Nucleotide Substrate Specificity of Anti-Hepatitis C Virus Nucleoside Analogs for Human Mitochondrial RNA Polymerase

Ehteshami, Maryam; Zhou, Longhu; Amiralaei, Sheida; Shelton, Jadd R.; Cho, Jong Hyun; Zhang, Hongwang; Li, Hao; Lu, Xiao; Öztürk, Tuğba; Stanton, Richard A.; Amblard, Franck; McBrayer, Tamara R.; Coats, Steven J.; Schinazi, Raymond F.

doi:10.1128/aac.00492-17

Cited by 8 publications

(8 citation statements)

References 22 publications

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“…Nucleoside analogs are widely used for the prevention and treatment of several viral infections, including human immunodeficiency virus, hepatitis B virus, and herpes infections ( Albrecht et al, 1970 ; Burchenal et al, 1975 ; Aguirrebengoa et al, 1996 ; Adams et al, 1997 ; Bavoux et al, 2000 ; Quan and Peters, 2004 ; Anderson, 2008 ; Angusti et al, 2008 ; Ambrose et al, 2009 ; Billioud et al, 2011 ; Agarwal et al, 2015 ; Cao et al, 2015 ; Ehteshami et al, 2017 ). Due to their hydrophilic nature, nucleoside analogs depend on a carrier-mediated transport process to permeate through host cell membranes before exhibiting their antiviral activities ( Yao et al, 1996 ; Damaraju et al, 2011 ; Moss et al, 2012 ).…”

Section: Introductionmentioning

confidence: 99%

Identification of Structural and Molecular Features Involved in the Transport of 3′-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3

2018

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Combination antiretroviral drug treatments depend on 3′-deoxy-nucleoside analogs such as 3′-azido-3′-deoxythymidine (AZT) and 2′3′-dideoxyinosine (DDI). Despite being effective in inhibiting human immunodeficiency virus replication, these drugs produce a range of toxicities, including myopathy, pancreatitis, neuropathy, and lactic acidosis, that are generally considered as sequelae to mitochondrial damage. Although cell surface–localized nucleoside transporters, such as human equilibrative nucleoside transporter 2 (hENT2) and human concentrative nucleoside transporter 1 (hCNT1), are known to increase the carrier-mediated uptake of 3′-deoxy-nucleoside analogs into cells, another ubiquitously expressed intracellular nucleoside transporter (namely, hENT3) has been implicated in the mitochondrial transport of 3′-deoxy-nucleoside analogs. Using site-directed mutagenesis, generation of chimeric hENTs, and 3H-permeant flux measurements in mutant/chimeric RNA–injected Xenopus oocytes, here we identified the molecular determinants of hENT3 that dictate membrane translocation of 3′-deoxy-nucleoside analogs. Our findings demonstrated that whereas hENT1 had no significant transport activity toward 3′-deoxy-nucleoside analogs, hENT3 was capable of transporting 3′-deoxy-nucleoside analogs similar to hENT2. Transport analyses of hENT3-hENT1 chimeric constructs demonstrated that the N-terminal half of hENT3 is primarily responsible for the hENT3–3′-deoxy-nucleoside analog interaction. In addition, mutagenic studies identified that 225D and 231L in the N-terminal half of hENT3 partially contribute to the ability of hENT3 to transport AZT and DDI. The identification of the transporter segment and amino acid residues that are important in hENT3 transport of 3′-deoxy-nucleoside analogs may present a possible mechanism for overcoming the adverse toxicities associated with 3′-deoxy-nucleoside analog treatment and may guide rational development of novel nucleoside analogs.

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Section: Introductionmentioning

confidence: 99%

Identification of Structural and Molecular Features Involved in the Transport of 3′-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3

2018

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“…Recent biochemical data examining levels of off-target incorporation of nucleoside analogue triphosphates by the human mitochondrial RNA polymerase may help explain the phenotypic relationship we observed: both 2′- C -methyluridine triphosphate and 2′- C -methyl-2′- C -fluorouridine triphosphate (the active metabolite of sofosbuvir) show much lower levels of incorporation by this host enzyme than 2′- C -modified analogues with other nucleobases. [50]. We propose that this may account for the better safety profile observed for sofosbuvir compared to other 2′- C -modified compounds that were tested in clinical trials.…”

Section: Resultsmentioning

confidence: 96%

Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells

Bernatchez

Coste

Beck

et al. 2019

Preprint

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Zika virus (ZIKV), an emerging flavivirus which causes neurodevelopmental impairment 22 to fetuses and has been linked to Guillain-Barré syndrome, continues to threaten global health due 23 to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of 24 efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) 25 have been employed to improve the bioavailability of ribonucleoside analogues. Here, we 26 synthesized and tested a library of 13 ProTides against ZIKV in human neural stem cells. Strong 27 activity was observed for 2'-C-methyluridine and 2'-C-ethynyluridine ProTides with an aryloxyl 28 phosphoramidate masking group. Conversion of the aryloxyl phosphoramidate ProTide group of 29 2'-C-methyluridine to a 2-(methylthio)ethyl phosphoramidate completely abolished antiviral 30 activity of the compound. The aryloxyl phosphoramidate ProTide of 2'-C-methyluridine 31 outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and 32 protection from cytopathic effect, while the former compound's triphosphate active metabolite was 33 better incorporated by purified ZIKV NS5 polymerase over time. Molecular superpositioning 34 revealed different orientations of residues opposite the 2'-fluoro group of sofosbuvir. These findings 35 suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue 36 ProTides in a disease-relevant cell model. 37 38 cells, RNA-dependent RNA polymerase 39 40 1. Introduction 41The explosive spread of Zika virus (ZIKV) during the 2015-2016 epidemics in Latin America 42 attracted worldwide attention to this previously neglected disease. The lack of effective vaccines or 43 small molecules to prevent or treat this infection remains a cause for concern and emphasizes the 44 urgent need for new therapeutic options [1]. ZIKV, an emerging flavivirus infection, causes several 48 manifestations of the disease, and in rare cases, ZIKV infection has been linked to the 49 neuroinflammatory Guillain-Barré syndrome [2]. 50 Viral polymerases remain attractive drug targets for the development of selective antiviral 51 therapies [3-5]. Generally, clinically-approved inhibitors that target these proteins fall into two broad 52 classes [6,7]. The first class consists of nucleoside analogues that mimic the natural substrate of the 53 enzyme. Upon analogue incorporation by the virally-encoded polymerase, DNA or RNA synthesis 54 is abrogated by preventing further nucleotide incorporation (chain termination), thereby arresting 55 viral replication. The second class is known as non-nucleoside inhibitors, which bind allosterically 56 and arrest viral nucleic acid synthesis by distorting the polymerase active site geometry to interfere 57 with nucleotide binding or nucleotide incorporation. 58 A common prodrug strategy used for antiviral ribonucleoside analogues involves the chemical 59 synthesis of nucleoside analogue monophosphates with metabolically-removable masking groups 60 [8...

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“…Our data showed, for example, that CTP mispairing with deoxyadenosine in the template has the highest misincorporation frequency, (7.69 ± 0.97) × 10 −5 [the discrimination value for this wrong CTP was (1.31 ± 0.17) × 10 4 ; Table 3 ]. Based on these data and references from other studies ( 12 , 13 , 16 ), we have proposed the following criteria for evaluating the potential mitochondrial toxicity of ribonucleotide analogs: 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 , where the D value is the value of discrimination of a ribonucleotide analog 5′-triphosphate versus a natural rNTP measured in the POLRMT assay. These criteria are applicable to ribonucleotide analog 5′-triphosphates that do not cause chain termination; these threshold values would be expected to be lower for ribonucleotide analog 5′-triphosphates causing chain termination.…”

Section: Discussionmentioning

confidence: 92%

“…There is a growing pool of evidence showing that ribonucleotide analog 5′-triphosphates can serve as substrates for human POLRMT and can be incorporated into newly synthesized RNA by POLRMT. Consequently, inhibition of mitochondrial RNA synthesis can lead to the disruption of mitochondrial DNA replication and/or mitochondrial function, causing mitochondrial toxicity ( 11 – 16 ). Therefore, characterization of the incorporation of ribonucleotide analog 5′-triphosphates by POLRMT could provide a useful tool for the evaluation of rNIs to determine their potential mitochondrial toxicity.…”

Section: Introductionmentioning

confidence: 99%

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

Bluemling

Mao

et al. 2018

Antimicrob Agents Chemother

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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.

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Nucleotide Substrate Specificity of Anti-Hepatitis C Virus Nucleoside Analogs for Human Mitochondrial RNA Polymerase

Cited by 8 publications

References 22 publications

Identification of Structural and Molecular Features Involved in the Transport of 3′-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3

Identification of Structural and Molecular Features Involved in the Transport of 3′-Deoxy-Nucleoside Analogs by Human Equilibrative Nucleoside Transporter 3

Activity of Selected Nucleoside Analogue ProTides against Zika Virus in Human Neural Stem Cells

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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