Development of Small-Molecule Inhibitors Against Zika Virus Infection

In recent years, the outbreak of infectious disease caused by Zika virus (ZIKV) has posed a major threat to global public health, calling for the development of therapeutics to treat ZIKV disease. Here, we have described the different stages of the ZIKV life cycle and summarized the latest progress in the development of small-molecule inhibitors against ZIKV infection. We have also discussed some general strategies for the discovery of small-molecule ZIKV inhibitors.


INTRODUCTION
Zika virus (ZIKV) is an arthropod-borne virus (arbovirus) belonging to the family Flaviviridae and genus Flavivirus. As a single-stranded positive RNA virus, the genome of ZIKV is approximately 10 kb and encodes three structural proteins and seven non-structural proteins (Wang et al., 2016). In 1947, ZIKV was discovered and isolated from a sentinel Rhesus monkey in the Zika Forest of Uganda (Dick et al., 1952). However, it was only in 2015 that the first outbreak of ZIKV-caused diseases was reported in Brazil (Petersen et al., 2016) with more than one million cases. Since then, it rapidly spread to 84 countries around world, particularly in South America, rendering ZIKV a public health threat (Han and Mesplede, 2018; Figure 1). Mild symptoms include fever, rash, headache, and joint pain, but the major concern involves the potential for severe neurological disorders, such as microcephaly, neurological disorders in newborns, meningo-encephalitis, Guillain-Barré syndrome, myelitis, and ocular abnormalities (Barros et al., 2018). Until now, neither a specific antiviral drug nor a vaccine has been developed to prevent or cure ZIKV infection. However, several well-characterized drug targets encoded by the virus, or presented in host cells, may help us prevent or treat ZIKV infection. In this review, we focus on current progress on the research and development of small-molecule ZIKV inhibitors, either viral or host cell inhibitors, targeting different stages of the ZIKV life cycle. Such data are essential to the design of drugs and drug delivery modalities against ZIKV and related viruses.

ZIKV LIFE CYCLE AND POTENTIAL TARGETS FOR THE DEVELOPMENT OF SMALL-MOLECULE INHIBITORS AGAINST ZIKV INFECTION
The life cycle of ZIKV can be divided into four stages, including virus entry, genome replication, virus assembly, and release. Mature ZIKV particles first adhere to host cells by interacting with FIGURE 1 | Summary of ZIKV morbidity statistics with country-and quarter-level panel data. The deeper the color, the higher the number of infected people. specific receptors on host cells, such as DC-SIGN, AXL, Tyro, and TIM-1 (Musso and Gubler, 2016;Nowakowski et al., 2016;Meertens et al., 2017). Several proteins, including DC-SIGN and TIM as well as some TAM proteins that belong to the phosphatidylserine receptor family, have been reported to act as receptors for entry of dengue virus (DENV) (Lozach et al., 2005;Meertens et al., 2012;Perera-Lecoin et al., 2013). To determine whether these receptors are also involved in ZIKV entry, a series of transfected HEK293T cells expressing DC-SIGN, TIM-1, or a TAM family member (AXL or Tyro3) could be infected by ZIKV at varying degrees (Hamel et al., 2015). DC-SIGN consists of group II (calcium-dependent with single carbohydrate recognition domain) transmembrane C-type lectins that can interact through their carbohydrate recognition domains to bind carbohydrates to viral protein E (Zelensky and Gready, 2005;Cruz-Oliveira et al., 2015). DC-SIGN also plays an important role in flavivirus binding and the infection of myeloid cells (Navarro-Sanchez et al., 2003) as it mediates attachment of viral particles on the cell surface and facilitates their interaction with primary receptors on the host cell (Chen et al., 1997;Germi et al., 2002). Tyro3 and AXL belong to the TAM family, a group of three receptor protein tryrosine kinases that mediate the clearance of apoptotic cells (Lemke and Rothlin, 2008). AXL is expressed in astrocytes and microglia in the human brain development and mediates ZIKV infection of glial cells (Nowakowski et al., 2016;Meertens et al., 2017). AXL consisting of two different Gas6binding epitopes, including the N-terminal Ig-like domain, and a second Ig domain exists in the dimeric form. Gas6, which is the ligand of AXL, connects ZIKV to glial cells. TIM-1, which is abundant on Th-2 T cells, mucosal epithelial cells, and mast cells, mediates the attachment of ZIKV particles on the cell surface to facilitate their interaction with AXL as well as the subsequent infection (Hamel et al., 2015). The availability of different entry receptors is likely to provide an evolutionary advantage for the virus, and, as a result, the virus is able to infect a wide range of human host cells.
