3.1. Direct-Acting Antivirals
Virus-directed drugs are those acting against the viral RNA-dependent RNA polymerase (RdRp) (NS5) catalytic domain, including nucleoside analogs and polymerase inhibitors. They target the methyltransferase catalytic domain of the NS5, responsible for transferring the mRNA cap; the NS2B-NS3 trypsin-like serine protease needed for proper processing of the viral polyprotein; and the NS3 helicase.
Nucleoside analogs/derivates, which target viral (not cellular polymerases) to terminate viral RNA replication after incorporation into the viral nascent RNA chain, are safe for use in humans [
44], because virus-encoded gene products required for RNA replication can significantly differ in substrate specificity from RNA polymerases involved in host cell replication. Consequently, they have been extensively evaluated against ZIKV in cell culture and in animal models. Eyer et al. tested 29 nucleoside analogues at a concentration of 50 µM for their ability to inhibit cytopathic effects (CPE) of ZIKV on Vero cells. They found that five of these compounds (7-deaza-2’-C-methyladenosine (7-deaza-2-CMA), 2’-C-methyladenosine (2-CMA), 2’-C-methylcythydine (2-CMC), 2’-C-methylguanisine (2-CMG), and 2’-C-methyluridine (2-CMU), significantly reduced cell death compared to non-treated ZIKV infected cells with 50% effective concentration (EC
50) values ranging from 5.3 to 45.5 µM [
45]. A similar observation was found when testing the effects of different 2’-C-methylated nucleosides on the in vitro activity of purified recombinant ZIKV RdRp [
46]. In addition, the viral polymerase inhibitor 7-deaza-2-CMA compound exhibited anti-ZIKV activity in Vero cells (EC
50 = 9.6 µM), decreasing viremia and delaying morbidity and mortality of ZIKV-infected AG129 (interferon (IFN)-α/β and IFN-γ receptor knockout) mice treated once a day with 50 mg/kg/day of the drug [
47]. Although 7-deaza-2-CMA inhibitor failed in human clinical trials for chronic hepatitis C treatment, probably due to mitochondrial toxicity [
48], this compound could still be suitable and safe for short-term therapy of acute flaviviral diseases, including WNV [
49], and thus represents one of the most promising candidates for the treatment of flaviviral infections to date. Favipiravir (6-fluoro-3-hydroxy-2-pyrazinecarboxamide), originally known as T-705, is a novel antiviral compound that selectively and potently inhibits the RdRp of flavi-, orthomyxo-, alpha-, filo-, bunya-, arena-, calici-, noro-viruses, and other RNA viruses [
50]. Although the exact mechanism of the interaction of favipiravir with the RdRp molecule has not been fully elucidated, it is hypothesized that favipiravir may be misincorporated in a nascent viral RNA, or it may act by binding to conserved polymerase domains, thus preventing incorporation of nucleotides for viral RNA replication and transcription. Our group and others have demonstrated the ability of favipiravir to inhibit several geographically and temporally different ZIKV strains in in vitro assays [
51,
52]. The genetic barrier of resistance against favipiravir has been shown to be high for several viruses. In clinical trials of healthy volunteers and in influenza virus-infected patients, favipiravir has been well tolerated. However, caution is needed because of the teratogenic risks of this molecule [
53].
A pyrimidine synthesis inhibitor such as NITD008 exhibited antiviral activity against ZIKV in vitro (EC
50 = 0.28–0.95 µM) and in A129 mice treated with 50 mg/kg/day of the drug. Also, 50% of NITD008-treated mice survived without developing any neurological signs [
54]. However, due to toxic effects in pre-clinical evaluation, this drug was dismissed [
55].
The prodrug sofosbuvir is a nucleoside analog that is a RdRp inhibitor commercially available for the treatment of chronic HCV infection. The sofosbuvir phosphoramidate prodrug is converted to its triphosphate analog in the cellular environment to become active. The active metabolite, 2’-fluoro-2-C-methyl-UTP, binds to the active site of NS5 [
56] and was shown to inhibit ZIKV infection and replication in human hepatocellular carcinoma (Huh-7) cells, human placental choriocarcinoma (Jar) cells, and SH-Sy5y neuroblastoma cells with EC
50 values in the range of 0.4–5 μM as well as in human fetal-derived hindbrain and cerebral cortex neuronal stem cells (NSCs) with EC
50 of about 32 μM [
56,
57,
58]. However, it did not exhibit anti-ZIKV inhibitory activity in Vero cells. Therefore, the inhibitory activity of sofosbuvir varied among different cell types [
58]. Interestingly, sequence analysis of ZIKV isolated from infected cells treated with sofosbuvir showed higher frequency of mutations compared with untreated cells [
58] suggesting that, besides its inhibitory effect, the drug also increases the incorporation of mutations in the viral genome, increasing error-prone replication [
59]. In wild-type (WT) C57BL/6 mice treated with an anti-IFN-α receptor 1 (IFN-αR1) blocking antibody [
60], therapeutic oral administration of sofosbuvir with a physiologically relevant dose of 33 mg/kg/day for seven days, protected the animals against ZIKV-induced weight loss and death in 50% of the treated mice. However, higher concentrations of the drug were not effective and resulted in toxicity [
57]. Importantly, in clinical phase II and III studies, sofosbuvir was found to be clinically safe and efficacious in patients treated for HCV infection [
61]. Sofosbuvir is a class B drug and can be used in men and non-pregnant women to prevent tissue damage.
