The thiopurine nucleoside analogue 6-methylmercaptopurine riboside (6MMPr) effectively blocks Zika virus replication

https://doi.org/10.1016/j.ijantimicag.2017.08.016Get rights and content

Highlights

  • 6-Methylmercaptopurine riboside (6MMPr) was identified as a potential antiviral drug against Zika virus (ZIKV).

  • All assays used the epidemic ZIKV strain circulating in Brazil.

  • Antiviral activity was tested both in epithelial and human neuronal cells.

  • 6MMPr was 1.6 times less toxic to neuronal cells compared with epithelial cells.

  • 6MMPr decreased ZIKV production in both cell lines by >99%.

Abstract

Since the emergence of Zika virus (ZIKV) in Brazil in 2015, 48 countries and territories in the Americas have confirmed autochthonous cases of disease caused by the virus. ZIKV-associated neurological manifestations and congenital defects make the development of safe and effective antivirals against ZIKV of utmost importance. Here we evaluated the antiviral activity of 6-methylmercaptopurine riboside (6MMPr), a thiopurine nucleoside analogue derived from the prodrug azathioprine, against the epidemic ZIKV strain circulating in Brazil. In all of the assays, an epithelial (Vero) and a human neuronal (SH-SY5Y) cell line were used to evaluate the cytotoxicity and effective concentrations of 6MMPr against ZIKV. Levels of ZIKV-RNA, viral infectious titre and the percentage of infected cells in the presence or absence of 6MMPr were used to determine antiviral efficacy. 6MMPr decreased ZIKV production by >99% in both cell lines in a dose- and time-dependent manner. Interestingly, 6MMPr was 1.6 times less toxic to SH-SY5Y cells compared with Vero cells, presenting a 50% cytotoxic concentrations (CC50) of 460.3 µM and 291 µM, respectively. The selectivity index of 6MMPr for Vero and SH-SY5Y cells was 11.9 and 22.7, respectively, highlighting the safety profile of the drug to neuronal cells. Taken together, these results identify, for the first time, the thiopurine nucleoside analogue 6MMPr as a promising antiviral candidate against ZIKV that warrants further in vivo evaluation.

Introduction

Zika virus (ZIKV) is a member of the Flavivirus genus within the family Flaviviridae. This genus comprises other important arboviruses such as dengue virus (DENV), yellow fever virus (YFV) and West Nile virus (WNV) [1]. The virus was initially isolated in 1947 from a rhesus monkey in the Zika Forest in Uganda. In March 2015, Brazil reported autochthonous transmission of ZIKV and since then the virus has spread throughout the Americas. ZIKV infection has already been reported in ca. 60 countries on different continents [2], [3]. ZIKV transmission occurs through the bite of an infected Aedes mosquitoes, although recent findings have also indicated sexual, congenital, perinatal and blood transfusion transmission. Clinically, ZIKV disease is manifested by rash, fever, arthralgia and conjunctivitis [4]. Importantly, infection of pregnant women may result in microcephaly of the fetus and other severe congenital defects such as intracranial calcifications, ventricular system dilation and neuronal migration disorders [5]. In adults, severe neurological complications such as myelitis, meningoencephalitis and Guillain–Barré syndrome have been associated with ZIKV infection and in some patients the infection can lead to death [6], [7], [8].

Like other members of the Flavivirus genus, ZIKV is an enveloped, single-stranded, positive-sense RNA virus with a genome of ca. 11 kb. Its genome encodes three structural and seven non-structural proteins. The structural proteins mediate the initial steps of virus–host interaction, including receptor binding and entry, whereas the non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) play different roles in viral replication, virion assembly and evasion of immune defence mechanisms [9]. The proteins NS3 and NS5 are targets for antiviral development because of their vital role in viral replication. NS5 is the most conserved protein across the Flavivirus genus, possessing N-terminal RNA methyltransferase and C-terminal RNA-dependent RNA polymerase (RdRp) activities [10]. Several non-structural proteins have been implicated as potential targets for drugs against flaviviruses [11].

Despite the burden of ZIKV-associated diseases, no vaccines or treatments are available to block viral replication. Recent studies have suggested that the specificity of ZIKV biology compared with other flaviviruses may reveal new challenges for antiviral therapy as well as new drug targets [1]. Nevertheless, various attempts to discover anti-ZIKV inhibitors are underway [12], [13], [14], [15], [16].

Many nucleoside and non-nucleoside analogues are inhibitors of viral polymerases and have been described as promising molecules against RNA viruses [10]. RdRp is responsible for viral RNA synthesis and there is no similar enzyme in the host. Compounds that bind to RdRp and inhibit de novo initiation of viral replication have already been shown to be active against DENV [17] and ZIKV [14], [16]. In general, they have high selectivity and broad-spectrum activity, making them attractive candidates for Flavivirus antiviral development [11].

Azathioprine belongs to the class of thiopurine-modified nucleosides and has shown activity against ZIKV [12]. It is a prodrug that is metabolised to 6-methylpurine (6MP) (inactive metabolite) by glutathione S-transferase enzyme; 6MP, in turn, is processed to 6-thioinosine and 6-methylmercaptopurine by the action of thiopurine methyltransferase enzyme, both active metabolites that can be converted to 6-methylmercaptopurine riboside (6MMPr) [18]. 6MMPr is also a nucleoside analogue thiopurine metabolite and its antiviral properties have already been demonstrated against flaviviruses such as hepatitis C virus (HCV) RNA replicon, bovine viral diarrhoea virus (BVDV) [19], [20], YFV, DENV-2 and WNV [21].

In this work, the antiviral activity of 6MMPr against the epidemic ZIKV strain that has recently emerged in Brazil was evaluated. For the first time, we demonstrate potent in vitro inhibitory activity of 6MMPr against ZIKV infection both in epithelial and neuronal cell lines. Together, these results identify the thiopurine analogue 6MMPr as a promising candidate for further clinical evaluation against ZIKV.

Section snippets

Cells, virus and drug preparation

Vero cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Carlsbad, CA) supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco), 2 mM l-glutamine (Gibco) and 100 U/mL penicillin/streptomycin (Gibco). Human neuroblastoma SH-SY5Y cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were grown in Minimum Essential Medium (MEM) (Inlab Diagnóstica, Sao Paulo, Brazil) supplemented with Ham's F12 Nutrient Mixture (Sigma, St Louis, MO), 1 mM

The thiopurine drug 6-methylmercaptopurine riboside (6MMPr) has a distinct toxicity profile in epithelial and neuronal cells

The MTT assay was performed to determine the cell toxicity of the thiopurine nucleoside analogue 6MMPr (Fig. 1) to Vero and SH-SY5Y cells. The CC20 and CC50 values for Vero cells were 60.5 µM and 291 µM, respectively (Table 1; Fig. 2A). For the SH-5YSY cell line, the CC20 was 157 µM and the CC50 was 460.3 µM (Table 1; Fig. 2B). To determine the relative drug efficacy in inhibiting viral replication compared with cellular toxicity, the SI was established for each cell line (Table 1). Thus, 6MMPr

Discussion

Since its emergence in Brazil in 2015, ZIKV has rapidly spread in the Americas, resulting in an epidemic of great public health concern. Despite some serious complications caused by the virus, including neurological disorders and congenital malformations, there are currently no available vaccines or specific antiviral drugs against this feared pathogen [26]. Therefore, the discovery and development of therapeutic strategies able to effectively control ZIKV is an urgent need. Here we showed that

Acknowledgment

The authors would like to thank all of the members from the Virology Laboratory at Fiocruz Pernambuco (Recife, Pernambuco, Brazil) for their assistance.

Funding: This work was supported in part by grants from CNPq (National Council for Scientific and Technological Development, Brazil) and FACEPE (Pernambuco State Foundation for Science and Technology, Brazil). OVC is the recipient of a postdoctoral fellowship from FACEPE. DMF is the recipient of a doctoral fellowship from CAPES. LRM is the

References (37)

  • M. Sironi et al.

    Nonstructural proteins are preferential positive selection targets in Zika virus and related Flaviviruses

    PLoS Negl Trop Dis

    (2016)
  • XuM. et al.

    Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

    Nat Med

    (2016)
  • M. Atif et al.

    Zika virus disease: a current review of the literature

    Infection

    (2016)
  • A.R. Plourde et al.

    A literature review of Zika virus

    Emerg Infect Dis

    (2016)
  • Y.R. Ramalho Rocha et al.

    Radiological characterization of cerebral phenotype in newborn microcephaly cases from 2015 outbreak in Brazil

    PLoS Curr

    (2016)
  • S.K. Saxena et al.

    Zika virus outbreak: an overview of the experimental therapeutics and treatment

    Virusdisease

    (2016)
  • W.M. Kok

    New developments in flavivirus drug discovery

    Expert Opin Dug Discov

    (2016)
  • D. Contreras et al.

    Zika virus infectious cell culture system and the in vitro prophylactic effect of interferons

    J Vis Exp

    (2016)
  • Cited by (33)

    • Evaluation of the maternal and developmental toxicity of 6-methylmercaptopurine riboside in rats

      2022, Reproductive Toxicology
      Citation Excerpt :

      The putative antiviral activity of 6MMPr against some viruses of Flaviviridae family, including the zika virus, has boosted the interest in obtaining more data on the potential developmental toxicity of this methylated 6-thiopurine nucleoside. Recent studies revealed that AZA, 6MP, and particularly 6MMPr –but not thioguanine and 6MMP– exhibited potent antiviral activities against several RNA viruses (flaviviruses and canine distemper virus) in cell culture test systems [17–21]. If intrauterine infections pose risks to the unborn child’s health, effective antiviral agents must cross the placenta to stop viral proliferation in embryonic tissues, and, in addition, they must not be developmentally toxic.

    • Synthesis of alkynylated 1,2,4-oxadiazole/1,2,3-1H-triazole glycoconjugates: Discovering new compounds for use in chemotherapy against lung carcinoma and Mycobacterium tuberculosis

      2021, European Journal of Medicinal Chemistry
      Citation Excerpt :

      Cytotoxicity using Vero cells: Vero cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Carlsbad, CA) supplemented with 10% inactivated fetal bovine serum (FBS) (Gibco), 2 mM l-glutamine (Gibco) and 100 U/mL penicillin/streptomycin (Gibco). The toxicity of the synthesized compounds was evaluated on growing cells using the MTT method (Sigma, St. Louis, USA) as previously described [58]. Briefly, Vero cells (1 × 104 cells/well) were plated in 96-well microplates 24 h in advance and thentreated with various concentrations of the compounds (800, 400, 200, 100, 50 μM) in max.

    • Advance of structural modification of nucleosides scaffold

      2021, European Journal of Medicinal Chemistry
      Citation Excerpt :

      To increase the lipophilicity of anti-HIV drugs and better penetrate the blood-brain barrier, 6-site of the purine can be usually modified. For example, adenosine deaminase active prodrugs 6-methylmercaptopurine riboside (6MMPr, 127) exhibits anti-Zika virus (ZIKV) potency [91]. 2′-F-6-Ethylaminoadenosine (128) and 2′-F-6-dimethylaminoadenosine (129) are much more lipophilic than their unmodified compound ddI (39) or its 2′-F analogue, although the anti-HIV activity is slightly reduced [74].

    • 7-Deaza-7-fluoro-2′-C-methyladenosine inhibits Zika virus infection and viral-induced neuroinflammation

      2020, Antiviral Research
      Citation Excerpt :

      Therefore, to analyze the neuron/ZIKV interaction and investigate potential antiviral drugs, neuronal-like immortal cell lines are being largely used. As an example, Sofosbuvir and a thiopurine nucleoside analogue (6MMPr) were both administrated in SH-SY5Y cell line during ZIKV infection and both showed reduced viral loads in a concentration or time dependency (Sacramento et al., 2017; Valério de Carvalho et al., 2017). Our findings are comparable to those found with other similar nucleoside analogs that presented anti-ZIKV activity such as (Zmurko et al., 2016) 2′-C-methyladenosine (2′-CMA) and 7-deaza-2′-C-methyladenosine (7DMA).

    View all citing articles on Scopus
    1

    These two authors contributed equally to this work.

    View full text