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

Abstract

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 >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.

Analysis of the Potential for N 4 -Hydroxycytidine To Inhibit Mitochondrial Replication and Function

“…2). Previously, it has been determined that POLRMT favors CTP over 3=-dCTP with a discrimination factor of 59.7 Ϯ 1.6 (7). By extrapolation, POLRMT favors CTP as a substrate over NHC-TP by a factor of approximately 740.…”

“…A nonradioactive primer extension assay was used to determine whether NHC-TP can be incorporated into RNA by POLRMT as a cytidine analog. The incorporation efficiency of a ribonucleoside analog is usually compared to the natural ribonucleoside; however, in the case of CTP analogs, CTP is incorporated much more efficiently by POLRMT than most CTP analogs which makes it difficult to directly compare their incorporation efficiencies under the same conditions (7). To facilitate the measurement, 3=-dCTP can be used as an intermediary (7).…”

“…The incorporation efficiency of a ribonucleoside analog is usually compared to the natural ribonucleoside; however, in the case of CTP analogs, CTP is incorporated much more efficiently by POLRMT than most CTP analogs which makes it difficult to directly compare their incorporation efficiencies under the same conditions (7). To facilitate the measurement, 3=-dCTP can be used as an intermediary (7). In a single nucleotide incorporation primer extension assay it was determined that POLRMT favors 3=-dCTP as a substrate over NHC-TP with a discrimination value of 12.4 Ϯ 1.2 (Fig.…”

“…polymerase (POLRMT) or by nuclear RNA polymerase (Pol) I, II, or III, resulting in undesired side effects (7,8). The development of several ribonucleoside analogs has been stopped as a result of toxicity observed during clinical trials.…”

“…To examine early in the drug development process the potential for nucleoside analogs to cause mitochondrial toxicity, a series of in vitro assays have been developed to characterize the effects of a compound on mitochondrial DNA copy number and mitochondrial function. Key among these are the measurement of mtDNA levels, lactate production, and mitochondrial protein expression (7,(11)(12)(13).…”