After binding with host cells, ZIKV is internalized by clathrin-mediated endocytosis and traffics to Rab5 + endosomes (Wang X. et al., 2017;Mottin et al., 2018). In the process of entering the host cell, AXL kinase activity is activated by the ZIKV/Gas6 complex, which downregulates interferon signaling and promotes infection. Then, the endosome membrane and virus envelope (E) are fused under the acidic environment of the endosome. The viral genomic RNA is then released into the cytoplasm (Wang X. et al., 2017;Mottin et al., 2018). In the process of virus entry, some inhibitors can block viral attachment, endocytosis, and fusion. The proteins E on ZIKV and the DC-SIGN, AXL, Tyro, and TIM-1 entry/adhesion factors on the host cell are involved in viral attachment, endocytosis, fusion, and entry (Hasan et al., 2017;Heinz and Stiasny, 2017;Shi and Gao, 2017). They all therefore serve as targets for the development of small molecule inhibitors. After virus entry, the genome of ZIKV is translated and cleaved into three structural proteins, including Capsid (C), Precursor of the FIGURE 2 | Zika virus infectious life cycle: Host cell membrane receptors bind the E protein of the mature Zika virion, triggering endocytosis. The acidic environment of the endosome induces fusion of the host endosome membrane with the viral envelope and the release of the RNA genome. The RNA is translated into a polyprotein complex, which is cleaved by the host and viral proteases in the ER lumen and cytoplasm, respectively. Following translation, a replication complex is assembled and associated with virus induced membranes where viral replication takes place. The methylated (+) ssRNA, C, E, and prM proteins assemble to form immature virions in the endoplasmic reticulum (ER). The immature virions bud out of the ER into the Golgi apparatus, and they then mature in the trans-Golgi network that are released by exocytosis. In the orange boxes are the names of the compounds that can inhibit the marked steps of the virus lifecycle and that are evaluated in animal models or clinical trials. membrane protein (prM)/membrane protein (M), Envelope (E), as well as seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is related to flavivirus replication and virion maturation. The NS2B protein activates the active region of the NS3 protein and forms an NS2B-NS3 complex with the NS3 protein to exert proteolytic enzyme activity. The NS4A and NS4B proteins comprise the endoplasmic reticulum (ER)-associated replication complex. The NS5 protein contains the C-terminal RNA-dependent RNA polymerase domain and the N-terminal methyltransferase domain, which cooperate during the initiation and extension of RNA synthesis. In addition, the NS5 protein is the largest NS protein in molecular weight and the most highly conserved. Then RNA is replicated with the actions of NS1, NS2B-NS3 proteinase and NS3 helicase, NS5 methyltransferase, and NS5RdRp. Viruses encode their own essential proteases in the viral replication process, which can serve as targets for therapeutic intervention. The methylated (+) ssRNA, C, E, and prM proteins are assembled to form immature virions in the ER. Then, the ER vesicles transport the virus particles to Golgi apparatus, and the virus particles undergo surface polysaccharide modification, prM-E protein trimer rearrangement, and Furin protease cleavage prM while mature virus particles with a smooth surface are produced, finally leaving the host cell by exocytosis as mature virus (Figure 2; Wang X. et al., 2017;Mottin et al., 2018). Some small molecule inhibitors that target the C protein or inhibit viral capsid formation are able to affect viral assembly and release. Meanwhile, correct expression and processing of nascent proteins in host cells are essential for efficient viral replication. Several host proteins, such as ER membrane complex, α-glucosidase, cyclophilin, and proteasome elements, are responsible for monitoring proper protein synthesis, folding, and degradation. Impairment of these functions results in reduced viral assembly and budding. Therefore, these host proteins may also serve as targets for the development of small molecule ZIKV inhibitors.
Most antiviral drugs are small-molecule inhibitors that target different stages of the viral life cycle by interacting with virus or host proteins critical for virus replication (De Clercq and Li, 2016). For example, inhibiting AXL function can protect cells from infection and, thus, may be a potential target for the production of entry inhibitors. However, destroying AXL function may also have many adverse consequences (Nowakowski et al., 2016). In addition, the proteases crucial for ZIKV replication are potential targets for developing ZIKV replication inhibitors. Therefore, more effective and appropriate targets need to be developed by researchers.

