Research Article

Potential Therapeutics for Dengue Virus Infection

Amrita Haikerwal1, Swatantra Kumar1, Ravi Kant1, and Shailendra K Saxena1,2*
1Department of Stem Cell/Cell Culture, King George’s Medical University (KGMU), India
2Department of Stem Cell/Cell Culture, CSIR-Centre for Cellular and Molecular Biology, India


*Corresponding author: Shailendra K Saxena, Department of Stem Cell/Cell Culture, Centre for Advance Research (CFAR), King George’s Medical University (KGMU), Chowk, Lucknow, 226003, India


Published: 12 Apr, 2017
Cite this article as: Haikerwal A, Kumar S, Kant R, Saxena SK. Potential Therapeutics for Dengue Virus Infection. Ann Pharmacol Pharm. 2017; 2(8): 1046.

Abstract

Dengue is a mosquito-borne, rapidly rising viral infection with around 400 million cases per year. The toll of this high number may be attributed to the lack of availability of vaccine and specific treatment. Therefore, the development of novel antivirals is of vital importance. All the 4 serotypes of Dengue virus (DENV) could actively infect the same population with different immune responses. Thus, various studies are focused towards finding potent antiviral therapeutics to target various DENV serotypes, and further inhibit cross-reactivity among similar viruses and antibody dependent enhancement of infection. Antiviral therapeutics targeting potential restriction sites during different stages of virus life cycle were studied. Several of them were conducted using small compounds like a tetracycline derivatives, polysaccharides, lectins, or monoclonal antibodies as antiviral therapeutics for DENV infection. In this paper, we are elucidating potential antiviral therapeutics as well as immune-therapies against DENV infection.
Keywords: DENV; Dengue fever; Immunopathogenesis; Antiviral drugs; Cytokines and Chemokines


Abbreviations

DENV: Dengue Virus; DF: Dengue Fever; DHF: Dengue Haemorrhagic Fever; ADE: Antibody Dependent Enhancement; DSS: Dengue Shock Syndrome; RdRp: RNA dependent RNA polymerase; CSP: Castanospermine; DNJ: Deoxynojirimycin


Introduction

Dengue is an acute viral infection, transmitted predominantly by a vector Aedes aegypti mosquitoes to their human host. Dengue virus (DENV) belongs to genus Flavivirus and family Flaviviridae. Geographically dengue virus infection is escalating in tropical and sub-tropical regions with around 400 million cases reported per year [1]. DENV-2 serotype is the most lethal among four related serotypes of DENV (DENV1-4). Mostly dengue virus infection is asymptomatic, thus symptoms can vary from dengue fever (DF) to dengue haemorrhagic fever (DHF) or dengue shock syndrome (DSS). DF shows flu-like symptoms like mild fever, malaise and vomiting, whereas DHF comprises of DF symptoms as well as increased vascular permeability and thrombocytopenia which might be fatal.
In spite of various research studies globally with the sole motive of eradication of dengue virus still there are no licensed vaccines or a specific drug available for the treatment of DENV. The reason behind this gap may be due to antigenic variability among all the four serotypes of DENV, crossreactivity between DENV and other Flaviviruses such as Zika virus, and specific antibody primed to one serotype of DENV unable to neutralize other serotypes. During subsequent infections, existing antibodies will opsonize the viral particle via Fcγ-receptor mediated endocytosis in macrophages and monocytes, which may result in viral load augmentation. This antibody-dependent enhancement (ADE) of infection causes a severe secondary infection. Hence, the current requirement for the treatment of DENV is to development of ideal therapeutics which can target all the four serotypes of DENV [2].


Life Cycle of DENV

DENV is a smooth spherical Flavivirus with positive sense single stranded capped RNA of 10.7 kb which translates into a single polyprotein comprised of three structural proteins as capsid (C), pre-membrane (pre-M) and envelope (E); and seven non-structural proteins as NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 [3]. The life cycle of virus involves seven stages: attachment, fusion, translation, replication, assembly, maturation and egress (Figure 1). Monocytes, macrophages, and skin dendritic cells are the primary target host cells for entry of dengue viruses. DENV attaches with various group of receptors present on the host cell surface like GAGs (heparan sulphate); Lectins (mannose residues, DC-SIGN, GRP78/BiP receptors); and TIM/TAM receptors [4]. Binding of virus with receptors triggers its internalization by clathrin mediated endocytosis. Subsequently, due to the acidic pH in endosome, rearrangement of E proteins occurs as the dimer dissociates to monomer and finally to a stabilized trimeric state. This rearrangement induces fusion of envelope with endosomal membrane allowing uncoating and release of ssRNA into the cytoplasm of host cell. ssRNA migrates to rough endoplasmic reticulum (RER) to get translated into a single polyprotein. This polyprotein is co- and post-translationally processed by host and viral encoded proteases into structural and non-structural proteins where E, prM and NS1 lies in the ER lumen while capsid, NS3 and NS5 are present in the cytosol (Figure 1) [5]. These non-structural proteins are also known to play a significant role in replication of viral RNA as NS3 has RNA helicase activity whereas NS2 involve in processing of viral polyprotein. NS5 is known to have RNA dependent RNA polymerase (RdRp) activity and it also helps in capping of progeny RNA [6]. The next event is the replication of viral RNA occurs in the vesicular spherules in ER cisternae opposite to the nuclear envelope [7]. The replication complex comprises of viral proteins, viral RNA and host factors commences the RNA synthesis. The entry of nucleotides and desertion of progeny (+) ssRNA occurs through the vesicular pore. Subsequently, assembly of viral particles occurs in the peripheral cisternae of ER mediated by binding of capsid, prM-E and the progeny RNA to form enveloped immature virions. These immature virions bud off from the distal cisternae of ER and will be transported through the secretory vesicles to the Golgi complex, where pH change results in furin mediated cleavage of prM to M followed egression of mature virions via exocytosis (Figure 1).


Figure 1

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Figure 1
The potential therapeutics targeted at various stages of Dengue virus life cycle. Various potential therapeutics against DENV (A - Suramin, B - Pentosan polysulphate, C - PI88, D - Lovastatin, E - Chloroquine, F - SA-17, G - 2’CMA, H - JG 40, I - Balapiravir, J - Castanospermine, and K – Deoxynojirimycin) have been exhibited at various stages of replication.

Inhibitors of DENV

Inhibition of virus attachment to the target cell is critical for the development of antiviral drugs because it forms the first barrier to prevent the viral infection (Figure 1). The E protein plays a significant role in attachment and fusion of DENV in host cell. Thus, the arrangement of E protein is of prime importance as it has various potential restriction sites. E protein is composed of β barrel organized in three structural domains. The central domain consists of amino terminus and two disulphide bridges, whereas domain II is an extended finger-like domain composed of fusion peptide which stabilizes the dimer form of E proteins. A binding pocket exists between domain I and domain II that can interact with a hydrophobic ligand, which could be an important target for the development of antiviral drugs against DENV. Domain III is an immunoglobulin-like domain which contains the receptor binding motif, one disulphide bond and Cterminal motif. Monoclonal antibody targeting domain III is the most efficient in arresting DENV infection [8]. Small molecules and peptide drugs targeting the hydrophobic binding pocket are characterized as the entry inhibitors of DENV. Tetracycline derivatives have been shown to interact with the hydrophobic binding pocket of the E protein which prevents the conformational rearrangement of E protein followed by the inhibition of viral fusion to the target host cell membrane (Table 1) [9]. NITD488 has been demonstrated to inhibit the DENV2 fusion which can bind to the hydrophobic pocket of the E protein, via in silico docking experiments [10].
Doxorubicin derivative SA-17
Doxorubicin is a broad spectrum anti-neoplastic antibiotic extracted from Streptomyces peucetius and its derivative, SA-17 is a potent inhibitor of DENV-2 replication. It has a structural similarity to the tetracycline which interferes with the entry of DENV serotypes 1, 2 and 3 [11]. It is less cytostatic than doxorubicin. Time-of-drugaddition assays had shown that SA-17 acts at very early stages of viral replication such as the time of viral attachment or entry. SA-17 changes the conformation of E- protein by binding to the hydrophobic pocket, was confirmed using Renilla luciferase -expressing dengue reporter.
Glycosidase Inhibitors
Inhibition of glycosylation of viral proteins in the lumen of endoplasmic reticulum (ER) can also be one of the target sites for reducing the number of mature viruses. Compounds like castanospermine (CSP) and deoxynojirimycin (DNJ) are imino sugars which mimics glucose in glycosylation process [12]. Therefore, the secreted viral proteins are inappropriately folded that would decreases the infectivity of DENV particles. This is an effective technique as it works on all the four serotypes of DENV and in turn, minimizes the risk of ADE.
Carbohydrate-binding proteins
Some proteins extracted from natural sources such as Concanavalin A and wheat germ agglutinin (WGA) has been reported to decrease the number of DENV. The binding of Concanavalin A with mannose residues and WGA with N-acetyl glucosamine present on viral proteins inhibit the absorption of DENV results in low level of plaque formation [13]. In another study, three lectins isolated from Galanthus nivalis (snowdrop), Hippeastrum hybrid (amaryllis) and Urtica dioica (stinging nettle) have been shown to inhibit DENV-2 infection via disruption of interaction between DC-SIGN and viral envelope protein [14].
Heparan sulphate receptors
Heparan sulphate (HS) receptors are the binding site for DENV, hence, targeting HS may provide potent entry inhibitors of DENV. GAGs and heparan mimics HS and inhibits the interaction between the domain III of E protein and HS. Some pharmaceutical polyanion products like suramin, pentosan polysulphate and sulphated polysaccharide, PI-88 are under clinical trials as they prevent DENV infection [15].
Balapiravir
Balapiravir acts as a potent inhibitor of RdRp when subjected to host cells it gets phosphorylated to 5’-triphosphate compound results in prevention of RNA replication in DENV serotypes. Furthermore, in a therapeutic trial, Balapiravir was administered in a dose dependent manner in cells but did not efficiently decrease viral loads and cytokine level as expected. The reason behind this lack of activity might be different levels of phosphorylation in cells. Hence, it was not suitable drug for treatment of DENV infection [16].
Chloroquine
Chloroquine acts as an alkalinizing agent, decreases the acidic conditions of organelles like endosome, lysosome and secretory vesicles of Golgi apparatus [17]. Chloroquine was suitable for hampering the release of DENV in the cytoplasm of host cell as fusion of virus and endosome was pH dependent as well as it inhibits furin dependent maturation of virus in Golgi [18]. It also acquires anti-inflammatory properties which helps in reducing the levels of cytokines released during DENV infection [19]. In a study it has been shown that Chloroquine administered in dose-dependent manner inhibits replication of DENV-2 serotype. Thus in clinical trial studies, efficacy of Chloroquine against DENV infection is insignificant.
Lovastatin
Lovastatin acts as inhibitor on various stages of DENV life-cycle. It has been shown that Lovastatin inhibits cholesterol synthesis, thus, it restricts replication of DENV-2 serotype as cholesterol is ultimate requirement of virus replication [20]. It also inhibits entry, maturation and egress of virus [21].


Table 1

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Table 1
Antiviral compounds against dengue virus infection.

