Zika virus: a race in search for antivirals

25 Pages·2017·0.48 MB·English

AAC Accepted Manuscript Posted Online 27 March 2017
Antimicrob. Agents Chemother. doi:10.1128/AAC.00411-17
Copyright © 2017 American Society for Microbiology. All Rights Reserved.
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1 Zika virus: a race in search for antivirals
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3 Running title: antivirals for ZIKV
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5 Juan-Carlos Saiz# and Miguel A. Martín-Acebes# lo
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7 Department of Biotechnology, Instituto Nacional de Investigación y Tecnología Agraria o
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8 y Alimentaria (INIA), Madrid, Spain h
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10 Abstract: 137 words c
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11 Text: 2,833 words m
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13 * J.-C.S. and M.A. M.-A. Contributed equally to this work n

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15 #Address correspondence to Miguel A Martín-Acebes, [email protected], or Juan- ,
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16 Carlos Saiz, [email protected] 9

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18 ABSTRACT
19 Zika virus (ZIKV), a flavivirus transmitted by mosquitoes, was an almost neglected
20 pathogen until its introduction in the Americas in 2015, and its subsequent explosive
21 spread throughout the continent, where it has infected millions of people. The virus has
22 caused social and sanitary alarm, mainly due to its association with severe neurological D
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23 disorders (Guillain-Barré syndrome, and microcephaly in fetus and newborns). n
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24 Nowadays, no specific antiviral therapy is available against ZIKV. However, during the a
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25 past months, a great effort has been made in search for antiviral candidates by using
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26 different approaches and methodologies, from testing specific compounds with known m

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27 antiviral activity to screenings of libraries with hundreds of bioactive molecules. The tp
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28 identified antiviral candidates include drugs targeting viral components, as well as a
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29 cellular ones. Here, an updated review of what has been done in this line is presented. a
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31 INTRODUCTION
32 Zika virus (ZIKV) is a flavivirus (Flaviviridae family) transmitted by mosquitoes,
33 mainly by those of the Aedes genus (1). The virus was first isolated in Uganda in 1947
34 (2) and has since been confined in Africa until it was detected in Asia in the 1980s.
35 Subsequently, the first large human outbreaks were reported in 2007 in the Micronesia D
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36 and in 2013 in the French Polynesia (1). However, ZIKV was an almost neglected n
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37 pathogen until the virus jumped into the Americas, which most probably occurred by a d
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38 single introduction of an Asian viral strain during the second half of 2013 (3). Along f
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39 2015, the association of ZIKV with severe neurological disorders, including a striking

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40 increase in the number of cases of microcephaly in fetus and newborns and an unusual tp
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41 upsurge in Guillain-Barré syndrome (GBS) cases, drove the World Health Organization a
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42 (WHO) to declare a public health emergency of international concern (4). The s
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43 neurotropism of ZIKV has been confirmed experimentally by virus isolation from fetal .o
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44 brain tissue from miscarriages, biopsies from affected children, and the reproduction of o
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45 developmental disorders in animal models (5-9). Current data from ZIKV epidemics in M
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46 the Americas indicates 205,500 confirmed cases, 598,960 suspected, and 2,767 3
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47 confirmed congenital syndrome associated with ZIKV infection 0
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48 (http://www.paho.org/hq/index.php?option=com_content&view=article&id=12390%3A b
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49 zika-cumulative-cases&catid=8424%3Acontents&Itemid=42090&lang=en). u
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50 Nowadays, not a single specific antiviral agent against any flavivirus has been
51 approved (10), and treatment, when applied, is generally directed to symptom relief
52 with analgesics and anti-pyretic. However, a great effort has been lately carried out to
53 assay several drug candidates directed to viral targets (direct-acting antivirals) or against
54 cellular targets (host-targeting antivirals). This vast work has been performed through
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55 different approaches that include the screening of different compounds libraries and the
56 repurposing of drugs already used in clinical practice for other diseases, many of these
57 molecules being broad spectrum drugs (Table 1). For instances: nucleoside
58 analogs/derivatives, nucleoside synthesis inhibitors and polymerase inhibitors,
59 immunomodulators, antibiotics, and anti-inflammatory, anti-malaria, and anti-
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60 helminthic drugs, among others, have been tested. w
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61 DIRECT-ACTING ANTIVIRALS d
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62 Antivirals targeting the ZIKV polymerase. The NS5 protein of ZIKV is the RNA ro
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63 dependent RNA polymerase (RdRp) in charge or viral genome replication. Remarkably, h
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64 its structure has been recently determined, which would positively contribute the :/
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65 structure-based design of antiviral compounds against ZIKV (11). In fact, nucleoside c
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66 analogs/derivatives, which target viral but not cellular polymerases to terminate viral m
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67 RNA replication after incorporation into the viral nascent RNA chain, are usually safe rg
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68 for use in humans (12) and, thus, they have been extensively assayed against ZIKV in n

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69 cell culture and, in some instance, in animal models. Thereby, Eyer and coworkers (13) a
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70 tested several nucleoside analogues for their capability to inhibit ZIKV replication in ,

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71 Vero cells, finding that five [7-deaza-2´-C-methyladenosine (7-deaza-2´-CMA), 2´-C- 1
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72 methyladenosine (2´-CMA), 2´-C-methylcytidine (2´-CMC), 2´-C-methylguanosine (2´- y

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73 CMG), and 2´-C-methyluridine (2´-CMU)] reduce significantly virus-induced cell death e
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74 with 50% effective concentration (EC50) values ranging from 5.3 to 45.5 μM.
75 Comparable results were previously obtained by testing the effect of different 2′-C-
76 methylated nucleosides in the in vitro activity of purified recombinant ZIKV RdRp (14).
77 Similarly Zmurko and coworkers (15) showed that 7-deaza-2’-CMA exhibits anti-ZIKV
78 activity in Vero cells (EC50=9.6 μM for a Selectivity Index, SI, of 7) and also delays
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79 disease progression and reduced viral RNA loads in the serum of ZIKV-infected AG129
80 (IFN-α/β and IFN-γ receptor knock-out) mice treated once daily with 50 mg/kg/day of
81 the drug.
82 Likewise, the nucleoside analog BCX4430 also inhibited ZIKV multiplication in
83 Vero cells with EC50=3.8-11.7 and SI=5.5-11.6, depending of the viral strain tested D
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84 (16). Even more, 7 out of 8 ZIKV infected AG129 mice treated with 300 mg/kg/day n
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85 showed a significant reduction in viremia and were protected when compared with d
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86 vehicle treated animals (100% mortality). BCX4430 treatment also protected AG129
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87 mice even when administered after infection, although in this case RNA viral load in m

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88 serum were similar to that of vehicle treated animals. tp
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89 Sofosbuvir is a nucleotide analog that is an RdRp inhibitor approved by the US c
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90 Food and Drug Administration (FDA) for the treatment of HCV infection. This drug m
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91 was proposed as a ZIKV antiviral after showing that it reduced viral NS1 staining in rg
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92 human neuroepithelial stem cells (17). A further study (18) demonstrated that n

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93 Sofosbuvir efficiently inhibits replication and infection of ZIKV in cell lines of different a
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94 origin, like hepatoma (Huh-7) and human placental choriocarcinoma (Jar) cells ,

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95 (EC50=1-5 µM, SI≥40), as well as in hindbrain and cerebral cortex-derived neural stem 19

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96 cells (NSCs) (EC50~32 µM). Moreover, the study (18) showed that when Sofosbuvir y

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97 was orally administered (33 mg/kg/day) for 7 days to ZIKV infected mice, greater e
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98 overall survival rates against ZIKV-induced death was recorded when compared with
99 vehicle treated mice (50% vs 20%, respectively). Remarkably, these experiments were
100 performed using a recently developed model, WT C57BL/6 mice treated with an anti-
101 Ifnar1 blocking antibody (19). Another study (20) also reported that Sofosbuvir
102 inhibited ZIKV replication in Huh-7 hepatoma cells (EC50=0.4 µM, SI=1,191), as well
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103 as in neuroblastoma cells, SH-Sy5y (EC50=1.1 µM, SI=384), and, to a lesser extent, in
104 baby hamster kidney cells, BHK (EC50=1.9 µM, SI=184), but it did not exhibit anti-
105 ZIKV inhibitory activity in Vero cells, indicating that its inhibitory efficiency varied
106 among different cell types. Even more, the same study (20) also reported the reduction
107 of viral replication in treated human induced pluripotent stem (iPS) cell-derived NSCs
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108 by inducing cell death and impairing ZIKV-mediated neuropathogenesis, as well as in w
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109 brain organoids, which have been used to address brain development and microcephaly lo
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110 (21). Additionally, further analysis of ZIKV sequences from infected cells treated with e
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111 Sofosbuvir (20) showed an increase in the frequency of transition mutations when o
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112 compared with untreated cells, thus suggesting that, beside its direct inhibitory effect, h
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113 the drug also increase the incorporation of mutations in the viral genome, increasing //
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114 error-prone replication (22). c
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115 Adcock and coworkers (23) used a cell-based assay for high-throughput .o
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116 screening of broad-spectrum antiviral compounds as ZIKV inhibitor. They informed /
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117 that drugs that inhibit the purine synthesis (ribavirin and mycophenolic acid, MPA) M
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118 were toxic or did not reduce ZIKV multiplication in Vero cells. On the contrary, the 3
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119 pyrimidine synthesis inhibitors tested (NITD008, CID 91632869, finasteride, brequinar, 0
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120 and 6-azauridine) were capable to reduce viral multiplication to different levels, with b
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121 EC50 ranging from submicromolar (brequinar) to 3.2 μM (6-azauridine). Remarkably, 6- gu
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122 azauridine, an anticancer drug and viral inhibitor, was also identified (EC50=2.3; t
123 SI>33.3) in another screening (24). Regarding NITD008, this compound also exhibited
124 antiviral activity in A129 mice deficient in type I interferon receptor treated with 50
125 mg/kg/day of the drug, as all vehicle-treated mice died within 12 days after ZIKV
126 infection, whereas 50% of the NITD008 treated animals survived without developing
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127 any neurological signs (25). In this line, it has been also reported that Gemcitabine, a
128 nucleoside that interferes with de novo pyrimidine biosynthesis, inhibited ZIKV
129 multiplication (EC50=1 μM and SI >1,000) by interfering with the transcription of viral
130 RNA (26). Similarly, another screening (24) also identified the thymidylate synthetase
131 inhibitor 5-fluorouracil, an anticancer drug, as a potent inhibitor of ZIKV multiplication
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132 (EC50=14.3 μM; SI>2.5), further supporting pyrimidine synthesis inhibitors as potential w
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133 antiviral candidates against ZIKV. lo
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134 Methyltransferase and protease inhibitors. The NS5 protein of ZIKV exhibits not
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135 only the RdRp activity, but also displays a methyltransferase domain responsible for m