BCX4430, an adenosine nucleoside analog, is a selective inhibitor of the viral RdRp. This compound has already been shown to have broad-spectrum activity against a wide range of RNA viruses including WNV, YFV, Marburg and Ebola viruses [
62,
63]. BCX4430 acts on NS5 polymerase, promoting chain termination of viral RNA synthesis [
62]. It was found to inhibit ZIKV replication in Vero cells with EC
50 values in the range of 3.8–11.7 µg/ml with selective index values of 5.5 and 11.6 depending on the viral strain [
64]. In addition, treatment of AG129 mice infected with ZIKV (Malaysian strain, P-6-740) twice daily (BID) with an intramuscular dose of 300 mg/kg of BCX4430 significantly reduced viremia and protected 87.5% of treated mice from mortality. This protection was also observed when treatment was initiated 24 h after infection [
64]. This compound is currently in Phase 1 clinical trials to evaluate its safety, tolerability and pharmacokinetics in 94 healthy subjects aged 18–50 years. The results of this study have not been disclosed yet.
The methyltransferase catalytic domain of the NS5 is responsible for capping the 5’ end of viral genomic RNA. The metal binding pocket of RdRp and SAH/SAM (S-adenosyl-L-homocysteine/S-adenosyl-L-methionine) binding pocket of MTase are classically used in drug screening. Our group has performed virtual screening with a library of 28,341 compounds using a hydrophobic site close to the SAM pocket and identified 10 candidates showing decisive contacts with the MTase. Plaque reduction assay revealed EC
50 values in the range of 4.8–17.6 µM. However, their in vivo efficacy has not yet been evaluated [
65]. Hercik et al. reported the crystal structure of the ZIKV methyltransferase in complex with the pan-methyltransferase inhibitor sinefungin, an adenosine derivative, originally isolated from
Streptomyces griseoleus as a potential antifungal drug [
66]. This compound competes with SAM, the natural substrate of numerous MTases [
67]. Sinefungin attaches to GTP and GDP analogs and might be useful in enhancing their affinity toward the enzyme for better selectivity and inhibition of ZIKV replication [
68]. However, when this drug was used as anti-parasitic agent in studies conducted in dogs and goats, it was toxic, which has hampered its clinical use [
69].
NS2B-NS3 trypsin-like serine protease plays a key role in virus replication by contributing to viral polyprotein processing. Studies done by Lee et al. identified 10 compounds with inhibitory activity (EC
50 < 50 µM) and binding activity (K
D of ∼5–10 μM) against the Zika NS2B-NS3 protease from testing 71 HCV NS3/NS4A inhibitors that were initially discovered by high-throughput screening of ∼40,000 compounds [
70]. Many natural products such as polyphenols, which have antiviral activity against different viruses (influenza virus, DENV, coronaviruses, HIV-1, hepatitis B virus, etc.) [
71,
72,
73], have been tested against NS2B-NS3 protease, and some of them have been found to inhibit ZIKV protease activity. Lim et al. evaluated 22 polyphenol compounds and found that seven had an EC
50 ranging from 22 to 113 μM [
74]. Roy et al. identified five flavonoids (myricetin, quercetin, luteolin, isorhamnetin, apigenin) and one natural phenol (curcumin) which were shown to inhibit Zika NS2B-NS3 protease by binding to a pocket on the back of the active site and allosterically affect the structure-activity property of Zika NS2B-NS3 protease. The EC
50 from the flavonoids ranged between 1.3 and 56.3 μM whereas the curcumin EC
50 was 3.5 μM [
75]. Another group screened a total of 2816 Food and Drug Administration (FDA)-approved drugs and investigational drugs and found that 23 compounds had EC
50 below 15 μM. However, 12 of those compounds were considered Pan-Assay Interference Compounds (PAINS). Three (temoporfin, niclosamide, and nitazoxanide) of the 12 remaining compounds had an EC
50 value ranging from 1.1 to 15.9 μM. Temoporfin displayed a very low EC
50 value (nanomolar range) and, when tested in a lethal mouse model, was able to inhibit viremia and protect 83% of infected mice. In addition, mice that survived did not present any signs of neurological disorder [
76]. Similarly, a study done by Yuan et al. using an in-silico structure-based approach to rapidly screen a large chemical library of 8277 compounds, successfully identified eight clinically approved drugs with inhibitory activity on the ZIKV NS2B-NS3 protease [
77]. In addition, the authors further validated the anti-ZIKV activity of novobiocin, an aminocoumarin antibiotic, using in vitro antiviral assays and in an immunodeficient mouse model. In vitro, novobiocin had an EC
50 value of 24.82 μM and treatment of mice with 100 mg/kg of the drug BID from day 1 to 13 post-infection, significantly (
p < 0.05) increased survival rate (100% vs. 0%), decreased mean blood and tissue viral loads, and produced less severe histopathological changes than untreated controls [
77].
