Novel strategies for discovering inhibitors of Dengue and Zika fever

KEYWORDS:

• Dengue virus
• Zika virus and antivirals

1. Introduction and burden of dengue virus

Given the persistent and unanswered demand for therapeutic interventions for the many flaviviral diseases, the past decade has witnessed a surge in Flavivirus antiviral research. Dengue virus (DENV) alone poses a global threat affecting 3.9 billion people in 128 countries with an estimated 2.1 million cases of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) and 21,000 deaths per year worldwide [1]. The current Zika virus outbreak has prompted the Centers for Disease Control and Prevention to declare a ‘global health alert’, while West Nile Virus continues its march across the globe. Several target-based approaches have been deployed against DENV using viral, host, structure and systems biology. Herein we briefly summarize novel candidates of antivirals targeting DENV replication proteins, broad-spectrum antivirals, and other key strategies for combatting DENV infection. We then focus on the potential of repurposing drugs and using artificial intelligence for accelerating bench to bedside therapeutics.

2. DENV pathology

DENV is an enveloped, single-stranded positive-sense RNA virus, with an 11-kb genome comprised of genes encoding structural proteins Capsid (C), membrane (prM/M) and envelope (E); and nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. The E protein mediates cellular entry and nucleocapsid release of the virus, whereas the NS proteins facilitate viral replication and immune evasion. DENV infection can be asymptomatic or causes symptoms ranging from mild febrile illness to DHF/DSS. Unpredictable epidemiology, diagnostic difficulties, various host factors influencing disease severity, and variable timing of therapy have all added to the complexity of developing an antiviral drug.

DENV’s utilization of an error-prone RNA-dependent RNA polymerase with a high mutational frequency turns the virus’ proteins into moving targets for antiviral therapy. Viral escape mutants will occur, and countering viral resistance and evasion is required for effective therapy. Therefore monotherapy with directly acting antivirals that target a single DENV protein will unlikely be effective; however, deployment of combination therapy regimes could prove more successful, as has been demonstrated with HIV and hepatitis C virus treatments [2,3]. Due to the presence of an elevated genetic barrier to resistance in host-based antiviral therapies [4], host cell-based therapy may also be a more desirable approach than a directly acting monotherapy. No host antiviral target has been validated in dengue human trials as of yet, great challenges remain.

3. Broad spectrum antivirals

DENV drug discovery prospects and progress have been reviewed in considerable depth. To date, several strategies have been pursued in targeting DENV and host proteins, including structure based in silico, docking and rational design, high-throughput viral replication and enzymatic assays, and repurposing of hepatitis C antivirals. However, almost all strategies have proven futile due to diminished selectivity or pharmacokinetic properties [5].

Despite the challenges in developing a broad-spectrum antiviral drug with favorable pharmacokinetics, low toxicity, and good efficacy, many putative pan-viral candidates are being explored.

3.1. Alpha-glucosidase I inhibitors and celgosivir

Celgosivir, also known as 6-O-butanoyl castanospermine, is an endoplasmic reticulum (ER) resident host alpha-glucosidase I enzyme inhibitor that is required for the proper processing of DENV glycoproteins NS1, E and prM. It indicated good efficacy in a lethal antibody dependent enhancement DENV mouse model. It was tested in a Phase 1b clinical trial in 50 dengue patients in Singapore, but failed to reduce serum viral loads when administered twice daily in viremic patients. This led to a new clinical investigation with altered dosing regimen [6].

3.2. The iminosugars UV-4B and UV-12

The iminosugars UV-4B and UV-12 are similar to celgosivir and competitively inhibit glycoprotein processing enzymes alpha-glucosidases I and II. UV-4B and UV-12 mediated potent inhibition of DENV infection in mouse models are expected to enter clinical trials soon [4,7].

3.3. Other protein targets

Broad-spectrum antivirals targeting other host proteins show promise against DENV. The inhibition of Sec61, an ER membrane protein translocator, altered HIV, DENV and influenza proteostasis and inhibited viral replication [8]. Additionally, a drug inhibitor of Hsp70 chaperone network, JG40, potently blocked DENV infections in human primary blood cells without resulting in viral resistance or toxicity [9].