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136 capping the 5’ end of the viral genomic RNA. This enzymatic activity has just started to tp
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137 be explored as a potential antiviral target to combat ZIKV (27-29). ZIKV proteins other aa
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138 than NS5 also constitute potential druggable antiviral targets. This is the case of the a
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139 N2B-NS3 trypsin-like serine-protease, which plays a key role on virus replication by .o
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140 contributing to viral polyprotein processing (30), or through the NS3 helicase activity /
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141 (31). Due to the relevance of NS2B-NS3 function in the ZIKV life cycle, the search for M
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142 inhibitors of the enzymatic activity of this complex is at the front line of antiviral 3
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143 discovery against ZIKV (30, 32-34). For instance, taking advantage of a previous work 0
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144 (35) that identified inhibitors of the HCV protease by high-throughput screening of over b
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145 40,000 compounds, the same group (36) have recently analyzed 71 of these nonpeptidic g
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146 small molecules against ZIKV and found that 10 of them showed IC50 values lower than t
147 50 μM, with IC50=5.2 μM and 4.1 μM for compounds 2 and 3, respectively.
148 Additionally, the structure of NS2B-NS3 complex has been resolved under different
149 circumstances, including in complex with a peptidomimetic boronic acid inhibitor (37).
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150 Therapeutic antibodies and virucidal compounds. The administration of specific
151 potent neutralizing antibodies appears as a potential strategy for the treatment of
152 flavivirus infections (38). In the case of ZIKV, it has been recently showed that passive
153 transfer of human neutralizing antibodies through intraperitoneal inoculation of
154 pregnant mice suppressed ZIKV replication, inhibited cell death and reduction of
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155 neural progenitor cells (NPC) in infected fetal brains, and prevent microcephaly (39, w
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156 40). Furthermore, treatment of mice with a monoclonal antibody against the Domain III lo
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157 of the envelope protein of ZIKV is sufficient to protect mice from lethal ZIKV infection e
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158 (41). Overall, these studies support the potential of anti-ZIKV strategies based on the o
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159 usage of therapeutic antibodies. h
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160 In addition to therapeutic antibodies, other compounds targeting the viral particle aa
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161 like epigallocatechin gallate (EGCG), a polyphenol present in many natural products, a
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162 exhibit anti-ZIKV activity (EC50=21.4 μM) probably due to a virucidal effect (42). .o
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163 Similar results have been obtained with EGCG and delphinidin, other different /
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164 polyphenol (A. Vázquez-Calvo, N. Jiménez de Oya, M. A. Martín-Acebes, E. Garcia- M
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165 Moruno, J.-C. Saiz, submitted for publication). 3
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166 HOST-TARGETING ANTIVIRALS 1
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167 Apart from drugs targeting viral components, those targeted to cellular factors directly g
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168 involved in viral life cycle may also be useful, since their effect is less prone to being s
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169 evaded by mutations in the viral genome that frequently appear in RNA viruses. In this
170 line, 2,000 compounds from a library of FDA-approved drugs, as well as molecules
171 known to be bioactives, have been tested by means of a microscopy-based assay to
172 uncover inhibitors of ZIKV infection (43). By using human osteosarcoma cells (U2OS),
173 it was shown that up to 38 molecules blocked flavivirus infection, including
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174 nanchangmycin (half maximal inhibitory concentration, IC50=0.1 µM), a natural product
175 of Streptomyces nanchangensis that was shown to have insecticidal activity against
176 silkworms and anti-bacterial activity in vitro; tenovin-1 (IC50=0.7 µM), that protects
177 against MDM2-mediated p53 degradation; MPA (IC50=0.4 µM); and Gemcitabine
178 (IC50=0.3 µM). The last two have also been reported to be ZIKV inhibitors by others
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179 (26, 44). A less potent effect was also observed in human brain microvascular w
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180 endothelial cells (HBMECs), an immortalized model of the human blood brain barrier lo
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181 microvasculature that may be involved in ZIKV access to fetal brain. Even more, some e
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182 of these molecules were also active on Jeg-3 human placental cells. o
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183 Chloroquine is an anti-inflammatory FDA approved 4-aminoquinoline widely tp
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184 used as antimalaria drug and also administered to pregnant women at risk of exposure to aa
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185 Plasmodium parasites, which has also shown antiviral activity against several viruses a
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186 through the inhibition of pH-dependent steps of viral replication. This drug has been .o
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187 described to exhibit anti-ZIKV activity in Vero cells (45). The agent also affected ZIKV /
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188 infected hBMECs, as well as human NSCs, which depletion is one of the main M
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189 mechanisms responsible for primary microcephaly (46). Chloroquine reduced the 3
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190 number of ZIKV-infected cells, inhibiting virus production (including defective viral 0
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191 particles), and cell death promoted by ZIKV infection, thus interfering with the early b
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192 stages of the ZIKV replication cycle, possibly during fusion of the envelope protein to g
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193 the endosomal membrane (45). The reported EC50 were 9.8 µM, 14.2 µM, and 12.3 µM t
194 (for therapeutic index, TI, of 13.7, 8.2, and 7.7) for Vero, hBMECs, and NSCs,
195 respectively. Saliphenylhalamide (SaliPhe), which targets vacuolar-ATPase and blocks
196 the acidification of endosomes, also inhibit ZIKV replication in human retinal pigment
197 epithelial (RPE) cells, which are natural targets for ZIKV infection (47), with EC50=1
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198 μM and SI>200 (26). Interestingly, SaliPhe, chloroquine, and other compounds
199 interfering with the endocytic pathway, such as Dynasore and monesin, were also
200 identified as potential anti-ZIKV compounds in a different screening (23). In this line, it
201 has also been reported (26) that several agents used for the treatment of cancers, and
202 with already known capacity to inhibit multiplication of other viruses, such as
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203 Obatoclax, also known as GX15-070 (an inhibitor of the Bcl-2 family of proteins that w
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204 targets cellular Mcl-1 and inhibits endocytosis inducing apoptosis), presented EC50=0.3 loa
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205 μM and SI=65, and could impair ZIKV endocytic uptake. e
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206 Barrows and coworkers (44), after assaying a FDA approved library of m

