Exploring Host Factors of the Human Metabolism as Promising Targets for Dengue Treatment

Abstract

The absence of specific therapy and the challenges posed by currently available palliative drugs, such as paracetamol, underscore the urgent need for targeting medications against dengue. Extensive research in the field of antiviral therapies has primarily focused on investigating viral proteins as potential targets. However, despite these efforts, finding an effective therapy for dengue fever remains a daunting task. Importantly, like all viruses, Dengue virus relies on human host proteins to enable infection. Recognizing this fact has prompted the consideration of host factors as viable targets for intervention strategies to combat the infection. This chapter aims to provide an overview of host-virus interactions during Dengue virus infection, emphasizing the importance of metabolic pathways, as well as molecular and cellular processes such as lipid metabolism, autophagy, apoptosis, and the immune system, which are critical for virus propagation. The main goal here is to expand the list of human factors that could serve as potential drug targets. Additionally, molecules that interact with these factors are explored for their therapeutic potential. This comprehensive exploration of host-virus interactions lays the groundwork for more effective dengue treatments. The molecules highlighted here hold promise as antiviral agents, and their inclusion in repurposing research could expedite the development of therapies for dengue fever.

1. Introduction

Several arthropod-borne viruses, also known as arboviruses, are transmitted between vertebrate hosts and arthropod vectors, and pose a major threat to human health worldwide since they can cause significant morbidity and mortality, making it crucial to combat their spread and protect individuals globally [1, 2]. Most arboviruses causing human disease belong to four families: Flaviviridae, Togaviridae, Peribunyaviridae, and Phenuiviridae [3].

Among them, Dengue virus (DENV)—genus Flavivirus, