The thiopurine nucleoside analogue 6-methylmercaptopurine riboside (6MMPr) effectively blocks Zika virus replication
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)
- et al.
Guillain–Barre syndrome associated with Zika virus infection
Lancet
(2016) - et al.
Acute myelitis due to Zika virus infection
Lancet
(2016) - et al.
Fatal encephalitis associated with Zika virus infection in an adult
J Clin Virol
(2016) - et al.
In-silico screening for anti-Zika virus phytochemicals
J Mol Graph Model
(2016) - et al.
A screen of FDA-approved drugs for inhibitors of Zika virus infection
Cell Host Microbe
(2016) - et al.
Modulation of influenza virus replication by alteration of sodium ion transport and protein kinase C activity
Antiviral Res
(2008) - et al.
Evaluation of anti-Zika virus activities of broad-spectrum antivirals and NIH clinical collection compounds using a cell-based, high-throughput screen assay
Antiviral Res
(2017) - et al.
Efficacy of the broad-spectrum antiviral compound BCX4430 against Zika virus in cell culture and in a mouse model
Antiviral Res
(2017) - et al.
Excision of incorporated nucleotide analogue chain-terminators can diminish their inhibitory effects on viral RNA-dependent RNA polymerases
J Mol Biol
(2004) - et al.
Review of the potential effects of three commonly used antineoplastic and immunosuppressive drugs (cyclophosphamide, azathioprine, doxorubicin on the embryo and placenta)
Reprod Toxicol
(2004)
Nonstructural proteins are preferential positive selection targets in Zika virus and related Flaviviruses
PLoS Negl Trop Dis
Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen
Nat Med
Zika virus disease: a current review of the literature
Infection
A literature review of Zika virus
Emerg Infect Dis
Radiological characterization of cerebral phenotype in newborn microcephaly cases from 2015 outbreak in Brazil
PLoS Curr
Zika virus outbreak: an overview of the experimental therapeutics and treatment
Virusdisease
New developments in flavivirus drug discovery
Expert Opin Dug Discov
Zika virus infectious cell culture system and the in vitro prophylactic effect of interferons
J Vis Exp
Cited by (33)
Copper Catalyzed Three-Component Ullmann C-S Coupling in PEG for the Synthesis of 6-Aryl/alkylthio-purines
2024, Journal of Organic ChemistryEvaluation of the maternal and developmental toxicity of 6-methylmercaptopurine riboside in rats
2022, Reproductive ToxicologyCitation 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 ChemistryCitation 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 ChemistryCitation 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].
Bisbenzylisoquinoline alkaloids of Cissampelos sympodialis with antiviral activity against dengue virus
2021, Natural Product Research7-Deaza-7-fluoro-2′-C-methyladenosine inhibits Zika virus infection and viral-induced neuroinflammation
2020, Antiviral ResearchCitation 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).
- 1
These two authors contributed equally to this work.