DENV Specific Molecular Medicine

The molecular medicine has revolutionised the translational research by exploring the DENV and associated disease condition at the cellular and molecular level, allowing us to design various potential therapeutic molecules specific to the DENV, such as monoclonal antibodies targeting various antigenic epitopes specific to DENV serotypes, si-RNA targeting virus specific genes, molecules targeting chaperons etc., as mentioned below
HMab 2D22
Human monoclonal antibody (HMab) 2D22 has been shown to prevent the antibody-dependent enhancement of viral infection. It binds to the E-protein dimer epitopes which blocks the rearrangement of E proteins required for the fusion with the host target cell. It has been shown that a mutant HMab 2D22 (L234A, L235A) LALA variant, prevents Fcγ receptor binding and leads to the suppression of ADE. Mutant HMab 2D22 can be used for the development of a vaccine for DENV-2 [22].
Ab513
A novel strategy has been employed for the generation of antibodies based on non-immunodominant but functionally relevant epitope of E-protein domain III (EDIII). Characterization of the epitope-paratope interface on EDIII of a known antibody, 4E5A by using structure-guided connectivity network analysis helps in the elucidation of complementarity-determining region (CDR). Furthermore, an optimized antibody Ab513 using this strategy has been developed. Ab513 has high-affinity binding towards EDIII and it neutralizes all four serotypes of DENV. In mice, Ab513 prevents various outcomes of DENV infection such as thrombocytopenia, vascular permeability, viremia and the maternal-transfer model of lethal ADE [23].
RNAi against DENV
RNA interference is a promising technique for silencing gene expression. si-RNA targeted genes responsible for attachment to receptors and clathrin-mediated endocytosis inhibit the entry and replication of DENV in target host cells [24]. si-RNAs targeting NS4B and NS5 might inhibit viral replication in all 4 serotypes of DENV [25]. Human adenovirus type-5 vector transfected short-hairpin RNA in cells for targeting conserved sites in the viral genome. This short-hairpin RNA matures to si-RNA and subsequently inhibits antigen secretion and viral replication of all DENV serotypes [26].
Hsp70 inhibitors
Virus is completely dependent on host cell for its survival. Chaperons plays an important role in folding of proteins in both human and viruses. Chaperons are more utilized by viruses than by the host cells therefore, targeting Hsp70 will restrict the entry of virus, replication as well as generation of correctly folded proteins. Inhibition of Hsp70 would result in DENV life cycle arrest. Inhibitors such as JG40 and 2’CMA (2’C- methyladenosine) has been used to target Hsp70. The advantage of using Hsp70 inhibitors provides strategy where virus could not produce a resistant strain against the drugs [27].
Dengvaxia (CYD-TDV)
WHO reported in Weekly Epidemiological Record, 2016 about the Dengue vaccine, Dengavaxia (CYD-TDV). CYD-TDV is a tetravalent live attenuated vaccine which contains 4 recombinant virus for all the 4 serotypes of DENV. The CYD recombinants were obtained by replacing specific genes of attenuated yellow fever virus with 4 wild serotypes of DENV. This vaccine is registered for individuals suffering from of age 9-45 years in Phase III trials. The effectiveness and availability of CYD-TDV vaccine is yet to be evaluated [28].


Conclusion and Perspectives

Survival of virus is dependent on the host cell which in turn makes the life cycle of the virus very complex. In the human host, a DENV can primarily target monocytes, macrophages, and dendritic cells but now it is also known that dengue virus could also infect lymphocytes, hepatocytes, endothelial cells and epithelial cells. Thus, complexity of DENV is because of the variability between different serotypes enables them to interact with various receptors of different host cells. Consequently, the virus can take numerous path to infect cells that makes it challenging to target viral epitopes. Viral resistance against antiviral drugs is another important issue. The factors released from host cells which help the virus in infecting cells are also investigated and characterized for better understanding their mechanism of action. Currently, with technologies like live imaging, we could also track the viral trafficking inside the cell but this is not yet very commonly used.
Cases of dengue infection are escalating every year with many severe cases of DHF. This is an alarming state which requires prime attention towards the development of a potential vaccine which could target all the 4 serotypes of DENV. Antiviral drugs could terminate primary symptoms like dengue fever which will prevent intense infection. Thus early diagnosis of DENV infection will decrease the viral loads. Countries like India, Brazil, Mexico, Indonesia and some of the African countries have reported a large number of cases of dengue infection in last few years. Hence dengue is a global burden as its distribution is expanding geographically. Health agencies such as WHO and CDC should provide immense funding so that extensive research could be conducted to defeat dengue. The demand for the therapeutic is high as the reported numbers of cases are 400 million per year.
In this review various small molecules belonging to different categories like carbohydrates, proteins, tannins, tetracycline derivatives etc. are discussed as they inhibit several stages of viral life cycle still there is no licensed vaccine or a therapeutic available. Some chemical compounds like Chloroquine and Balapiravir inhibited entry and replication of the virus, respectively, subsequently undergone in vivo studies but showed very insignificant efficacy. The problem with most of the drugs is that they show reliable results during in vitro analysis but further they could not be taken for a clinical trial because of lack of a simple animal model for testing. Presently, in silico studies are the most promising technique to predict the efficacy of antiviral drugs on their target sites. To understand the chemistry, physicochemical and pharmacological properties, bioavailability and binding patterns of these compounds, an expert personnel is required. As dengue is a global threat, all the nations’ should participate and play a significant role in the eradication of dengue.


Acknowledgment

The authors are grateful to the Vice Chancellor, King George’s Medical University (KGMU), Lucknow and Director, Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research (CSIRCCMB), India for the encouragement and support for this work. SK Saxena is also supported by CCRH, Government of India, and US NIH grants: R37DA025576 and R01MH085259. The authors have no other 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 apart from those disclosed.


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