nucleotide analog inhibitors of dengue virus

Accepted Manuscript The search for nucleoside/nucleotide analog inhibitors of dengue virus Yen-Liang Chen, Fumiaki Yokokawa, Pei-Yong Shi PII: DOI: Reference:

S0166-3542(15)00173-4 http://dx.doi.org/10.1016/j.antiviral.2015.07.010 AVR 3670

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Antiviral Research

Received Date: Revised Date: Accepted Date:

9 April 2015 29 July 2015 31 July 2015

Please cite this article as: Chen, Y-L., Yokokawa, F., Shi, P-Y., The search for nucleoside/nucleotide analog inhibitors of dengue virus, Antiviral Research (2015), doi: http://dx.doi.org/10.1016/j.antiviral.2015.07.010

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The search for nucleoside/nucleotide analog inhibitors of dengue virus Yen-Liang Chen*, Fumiaki Yokokawa, and Pei-Yong Shi*

Novartis Institute for Tropical Diseases, Singapore

*Correspondence could be addressed to YLC or PYS Yen-Liang Chen: Phone: +65 6722 2975; Fax : +65 6722 2916; Email: [email protected] Pei-Yong Shi: Phone: +65 6722 2909; Fax: +65 6722 2916; Email: [email protected] Mailing address: Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos Building, Singapore 138670, Singapore

Keywords: Nucleoside analogs, dengue virus, antiviral therapy, monophosphate prodrug

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Abstract Nucleoside analogs represent the largest class of antiviral agents and have been actively pursued for potential therapy of dengue virus (DENV) infection. Early success in the treatment of human immunodeficiency virus (HIV) infection and the recent approval of sofobuvir for chronic hepatitis C have provided proof of concept for this class of compounds in clinics. Here we review (i) nucleoside analogs with known anti-DENV activity; (ii) challenges of the nucleoside antiviral approach for dengue; and (iii) potential strategies to overcome these challenges. This article forms part of a symposium in Antiviral Research on flavivirus drug discovery.

Contents 1. Introduction 2. Known nucleoside inhibitors of dengue virus 3. Challenges of nucleoside analog development and overcoming strategies 3.1. Intracellular triphosphate nucleotide as a key surrogate parameter for efficacy measurement 3.2. Monophosphate prodrug to improve intracellular triphosphate nucleoside conversion 3.3. Kinetics of intracellular triphosphate nucleoside formation 3.4. Effect of cell types and viral infection on intracellular triphosphate nucleoside conversion 3.5. Nucleoside toxicity and drug-drug interactions 4. Conclusions

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1. Introduction Dengue virus (DENV) non-structural protein 5 (NS5) possesses multiple enzymatic activities, including a guanylyltransferase (Bollati et al., 2009; Egloff et al., 2007; Issur et al., 2009), RNA cap methyltransferase (MTase) (Egloff et al., 2002; Ray et al., 2006; Zhou et al., 2007), internal RNA MTase (Dong et al., 2012), and RNA-dependent RNA polymerase (RdRp) (Ackermann and Padmanabhan, 2001; Lim et al, 2013; Zhao et al., 2015). Among these enzymatic activities, the RdRp plays a central role in viral replication and represents the most attractive target for antiviral research. The preference of targeting viral replication machinery has been clearly evident from the recent success of sofosbuvir in the treatment of chronic hepatitis C. For HIV therapy, even with the emergence of many new classes of inhibitors, nucleos(t)ide analogs remain the backbone of first-line treatments. The structure of RdRp adopts the classical half-closed right hand configuration consisting of a palm, thumb, and finger domains. Flavivirus RdRp contains additional finger-loop motif connecting finger and thumb subdomains (Haudecoeur et al., 2013; Zhao et al., 2015). Two broad approaches have been pursued to inhibit RdRp for antiviral therapy: non-nucleoside and nucleos(t)ide inhibitors. Non-nucleoside inhibitors bind to an allosteric sites away from the active site and affect the enzymatic activity of RdRp (Niyomrattanakit et al., 2010). Nonnucleoside inhibitors could also inhibit viral replication through blocking the binding between RdRp and other viral replication components as well as host factors (Fraser et al., 2014). In the case of HCV, several allosteric sites (such as Palm I, Palm II, Thumb I and Thumb II sites) have been identified and explored for the development of non-nucleoside inhibitors (Beaulieu 2009). Nucleoside analogs are historically used for anti-cancer therapy through inhibiting cellular polymerases (Jordheim et al., 2013). The field of antiviral nucleoside development blossomed after the successful introduction in the HIV field. Unlike non-nucleoside inhibitors 3

which directly bind to viral polymerase to exert their antiviral effect, nucleoside inhibitors have to be converted into their corresponding triphosphate forms inside cells by various host kinases (Stein and Moore, 2001). Even a small modification of the nucleoside structure could significantly affect the host kinases’ activity on the nucleoside analogs because of the tight substrate specificity of some kinases (Golitsina et al., 2010). Since the substrate specificity of various kinases is not well defined, the kinases responsible for phosphorylating nucleoside analogs are often determined retrospectively. After conversion of nucleoside analogs into corresponding nucleoside trisphosphates, they are incorporated into viral DNA/RNA ((De Clercq, 2012). Such insertion of nucleoside often leads to inability of the viral polymerase to carry out further incorporation, leading to chain termination (Olsen et al, 2004). Therefore, two criteria are required for any nucleoside analogs to achieve antiviral activity: (i) efficient conversion to its triphosphate nucleotide inside cell; and (ii) inhibition of viral polymerase by the triphosphate nucleotide analog.

2. Known nucleoside inhibitors of DENV Many nucleoside inhibitors of DENV were originated from HCV drug discovery due to the similarity between these two viruses, from different genera within the same family Flaviviridae. In particular, the 2’-C-methyl substitution was first reported to be active in HCV and it was soon found to have anit-DENV activity as well (Migliaccio et al., 2003). In addition, several other nucleoside analogs such as balapiravir and INX-189 were active in both DENV and HCV (Yeo et al., 2015). It is of interest to note that all DENV-active nucleosides are potent inhibitors of HCV. However, the reverse is not true, as not all anti-HCV nucleoside had antidengue activity (Feng et al., 2014; Stuyver et al., 2006). Therefore, the chemical space for antiDENV compounds seems to be smaller than the anti-HCV nucleosides. The reason behind this 4

discrepancy is not well understood. Table 1 summarizes the nucleoside analogs with anti-DENV activities. Modifications of each of the four naturally occurring nucleoside types could lead to anti-DENV compounds. In the case of adenosine analogs, 2’-C-methyladenosine was first reported to have antiDENV activity, with an EC50 of 4 µM (Migliaccio et al., 2003). This compound was not suitable for drug development because of its rapid conversion to 2’-C-methylinosine by host deaminase (Eldrup et al., 2004). Substitution of N7 nitrogen for C7 of 2’-C-methyladenosine reduced deamination of the compound by the host deaminase, resulting in the discovery of MK-0608 (Olsen et al., 2004). However, this substitution reduced the antiviral activity (EC50 of 15 µM). For regaining potency, a 2’-ethynyl group was introduced to replace 2’-C-methyl, resulting in NITD008 with an improved potency of EC50 of 0.7 µM (Yin et al., 2009). Further modification on the C7-position of adenosine base led to the discovery of NITD449, but NITD449 exhibited poor pharmacokinetic properties. To overcome this problem, NITD203 containing an ester prodrug moiety was made to improve the pharmacokinetic properties and potency of NITD449 (Chen et al., 2010). Although both NITD008 and NITD203 showed potency in the AG129 mouse model (even when the treatment was delayed after DENV infection), both compounds failed in the pre-clinical in vivo toxicity studies and were terminated for further development (Chen et al., 2010; Yin et al., 2009). For guanosine analogs, 2’-methylguanosine exhibited a weak anti-DENV activity with an EC50 of 13.6 µM (Migliaccio et al., 2003). Addition of a phosphoramidate prodrug moiety and a 6’-methoxy at guanine (INX-08189) significantly improved the EC50 to 14 nM (Table 1). Such monophosphate prodrug approach has been now commonly used to improve nucleoside analog potency (see details in section 3.2). Besides adenosine and guanosine analogs, analogs of other two nucleoside types have also been reported to have anti-DENV activities, including balapiravir 5

(an ester prodrug of cytidine analog 4’-azidocytidine) and 3’,5’-di-O-trityl-5-fluoro-2’deoxyuridine (Table 1). Balapiravir represents the first direct antiviral agent that had been tested in dengue patients (Nguyen et al., 2013). Balapiravir was originally developed for treatment of HCV infection (Klumpp et al., 2006). Its clinical development for HCV was discontinued due to unacceptable toxicity (Roberts et al., 2008). Since balapiravir has anti-DENV activity in vitro, it was repurposed for a phase II trial for DENV. Surprisingly, balapiravir treatment did not show any viremia reduction in dengue patients, even though the plasma concentration of the drug well surpassed its EC50 value derived from the DENV-infected cell culture (Nguyen et al., 2013). Although multiple reasons may account for the efficacy failure, one explanation could be the decreased potency of balapiravir when treating human PBMC (peripheral blood mononuclear cells) that were pre-infected with DENV (see details in Section 3.4) (Chen et al., 2014).

3. Challenges of ncuceloside analog development and overcome strategies Despite being the major class of antiviral drugs, development of nucleoside antiviral inhibitors has a number of challenges. Many of these challenges are intrinsically determined by the nature of nucleoside inhibitors (i.e., requirement of host kinases for conversion to the triphosphate form). In the following sections, we outline the challenges of nucleoside approach for DENV drug discovery and provide potential strategies to overcome these challenges. Most of the strategies have been derived from the wisdom of HIV and HCV nucleoside drug discovery and development.

3.1. Intracellular triphosphate nucleotide as a key surrogate parameter for efficacy measurement

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Since different cell types have different levels of kinase activities, the efficiency of nucleoside triphosphate formation could vary significantly, leading to different EC50 values after the same nucleoside compound has been tested in different cell types. The variation in EC50 values could be confusing when trying to connect the potencies derived from in vitro and in vivo experiments. Since the triphosphate form of a nucleoside analog is the active species that inhibits viral polymerase, the concentration of nucleoside triphosphate inside cell could be the universal indicator for antiviral activity (Bazzoli et al., 2010; Fletcher et al., 2000). In other words, although a compound could show distinct EC50 values in assays using different cell types, an equivalent intracellular concentration of nucleoside triphosphate is required to suppress 50% of viral replication among these cells, provided the natural nucleoside triphosphate concentrations are similar among the cell lines studied. This concept has been successfully used to gauge nucleoside inhibitors for HIV, HCV, and RSV (respiratory syncytial virus) pre-clinical and clinical development (Sofia et al., 2010; Wang et al., 2004, 2015). Besides intracellular nucleoside triphosphate, it is important to point out that the half-life and pharmacokinetic properties of nucleoside analogs differ significantly from that of the natural nucleoside (Stein and Moore, 2001). The exposure and half-life of nucleoside triphosphate (rather than those of the nucleoside itself) were successfully used to support the once daily dosing regimen of emtricitabine (a deoxycytidine nucleoside inhibitor of reverse transcriptase) for treatment of HIV patients (Wang et al., 2004). Such practice could be applicable to DENV when developing a nucleoside inhibitor. Despite the fact that it is an important parameter to monitor, direct measurement of intracellular nucleoside triphosphate has been tricky due its extremely polar phosphate groups and interference from the natural nucleoside triphosphates. At the beginning, quantification of nucleotide triphosphates were accomplished enzymatically by first isolating the nucleoside 7

triphosphate analogs, removal of phosphate group by phosphatase, and then quantification of nucleoside itself (Robbins et al, 1994). More recently, direct measurement of nucleoside triphosphate by mass-spectrometry becomes possible after improving the separation of nucleoside analog triphosphates from natural nucleoside triphosphates and the sensitivity of the mass-spectrometry instrumentation (Jansen et al, 2010).

3.2. Monophosphate prodrug to improve intracellular triphosphate nucleoside conversion For most nucleoside analogs, the first phosphorylation is often the rate-limiting step of triphosphate conversion (Stein and Moore, 2001), although in some cases, the second phosphorylation step can be rate-limiting (Lavie et al., 1997; Mukherji et al., 1994). To overcome the bottleneck of first kinase phosphorylation, various phosphate/phosphonate prodrugs have been developed (Cho et al., 2012; McGuigan et al., 2009a; Perrone et al., 2007; Ray and Hostetler, 2011). In the past decade, there is a trend towards developing nucleoside prodrugs with phosphonate/phosphate group to overcome the limitations associated with the first phosphorylation (McGuigan et al., 2009b, 2005). This approach has turned some inactive nucleosides into potent inhibitors or has improved compound efficacy by increasing the intracellular nucleoside triphosphate concentration. The drawback of this approach is the increased complexity of compound synthesis, formulation, and pharmacokinetic analysis, all of which could lengthen the time of drug development. As exemplified by HCV nucleoside development, early clinical candidates were all nucleoside inhibitors, but none of them progressed beyond phase III clinical trials due to unexpected toxicity or insufficient potency (Brandl et al., 2008). A phosphoramidate prodrug approach was used to overcome these issues and ultimately led to the approval of sofosbuvir for HCV treatment with excellent safety and efficacy profile (Sofia, 2013). As described above for DENV (section 3.2), the phosphoramidate 8

prodrug approach has improved the EC50 value by almost 1,000-fold when applied to 2’methylguanosine inhibitor (Table 1). While the monophosphate prodrug approach is attractive, one major difference between DENV nucleoside inhibitors compared to HCV nucleoside inhibitors is that the DENV drugs have to be systematically distributed as opposed to liver-targeting for the HCV inhibitors. For DENV inhibitors, it is therefore essential to test whether the particular prodrug can reach circulation to ensure proper exposure to infected tissues and organs.

3.3. Kinetics of intracellular triphosphate nucleoside formation DENV infection is acute, with disappearance of viremia in ten days after fever appearance (Simmons et al., 2012). Like other acute viral infection, an effective anti-DENV drug should be fast-acting to meet the narrow time of treatment window. Unlike most other classes of antivirals (such as protease inhibitors that can readily act on viral protease in infected cells), some nucleoside analogs have a prolonged lag time to achieve optimal activity due to the need for conversion into the triphosphate form. A comparison has been made for HCV drugs administered within the first two days suggests that some drugs such as mericitabine and interferon (IFN) had modest therapeutic effect as compared to fast acting protease inhibitor telaprevir (Perelson and Guedj 2015). In another example, it took 4.5 days, 0.8 days and 0.25 days for mericitabine, sofosbuvir, and GS-938, respectively, to reach >90% of final effectiveness when given to HCV patients (Guedj et al., 2014). It should be noted that both sofosbuvir and GS938 have monophosphate prodrug moieties, whereas mericitabine does not. In this regard, monophosphate nucleotide prodrug clearly has advantage over nucleoside inhibitors which require additional (often rate-limiting) phosphate addition.

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3.4. Effect of cell types and viral infection on triphosphate nucleoside conversion Cell type-specific activities of nucleoside analogs are commonly observed due to the difference in kinase expression (Becher et al., 2004; Gao et al., 1993). It is therefore essential to evaluate the nucleoside compound in cells relevant to disease indications. Along this line, human PBMC and liver Huh7 cells have been successfully used to evaluate the efficacy of nucleoside analogs for HIV and HCV, respectively (Balsitis et al., 2009; Martina et al., 2009). For DENV, since the tropism of dengue has yet to be conclusively determined (Durbin et a., 2008; Jessie et al., 2004, Rosen et al., 1999), a pan-cellular active nucleoside would be highly desirable. Besides cell types, primary cells and immortalized cell lines could have different levels of kinase activities, leading to different EC50 values for the same nucleoside inhibitor. For instance, where is a significant difference between the efficiency of triphosphate conversion when comparing Huh7 (human liver cell line) and human primary hepatocytes (Berke et al, 2011). Furthermore, viral infection could alter the cellular kinase level and compound efficacy. In support of the latter point, we recently showed that DENV infection triggered PBMCs to generate cytokines which decreased their efficiency to convert balapiravir to its triphosphate form, resulting in decreased antiviral potency (Chen et al., 2014). In contrast to the cytidinebased balapiravir, the potency of an adenosine-based inhibitor of DENV (NITD008) was much less affected by pre-infection of DENV or cytokine treatment. These results demonstrate that viral infection in patient before treatment could significantly affect the prodrug conversion to its active form; such effect is nucleoside class-dependent (Chen et al., 2014). Similarly, HIV infection of PBMCs was also reported to affect both the nucleoside analog triphosphate conversion and the concentration of corresponding natural nucleoside triphosphate, leading to reduced compound efficacy (Gao et al., 1993; García-Lerma et al., 2011).

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3.5. Nucleoside toxicity and drug-drug interactions Another major challenge of nucleoside drug discovery is the unpredictable in vivo toxicity. Apart from the usual CYP mediated drug-drug interactions, nucleoside analogs deserve special considerations due to their peculiar activation process inside cells. Although toxicity of nucleoside analogs have often been attributed to inhibition of mitochondrial polymerases (Arnold et al., 2012; Khungar and Han, 2010; Wang et al., 2015), other perturbations of mitochondrial dysfunctions are also possible (Amacher, 2005; Selvaraj et al., 2014). It is often difficult to predict the side effects of nucleosides using in vitro assays and thus many nucleosides failed at the clinical stage (Coats et al., 2014). Nucleosides are excreted primarily through kidney due to their polar nature. This could cause renal toxicity, especially when two drugs use the same transporter for elimination (Cihlar et al., 2009; Ronaldson and Bendayan, 2002). Drug-drug interaction is also likely to occur when two nucleoside inhibitors are phosphorylated by the same enzyme (Kewn et al., 1997). In addition, toxicity can arise through blocking the degradation of nucleotide triphosphate inside cells (Okuda et al., 1998). Although no compound has been approved for clinical use for DENV, it is conceivable that a combinatory therapy of two inhibitors could not only reduce the dose regimen and improve safety, but also prevent the emergence of resistant virus. Indeed, antiviral synergy could be achieved in DENV cell culture when a nucleoside analog is paired with an inhibitor of the synthesis pathway for the corresponding natural nucleoside. For example, synergy was observed between ribavirin (a guanosine analog with several antiviral mechanisms, one of which is to inhibit de novo biosynthesis of guanine nucleotide through direct binding to cellular IMP dehydrogenase (Malinoski and Stollar, 1981) and INX-08189 (a guanosine analog) in cell culture assay system for DENV (Yeo et al., 2015). Similar antiviral synergies were observed for combination therapy in HIV and HCV (Gao et al., 2000). 11

4. Conclusions Three approaches could be pursued for antiviral therapy. The first approach is to develop inhibitors of viral targets that are essential for pathogen replication. Most of the clinically approved antiviral drugs inhibit viral proteins. The second approach is to develop inhibitors of cellular proteins that are essential for viral replication. Such an approach successfully delivered Maraviroc, an antagonist of CCR5 co-receptor for HIV entry (Abel et al., 2009). For DENV, Celgosivir (an inhibitor of cellular α-glucosidase required to break down complex carbohydrates of viral NS1, prM, and E proteins) and chloroquine (a malaria drug that inhibits DENV entry/fusion and also has immunomodulatory activities) inhibited DENV in cell culture; however, neither reduced viremia or exhibited clinical benefits in dengue patients (Low et al., 2014; Tricou et al., 2010). The third approach is to develop inhibitors of pathological pathways that lead to disease symptoms. In the case of dengue, the severe disease symptoms include dengue hemorrhagic fever (DHF) and shock syndrome (DSS). Unfortunately, the disease pathways that lead to DHF/DSS have not yet been well defined at a molecular level. Therefore, it is currently challenging to pursue this approach. Nevertheless, prednisolone, a corticosteroid drug with immune modulation activities that were believed to alleviate plasma leakage in dengue patients, has been tested in clinical trial; no clinical benefit was observed in dengue patients (Tam et al., 2012). Among the three therapeutic approaches discussed above, inhibitors targeting viral proteins are most promising and relatively straightforward for development. The ideal treatment for dengue is a safe, pan-serotype active, fast-acting direct antiviral with a high resistance barrier. Identifying a compound with pan-serotype activity is challenging because of the 30-35% amino acid variation among the four serotypes of DENV (Green and Rothman, 2006). The same 12

challenge to develop pan-genotype inhibitors has confronted HCV antiviral discovery. This is because a similar level of amino acid variation exists for the seven genotypes of HCV. Among the various HCV inhibitors, nucleoside analogs have been proven to be the most effective mechanism to achieve the pan-genotypeactivity. Therefore, due to the high conservation of RdRp active site and close structural mimicry of the natural substrates, nucleoside analogs are expected to act on all four serotypes of DENV, which may not be the case for other types of inhibitors (van Cleef et al., 2013; Wang et al., 2015; Xie et al., 2015). Indeed, all known DENV nucleoside inhibitors were shown to be pan-serotype active (Chen et al., 2010; Yin et al., 2009). Besides the advantage of pan-serotype activity, nucleoside analogs have high barrier of resistance compared to other classes of inhibitors (Lawitz et al., 2011; McCown et al., 2008; Svarovskaia et al., 2012). In the case of HCV, nucleoside analogs and host targets have the highest barrier of resistance compared to other types of inhibitors (Delang et al., 2011). In the case of DENV, our attempts to select resistant DENV against adenosine analog NITD008 failed after various selection procedures (unpublished results), confirming the high resistance barrier of nucleoside inhibitor in DENV. Some nucleoside analogs have broad antiviral spectrum and have been used for treatment of different viral infections. For instance, tenofovir disoproxil fumarate was initially used in HIV treatment, and was later found to be active against HBV and, therefore, was expanded for treatment of HBV patients. Apart from usual nucleoside analogs, the nucleobase analog Favipiravir T-705 was also demonstrated to have pan-antiviral activities against influenza virus and many other viruses including Ebola virus (Furuta et al., 2013; Oestereich et al., 2014; Sangawa et al., 2013). Therefore, testing nucleoside inhibitors of other viruses in DENV is a sensible approach for dengue drug discovery. This is especially true for testing nucleoside inhibitors targeting HCV where some of which have already been shown to be active in DENV. 13

In summary, nucleoside inhibitors represent an attractive approach for DENV drug discovery. Nucleoside analogs with chemical space to allow host kinases for triphosphate formation as well as viral RdRp to incorporate them into viral RNA are prerequisites for cellular antiviral activity. Despite many challenges, the recent advances in nucleoside inhibitors for HCV, RSV, and other viral infection treatment will enhance the potential of this approach for DENV antiviral development.

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Acknowledgements We thank our colleagues at the Novartis Institute for Tropical Diseases for their contributions to dengue drug discovery and for helpful discussions during the course of this work.

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Table 1 Adenosine based Nucleoside inhibitors of dengue RNA dependent RNA polymerase. These adenosine based analogs do not need monophosphate prodrugs as they are efficiently converted to their corresponding monophosphate by host adenosine kinase. However, Name

Nucleoside

2’-methyladenosine

Structure

In vivo mouse activity

Reference

Adenosine

Cellular activity (EC50/CC50) µM 4/18

No

(Migliacci o et al., 2003)

MK-0608

Adenosine

15/>320

Yes

(Olsen et al., 2004)

NITD008

Adenosine

0.7/>100

Yes

(Yin et al., 2009)

NITD449

Adenosine

1.626.99/>50

No

( Chen et al., 2010b)

NITD203

Adenosine

0.10.71/>50

Yes

( Chen et al., 2010b)

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Table 2: Dengue nucleoside inhibitors with different nucleosides other than adenosine. Some of these nucleosides required monophosphate prodrug as conversion from nucleosides into nucleoside monophosphates are not efficient. Name

Nucleoside

2’-methylguanosine

Structure

In vivo mouse activity

Reference

Guanosine

Cellular activity (EC50/CC50) µM 13.6/>60

No

(Migliacci o et al., 2003)

INX-08189

Guanosine

0.014/>1

No

(Yeo et al., 2015)

Balapiravir

Cytidine

1.911/>2000

No

(Nguyen et al., 2013)

3’,5’-di-Otrityl-5fluoro-2’deoxyuridine

Uridine

1.2/>50

No

(Saudi et al., 2014)

F Ph Ph Ph

O

O

N

O NH O

Ph Ph

O

OH

Ph

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CHEN Revised highlights 31 July 15

• Nucleos(t)ide inhibitors are the most attractive class of anti-dengue drug. • The RdRp of dengue virus is the most attractive target due to proven track record in other antiviral treatments. • Most recent nucleos(t)ide drugs are monophosphate prodrugs with better potency and shorter time to maximum activity. • Toxicities associated with nucleoside analogs are difficult to predict and remain one of the main reasons for clinical failure. • Combination therapies could minimize resistance, improve therapeutic index, and reduced unwanted side effects.

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