Drugs for the Treatment of Zika Virus Infection

Abstract

Zika virus is an emerging flavivirus that causes the neurodevelopmental congenital Zika syndrome and that has been linked to the neuroinflammatory Guillain–Barré syndrome. The absence of a vaccine or a clinically approved drug to treat the disease combined with the likelihood that another outbreak will occur in the future defines an unmet medical need. Several promising drug candidate molecules have been reported via repurposing studies, high-throughput compound library screening, and de novo design in the short span of a few years. Intense research activity in this area has occurred in response to the World Health Organization declaration of a Public Health Emergency of International Concern on February 1, 2016. In this Perspective, the authors review the emergence of Zika virus, the biology of its replication, targets for therapeutic intervention, target product profile, and current drug development initiatives.

Graphical Abstract

INTRODUCTION. ZIKA VIRUS (ZIKV) EMERGENCE AND PATHOLOGY

The first isolation of ZIKV was reported in 1947 in the Zika forest region of Uganda from an infected rhesus macaque (Figure 1).¹ The following year, the virus was detected in Aedes africanus mosquitos in the same area, suggesting a vector route of transmission for the infection.² It was not until 1952 that the first human cases of ZIKV infection were reported in the United Republic of Tanzania and Uganda: neutralizing antibodies against the virus were detected in the sera of patients in the study.³ ZIKV garnered little attention in the intervening years between these early case reports and the recent explosive outbreaks in Asia and Latin America, largely due to the mild nature of symptoms during infection and the rarity of clinically significant disease.

Figure 1.

Discovery of ZIKV and its emergence as a global health threat.

The first reemergence of the virus occurred on the Yap Islands in Micronesia in 2007, with 49 individuals reported as having clinical symptoms.⁴ Subsequently, an outbreak of ZIKV was identified in French Polynesia in 2013, which manifested in over 8000 clinical cases of disease. Reports were received of symptoms that would become characteristic of the outbreaks to come: birth defects in children born to infected mothers and a correlation of viral infection with the development of the neuroinflammatory Guillain–Barré syndrome (GBS).^5–10 Spreading rapidly throughout southeast Asia, ZIKV emerged in Brazil in late 2013 or early 2014, and continued propagation of the virus through South and Central America proceeded throughout 2014.¹¹ Large numbers of cases of microcephaly and symptoms such as rash, arthralgia, and ocular malformations were reported in 2015 and 2016, with peak infection occurring in the first quarter of 2016.¹² On February 1, 2016, the World Health Organization (WHO) declared a Public Health Emergency of International Concern (PHEIC) as a consequence of the ZIKV outbreak and its associated clinical manifestations. Brazil was particularly affected by this outbreak, with 1950 cases of ZIKV-linked microcephaly alone reported during this period, with over 70% of these cases occurring in the northeastern region of the country.¹³ In the wake of this alarming outbreak, intense scientific activity in the past few years to uncover mechanisms of viral pathogenesis, epidemiology, and therapeutic targets for ZIKV has occurred. This research has broadened our understanding of this neglected disease, with the hope of finding suitable prophylactics and treatments before the virus reemerges once again.

ZIKV is a member of the Flaviviridae, a family of single-stranded, positive-sense enveloped RNA viruses, and is of the genus Flavivirus, which include West Nile virus (WNV), Dengue virus (DENV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV).¹⁴ The virus can be transmitted via Aedes spp. mosquitos through sexual intercourse or by vertical transmission from mother to fetus.^15–21 Numerous studies have investigated possible reservoirs for ZIKV persistence, with evidence supporting the eyes, testes, and whole-blood as sites for harboring virus.^22–29 There are currently no specific treatments that are clinically approved for ZIKV infection, highlighting the urgency for the development of new therapeutics. Current guidelines from the Centers for Disease Control and Prevention (CDC) suggest symptom treatment, rest, hydration, and taking acetaminophen for fever and pain for management of ZIKV infection.

One of the recent great successes in antiviral drug development has been the deployment of effective, curative, pan-viral genotype drug treatment regimens for a Flaviviridae member related to ZIKV, hepatitis C virus (HCV).^30,31 Treatment regimens commonly use a 12-week course of a once-a-day pill combining drugs targeting two or more of the following: the viral polymerase NS5B, the viral RNA binding protein NS5A, or the viral protease NS3. Given the success of targeting these proteins in HCV infections and the presence of homologs in ZIKV, the repurposing of HCV inhibitors for ZIKV is an attractive starting point for new therapies. Examples of drug repurposing will be discussed in later sections of this Perspective.

FLAVIVIRUS VACCINE DEVELOPMENT AND DIAGNOSTIC TESTS

The development of a live, attenuated vaccine against YFV marks one of the landmarks in the effort to contain the spread of flavivirus infection (Table 1).³² The vaccine is considered safe and effective. However, contraindications exist for this vaccine, particularly in individuals that have allergies to eggs, that are immunocompromised, that are pregnant, or that are under 9 months of age.

Table 1.

List of Approved Flavivirus Vaccines and ZIKV Vaccines under Development

virus	vaccine name	immunogen	manufacturer or sponsor
DENV (approved)	CYD-TDV (Dengvaxia)	attenuated virus	Sanofi-Pasteur
YFV (approved)	17D-204 substrain	attenuated virus	Wuhan Institute of Biological Products, China Sanofi Pasteur, France and USA Institut Pasteur, Dakar, Senegal
YFV (approved)	17D-213 substrain	attenuated virus	Federal State Unitary Enterprise of Chumakov Institute, Russia
YFV (approved)	17DD substrain	attenuated virus	Bio-Manguinhos, Brazil
ZIKV (phase I)	GLS-5700	prME (DNA vaccine)	GeneOne Life Science, Inc./Inovio Pharmaceuticals
ZIKV (phase II)	mRNA-1325	prME (mRNA vaccine)	Moderna Therapeutics
ZIKV (phase I)	MV-Zika	prME	Themis Bioscience
ZIKV (phase I)	PIZV or TAK-426	attenuated virus	Takeda
ZIKV (phase I)	VLA1601	attenuated virus	Valneva Austria GmbH
ZIKV (phase I)	VRC-ZKADNA085-00-VP	prME	NIH/NIAID
ZIKV (phase II)	VRC-ZKADNA085-00-VP	prME	NIH/NIAID
ZIKV (phase I)	ZIKV PIV	prME	NIH/NIAID
ZIKV (phase I)	BBV121	attenuated virus	Bharat Biotech

Recently, a chimeric, live, attenuated vaccine against DENV (Dengvaxia from Sanofi-Pasteur³³) was developed using the YFV vaccine strain as backbone with prM and E structural proteins (prME) of DENV from serotypes 1–4 (tetravalent) inserted (Table 1).³⁴ While this represents another major milestone in the effort to prevent and control flavivirus infections, variability in immune response based on viral serotype, age of the patient, and preexisting DENV exposure are persistent problems in the use of this therapeutic agent.^35,36 When Dengvaxia use was approved in the Philippines, the government promoted a campaign to mass-vaccinate children. Some scientists did not support this action, as clinical safety data indicated that there was an increased risk of cytoplasmic leakage syndrome after vaccination for certain individuals, particularly those that did not have prior DENV exposure.³⁷ After performing further safety studies, Sanofi-Pasteur confirmed these concerns and announced on its Web site: “For individuals who have not been previously infected by DENV, vaccination should not be recommended.”^38,39 The lessons learned with this DENV vaccine must be considered for ZIKV vaccine development. DENV vaccines may have direct implications for ZIKV severity, as it is expected that there are conserved antigens and therefore cross-reactivity. In the same way, a ZIKV vaccine could increase the probability of severe dengue fever in individuals who have not been previously exposed to DENV.⁴⁰

During the 2015–2016 ZIKV outbreak, soon after WHO director Dr. Chan declared ZIKV and its associated clinical complications a PHEIC, the scientific community responded with the introduction of 45 vaccine candidates, an unprecedented reaction in such a short time to address the serious unmet clinical need posed by this emerging virus.⁴¹ An effective and safe ZIKV vaccine has the potential to protect over 2 billion people living in endemic areas. The most current report from the WHO Global Observatory on Health R&D⁴² lists eight projects still ongoing for ZIKV vaccine development (we have also listed a ninth ongoing trial in this Perspective) (Table 1). From the WHO-listed projects, six target the prME and two used live attenuated viruses as immunogen. Only two projects have advanced so far to clinical phase II trials to assess efficacy of the vaccine: the mRNA-1325 sponsored by Moderna Therapeutics (NCT03014089) and VRC-ZKADNA085–00-VP sponsored by NIH-NIAID (NCT03110770). The former trial uses an mRNA delivery system targeting ZIKV prME as immunogen; this trial is open but is not yet recruiting. The latter trial uses a DNA delivery system, targets ZIKV prME as immunogen, and is open and recruiting volunteers. Unfortunately, there is very little information about the projects that have already been concluded. There are only three peer-reviewed publications reporting results (final or preliminary) about the vaccine development projects. All three report the safety data and immunogenicity of vaccine candidates.^43–45 One challenge that the vaccine development projects have been facing in clinical phase II (that will likely be the case for a future clinical phase III as well) is the difficulty in recruiting subjects for the trials. The current low number of ZIKV infection cases combined with unpredictable geographical epidemics and the broad spectrum of clinical manifestations (complicating a single end point definition) have the potential to jeopardize the current and future clinical studies with ZIKV.⁴⁶

Sanofi-Pasteur stopped the development of a ZIKV vaccine despite promising preliminary data.⁴⁴ The ZPIV vaccine, initially developed by the Walter Reed Army Institute of Research (WRAIR) with public funding, was licensed to Sanofi-Pasteur to complete testing and bring the product to market. U.S. lawmakers exerted pressure on Sanofi-Pasteur to charge an affordable price for the vaccine, given the initial development funded by taxpayers and target population in endemic regions. The company did not agree with the proposal and decided to stop the project. The Biomedical Advanced Research and Development Authority (BARDA) stepped in and decided to further fund the project.

The lack of sensitive and virus-specific diagnostic assays implemented at the point-of-care (PoC) contributes to the complexity of the clinical studies for vaccine development. Available serological tests do not present adequate specificity. Given the evolutionary similarity of flaviviruses, combined with the presentation of common clinical symptoms and overlapping geographical distribution in endemic areas (especially in Latin America and Southeast Asia), this creates an unfavorable scenario for developing vaccines and specific chemotherapies. The only test that is capable of differentiating infection by DENV and ZIKV (reverse transcription polymerase chain reaction (RT-PCR)) requires highly trained personnel in specialized laboratory facilities, making it logistically impractical for clinical trials in remote endemic areas.⁴⁶ It is nonetheless of utmost importance that research continues to address this unmet clinical need, with both a vaccine candidate and an effective chemotherapy, ideally with an appropriate diagnostic assay to be used at PoC with excellent sensitivity and specificity. We will focus and discuss in this Perspective the repurposing of existing antiviral strategies for ZIKV, as well as possible novel approaches that have been explored in the past few years for the development of new antiviral chemotherapies.

CO-INFECTIONS AND IMPLICATIONS FOR ZIKV DRUG DEVELOPMENT

It is also important to highlight that ZIKV cocirculates in some endemic regions with other diseases including chikungunya, DENV, YFV, Mayaro virus, and Oropouche virus, among others. Many of the symptoms of ZIKV like fever, rashes, joint pain, and ocular manifestations are similar to other diseases that coexist with ZIKV, especially in Southeast Asia and Latin America.⁴⁷ Since some of these viruses’ antigens are highly conserved, differential serological diagnosis is challenging due to antigen cross-reactivity. In addition, pre-existence or coexistence of these diseases with ZIKV will lead to complications in the immunological response based on antigen cross-reactivity. Co-infection of ZIKV and human immunodeficiency virus (HIV) can also affect the clinical manifestation of the arboviral disease.⁴⁸ These elements have direct implications for treatment options, particularly if drugs are developed individually for each flavivirus present in endemic areas. A better option to bypass issues associated with diagnosis and vaccine cross-reactivity would be the development of a pan-flavivirus chemotherapy with a single dose regimen. Fortunately, several of the ZIKV preclinical compounds discussed herein have activity against multiple flaviviruses and may hold promise as multi- or pan-flavivirus inhibitors.

TARGET PRODUCT PROFILE (TPP)

While a definitive TPP is available for ZIKV vaccine development through the WHO, such a description is yet unavailable for the development of small molecule therapeutics for the infection. We hope to provide here an outline of possible requirements and challenges for the development of drugs for treatment of acute ZIKV infection and for the treatment of pregnant women with the infection.

An important first point to include in the TPP for ZIKV drugs is the need for blood–brain barrier penetrance of the drug for acute infection to reach the ultimate target cells of the viral infection. Second, for drugs indicated for pregnant women, placental penetrance of prospective therapeutics is essential for adequate access to the placental epithelium and the neural progenitor cells of the developing fetus to prevent vertical transmission of the virus. Third, a high level of safety is required for any drug that will be used for the treatment of pregnant women with ZIKV infection due to the risk of harming the fetus with the therapeutic regimen. A comprehensive listing of our proposed TTP is found in Table 2.

Table 2.

Target Product Profile (TPP) for a Potential ZIKV Drug

	optimum/ideal	minimum/acceptable
Target population	1. Population living in endemic area presenting clinical symptoms of ZIKV infection.	Pregnant women living or who have recently visited an endemic area and are presenting clinical manifestation of ZIKV infection
	2. Pregnant women living in endemic area without clinical manifestation (prophylaxis)
Efficacy	1. Complete elimination of circulating ZIKV from the bloodstream assessed by PCR specific test	Prevents or reduces the risk of neurological impairment of the fetus
	2. Prevents neurologicalimpairment of the fetus
Dosage and administration	Single oral dose	Treatment length extended throughout the course of pregnancy, oral doses
Pharmacokinetic profile	Permeable to the placenta barrier and to the blood—brain barrier	If given prophylactically throughout pregnancy, permeability to placenta barrier and blood—brain barrier is not required.
Safety, tolerability	Safe to be taken prophylactically during pregnancy (category Aa)	Safe to be taken prophylactically during pregnancy (category Bb)
Adverse reactions	NOAR (no observed adverse reaction)	Mild and reversible adverse reactions, as long as the fetus is not affected in case of pregnancy
Contraindications	No contraindications
Warnings and precautions	No warnings or precautions	No genotoxicity, no teratogenicity
Drug interactions	No reported drug interactions	No interaction with commonly used
		Drugs during pregnancy against diabetes (insulin, glyburide, or metformin), depression (SSRIs, SNRIs, bupropion, or tricyclic antidepressants) and high blood pressure
Storage and handling	Stable for 1 year at room temperature, climatic zone IV	Require refrigeration for stability
Cost of goods (CoG)	Less than $10/treatment course	Less than $100/treatment course

^aCategory A: Adequate and well-controlled studies have failed to demonstrate a risk to the fetus in the first trimester of pregnancy (and there is no evidence of risk in later trimesters).

^bCategory B: Animal reproduction studies have failed to demonstrate a risk to the fetus, and there are no adequate and well-controlled studies in pregnant women.

TARGETS AND CHEMICAL CLASSES OF INHIBITORS

A. Entry (Receptors).

The identification of the viral and host factors involved in ZIKV entry continues to be an active area of scientific enquiry, and some controversy in the literature surrounds the exact receptors used during this process. Initially, AXL was proposed as a host factor involved in virus entry in human glial cells, but subsequent work has cast doubt on whether this is a necessary factor for virus internalization: genetic knockdown of the receptor in human neural stem cells and cerebral organoids was insufficient in protecting these systems from ZIKV infection (Figure 2).^49,50 TIM1 has been demonstrated to be important in binding of viral phospholipid during viral entry in ZIKV and related flaviviruses (DENV-2 and WNV) and may be a possible target for therapeutic intervention.⁵¹ Inhibitors that interfere with binding of the viral envelope (E) protein and host receptors or that disrupt the viral membrane clearly represent an attractive avenue for drug development for ZIKV. However, additional in vitro biological studies of ZIKV entry in physiologically relevant cell and organoid models and in vivo validation of putative targets must be performed to better assess which are the best therapeutic targets for drug development in this class. Natural products, commercially available small molecule libraries, and synthetic peptide-based strategies that disrupt the viral membrane have been profiled to identify hits associated with this drug class.^52–56

Figure 2.

Overview of the ZIKV lifecycle.

B. RNA-Dependent RNA Polymerase (RdRp).

Viral polymerases represent a well-characterized and thoroughly explored drug target class with a wealth of literature to support the efficacy and safety of developing drugs for this indication.⁵⁷ Essential for the replication of the viral genetic material, inhibition of this class of proteins results in abrogation of both viral replication and the production of viable viral particles. Flaviviruses, including ZIKV, utilize a single-stranded, positive-sense RNA genome as a template for synthesis of a complementary, negative-strand intermediate to act as a template for subsequent rounds of positive-strand RNA synthesis, used for mRNA production and copying of the viral genome for packaging into mature viral particles (Figure 2). In the case of ZIKV, NS5 protein, which possesses both RdRp and methyltransferase (MT) activities essential for viral replication, is the enzyme that executes the biochemical function of viral RNA synthesis, N-7 and 2′-O-methylation of the viral mRNA cap, and 2′-O-methylation of internal adenosine residues.^58,59 NS5 has also been shown to be involved in suppression of interferon signaling through engagement of STAT2, indicating that it may play other roles in viral pathogenesis distinct from its polymerase function.^60,61 Several studies have examined the activity of nucleoside analogues, and other non-nucleoside RdRp and MT inhibitors in biochemical assays using recombinantly expressed and purified ZIKV NS5 protein, against ZIKV in a variety of cellular systems and/or in animal models.

C. Protease.

The virally encoded NS3 protein and its NS2B cofactor of ZIKV are responsible for proteolytic processing of viral polyproteins required for proper viral maturation (Figure 2). Inhibition of functionally equivalent proteins in other viruses, such as HIV protease and NS3 protease of HCV, has been used as a successful antiviral strategy in treating these infections clinically.^62,63 Classically, synthetic peptide analogs that mimic the transition state of the proteolytic cleavage reaction have been employed in the rational design of these types of drugs; however, several ZIKV protease inhibitors that have been reported as active against ZIKV are structurally distinct from this archetypical chemical form and are at various stages of development (from in vitro testing through preclinical animal studies).

D. Assembly.

Limited research efforts have been made to target ZIKV viral particle assembly, most notably though targeting the viral capsid protein. This dimeric protein is needed to provide stability for maturing viral particles; disruption of its interactions results in impaired ability of the virus to assemble during egress and disassemble during entry (Figure 2).⁶⁴ High-throughput screening has identified a candidate molecule that has been shown to affect capsid assembly in related flaviviruses (DENV 1–4, YFV, and HCV). Despite the paucity of compounds specifically targeting ZIKV by this mechanism, this remains a promising target given its essentiality for viral maturation.

E. Endosomal Fusion and Autophagy.

Flavivirus entry is mediated by acidification of the virus-containing endosome; the decrease in pH in the resulting fusion compartment triggers a conformational change in viral surface proteins. This results in fusion of the viral membrane with the membrane of the endosome and the eventual release of the viral genome into the cell (Figure 2).^65,66 Several studies have been conducted recently exploring inhibitors of this fusion process, as it is an essential step for viral entry into the cell to commence the replication cycle. Proapoptotic agents, endosomal pH modulators, and small molecules that interfere with viral E protein–host membrane interactions have all been explored as possible drug candidates within this target class.^67–69

DRUG DISCOVERY INITIATIVES

A. Entry (Receptor) Inhibitors.

The natural product 1 (nanchangmycin), which is generated by Streptomyces nanchangensis, has been proposed to act through inhibition of AXL receptors and abrogation of viral internalization through clatherin-coated vesicles. However, the mechanism of action remains to be further explored (Figure 3).⁵⁵ In silico screening of compound libraries for inhibitors of the viral E protein interactions with putative viral receptors and subsequent testing against the virus has revealed 2 (ZINC33683341) as a possible inhibitor of viral entry.⁵⁶ Recent work has identified synthetic carbohydrate receptors that interfere with the early stages of ZIKV infection in Vero and HeLa cells; it is postulated that these compounds act by inhibiting the interaction between the virus and cell surface glycans.⁷⁰ Given the limited biological understanding of ZIKV entry, more target validation research and insight into the mechanism of action of these compounds would need to occur. In addition, animal studies for these leads must be performed. Nevertheless, these molecules represent a starting point for future understanding of the details of ZIKV binding and entry into their target cells.

Figure 3.

ZIKV entry inhibitors.

B. RdRp Inhibitors.

Several collections of nucleoside analogues and/or their phosphoramidate prodrugs (ProTides) have been examined for activity against ZIKV in purified enzyme assays, cell-based assays, and mouse models of infection (Figure 4). This class of compounds has historically been highly successful against a number of different families of viruses and forms the backbone of antiviral strategies against HCV, HIV, and herpes simplex virus (HSV), among others. We feel that these compounds may represent some of the best candidates for the clinical development of ZIKV inhibitors, given their safety and efficacy profiles. Extensive research activity has taken place in this area in recent years for ZIKV.

Figure 4.

Nucleoside analogue inhibitors of ZIKV RdRp.

Among the first studies to assess the activity of these molecules against ZIKV, Eyer and colleagues demonstrated that 2′-C-methylated ribonucleosides and 3′-O-methyl-ribonucleosides had activity in vitro against ZIKV in Vero cells. No cellular toxicity was reported.⁷¹ Further reports by Lu et al., Hercík et al., and Bernatchez et al. confirmed that the former class of compounds had activity in purified enzyme and cell-based assays through different end point readouts.^72–74 Deng and co-workers have also shown that the adenosine analogue 3 (NITD008) has potent anti-ZIKV activity in cell culture while reducing viremia and preventing death in an A129 mouse model of infection.⁷⁵ The compound has also been shown to inhibit DENV in vitro and in vivo.⁷⁶ Despite this promising data, 3 was found to be too toxic in animal studies to move the compound forward to clinical trials.⁷⁷ The nucleoside analogue 4 (7-deaza-2′-C-methyladenosine (7DMA)), which was previously investigated as a compound for the treatment of HCV and which subsequently failed in clinical trials, was shown to be potent as an inhibitor of ZIKV infection in Vero cells and in a mouse model of ZIKV-infection by Zmurko and colleagues.⁷⁸ Unfortunately, the compound was unsuccessful in a phase I clinical trial.⁷⁷

Recently, the clinically approved HCV nucleoside analogue ProTide 5 (sofosbuvir) was the subject of several studies demonstrating its efficacy and safety as an anti-ZIKV compound in neural cell lines and in vivo mouse models of infection (including prevention of ZIKV vertical transfer), with a reduction in the severity of disease and mortality associated with dosing of the drug in the latter system.^79–82 Recent in vitro studies of related nucleoside analogue ProTides have revealed that 2′-C-methyl and 2′-C-ethynyluridine aryoxyl phosphoramidate ProTides (6 and 7) show improved anti-ZIKV activity compared to sofosbuvir.⁸³ The authors note both a nucleobase and ProTide masking group bias in compound activity, the basis of which remains to be explored mechanistically. Sofosbuvir is FDA-approved for the treatment of HCV and is classified as a pregnancy category B drug, suggesting it may be an ideal candidate for acute dosing clinical trials for pregnant women. Published clinical trial data on this inhibitor in the context of safety and efficacy in the prevention of ZIKV vertical and sexual transfer in humans would be of significant interest to the ZIKV community. Furthermore, evaluation of closely related analogues of sofosbuvir with greater potency against ZIKV in animal models could yield a successful alternative treatment if sofosbuvir fails to adequately prevent vertical and sexual transmission in clinical trials or is found be unsafe in pregnant women.

The broad-spectrum inhibitor 8 (galidesivir, BCX4430), an adenosine analogue that has antiviral activity against Ebola virus (EBOV), Marburg virus (MARV), and YFV, is effective at suppressing ZIKV in vitro and vivo.^84,85 This compound has been the subject of a phase I clinical trial for filoviruses (results have yet to be published), and a phase I clinical trial for YFV is planned (but is not yet recruiting). Like sofosbuvir, clinical trial data for BCX4430 in the context of the prevention of ZIKV vertical and sexual transmission would be of particular interest to the scientific community. Furthermore, the broad-spectrum activity of BCX4430 makes it an attractive drug candidate, as it displays both ZIKV and YFV activity.

The anti-HCV drug 9 (ribavirin) has been reported to have potent anti-ZIKV activity in vitro and in a STAT-1 deficient mouse model of ZIKV infection.^74,86 While the mechanism of action of ribavirin remains under some debate, it is believed to act through a combination of RNA-synthesis abrogation upon incorporation by the RdpRp and through the induction of lethal mutagenesis via mismatch incorporation opposite the incorporated inhibitor in the template strand.

Additional studies have put forward putative non-nucleoside inhibitors of ZIKV RdRp, which can inhibit the enzyme either by binding to the polymerase active site or an allosteric site and thereby interfering with the chemical step of nucleotide incorporation or by blocking the MT activity of the enzyme (Figure 5). 10 (TPB), identified through a high-throughput screen, was reported by Pattnaik and colleagues as an inhibitor of ZIKV in vitro.⁸⁷ Subsequently, molecular modeling work by the group suggested that the compound bound to the RdRp active site and may act by interfering with either nucleotide or RNA binding. Evaluation of the compound in a Balb/C mouse model of ZIKV infection revealed that compound was able to reduce viral load by 40-fold compared to vehicle controls.

Figure 5.

Non-nucleoside analogue inhibitors of ZIKV RdRp.

Another study by Yang et al. reported the FDA-approved antiprotozoal drug 11 (emetine) as having anti-ZIKV activity through binding to the NS5 active site, with reported effective concentration 50 (EC₅₀) values of between 0.009 and 0.053 μM. In an Ifnar1^−/− immunocompromised mouse model of ZIKV infection, emetine was shown to significantly reduce NS1 protein and viral RNA concentrations in serum and liver.⁸⁸

Kaptein and colleagues built on previous work using purine and quinoline scaffolds in the development of non-nucleoside inhibitors which bound to an allosteric site (cavity B) of DENV NS5.^89,90 Through further derivatization, the authors were able to generate dual DENV/ZIKV inhibitors of this series which were predicted to bind to cavity B of NS5 for both viruses. A follow-up study employing tandem phenotypic screening was able to identify three new chemotypes derived from the original purine series: (i) 5H-pyrrolo[3,4-d]-pyrimidin-7(6H)-one; (ii) furo[3,4-d]pyrimidin-7(5H)-one; and (iii) 2,4,5,6-tetrasubstituted pyridines.⁹¹ The most promising compound, 12, derived from this third new class of chemotype, was 3 times more potent than the nucleoside analogue inhibitor 7DMA against ZIKV with a good selectivity index (>41).

Xu et al. provide evidence that a non-nucleoside analogue compound, 13 (DMB213), competes with the natural nucleotide substrate at the polymerase active of NS5 to inhibit RNA synthesis.⁹² The S604T mutation in ZIKV NS5, which is functionally equivalent to the S282T mutation in HCV NS5B which confers resistance to sofosbuvir, caused resistance to this drug but not 13. Biochemical and cell-based assays used by the authors report an inhibitory concentration 50 (IC₅₀) of 5.2 μM, EC₅₀ of 4.6 μM, and cytotoxicity concentration 50 (CC₅₀) of 22 μM for 13.

By use of molecular modeling and small-molecule docking of a library of 28 341 compounds against the ZIKV MT, four compounds were confirmed to have activity against the virus via plaque assay.⁹³ The most potent of these, 14 (F3043–0013), was found to have an EC₅₀ of 4.8 μM in this assay. The authors have proposed testing these compounds in rodent models and suggest that their in silico method can be further applied to other emerging viruses.

A structural analogue of S-adenosylmethionine (SAM), the methyl donor required for ZIKV MT activity, has been synthesized and cocrystallized with ZIKV NS5. The compound, 15 (MS2042), has a reported K_D of 24.3 μM (as compared to 2.58 μM for SAM) and is shown crystallographically to extend into the RNA binding tunnel usually occupied by the 2′-OH of the RNA cap. This molecule may represent the starting point for the synthesis of further competitive inhibitors of ZIKV MT activity.

The natural product 16 (sinefungin), isolated from Streptomyces griseoleus, is a competitor of SAM and has been cocrystallized with ZIKV MT.⁹⁴ This inhibitor and its analogues are of particular interest for antiviral drug development in that 16 has been shown to have broad flavivirus activity against WNV, DENV-2, and YFV.⁹⁵ Although 16 has shown activity against purified ZIKV MT in enzymatic assays,⁵⁹ it surprisingly was found to be inactive against ZIKV in a follow-up study that tested the parent compound in parallel with an analogue series.⁹⁵ The synthesis of 3-fluoro- or chlorobenzyl derivatives at the N-6 position of the adenine moiety in 16, a strategy used effectively to generate potent inhibitors of DENV-3 MT,⁹⁶ was employed to generate additional putative ZIKV MT inhibitors.⁹⁵ The best compound from this optimization, 17, showed both activity (IC₅₀ = 29.98 μM) and acceptable toxicity (CC₅₀ > 200 μM).

X-ray crystallography and molecular docking were used to optimize compounds targeting ZIKV MT via a fragment-based approach.⁹⁷ The best compounds from these optimizations, 18 and 19 (with respective IC₅₀ values in enzymatic MT inhibition assays of 26 μM and 23 μM (DENV), and 28 μM and 19 μM (ZIKV)), are another class of compounds that form an attractive starting point for ZIKV drug development. These compounds were found to be inactive in cell culture systems at concentrations up to 20 μM, and while the CC₅₀ for 19 was >100 μM, 18 had a CC₅₀ of 37.5 μM. The authors speculate that this lack of activity may be due to low activity ex vivo or poor membrane penetrance. Further structural optimization of these compounds for improved bioavailability may help advance these compounds through the preclinical pipeline.

Other inhibitors tested in enzymatic assays for their activity against the MT activity of ZIKV include SAM analogues, cap analogues, and allosteric inhibitors DENV MT activity.^59,98 The most potent of the reported allosteric inhibitors of ZIKV 2′-O-MT activity (IC₅₀ = 24.0 μM for internal methylation and IC₅₀ = 79.0 μM for cap methylation), 20, was also active against the equivalent DENV enzyme. This is promising feature of this compound set, as more than one flavivirus enzyme is inhibited in these assays.

In all, the diversity of approaches, wealth of active compounds throughout the preclinical pipeline, and historical success associated with RdRp inhibitors make this one of the most promising and attractive areas for ZIKV drug development.

C. Protease Inhibitors.

Among all targets, NS2B-NS3 protease of ZIKV has been one of the most actively studied due to its key role in viral maturation and replication.⁹⁹ Conveniently, there are many available crystal structures of free or inhibitor-bound ZIKV NS2B-NS3 protease that can be used for drug design and discovery.^100–103 Substrate-mimic peptide or peptidomimetic compounds are a logical drug class for designing novel inhibitors against ZIKV NS2B-NS3 protease, given the success of protease inhibitors in clinical regimens to treat other viral infections, such as HCV and HIV. Some of the challenges associated with the rational design of substrate mimics for ZIKV protease derive from the flat nature of the protease active site and the charges associated with the ligands that bind to it.¹⁰⁴ Despite this, compounds have been developed using this mechanism of action.

One such compound is 21 (cn-716), a boronic acid containing dipeptide inhibitor that displays strong inhibitory activity to ZIKV NS2B-NS3 protease with IC₅₀ = 0.25 μM and K_i = 0.040 μM (Figure 6). The crystal structure of ZIKV NS2B-NS3 protease in complex with 21 was disclosed by Lei et al.,¹⁰⁵ in which the boronic acid fragment forms a 1.5 Å covalent bond with the Oγ of Ser135 in the active site. Following this work, several novel boronic acid-containing peptidomimetic inhibitors against NS2B-NS3 protease have been reported.¹⁰⁶ These compounds are active against not only ZIKV but also DENV and WNV, suggesting that these could be developed as effective broad-spectrum anti-flavivirus agents.

Figure 6.

Small-molecule ZIKV protease inhibitors.

A crystal structure of the pyrazole ester derivative 22 (5-amino-1-((4-methoxyphenyl)sulfonyl)-1H-pyrazol-3-yl benzoate) as a covalent inhibitor of ZIKV protease has been published.¹⁰³ While 22 had an IC₅₀ of 1.5 μM against the purified ZIKV enzyme and shows activity against both WNV and DENV proteases, the related compound 23 had an IC₅₀ of 0.1 μM against ZIKV protease.¹⁰³ Although this series of compounds has known limitations deriving from poor stability in buffer, these molecules may overcome problems with cell penetration that come with the design of charged substrate analogues. Perhaps the design of isosteres of these compounds with improved stability may overcome their biological limitations.

The competitive serine protease inhibitor aprotinin has been characterized as an inhibitor of ZIKV NS2B-NS3 protease (K_i = 361 nM, IC₅₀ = 70 nM)^107,108 as well as DENV protease^109,110 and WNV protease.^111,112 This is the only classical broad spectrum serine protease inhibitor to show activity against multiple flaviviruses (as well as host proteases), which might on the surface suggest that it may be challenging to design a pan-flavivirus protease inhibitor of this type. On the other hand, a higher degree of selectivity might be developed for flavivirus protease inhibitors using this strategy based on active site differences. This could reduce host-side toxicity and make for safe, effective inhibitors with potential for administration in pregnant women.

Lee and co-workers have identified, using biophysical approaches, small molecule inhibitors of the ZIKV NS2B-NS3 complex. In the process, the authors also identified a “preopen conformation” of the enzyme that may be useful for the rational design of new inhibitors for this target.¹¹⁰

Work by Lim et al. profiled 22 polyphenol-based compounds against recombinantly expressed NS2B-NS3; the authors reported in vitro activity against the enzyme in a Förster resonance energy transfer (FRET) assay for 24 (myricetin), 25 (luteolin), 26 (epicatechin gallate), 27 (astragalin), 28 (rutin), 29 (gallocatechin gallate), and 30 (epigallocatechin gallate).¹¹³

Recent work by Shiryaev et al. revealed 31 (NSC157058) as an allosteric inhibitor of ZIKV protease through in silico docking studies.¹⁰⁸ Testing of the compound in human fetal neural stem cells (hfNSCs) revealed that the molecule was protective against ZIKV infection, with an EC₅₀ of 820 nM without cellular toxicity up to concentrations of 50 μM inhibitor. Despite rapid plasma clearance (~20 min half-life), a dosing regimen of 30 mg per kg per day, 5 times a day (administered through drinking water) of the compound was able to decrease by 10-fold circulating ZIKV in treated mice compared to controls.