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207 compounds, showed that over 20 of them reduced ZIKV infection in hepatoma-derived tp
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208 HuH-7 cells, including Bortezomib (a selective inhibitor of proteasome activity used in aa
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209 patients with multiple myeloma), daptomycin (a lipopeptide antibiotic), MPA sertraline, a
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210 pyrimethamine, cyclosporine A, azathioprine, and mefloquine. Next, selected drugs .o
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211 were tested in human cervical [as ZIKV can be sexually transmitted, probably when /
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212 infected semen enters in contact with the vaginal mucosa or the cervix (48)], placental, M
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213 neural stem cell lines, and primary human amnion cells, showing EC50 of 1 μM 3
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214 (daptomycin), 0.1 μM (MPA) and =1-10 μM (ivermectin), respectively. 0
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215 Another recent study of drug repurposing (49), based in the expression of the y

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216 viral NS1 protein as a read-out of anti-ZIKV activity for primary screening, tested over e
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217 5,000 compounds from the LOPAC (Library of Pharmacologically Active Compounds),
218 the NCATS (National Center for Advancing Translational Sciences Pharmaceutical
219 Collection), and a collection of clinical candidates. The study was conducted in ZIKV-
220 infected glioblastoma SNB-19 cells, as well as in human NPCs and astrocytes, both of
221 which are target cells for ZIKV infection in the fetal brain (50, 51). Emricasan, a pan-