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Author Notes:

Address correspondence to Alexander A. Kolykhalov, a.kolykhalov@emory.edu.

Subject:

Research Funding:

Partial funding for this project was provided by Georgia Research Alliance (GRA) grant GRA.VL16.C3.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Microbiology
  • Pharmacology & Pharmacy
  • discrimination value
  • POLRMT
  • RNA-dependent RNA polymerase
  • RdRp
  • human mitochondrial RNA polymerase
  • mitochondrial polymerase
  • nonradioactive assay
  • nucleoside analogs
  • primer extension
  • ribonucleotide
  • HEPATITIS-C VIRUS
  • TRANSCRIPTION MACHINERY
  • ANTIVIRAL THERAPY
  • INHIBITORS
  • TOXICITY
  • SCAFFOLDS

Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5'-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase

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Journal Title:

Antimicrobial Agents and Chemotherapy

Volume:

Volume 62, Number 2

Publisher:

Type of Work:

Article | Final Publisher PDF

Abstract:

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. There is a growing body of evidence suggesting that some ribonucleoside/ribonucleotide analogs may be incorporated into mitochondrial RNA by human mitochondrial DNA-dependent RNA polymerase (POLRMT) and disrupt mitochondrial RNA synthesis. An assessment of the incorporation efficiency of a ribonucleotide analog 5=-triphosphate by POLRMT may be used to evaluate the potential mitochondrial toxicity of the analog early in the development process. In this report, we provide a simple method to prepare active recombinant POLRMT. A robust in vitro nonradioactive primer extension assay was developed to assay the incorporation efficiency of ribonucleotide analog 5=-triphosphates. Our results show that many ribonucleotide analogs, including some antiviral compounds currently in various preclinical or clinical development stages, can be incorporated into newly synthesized RNA by POLRMT and that the incorporation of some of them can lead to chain termination. The discrimination (D) values of ribonucleotide analog 5=-triphosphates over those of natural ribonucleotide triphosphates (rNTPs) were measured to evaluate the incorporation efficiency of the ribonucleotide analog 5=-triphosphates by POLRMT. The discrimination values of natural rNTPs under the condition of misincorporation by POLRMT were used as a reference to evaluate the potential mitochondrial toxicity of ribonucleotide analogs. We propose the following criteria for the potential mitochondrial toxicity of ribonucleotide analogs based on D values: a safe compound has a D value of > 10 5 ; a potentially toxic compound has a D value of > 10 4 but < 10 5 ; and a toxic compound has a D value of < 10 4 . This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

Copyright information:

© 2018 Lu et al.

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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