016/j.bmcl.2018.01.022

Crystal structure of the RNA component of bacterial ribonuclease P
journal, August 2005

  • Torres-Larios, Alfredo; Swinger, Kerren K.; Krasilnikov, Andrey S.
  • Nature, Vol. 437, Issue 7058
  • DOI: 10.1038/nature04074

Canonical sampling through velocity rescaling
journal, January 2007

  • Bussi, Giovanni; Donadio, Davide; Parrinello, Michele
  • The Journal of Chemical Physics, Vol. 126, Issue 1
  • DOI: 10.1063/1.2408420

LINCS: A linear constraint solver for molecular simulations
journal, September 1997

Properties of purified ribonuclease P from Escherichia coli
journal, March 1981

Particle mesh Ewald: An N ⋅log( N ) method for Ewald sums in large systems
journal, June 1993

  • Darden, Tom; York, Darrin; Pedersen, Lee
  • The Journal of Chemical Physics, Vol. 98, Issue 12
  • DOI: 10.1063/1.464397

Antibiotics that target protein synthesis: Antibiotics that target protein synthesis
journal, November 2010

  • McCoy, Lisa S.; Xie, Yun; Tor, Yitzhak
  • Wiley Interdisciplinary Reviews: RNA, Vol. 2, Issue 2
  • DOI: 10.1002/wrna.60

Riboswitches: Discovery of Drugs That Target Bacterial Gene-Regulatory RNAs
journal, December 2011

  • Deigan, Katherine E.; FerrÉ-D’AmarÉ, Adrian R.
  • Accounts of Chemical Research, Vol. 44, Issue 12
  • DOI: 10.1021/ar200039b

A Bright Future for Antibiotics?
journal, June 2017

Transition-state stabilization in Escherichia coli ribonuclease P RNA-mediated cleavage of model substrates
journal, October 2013

  • Wu, Shiying; Chen, Yu; Mao, Guanzhong
  • Nucleic Acids Research, Vol. 42, Issue 1
  • DOI: 10.1093/nar/gkt853

MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009

  • Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
  • DOI: 10.1107/S0907444909042073

Molecular dynamics with coupling to an external bath
journal, October 1984

  • Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.
  • The Journal of Chemical Physics, Vol. 81, Issue 8
  • DOI: 10.1063/1.448118

Coot model-building tools for molecular graphics
journal, November 2004

  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

RNase P RNA mediated cleavage: Substrate recognition and catalysis
journal, October 2007

PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

An introduction to data reduction: space-group determination, scaling and intensity statistics
journal, March 2011

  • Evans, Philip R.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 67, Issue 4
  • DOI: 10.1107/S090744491003982X

Polymorphic transitions in single crystals: A new molecular dynamics method
journal, December 1981

  • Parrinello, M.; Rahman, A.
  • Journal of Applied Physics, Vol. 52, Issue 12
  • DOI: 10.1063/1.328693

Structural Studies of RNase P
journal, May 2013

Chemistry: Chemical con artists foil drug discovery
journal, September 2014

  • Baell, Jonathan; Walters, Michael A.
  • Nature, Vol. 513, Issue 7519
  • DOI: 10.1038/513481a

Antibacterial drug discovery in the resistance era
journal, January 2016

A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays
journal, April 1999

Bacterial RNase P: a new view of an ancient enzyme
journal, October 2006

  • Kazantsev, Alexei V.; Pace, Norman R.
  • Nature Reviews Microbiology, Vol. 4, Issue 10
  • DOI: 10.1038/nrmicro1491

Inhibition of RNase P RNA cleavage by aminoglycosides
journal, May 1999

  • Mikkelsen, N. E.; Brannvall, M.; Virtanen, A.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 11
  • DOI: 10.1073/pnas.96.11.6155

Roles of Regulatory RNAs for Antibiotic Resistance in Bacteria and Their Potential Value as Novel Drug Targets
journal, May 2017

A Divalent Cation Stabilizes the Active Conformation of the B. subtilis RNase P·Pre-tRNA Complex: A Role for an Inner-Sphere Metal Ion in RNase P
journal, July 2010

  • Hsieh, John; Koutmou, Kristin S.; Rueda, David
  • Journal of Molecular Biology, Vol. 400, Issue 1
  • DOI: 10.1016/j.jmb.2010.04.050

RNase P RNAs from some Archaea are catalytically active
journal, July 1999

  • Pannucci, J. A.; Haas, E. S.; Hall, T. A.
  • Proceedings of the National Academy of Sciences, Vol. 96, Issue 14
  • DOI: 10.1073/pnas.96.14.7803

The PDB_REDO server for macromolecular structure model optimization
journal, May 2014

Phaser crystallographic software
journal, July 2007

  • McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
  • Journal of Applied Crystallography, Vol. 40, Issue 4
  • DOI: 10.1107/S0021889807021206

Linked Folding and Anion Binding of the Bacillus subtilis Ribonuclease P Protein †
journal, March 2001

  • Henkels, Christopher H.; Kurz, Jeffrey C.; Fierke, Carol A.
  • Biochemistry, Vol. 40, Issue 9
  • DOI: 10.1021/bi002078y

Protein activation of a ribozyme: the role of bacterial RNase P protein
journal, September 2005

How to Triage PAINS-Full Research
journal, April 2016

  • Dahlin, Jayme L.; Walters, Michael A.
  • ASSAY and Drug Development Technologies, Vol. 14, Issue 3
  • DOI: 10.1089/adt.2015.674

Protein Component of Bacillus subtilis RNase P Specifically Enhances the Affinity for Precursor-tRNA Asp   †
journal, February 1998

  • Kurz, Jeffrey C.; Niranjanakumari, S.; Fierke, Carol A.
  • Biochemistry, Vol. 37, Issue 8
  • DOI: 10.1021/bi972530m

Eukaryotic RNase P RNA mediates cleavage in the absence of protein
journal, February 2007

  • Kikovska, E.; Svard, S. G.; Kirsebom, L. A.
  • Proceedings of the National Academy of Sciences, Vol. 104, Issue 7
  • DOI: 10.1073/pnas.0607326104

Characterization of RNase P from Thermotoga maritima
journal, February 2001

    [ × clear filter / sort ]

    Similar Records in DOE PAGES and OSTI.GOV collections: