Nucleotide substrate specificity of anti-hepatitis C virus nucleoside analogs for human mitochondrial RNA polymerase

Article abstract of DOI:10.1128/AAC.00492-17

Nucleoside analog inhibitors (NAIs) are an important class of antiviral agents. Although highly effective, some NAIs with activity against hepatitis C virus (HCV) can cause toxicity, presumably due to off-target inhibition of host mitochondrial RNA polymerase (POLRMT). The in vitro nucleotide substrate specificity of POLRMT was studied in order to explore structure-activity relationships that can facilitate the identification of nontoxic NAIs. These findings have important implications for the development of all anti-RNA virus NAIs.

Full text of DOI:10.1128/AAC.00492-17

AAC Accepted Manuscript Posted Online 30 May 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00492-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
Antimicrob. Agents Chemother. Short communication. AAC00492-17_revised
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Nucleotide Substrate Specificity of Hepatitis C Analogs for Human Mitochondrial RNA
Polymerase
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Maryam Ehteshamia, Longhu Zhoua, Sheida Amiralaeia, Jadd R. Sheltona, Jong Hyun Choa,
Hongwang Zhanga, Hao Li, Xiao Lua, Tugba Ozturka, Richard Stantona, Franck Amblarda,
Tamara R. McBrayerb, Steven J. Coatsb, and Raymond F. Schinazia #
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a Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of
Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
b Cocrystal Pharma, Inc., Tucker, GA, USA
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Running title: Nucleoside analogs as substrates of POLRMT
# - Address correspondence to Raymond F. Schinazi, Emory University, HSRB, Room E-420,
15 1760 Haygood Drive, Atlanta, GA 30322, USA.
16 Email: rschina@emory.edu
17 Tel: +1-40-727-1414, Fax: +1-404-727-1330
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19 ABSTRACT (75 words maximum)
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Nucleoside analog inhibitors (NAI) are an important class of antiviral agents. Although highly
effective, some NAI with anti-hepatitis C virus (HCV) activity can cause toxicity presumably
22 due to off-target inhibition of host mitochondrial RNA polymerase (POLRMT). The in vitro
23 nucleotide substrate specificity of POLRMT enzyme was studied in order to explore structure-
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Antimicrob. Agents Chemother. Short communication. AAC00492-17_revised
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activity relationships that can facilitate the identification of non-toxic NAI. These finding have
25 important implications for all anti-RNA virus NAI development.
26 TEXT
27 Nucleoside analog inhibitors (NAI) have been an important component of antiviral therapy for
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decades. 2’-Deoxyribonucleoside analogs are regularly used for treatment of viral infections such
29 as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus
30 (HSV) (reviewed in (1, 2)). Upon cellular entry, NAIs must be phosphorylated by viral or
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cellular kinases into their active 5’-triphosphate (TP) form. NAI-TPs are substrates for the viral
polymerase and their incorporation often results in chain-termination of the growing genome and
subsequent inhibition of virus replication. Recently, sofosbuvir became the first ribonucleoside
analog inhibitor to receive approval for treatment of hepatitis C virus (HCV), an RNA virus (3).
Sofosbuvir is administered as a phoshoramidate prodrug in order to target the liver, improve cell
permeability and bypass the first phosphorylation step by host cell kinases (4, 5). Despite the
excellent safety profile of sofosbuvir, developments of several preceding anti-HCV
ribonucleotide prodrugs were halted due to toxicity (reviewed in (6)). For example, phase II
clinical trials with BMS-986094 (formerly known as INX-189) were terminated after reports of
40 severe adverse events and one death (7-9). Although the exact mechanism of toxicity is not yet
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fully elucidated, it has been shown that treatment with NAI can sometimes lead to off-target
inhibition of host polymerases. For example, off-target inhibition of mitochondrial DNA
polymerase γ has been well documented for zidovudine triphosphate (AZT-TP), the first anti-
HIV 2’-deoxyribonucleoside analogs to received FDA approval (10-13). Importantly, it was
recently shown that ribonucleoside analog triphosphates could be substrates for recombinant
human mitochondrial RNA polymerase (POLRMT) (14-16). Unlike the nuclear counterpart
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RNA POL II, POLRMT lacks exonuclease activity and, therefore, incorporation of chain-
48 terminating nucleotides was shown to be irreversible in vitro (14). Consistent with these
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observations, Arnold et al. reported that increased POLRMT incorporation of NAI-TPs, as well
as high intracellular NAI-TP levels, correlated positively with reductions in mitochondrial RNA
levels and cytotoxicity (14). Similarly, we recently showed that treatment of Huh-7 cells with
BMS-986094, which generates high levels of 2’-C-methyl-GTP intracellularly, inhibited
53 mitochondrial RNA transcription (17). Interestingly, treatment with a 2,6-diaminopurine
54 nucleotide (DAPN) prodrug, an investigational anti-HCV agent that also generates 2’-C-methyl-
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GTP (18), did not have similar deleterious effects. Lower intracellular NAI-TP accumulation was
proposed to account for the distinct cytotoxic profiles of these compounds (17).
Considering the importance of safety in developing novel ribonucleoside analogs as inhibitors of
HCV and other RNA viruses, we explored the nucleotide substrate specificity of POLRMT. Here
we report on the in vitro POLRMT incorporation profiles of over forty ribonucleoside analog 5’-
triphosphates in order to shed light on structure-activity relationships that lead to the
identification of non-toxic NAI. NAI-TPs that were not substrates for this enzyme were further
examined for antiviral activity. Knowledge gained from this study has important implications not
64 only for anti-HCV, but all anti-RNA virus antiviral NAI development.
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66 Incorporation of nucleoside analogs by POLRMT. In order to examine the nucleoside
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substrate specificity of POLRMT, we evaluated the incorporation profile of 41 nucleosides with
various chemical modifications on the ribose or base moiety (chemical structures summarized in
Figure 1). As previously described (14, 17), 125 nM of purified POLRMT enzyme was
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incubated with 500 nM of 5’-radiolabeled RNA/DNA primer/ template that allowed for the
71 incorporation of A, C, G, or U nucleotide analogs at position +1, respectively. 100 μM of each
72 nucleoside 5’-triphosphate analog was incubated with the POLRMT:RNA/DNA complex
73 containing the appropriate DNA template for two hours at 30 °C. The amount of NAI-TP
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incorporation was normalized to the corresponding natural rNTP substrate (Figure 2). We
determined that most modifications on the ribose ring of nucleotide analogs did not completely
76 neutralize incorporation by POLRMT. As depicted in Figure 2a, addition of 2’-O-methyl, 2’-
77 fluoro, or 2’-amino modifications did not consistently affect POLRMT incorporation for all
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nucleotides tested. Consistent with previous findings (14, 15), addition of a C-methyl group at
79 the 2’ position generally led to notable reductions in incorporation for C, G, and U analogs.
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Nucleotides harboring this modification have been examined for their antiviral activity against
HCV (19, 20) and dengue virus (21). Interestingly, the effect of the 2’-C-methyl modification
was especially pronounced for 2’-C-methyl-UTP whose incorporation was reduced to 10 % of
83 natural UTP, suggesting that U analogs may be particularly vulnerable to chemical modifications
84 with regards to POLRMT incorporation. This is in agreement with the observations that 2’-C-
85 methyl-2’-F-UTP, the active metabolite of sofosbuvir, is an exceedingly poor substrate for
86 PORLMT (Figure 2a). Under the conditions tested, we found 3’- or 2’-deoxynucleoside
87 triphosphates were generally good substrates for POLRMT (with the exception of 3’-dCTP)
88 (Figure 2b). However, the simultaneous removal of both hydroxyl groups (2’,3’-ddNTPs)
89 completely abrogated incorporation, suggesting that the presence of at least one OH group is
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essential for NAI-TP incorporation. Our data are consistent with previous observations that little
discrimination exists against sugar-modified nucleotides (22). The observed promiscuity of
92 POLRMT has implications for fidelity of this enzyme during mitochondrial RNA transcription.
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Considering that 2'-deoxynucleoside triphosphate analogs were substrates for POLRMT, we next
95 asked whether dNAI-TPs active against DNA viruses or retroviruses might also be substrates for
96 POLRMT (see Figure 1b for chemical structures). As expected, DAPD-TP, DXG-TP, AZT-TP
97 and ETV-TP were not incorporated by POLRMT. Surprisingly, we found 30% and 36%
98 incorporation for 3TC-TP and GCV-TP, respectively at 100 µM of the NTP (Figure 2c).
99 Although extensive literature exists on the impact of dNAI-TPs on mitochondrial DNA
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synthesis, it is worth noting that little information is available on the role of these compounds
with regards to interference with mitochondrial RNA transcription in cells.
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In our search for nucleoside analogs that are not substrates for POLRMT, but active against the
viral RNA polymerase of HCV, we next investigated the incorporation profiles of nucleoside
analog 5’-triphosphates harboring various base moiety modifications (Figure 1c). Of the nine
compounds tested, we found that the majority of modifications at positions 4, 5, 6 and 8 of
purines and pyrimidines did not significantly reduced incorporation by POLRMT (Figure 2c).