4. Innate immunity targets

Targeting of host antiviral innate response elements is also being explored. M8, a 99 nucleotide, uridine-rich hairpin 5ʹpppRNA, is a RIG-I (retinoic acid-inducible gene I) agonist that stimulated a robust interferon response and inhibited DENV replication in vitro [10]. Additionally, C19orf66, a cellular gene named RyDEN, for repressor of yield of DENV, was upregulated by interferon treatment and inhibited replication of all DENV serotypes [11].

5. Mouse models of DENV infection

Finally, ensuring the use of available DENV mouse models that recapitulate the clinical manifestations of the human disease is key for determining the efficacy and safety of antiviral compounds, and for defining new therapeutics [12].

6. Prospects for Zika antivirals

Zika virus (ZIKV) causes Zika fever and has putatively been linked to fetal microencephaly and adult Guillain–Barre Syndrome (GBS). The pathogenesis of fetal central nervous system (CNS) infection, and fetal pharmacology and vaccinology add layers of complexity to this problem, which are beyond the scope of this review. Further complicating matters is that DENV and ZIKV share geographic ranges, vectors, and symptoms. Indeed coinfections with DENV and ZIKV have already been documented. If ZIKV’s etiologic role is proven, antiviral therapy in nonpregnant patients will most likely be prioritized toward preventing GBS. Given the two viruses’ similar structure, compounds that inhibit DENV replication proteins, as well as broadly neutralizing antibodies and small molecules that inhibit DENV entry and fusion, may be effective against ZIKV. Although we would hope that DENV and ZIKVs’ similar virology would provide a head start in ZIKV drug development, the difference in their clinical diseases adds complexity and emphasizes the importance of elucidating both viruses’ pathogeneses.

Both the continuous geographic spread of DENV and the ZIKV outbreak are testimony to the urgent need for antivirals. Changes in weather patterns and climate, along with the significant increase in international travel, will cause viral diseases to emerge and others to re-emerge around the globe. With the 2016 Olympics in Brazil fast approaching, public education programs to increase awareness are also needed. Having a systematic and methodological approach in conducting clinical trials, and collecting and sharing data through a collaborative space for industry partners, clinicians, academicians, government, will be crucial for the fruitful development of effective dengue and Flavivirus therapeutics to alleviate human suffering.

7. Expert opinion

7.1. Repurposing drugs

Although high tumor necrosis factor (TNF) levels are strongly implicated in the hemorrhagic clinical manifestations of DENV, there is little data on anti-TNF therapies from clinical studies. Based on a report of small number of cases of DENV patients who were taking a TNF blocker [13], drugs such as Remicade/infliximab, may alleviate hemorrhagic symptoms and save patient lives. Moreover, a clinical trial of ketotifen to treat the vascular leakage caused by dengue virus infection is underway. Ketotifen targets mast cells and prevents their degranulation. As these cells are strongly activated by DENV, targeting them may reduce vascular leakage in DENV patients [14].

7.2. Artificial intelligence and systems biology

A deeper understanding of the viral and host mechanisms that regulate the balance between immune-mediated pathology and protection is critical for the development of safe and effective treatments and vaccines against DENV. Similar to the fluomics approach in the influenza field, determination of the 3D configuration of the host genome architecture in response to virus infection, and identification and characterization of the underlying transcriptional changes in the human host genome in response to DENV infection will shed light on key promoter regulatory domains and chromatin interactions. Knowledge gained from patient studies should then be used to perform mechanistic studies that explore precise virus–host interactions using animal models and human cell culture models. CRISPR technology may accelerate antiviral drug discovery, by defining precisely the role of genetic elements in DENV infected cells.

Additionally, challenges can only be surpassed by using a biology systems approach [15]. The presence of anti-nuclear antibodies in infection, autoimmunity, and cancer, such as anti-histones autoantibodies in scleroderma, is just one example of potential hidden links between infection and autoimmune diseases that only artificial intelligence and systems immunology can help unravel. Artificial intelligence with effective dataset management in the context of patient genetic predispositions will be crucial in moving us into the era of personalized medicine and clinical trials of n = 1.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.