NS3 helicases display adenosine triphosphatase (ATPase) and RNA triphosphatase (RTPase) activities. NS3 inhibitors can be used to impede ZIKV infection. Suramin, an anti-parasitic drug used to treat trypanosomal human sleeping sickness, is available for prophylactic and therapeutic use in children. This drug was also shown to inhibit multiple DNA and RNA viruses including DENV, herpes simplex virus type 1, cytomegaloviruses human hepatitis B, hepatitis D, hepatitis C, bunyaviruses, enterovirus 71, and others [
78,
79,
80,
81]. Suramin was also able to inhibit enterovirus 71 by neutralizing virus particles prior to attachment and chikungunya virus in mice [
82,
83,
84]. Albulescu et al. showed that suramin has anti-ZIKV activity, with an EC
50 of 39.8 μM, by interfering with viral attachment and the release of infectious progeny from ZIKV-infected cells [
85]. When treatment was initiated post-entry, viral RNA synthesis was unaffected but both the release of genomes and the infectivity of ZIKV were reduced, suggesting that this drug also affects virus biogenesis probably by interfering with glycosylation and maturation of ZIKV during traffic through the secretory pathway [
85].
3.2. Host-Targeting Antivirals
Targeting host cell processes provides an attractive broad-spectrum strategy because they are often employed by multiple viruses and, in addition, they are less prone to develop drug resistance [
86]. These host-acting inhibitors can be directed to any molecule or pathway implicated in the different steps of the viral life cycle, from binding, entry and fusion, to the formation of the replication complex, viral maturation, and egress.
In order to maintain proper replication, viruses rely on the supply of nucleosides from the host cells. Ribavirin is a guanosine analogue that has broad-spectrum activity against several RNA and DNA viruses [
87,
88]. Different mechanisms have been proposed to explain ribavirin’s antiviral properties including indirect mechanisms such as inosine monophosphate dehydrogenase inhibition (IMPDH) and immunomodulatory effects as well as direct mechanisms such as interference with RNA capping, polymerase inhibition, and lethal mutagenesis [
87]. This antiviral is usually employed in combination therapies to treat chronic HCV infections. Our group and others demonstrated the inhibitory activity of ribavirin against ZIKV strains of different geographic origins in several cell lines such as Vero cells, human neural progenitor cells (hNPCs), human dermal fibroblasts (HDFs), and human lung adenocarcinoma cells (A549) [
51,
89]. Ribavirin was also shown to suppress viremia in ZIKV-infected STAT-1-deficient mice, which lack type I IFN signaling and are thus highly sensitive to ZIKV infection with a lethal outcome [
89]. Recent studies have shown that merimepodib (MMPD or VX-497) and mycophenolic acid (MPA), two IMPDH inhibitors, also inhibit ZIKV-RNA replication in different cell types, including Huh-7 cells, human cervical placental cells, and neural stem and primary amniotic cells [
90,
91,
92]. Azathioprine, another inhibitor of the purine synthesis and immunosuppressive compound, was shown to abolish ZIKV replication in HeLa (cervical cancer cells) and JEG3 (human choriocarcinoma cell line) cells; however, its use in pregnant women is not recommended [
92].
Similar to the purine synthesis inhibitors, compounds inhibiting the synthesis of pyrimidines have also been shown to affect ZIKV replication. Pascoalino et al. screened a library of 725 compounds from a collection of chemically diverse FDA-approved drugs with known and unknown mechanisms of action. The entire library was screened at 20 µM against ZIKV infecting Huh7 cells. The authors identified the 6-azauridine (EC
50 = 2.3 μM) and another pyrimidine biosynthesis inhibitor, 5-fluorouracil (EC
50 = 14.3 μM), which inhibit thymidylate synthase (the enzyme that catalyzes the final step of thymidine biosynthesis) [
93]. These compounds are classified in pregnancy category D by the FDA [
92] and thus have human fetal risk which is not unexpected because they deplete the cellular pool of nucleotides, affecting proper development of the fetus. In addition, the authors have identified lovastatin, a 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA) reductase inhibitor, whose activity against ZIKV was confirmed through a dose-response assay (EC
50 = 20.7 μM) [
93]. The antiflaviviral activity of lovastatin was also demonstrated against HCV and DENV [
94,
95]. In addition, Sarkey et al., demonstrated that a short-term parenteral course of high-dose lovastatin in mice markedly attenuated nervous system injury and, thus, could be eventually used in inflammatory peripheral nerve diseases such as Guillain–Barré syndrome (GBS), a consequence of ZIKV infection [
96].
Retallack et al. performed the screening of 2177 clinically approved compounds by monitoring inhibition of virus-dependent cell death at 72 h post-infection (hpi) in Vero cells. The screening identified several compounds that rescued cell viability, including antibiotics and inhibitors of nucleotide and protein synthesis with many that showed toxicity in Vero or U87 cells or were contraindicated during pregnancy. However, they identified the macrolide antibiotic azithromycin, which rescued ZIKV-induced cytopathic effect in glial cells with low toxicity and reduced ZIKV infection of U87 cells at an EC
50 of 2 to 3 µM [
97]. This drug is generally safe during pregnancy [
98] and was suggested as a potential option to prevent GBS and microcephaly [
97].
Chloroquine, an anti-inflammatory FDA-approved 4-aminoquinoline, is an old antimalarial drug which can be prescribed to pregnant women at risk of exposure to
Plasmodium parasites [
99,
100]. This drug has shown antiviral activity against several viruses, through the inhibition of pH-dependent steps of viral replication, including anti-ZIKV activity in Vero cells, human brain microvascular endothelial cells (hBMECs), and human neural stem cells (NSCs) with EC
50 values of 9.82, 14.2, and 12.36 μM, respectively. In addition, chloroquine was able to partially reverse morphological changes induced by ZIKV in mouse neurospheres [
101]. In vitro, chloroquine reduces the number of ZIKV-infected cells, and inhibits virus production and cell death promoted by ZIKV infection without cytotoxic effects [
101].
Saliphenylhalamide (SaliPhe), a viral entry blocker which targets vacuolar ATPase and prevents the acidification of endosomes, also inhibits ZIKV replication in human retinal pigment epithelial (RPE) cells, which are natural targets for ZIKV infection [
102] with an EC
50 of 1 μM [
103]. Similarly, obatoclax mesylate, also known as GX15-070, is an experimental drug for the treatment of different types of cancer. This drug is an inhibitor of the Bcl-2 family of proteins that targets cellular Mcl-1 and inhibits endocytosis, thus inducing apoptosis. Obatoclax mesylate displays an EC
50 of 0.3 μM against ZIKV. Niclosamide is an FDA-approved drug, formerly designated in pregnancy category B, that has been used for more than 50 years showing acceptable safety. It has been broadly used in the treatment of intestinal helminthiasis. This drug blocks the acidification of endosomes, using a mechanism that has not yet been fully elucidated [
104]. In glioblastoma SNB-19 cells, the EC
50 against ZIKV was 0.37 μM based on the measurement of intracellular viral RNA [
105]. PHA-690509, is an investigational cyclin-dependent kinase inhibitor which inhibited ZIKV infection with an EC
50 value of 1.72 μM [
105]. This drug was detected by Xu et al. as part of a large drug repurposing screen for ZIKV. The authors measured ZIKV-induced caspase-3 activity and cell viability from over 6000 approved drugs and drug candidate compounds using human neural cells. This study led to the identification of small molecules that either protect against cell death in multiple neural cell types or inhibit ZIKV replication. Among them, seliciclib (a purine analog) and RGB-286147 inhibited ZIKV infection at sub-micromolar concentrations [
105].
Studies from Costa et al. showed that ZIKV has tropism for the central nervous system (CNS) and replicates preferentially in neurons, inducing neurodegeneration, neuroinflammation, and ophthalmologic disorders [
106]. Neurodegeneration in ZIKV disease possibly occurs due to the excitotoxicity of glutamate. FDA-approved
N-methyl-D-aspartate receptor (NMDAR) antagonistic drugs to treat Alzheimer disease such as memantine, MK-801, agmatine, and ifenprodil were found to prevent neuronal cell death caused by ZIKV under in vitro conditions without reducing viral titers [
106]. Blocking hyperactivation of NMDAR would therefore reduce rates of Zika virus-induced cell death and help ameliorate neuronal symptoms during infection. Costa et al. showed that memantine was very effective at preventing ZIKV-induced neuronal cell death and neurodegeneration in IFN-α/βR
−/−mice [
106]. Importantly, memantine is also listed in pregnancy category B drugs by the FDA. Therefore, it could be used safely to reduce neurological complications associated with ZIKV infection.