Similarly, nucleoside analog triphosphates containing bulky modifications (such as 8-azido-ATP,
4-thio-UTP and 5-bromo-UTP) were readily incorporated by POLRMT. The combined presence
of 2’-C-methyl and 2-fluoro modifications resulted poor POLRMT incorporation (12% of the
natural ATP substrate). We have previously reported on the anti-HCV activity of this compound
111 (23), where phosphoramidate prodrug of 2’-C-methyl-2-fluoro-ATP was found to inhibit HCV
112 replicons with submicromolar activity.
113 The addition of NHOH chemical group on position 4 of pyrimidine ring (4-N-OH-CTP) was also
114 observed to reduce incorporation by PORLMT to 50% of CTP (Figure 2c). Addition of 2’-C-
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methyl group further reduced incorporation to 20% of CTP. Both 4-N-OH-CTP and 2’-C-
116 methyl-4-N-OH-CTP demonstrated anti-HCV activity in cell culture (24, 25).
117 Incorporation of N1-methyl-GTP by POLRMT. We identified N1-methyl-GTP as a
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nucleoside analog 5’-triphosphate with minimal incorporation by POLRMT. Considering that the
addition of the N1-methyl group on the base moiety reduced POLRMT incorporation to 10% of
the natural GTP substrate (Figure 2c), N1-methyl-GTP was selected for further analysis with
regards to incorporation by viral NS5B RNA polymerase. NS5B incorporation assays were
122 performed as previously described (17). NS5B: RNA/RNA complexes were incubated with
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increasing concentrations of GTP or N1-methyl-GTP. Single nucleotide incorporation was
observed over time and visualized on a denaturing polyacrylamide gel (Figure 3) where the 5’-
125 radiolabeled 9mer primer was extended to a 10mer product. As expected, natural GTP substrate
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was rapidly incorporated with an apparent dissociation constant (Kd,app) value of 4.1 ± 1.6 μM
(Figure 3a). Conversely, at least 125 μM of N1-methyl-GTP was required for 50 % incorporation
128 by HCV NS5B enzyme (Kd,app > 100 μM), suggesting compromised incorporation when
129 compared to natural GTP (Figure 3b).
130 We next asked whether RNA extension could occur after N1-methyl-GTP incorporation by
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NS5B enzyme. NAI-TP incorporation was assessed in the presence of 10 μM CTP, ATP and
UTP and increasing amounts of N1-methyl-GTP (Figure 4a). We found that as N1-methyl-GTP
levels increased, an increase in full-length 20mer product was observed suggesting that
nucleotide extension can occur following N1-methyl-GTP incorporation (Figure 4b, left panel).
135 As expected full-length RNA synthesis was more readily observed with low levels of GTP
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(Figure 4b, right panel). Overall, these data suggested that although N1-methyl-GTP was a poor
substrate for NS5B enzyme, its incorporation does not result in RNA chain-termination. We next
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asked whether N1-methyl-GTP could inhibit RNA polymerization in the presence of competing
GTP. We found that when 1 μM of GTP was present in addition to 10 μM of CTP, ATP and
UTP, N1-methyl-GTP had no effect on full-length RNA synthesis (Figure 4c). This is in contrast
141 to control compound 2’-C-methyl-GTP, which inhibited RNA synthesis with an IC50 value of 3.3
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± 0.5 μM (Figure 4c). Finally, we asked whether N1-methyl-GTP might inhibit viral genome
synthesis in a manner that may not be detectable in our in vitro assay. To address this issue, we
chemically synthesized a phosphoramidate prodrug of N1-methyl-GTP for cell culture testing
(Scheme 1). We observed no inhibition when up to 200 μM of N1-methyl-G-phosphoramidate
prodrug was incubated with HCV genotype 1b replicon cells (see (24) for experimental
methods). Further experiments suggested that this prodrug is not efficiently triphosphorylated
intracellularly (data not shown). Together, these data suggest that although N1-methyl-GTP may
be a safe NAI-TP with regards to off-target incorporation by human POLRMT, it will likely not
be effective as an anti-HCV agent. We also did not detect any antiviral activity against
chikungunya, influenza A and respiratory syncytial virus when tested up to 100 µM. It remains
to be determined whether this compound can be of interest as an antiviral agent for other RNA
viruses.
154 In conclusion, in this report we examined the in vitro nucleotide substrate specificity of
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POLRMT. We found to our surprise that the POLRMT active site is relatively tolerant of
incorporating most of the nucleoside analog 5’-triphosphates tested. Several anti-HCV NAI-TPs
were identified to be poor substrates for POLRMT. In conclusion, the information on NAI-TP
incorporation profile of POLRMT described herein sheds light on the biochemical properties of
this enzyme active site and inform future ribonucleotide analog drug design for all RNA viruses.
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Acknowledgements
This work was supported in part by NIH grant 5P30-AI-50409 (Center for AIDS Research) (to
166 RFS). ME is the recipient of CIHR-NCRTP postdoctoral fellowship award and American Liver
167 Foundation postdoctoral fellowship award. We thank Sijia Tao for performing the cellular
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pharmacology studies and Louise McCormick for conducting the influenza and respiratory
syncytial virus assays.
Abbreviations
HCV, hepatitis C virus; POLRMT, human mitochondrial RNA polymerase; NAI, Nucleoside
analog inhibitors; TP, 5’-triphosphate.
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Figures
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FIG 1. Chemical structures of NAI-TPs. A) Structures of NAI-TPs with modifications on the
ribose moiety. Compounds are grouped according to base moiety. B) Chemical structures dNAI-
TPs with anti-HIV, anti-HBV or anti-HSV activity. C) Structures of ribonucleoside analog 5’-
triphosphates with modifications on the base moiety.
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283 FIG 2. Nucleoside analog 5’-triphosphate incorporation by POLRMT. A) Nucleoside analog
284 triphosphates with modifications at the 2’-position of the ribose moiety were assessed for
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incorporation by POLRMT. Incorporation reactions were allowed to proceed for 2 h in the
presence of 100 μM of each substrate. Percentage incorporation for each nucleoside analog
287 triphosphate was normalized to that of the corresponding natural nucleotide (ATP, CTP, GTP, or
288 UTP) substrate. B) POLRMT incorporation of 2’-deoxyribonucleoside analogs and NAI-TPs
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with anti-HBV, anti-HIV or anti-HSV activity was assessed as described above. C) POLRMT
incorporation was assessed for ribonucleoside analog 5’-triphosphates with modifications on the
291 base moiety. Error bars represent S.D. values for two to three separate experiments.
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FIG 3. Incorporation profile of N1-methyl-GTP. A) Increasing concentrations of GTP (0.7
μM to 180 μM) were incubated with a pre-formed NS5B: RNA/RNA complexes and nucleotide
extension was measured over time. The extended 10mer RNA product was visualized on a 20%
denaturing acrylamide gel. Rates of incorporation at various nucleotide concentrations were
plotted as described previously in order to obtain apparent dissociation constant (Kd,app) value of
4.1 ± 1.6 μM for GTP (17). B) Increasing concentrations of N1-methyl-GTP (15.6 μM to 500
μM) were incubated with the preformed NS5B:RNA/RNA complex as described above. Kd,app
value for N1-methyl-GTP was estimated to be > 100 μM.
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FIG 4. Effect of N1-methyl-GTP incorporation on NS5B-mediated RNA extension. A)
Increasing concentrations of N1-methyl-GTP were incubated with NS5B enzyme, 5’-
radiolabeled GG primer, and a 20mer RNA template in the presence of 10 μM ATP, CTP, and
UTP. RNA synthesis was allowed to proceed for 2 h at 30°C. In the absence of N1-methyl-GTP
(lane 0), a strong pausing site was observed at position 9 while small amounts of 20mer full-
length RNA product were accumulated as a result of nucleotide misincorporation. Increasing N1-
methyl-GTP concentrations correlated with the appearance of a 10mer band (site of G
incorporation) and full-length 20mer product. B) Amount of 20mer product accumulation was
plotted as a function of N1-methyl-GTP concentration (left). Parallel experiment with increasing
concentrations of GTP was also plotted (right). C) RNA synthesis was monitored as described
above, with the addition of 1 μM GTP in the presence of increasing concentrations of N1-
methyl-GTP (left) and control inhibitor 2’-C-methyl-GTP (right). No inhibition of RNA
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Antimicrob. Agents Chemother. Short communication. AAC00492-17_revised
325 synthesis was observed with up to 1,000 μM of N1-methyl-GTP while 2’-C-methyl-GTP
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inhibited RNA synthesis with an IC50 value of 3.3 ± 0.5 μM (average of two separate
experiments ±S.D.).
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Scheme 1. Chemical synthesis of N1-methyl-G-phosphoramidate prodrug. (a) p-
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Toluenesulfonic acid, 2,2-dimethoxypropane, acetone, rt, overnight, 93%; (b) NaH, MeI, DMSO,
334 rt, 89.7%; (c) phosphorochloridate, N-methylimidazole, THF/CH3CN, rt, 3 h, 90.7%; (d) 85%
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TFA, rt, 1 h, 86%.
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