Another innovative approach in the design of ZIKV protease inhibitors is the metallopeptides reported by Pinkham and co-workers.¹¹⁴ These inhibitors, which contain both a peptide binding motif and either a Cu- or Ni-containing motif, are also active against WNV protease and irreversibly inactivate the viral protease. The compounds also have a high barrier to the development of resistance, making them attractive as antiviral agents.

An allosteric inhibitor of DENV2 protease, 32 (NSC135618), was identified in a high-throughput screening assay tracking conformational switching in the molecular target (IC₅₀ = 1.8 μM) and was subsequently found to have antiviral activity in vitro against ZIKV (EC₅₀ = 1.0 μM), YFV (EC₅₀ = 0.28 μM), DENV2 (EC₅₀ = 0.81 μM), WNV (EC₅₀ = 1.27 μM), and a CC₅₀ of 48.8 μM.¹¹⁵ In addition to blocking ZIKV replication in human neural progenitor cells, the compound also inhibits the virus’ replication in human placental epithelial cells, suggesting that it may help in preventing transmission of the virus to the fetus, an important consideration for developing the drug as a potential prophylactic. With strong in vitro potency across multiple flaviviruses, this compound merits further testing in animal infection models.

The antibacterial agent 33 (novobiocin) has been reported as a ZIKV protease inhibitor with an IC₅₀ in the micromolar range.¹¹⁶ Using dexamethasone to immunosuppress their mice, the authors tested the efficacy and toxicity of their anti-ZIKV compound at high dose (100 mg per kg twice per day for 13 days) against the virus and achieved complete survival of their treated group compared to their control group, where there was 0% survival. Minimal adverse effects in the treated group were reported, suggesting that optimization of the pharmacokinetic properties of this molecule may yield a promising drug candidate.

34 (bromocriptine) has been characterized as an inhibitor of NS2B-NS3 by Chan and co-workers using docking models and a fluorescence-based protease assay; the authors propose that the compound be further evaluated for in vivo efficacy.¹¹⁷

Reversible peptide inhibitors have shown promising activity toward ZIKV NS2B-NS3 protease, such as compounds 35 and 36 reported by Nitsche et al. (Figure 7).¹¹⁸ Compounds 35 and 36 are both macrocyclic peptides with IC₅₀ values against ZIKV NS2B-NS3 protease of 1.5 and 0.25 μM, respectively.

Figure 7.

Cyclic peptide ZIKV protease inhibitors.

D. Assembly Inhibitors.

37 (ST-148), a small molecule inhibitor of the viral capsid of DENV 1–4, YFV, and HCV that was identified in a high-throughput screen, may be a promising candidate for similar drug development efforts for ZIKV (Figure 8).^52,53 Compound toxicity was not reported for the compound across multiple cell lines. As such, this compound should be tested against ZIKV to assess its activity.

Figure 8.

Potential ZIKV assembly inhibitor candidate.

E. Fusion Inhibitors.

The antimalarial drug 38 (chloroquine) has been reported in several drug repurposing screens as an anti-ZIKV compound (Figure 9).^68,119,120 It is believed that the drug interferes with virus internalization, possibly through blocking of ZIKV-mediated autophagy or through yet uncharacterized host or viral factors in the early replication process. Significant reduction in ZIKV burden is observed in Balb/C mice treated with 100 mg per kg of chloroquine administered intragastrically compared to control mice. Dosing of compound for 5 days postinfection using the same treatment regimen with A129 mice significantly reduced viral load.

Figure 9.

Inhibitors of ZIKV fusion. Single-letter codes for amino sequences are used for compounds 43 and 44.

The antimalarial amodiaquine, a known inhibitor of cellular autophagy, has also been reported as an inhibitor of ZIKV replication in vitro with EC₅₀ values ranging from 2.8 to 4.4 μM in hNPCs and Vero cells.¹²¹ 39 (hydroxychloroquine) has also been shown to inhibit ZIKV in cellular and mouse models of infection.¹²²

Further compounds from this class shown to have micromolar potency against ZIKV in vitro include 40 (quinacrine), 41 (mefloquine), and 42 (GSK369796).^123–125 Taken together, quinolones represent a promising class of antiviral agents which could be potentially exploited for the development of potent and selective agents against ZIKV.

Recent reports by Yu and colleagues have shown that a synthetic peptide, 43 (Z2), potently interacts with the E protein of ZIKV and disrupts the integrity of the viral membrane, leading to the inactivation of the viral particles.⁵⁴ This antiviral activity is demonstrated in Vero cells and in a pregnant mouse model of infection, with intraperitoneal administration of the peptide preventing mother-to-child transmission of the virus. In type I and type I/II interferon-deficient mice, the peptide protected against lethal viral challenge. Importantly, 43 could penetrate the placental barrier and was nontoxic in pregnant mice. Further clinical evaluation would be needed to assess the in-human activity of the compound and its safety profile in pregnant women. In another study focused on targeting the viral E protein, two synthetic peptides were designed, mimicking the conserved stem region of the E protein of JEV.¹²⁶ The peptide designated 44 (P5) inhibited ZIKV replication in Vero cells with an EC₅₀ of 3.27 μM. In a mouse model of infection using animals with deficiencies in type I and type II interferon, 44 was shown to lessen the brain and testes damage upon tissue histology examination.

Lastly, recent work suggests that the disaccharide 45 (trehalose) may prevent placental transmission of ZIKV by activating degradative autophagy.¹²⁷

F. Nucleoside Biosynthesis Inhibitors.

One effective method to suppress rapidly replicating viruses is to decrease the level of available nucleoside triphosphate pools. The conversion of inosine 5′-phosphate (IMP) to xanthosine 5′-phosphate (XMP), catalyzed by the enzyme inosine-5′-monophosphate dehydrogenase (IMPDH), is the rate-limiting step in the de novo biosynthesis of guanine nucleotides.¹²⁸ As such, IMPDH is an important regulator of the intracellular guanine nucleotide pool. The activity of IMPDH is tightly linked with cell proliferation and is a potential target for cancer and viral chemotherapy.¹²⁹ One inhibitor of IMPDH, 9 (ribavirin), is a broad-spectrum antiviral used to treat HCV infection.¹³⁰

46 (merimepodib) is another IMPDH inhibitor that has demonstrated activity against a variety of DNA and RNA viruses in vitro (Figure 10). With the aim of finding an alternative strategy for handling ZIKV infection, Tong et al. broadened the antiviral spectrum of merimepodib to ZIKV.¹³¹ The experimental results showed that merimepodib was able to inhibit ZIKV RNA replication with an EC₅₀ of 0.6 μM and EC₉₀ of 1.0 μM. Additionally, the CC₅₀ was determined to be >10 μM in the cytotoxicity assay, resulting in a selectivity index of greater than 17. ZIKV production in Vero cells was strongly inhibited by treatment with merimepodib at concentrations of 3.3 and 10 μM. Merimepodib inhibition was reversed with the addition of guanosine, which confirmed its mechanism of action as an IMPDH inhibitor. Finally, drug combination effects were tested with the treatment of merimepodib (3.3 and 10 μM) along with ribavirin (9) (33 and 100 μM). Compared with treatment by a single drug, reduction in virus production was significantly enhanced with the cotreatment of merimepodib and ribavirin without a significant change in cytotoxicity. These results demonstrated that merimepodib is a promising therapeutic agent for the treatment of ZIKV infection.

Figure 10.

Inhibitors of host nucleoside biosynthesis.

The dihydrofolate reductase (DHFR) inhibitor 47 (methotrexate) was identified as an anti-ZIKV compound in a high-throughput screen.⁵⁵ Subsequent mechanistic work revealed that addition of leucovorin, a downstream metabolite of the DHFR pathway, rescued ZIKV replication from methotrexate.¹³² Furthermore, addition of adenosine was able to rescue ZIKV replication from methotrexate, suggesting that the antiviral effect of the inhibitor occurs through suppression of de novo synthesis ATP pools. This effect is in line with the reported effect of methotrexate on other flaviviruses.¹³³ These results point to the targeting of the DHFR pathway as a possible strategy for therapeutic intervention for ZIKV infections.

The inhibitor 48 (RYL-634) was discovered during a phenotypic high-throughput screen as a potent, broad-spectrum antiviral agent that inhibited numerous viruses, including ZIKV. Through liquid chromatography/mass spectrometry (LC/MS) pulldown experiments, bioinformatic analysis, and reverse docking strategies, the target of this compound was identified as dihydroorotate dehydrogenase, a key enzyme in the de novo synthesis of pyrimidines.¹³⁴

G. Natural Products and Their Derivatives.

Bioactive-natural products are an important compound source for drug discovery. More than 50% of approved drugs are derived from natural products. We highlight here some of the numerous natural products with diverse structural backbones that have shown promising virucidal activity against ZIKV.

Six natural products belonging to flavonoid and natural phenol classes demonstrated significant inhibitory effects against ZIKV protease.¹³⁵ Among them, 24 (myricetin) was the most potent candidate, which inhibited ZIKV protease noncompetitively with an IC₅₀ of 1.3 μM and K_i of 0.8 μM. The docking result also showed that myricetin is an allosteric inhibitor of ZIKV protease with the plausible binding site behind the active site. Several H-bonds were formed between the phenol groups and the residues of ZIKV protease, which can explain the high binding affinity.

49 (berberine) and 50 (emodin), two natural products isolated from oriental medicinal herbs, demonstrated activity against ZIKV (Figure 11).¹³⁶ ZIKV infectivity was reduced by 77.6% and 88.3% when exposed to 160 μM berberine and 40 μM emodin, respectively. The IC₅₀ values of berberine and emodin were calculated to be 39.1 μM and 3.2 μM. In contrast, these two natural products did not show noticeable cytotoxicity to Vero E6 cells with CC₅₀ values of 221 μM for berberine and 68.6 μM for emodin. A mechanistic study demonstrated that emodin could effectively reduce cellular ZIKV entry, while both berberine and emodin directly impair virus particles. Considering their promising anti-ZIKV activity and favorable safety profiles, structure optimization should be conducted on berberine and emodin to further improve their activity.

Figure 11.

ZIKV inhibitors derived from natural products.

Natural products derived from marinopyrroles have been reported to be potent anti-methicillin-resistant Staphylococcus aureus (MRSA) and anticancer agents.^137–141 Testing of a library of 24 marinopyrrole derivatives in Vero and human neural stem cells uncovered one compound from the series, 51, which inhibited ZIKV in vitro (EC₅₀ = 5.95–8.56 μM, CC₅₀ > 100 μM).⁷⁴ While the biological target for this compound remains to be elucidated, this represents another starting point for the development of potentially novel anti-ZIKV agents based on marine natural products.

Lee and colleagues profiled a library of 483 flavonoid derivatives in human placental cells (JEG-3). The most potent compound, 52 (pinocembrin), which is found in honey and tea, had an IC₅₀ of 17.4 μM against ZIKV and was shown to inhibit both ZIKV and DENV serotype 2. Pinocembrin was shown to act against ZIKV postentry.¹⁴²

H. Host-Directed Antivirals.

In addition to viral proteases, host proteases also play important roles in the replication process of ZIKV. 53 (cavinafungin), an alaninal-containing lipopeptide isolated from Colispora cavincola,¹⁴³ is highly selective for and potently inhibits the growth of ZIKV-infected cells (IC₅₀ = 0.15 μM and CC₅₀ = 1.65 μM) (Figure 12).¹⁴⁴ An unbiased genome-wide CRISPR/Cas9 chemogenomic profiling approach identified the host endoplasmic reticulum-localized signal peptidase (ER-SPase) as the target of 53. Common to all flaviviruses including ZIKV, ER-SPase plays important roles in the processing of prME, which can be effectively inhibited by 53.¹⁴⁵ This work provides evidence for 53 as a first-in-class, selective, and cellularly active inhibitor against the ER-SPase. Furthermore, the study confirms the critical role of the ER-SPase in flavivirus polyprotein processing, paving the way for the development of novel ZIKV inhibitors.

Figure 12.

Host-directed ZIKV antivirals.

The adenosine monophosphate-activated protein kinase (AMPK) activator 54 (PF-06409577) has been reported to broadly inhibit flavivirus (including ZIKV) replication by inhibitory phosphorylation of the acetyl coenzyme A (acetyl-CoA) carboxylase (ACC).¹⁴⁶ Subsequent lipidomic studies carried out by the authors revealed that modulations in host lipid metabolism occur upon treatment with 54, most notably with the reduction of sphingolipids and glycerophospholipids essential for viral replication. The authors surmise that improper replication complex development occurs in the absence of these critical lipid components.

CONCLUSIONS AND FUTURE DIRECTIONS

While ZIKV cases have dropped dramatically from their peak during the 2015–2017 outbreak in Latin America, the threat of a resurgence of the virus due to virus mutation, human travel, and vector migration remains. Furthermore, outbreaks of DENV and YFV continue to affect broad swaths of the endemic areas for ZIKV, where vaccines for these two former viruses are usually administered in regions at highest risk for outbreak development. Given this situation, the development of inhibitors that could broadly treat multiple flavivirus infections, including ZIKV, may represent a cost-effective and efficient strategy for dealing with co-infections in endemic regions and reduce the risk of vaccine-mediated exacerbation of the symptoms of dengue fever. Several of the studies discussed herein utilize this approach and will hopefully yield a successful antiviral drug in the near future.

The amount of literature that has been produced in the past few years on ZIKV pathogenesis and therapeutic interventions has been truly astonishing: major advancements have been made in the understanding of the fundamental pathology of ZIKV infection. New therapeutic targets may provide for additional opportunities for prevention and treatment of this disease, and research advances in ZIKV may inform treatment strategies for other flaviviruses and vice versa. Given the effort that has been devoted to the development of inhibitors of ZIKV, it is only a matter of time before additional agents are brought to clinical trials for this infection. The development of broad-spectrum agents with activity against multiple flavivirus may be the key to developing a cost-effective and safe therapeutic strategy for ZIKV-endemic regions that will finally be able to efficiently control future outbreaks.

As discussed in this Perspective, extensive preclinical investigation of existing drugs and novel chemical entities in both in vitro and in vivo systems has been performed. However, limited nonbiologic, small-molecule candidates with anti-ZIKV activity in animal models have advanced to clinical trials. Therefore, there remains a continuing need for progression of additional candidates.

While several vaccine candidates against ZIKV have advanced to clinical trials, there is concern that immunization of individuals against ZIKV may exacerbate subsequent DENV infection symptoms, given the prevalence of multiple flaviviruses being present in ZIKV-endemic regions. This effect has been recapitulated in mouse models: maternally acquired ZIKV antibodies cross-react but do not neutralize DENV, enhance vertical transfer of the virus, and increase the severity of DENV infection in mouse pups.¹⁴⁷ Given that this scenario is likely to occur in ZIKV-endemic regions, an approach using targeted ZIKV or broad-spectrum flavivirus antivirals as prophylactics during viral outbreaks or as a therapeutic if administered early in the infection might be more effective at globally reducing flavivirus-associated disease in areas where multiple flaviviruses can be transmitted to the host. Numerous possible avenues toward developing new drugs for ZIKV are currently being explored, and advances in the understanding of ZIKV host–pathogen interactions will only improve our ability to generate targeted and selective therapeutics for this infection.

ABBREVIATIONS USED

7DMA

7-deaza-2′-C-methyladenosine

ACC

acetyl-CoA carboxylase

acetyl-CoA

acetyl coenzyme A

AMPK

adenosine monophosphate activated protein kinase

BARDA

Biomedical Advanced Research and Development Authority

CC₅₀

cytotoxicity concentration 50

CDC

Centers for Disease Control and Prevention

DENV

Dengue virus

DHFR

dihydrofolate reductase

E

envelope

EBOV

Ebola virus

EC₅₀

effective concentration 50

ER-SPase

endoplasmic reticulum localized signal peptidase

FRET

Förster resonance energy transfer

GBS

Guillain–Barré syndrome

HCV

hepatitis C virus

hfNSC

human fetal neural stem cells

HIV

human immunodeficiency virus

HSV

herpes simplex virus

IC₅₀

inhibitory concentration 50

IMP

inosine 5′-phosphate

IMPDH

inosine 5′-monophosphate dehydrogenase

JEV

Japanese encephalitis virus

LC/MS

liquid chromatography/mass spectrometry

MARV

Marburg virus

MRSA

methicillin-resistant Staphylococcus aureus

MT

methyltransferase

PHEIC

Public Health Emergency of International Concern

PoC

point-of-care

prME

prM and E structural proteins

ProTide

nucleotide prodrug

RdRP

RNA-dependent RNA polymerase

RT-PCR

reverse transcription polymerase chain reaction

SAM

S-adenosylmethionine

TPB

3-chloro-N-[(amino)carbonothioyl]-1-benzothiophene-2-carboxamide

TPP

target product profile

WHO

World Health Organization

XMP

xanthosine 5′-phosphate

WRAIR

Walter Reed Army Institute of Research

WNV

West Nile virus

YFV

yellow fever virus

ZIKV

Zika virus

Biographies

Jean A. Bernatchez completed his B.Sc. (First-Class Honors) in Biochemistry at McGill University in Montreal, Quebec, Canada, in 2008. He then pursued Ph.D. studies at McGill in Biochemistry as part of the Chemical Biology Interdepartmental Graduate Program and graduated in 2015. Currently, Dr. Bernatchez is a Postdoctoral Scholar at the Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego. His current research focus is on the development and use of phenotypic, cell-based high-throughput screening assays for the discovery of novel anticancer, antiviral, and antiparasitic agents.

Lana T. Tran received her B.A. degree in Molecular and Cell Biology at the University of California, Berkeley, and her Pharm.D. degree at the University of Michigan College of Pharmacy. She is currently a Drug Development Fellow in the Division of Pharmacotherapy and Experimental Therapeutics at the University of North Carolina Eshelman School of Pharmacy.

Jerry Li received his B.A. degree in Molecular and Cell Biology at the University of California, Berkeley, and his Pharm.D. degree at the University of Michigan College of Pharmacy. Dr. Li is currently a Clinical Pharmacology Oncology Fellow at Pfizer and the University of California, San Diego Skaggs School of Pharmacy and Pharmaceutical Sciences.

Yepeng Luan received his Ph.D. degree in 2010 from the Department of Medicinal Chemistry, School of Pharmacy, Shandong University, China. During this period, he did a year of research as a visiting student at the Department of Pharmaceutics at the University of Kansas. His postdoctoral training was at Ocean University of China (2010–2012) and the University of Georgia (2012–2015). Dr. Luan is a lecturer in the Department of Medicinal Chemistry, School of Pharmacy, Qingdao University, China. His research focuses on the design, synthesis, and activity evaluation of novel isoform-selective HDAC inhibitors and hybrid HDAC inhibitors. He is also interested in designing novel antagonists against other epigenetic targets, including HAT and protein arginine methyltransferases.

Jair L. Siqueira-Neto is an Assistant Professor at the Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, and the Director of the UCSD Screening Core. After completing his Ph.D. in Genetics and Molecular Biology at University of Campinas, SP, Brazil, he joined the scientific staff at the Institut Pasteur Korea, Seoul, South Korea, leading a team of multidisciplinary researchers to identify new candidates to treat infectious diseases. After a short time at UCSF, he was recruited to UCSD in 2014 as faculty. Dr. Siqueira-Neto’s lab research focuses on discovering and developing therapies for neglected tropical diseases (NTDs). He pioneered the development of phenotypic high-content screening methods against intracellular Leishmania sp. and Trypanosoma cruzi.

Rongshi Li is a Professor of Chemistry and Medicinal Chemistry at the University of Nebraska Medical Center (UNMC). After 14 years in industry, advancing from Scientist to Senior Vice President, Dr. Li moved to Moffitt Cancer Center in Tampa, FL. In 2013, he was recruited to UNMC, where he has received the Distinguished Scientist Award (2016) and New Inventions (2014, 2016, and 2018). Dr. Li has over 200 scientific contributions including peer-reviewed articles, patents, reviews/perspectives, book and book chapters, editorial, and published scientific presentations. Dr. Li has delivered more than 100 lectures as a keynote or invited speaker at numerous universities and research institutions and at national and international symposia, and he has been a Guest Editor for Medicinal Research Reviews.