ZIKV Inhibitors Targeting Viral E Protein
The structure of ZIKV envelope protein (E protein) is similar to that of other flaviviruses, and it has three characteristic domains: a central β-barrel-shaped domain I, a Slender fingerlike domain II, and a C-terminal immunoglobulin-like domain III (Dai et al., 2016). The recognition and binding of ZIKV E proteins to host cell receptors mark the beginning of ZIKV infection; therefore, some inhibitors designed for envelope proteins can effectively inhibit virus infection (Byrd et al., 2013).
Small molecule inhibitors that specifically target the ZIKV E protein have been reported. Peptide Z2 ( Table 1), derived from the stem region of the ZIKV E protein, inhibits vertical transmission of ZIKV in pregnant C57BL/6 mice and protects type I or type I/II interferon receptor-deficient mice against lethal ZIKV challenge (Yu et al., 2017). Peptide Z2 can interact with viral E proteins to form a membrane pore and disrupt the integrity of the viral membrane (Yu et al., 2017). ZINC33683341 [ Figure 3(1); Fernando et al., 2016], which can bind with the ZIKV E protein, is preferential when compared with glycan and can block the formation of glycoside bonds between ZIKV and host Vero cells at the concentration of 100 µM ( Table 2; Fernando et al., 2016).
Some small molecule inhibitors that non-specifically target the ZIKV E protein were also active against other medically relevant viruses that use a similar route of entry. P5, a peptide extracted from the stem of Japanese encephalitis virus E protein, can inhibit ZIKV entry into host cells by changing the conformation of the E protein under low pH. The hydrophobicity of the last seven amino acid residues is also considered to be the key to the binding of the viral membrane (Table 1; Chen et al., 2017). In vivo experiments in mice highly sensitive to ZIKV showed that P5 can inhibit spermatic tubule disorder and reproductive epithelial cell degeneration while also alleviating the circulatory constriction of blood vessels (Chen et al., 2017).
The molecular tweezer CLR01, which has potent inhibition activity to envelope viruses, can inhibit ZIKV strains in Vero E6 cells [ Figure 3(2) and Table 2] by destroying the intact membrane structure that is enriched with high levels of sphingolipid and cholesterol (Bavari et al., 2002;Chazal and Gerlier, 2003;Brugger et al., 2006;Lorizate et al., 2013;Rocker et al., 2018). In addition, it can inhibit ZIKV infection in semen, urine, saliva, cerebrospinal fluid, and other body fluids, but lose activity in serum (Rocker et al., 2018). Some studies have attributed this effect to the relatively high protein content in serum (Rocker et al., 2018). Baicalin [ Figure 3(3)], which has high affinity to the virus E protein and low toxicity to cells, can inhibit ZIKV from entering cells ( Table 2; Oo et al., 2019). (-)-Epigallocatechin gallate (EGCG), a polyphenol from green tea, was shown to inhibit many viruses [ Figure 3(4) and Table 2; Isaacs et al., 2008;Nance et al., 2009;Calland et al., 2012]. Accordingly, EGCG can bind to the ZIKV E protein to block ZIKV entry into host cells (Song et al., 2005). However, EGCG contains the catechol group that may non-specifically inhibit many different targets (Mottin et al., 2018). Curcumin can inhibit ZIKV infection in a dosedependent manner [ Figure 3(5)]. It is not only a replication inhibitor of ZIKV, but also prevents the viral E protein from  binding to the cell surface (Mounce et al., 2017;Roy et al., 2017). In Vero cells, the IC 50 and CC 50 value of curcumin inhibiting ZIKV is 1.90 and 11.6 µM, respectively ( Table 2; Mounce et al., 2017). Nanchangmycin [ Figure 3(6)], produced by Streptomyces nanchang fermentation, can inhibit gram-positive bacteria and has insecticidal and antibacterial activities against poultry in vitro (Rausch et al., 2017). For Zika virus, Nanchangmycin can inhibit ZIKV infection by blocking clathrin-mediated endocytosis with IC 50 s between 0.1 and 0.4 µM, and it has low toxicity in this range ( Table 2) in human U2OS cells, human brain microvascular endothelial cells (HBMEC), and human Jeg-3 cells, respectively (Rausch et al., 2017).

ZIKV Inhibitors Targeting Endosome
Endosomes provide a transport route for ZIKV to enter host cells. Ev37 (Table 1), an endosomal scorpion peptide inhibitor, can effectively inhibit ZIKV infection at a non-cytotoxic concentration (Li et al., 2019). Ev37 is a broad-spectrum and specific antiviral peptide, which can alkalize the pH value of endosomes, inhibit the release of a viral genome, and prevent it from entering the cytoplasm, thus blocking ZIKV infection (Li et al., 2019). In Huh-7 cells, Ev37 can reduce 87% of ZIKV infection at a concentration of 10 µM (Li et al., 2019). Chloroquine (Li et al., 2017a), Suramin (Albulescu et al., 2017), and 25-hydroxycholesterol [ Figure 3(7-9) and Table 2; Li et al., 2017a) demonstrated their ability to inhibit ZIKV internalization in vitro. Niclosamide is an FDA-approved drug broadly used in the treatment of intestinal helminthiasis [ Figure 3(10) and Table 2]. It can prevent endosomal acidification, but the mechanism is not fully elucidated (Fonseca et al., 2012;Jurgeit et al., 2012).

ZIKV Inhibitors Targeting AXL
AXL is a tyrosine kinase receptor (TKR), which can mediate viral attachment to host cells. Therefore, it is necessary to inhibit primary cells with high AXL content (Nowakowski et al., 2016). Cabozantinib and BMS-777607 are two kinase inhibitors that inhibit AXL [ Figure 3(11,12); Rausch et al., 2017]. In human U2OS cells, their IC 50 values are 0.2 and 0.6 µM, respectively, and the CC 50 values are greater than 10 µM ( Table 2; Rausch et al., 2017). However, the experiments showed that AXL inhibitors were effective only on AXL-rich cells (Rausch et al., 2017), indicating that the effect is cell-type specific.
Although several studies proclaimed that AXL is a receptor for ZIKV entry in vitro, a few reports showed the opposite results. The genetic ablation of AXL has no significant effect on ZIKV entry or ZIKV-mediated cell death in human-induced pluripotent stem cell (iPSC)-derived NPCs or cerebral organoids (Nyboe Andersen et al., 2017;Rausch et al., 2017) reported that Jeg-3 cells that show no detectable AXL expression were highly permissive to ZIKV infection, suggesting that AXL may not be essential for ZIKV infection. This hypothesis is corroborated by an in vivo study (Wang Z. Y. et al., 2017). Notably, the AXL receptor supports neural stem cell survival, proliferation and neurogenesis (Ji et al., 2014), and signaling; the AXL also regulates blood-brain barrier (BBB) integrity in the context of viral infections (Miner et al., 2015). Therefore, while blocking AXL may protect against ZIKV infecting or viral replication, perturbation of AXL function may also have multiple adverse consequences. Therefore, the use of the AXL receptor as an idea target for the inhibition of Zika virus infection remains to be confirmed. Efforts to elucidate the molecular mechanism for ZIKV infection, through both targeted TAM receptor knockout studies and unbiased screening for other binding factors that render cells resistant to ZIKV, will lead to the identification of new targets for development of anti-ZIKV therapeutics.

ZIKV Replication Inhibitors
ZIKV Inhibitors Targeting NS2B-NS3 Protease NS2B-NS3 protease of Zika virus plays an essential role in ZIKV replication and maturation. NS2B-NS3 processes the viral nonstructural proteins from the viral polyprotein into individual proteins. NS2B-NS3 is a serine protease that consists of the N-terminal domain of NS3 and a short cofactor from the hydrophilic core sequence of NS2B. Like the NS4A cofactor of the HCV protease, Flavivirus NS3 is inactive without the NS2B co-factor (Erbel et al., 2006).
Three different ZIKV NS2B-NS3 protease (ZIKVpro) constructs have been proposed. First, a covalent G 4 SG 4 linker peptide between NS2B and NS3 (gZiPro) construct was adopted based on previous West Nile and DENV protease constructs (Lei et al., 2016). The other two constructs include one bivalent protease consisting of two separate polypeptide NS2B and NS3 (bZiPro)  and one with its own NS2B C-terminal peptide (TGKR) binding NS2B to NS3 (eZiPro) . Remarkably, the single-chain enzyme gZiPro with an artificial linker that is commonly applied for the constructs of other flaviviruses has been widely used for screening inhibitors. Aprotinin, a 58 amino acid bovine trypsin inhibitor, inhibits ZIKV NS2B-NS3 protease with an IC 50 of 70 nM by blocking the interactions of NS3 and NS2B, as predicted by molecular modeling studies ( Table 1; Shiryaev et al., 2017). By using structure-based virtual screening, novobiocin and lopinavir-ritonavir can inhibit ZIKV NS2B-NS3 protease activity by using molecular docking analysis [ Figure 3(13-15); Yuan et al., 2017]. Novobiocin, an aminocoumarin antibiotic, inhibited protease activity by highly stable binding with ZIKV NS2B-NS3 protease to diminish its catalytic efficiency (Kirby et al., 1956;Yuan et al., 2017). It can inhibit ZIKV replication with an IC 50 of 26.12 ± 0.33 µg/ml and CC 50 of 850.50 µg/ml in Vero cells and an IC 50 of 38.14 ± 4.53 µg/ml and CC 50 of 1103.18 µg/ml in Huh-7 cells ( Table 3; Yuan et al., 2017). Lopinavir-ritonavir can inhibit protease activity of ZIKV replication with an IC 50 of 4.78 ± 0.41 µg/ml and CC 50 of 30.00 µg/ml in Vero cells and an IC 50 of 3.31 ± 0.36 µg/ml CC 50 of 32.12 µg/ml in Huh-7 cells ( Table 3; Yuan et al., 2017).
Temoporfin was tested in a viremia mouse model and a lethal mouse model, and it was able to inhibit viremia and protect 83% of the mice; the mice that survived did not present any signs of neurological disorder (Li et al., 2017d). These compounds inhibit the interaction between NS3 and the NS2B N-terminal fragment. By using an e-pharmacophore-based virtual screening assay, BAS 19192837 was chosen as a potent Zika NS2B-NS3 protein inhibitor [Figure 3(32)]. However, the experimental data of the IC 50 about this inhibitor was not shown (Table 3; Rohini et al., 2019).
Berberine, an FDA-approved drug against DENV, has shown high binding affinity of 5.8 kcal/mol, and it binds around the active site of the receptor [Figure 3(33) and Table 3; Sahoo et al., 2016]. Niclosamide, an FDA-approved category B anthelmintic drug for treating worm infections in both humans and domestic livestock, inhibited all three strains of ZIKV, which was measured by intracellular ZIKV RNA levels with IC 50 values of 1.72 µM in SNB-19 cells (Xu et al., 2016). PHA-690509, an investigational compound that functions as a cyclin-dependent kinase inhibitor (CDKi), inhibited three stains with IC 50 values of 0.37 µM as measured by intracellular ZIKV RNA levels in SNB-19 cells (Xu et al., 2016). According to experimental results, the mechanism of these two compounds occurs at post-entry stage, likely at the viral RNA replication step (Xu et al., 2016). Suramin [ Figure 3(8)], an approved polyanion antiparasitic drug, can be a potential inhibitor of Zika virus complex NS2B/NS3 proteinase with IC 50 of 47 µM ( Table 2; Coronado et al., 2018). Computational analysis showed that suramin suppressed NS2B/NS3 proteinase activity by blocking catalytical Ser135 residue and interacting with the catalytical histidine residue (Coronado et al., 2018). Erythrosin B [Figure 3(34)], a pregnancy category B food additive, inhibited ZIKV replication by targeting NS2B-NS3 proteases with an IC 50 of 0.62 ± 0.12 µM in A549 cells (Table 3) via a non-competitive mechanism by enzymatic kinetic experiments . Erythrosin B can also inhibit ZIKV RNA synthesis and protein expression in ZIKV-relevant neural progenitor and human placental cells .

STRATEGIES FOR DEVELOPING SMALL-MOLECULE ZIKA INHIBITORS
The development of anti-ZIKV drugs requires effective strategies. For example, using existing drug libraries to screen drug molecules that inhibit new targets is an effective method to develop new drugs Kumar et al., 2018). In silicon-based drug modeling, it is another economical and useful strategy to identify candidate drugs in a short amount of time (Pal et al., 2017;Kumar et al., 2018). In addition, virtual screening and electronic pharmacokinetic modeling also facilitate the discovery of effective drug molecules (Rohini et al., 2019). First, the virtual screening method based on electronic pharmacodynamics was adopted to screen effective inhibitors of ZIKV NS2B-NS3 protein from the ASINE database (including 467,802 molecules) (Rohini et al., 2019). Then, the complexes of known NS2B-NS3 protein and its inhibitor were used to establish a five-featured pharmacophore hypothesis, ADDRR, which consists of one hydrogen bond acceptor (A), two hydrogen bond donors (D), and two aromatic rings (R) (Rohini et al., 2019). The pharmacophore model was verified by enrichment analysis before the virtual screening process (Rohini et al., 2019). Active development of screening methods to assess the antiviral activity of compounds is a key step in the discovery of new drugs (Ekins et al., 2016). Virus replication relies on cellular mechanisms, which means that in vitro experiments use host cells for culture and virus replication. Since ZIKV can infect many different cells, multiple cell lines should be used to study ZIKV infection. The screening of effective drugs using multiple cells provides a good framework for drug discovery (Barr et al., 2016). Other strategies include RNA interference, long non-coding RNAs, miRNAs, interfering peptides, and compounds targeting viral RNA (Han and Mesplede, 2018), underexplored building blocks, and elements introducing into medicinal chemistry (Nitsche et al., 2017).

CONCLUSION
As an arthropod-borne single-stranded positive RNA virus, ZIKV utilizes a number of host viral proteins and cellular components to accomplish its replication cycle, including the steps of viral entry, genomic replication, structural and nonstructural protein processing, assembly, and budding of virions. Such actions result in a series of congenital abnormities like Guillain-Barré syndrome in adults, microcephaly in newborns, and fetal demise during pregnancy (Dick et al., 1952), and the viral and host proteins involved in the virus life cycle can serve as targets for development of small-molecule ZIKV inhibitors. For example, the ZIKV E protein is responsible for the binding of the virus to host cell receptors and mediating viral entry into the host cell; therefore, some small molecule inhibitors targeting the ZIKV E protein are effective in inhibiting virus attachment and entry (Byrd et al., 2013;Fernando et al., 2016;Oo et al., 2019). AXL expressed on human glial cells can permit ZIKV binding and entry into the host glial cells (Nowakowski et al., 2016;Meertens et al., 2017) and small molecule compounds targeting AXL may be effective in inhibiting ZIKV infection (Rausch et al., 2017). However, any compounds targeting host proteins may affect their normal functions and cause adverse effects.
Study has shown that, since the stem region of the ZIKV E protein has high sequence similarity to that of other flavivirues, such as DENV and yellow fever virus (YFV), the ZIKV inhibitor targeting this region is also highly effective against DENV and YFV infection (Yu et al., 2017). Therefore, it is essential to develop small molecule compounds with broad flavivirus inhibitory activity. Another important strategy is to develop small molecule ZIKV inhibitors targeting the different steps of ZIKV replication cycle with a synergistic antiviral effect when they are used in combination.
Numerous cases of ZIKV sexual transmission have been reported during recent ZIKV outbreaks, and studies have shown that ZIKV also replicates in human prostate cells (Spencer et al., 2018). However, little is known about what viral protein(s) and host factor(s) are involved in this event. Therefore, it is essential to identify these proteins as targets for development of smallmolecular inhibitors for preventing sexual transmission of ZIKV.
With the increasing understanding of viral protein structure, tremendous progresses have been made in structure-based discovery of inhibitors targeting the structure and non-structure protein of ZIKV, such as the E protein, three NS2B-NS3 proteinase constructs and helicase, NS5 methyltransferase and polymerase. Several series of small-molecule ZIKV inhibitors targeting these proteins have been reported. However, most of them were tested in vitro while only a small percentage of these compounds have been evaluated in animal models in vivo, and very few have advanced into clinical trials. Therefore, further studies should focus on exploiting novel strategies to identify new anti-ZIKV compounds, elucidating their mechanisms of action, improving the efficacy of anti-ZIKV compounds, and evaluating the in vivo efficacy and safety of these compounds in suitable animal models and patients. Further development of small-molecule ZIKV inhibitors with high-efficiency and low toxicity will bring promise for clinic treatment of ZIKV infection and related diseases in the near future.

AUTHOR CONTRIBUTIONS
LW, RL, YG, YL, XD, RX, YZ, and FY drafted the manuscript. TY, SJ, and FY revised and edited the manuscript.