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

Gaofei Lu,Shuli Mao,Abel De La Rosa,Michael Hager,Kasinath Sana,Damien Kuiper,Alexander A Kolykhalov,Paul Collop,Bharat P Gurale,Gregory R Bluemling,George R Painter

doi:10.1128/aac.01830-17

Abstract

ABSTRACTThere 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 >105; a potentially toxic compound has a D value of >104 but <105; and a toxic compound has a D value of <104. This report provides a simple screening method that should assist investigators in designing ribonucleoside-based drugs having lower mitochondrial toxicity.

Highlights

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
The failure of ribonucleoside/ribonucleotide analogs during clinical development has been due to toxicity that was not detected during preclinical studies
Our results showed that all five POLRMT variants can be successfully expressed in E. coli, and the expression levels and the solubilities of the recombinant proteins varied (Fig. 1B)

Summary

Introduction

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. 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. It has been shown that Pol II can quantitatively remove misincorporated nucleotides from the nascent transcript during rapid chain extension [6] This proofreading ability of Pol I, Pol II, and Pol III can mitigate the possibility of their inhibition by incorporated ribonucleotide analogs. Inhibition of POLRMT leads to the inhibition of both mitochondrial DNA transcription, resulting in decreased mitochondrial protein expression, and mitochondrial DNA replication, resulting in a decrease in the mitochondrial number

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Discussion

Conclusion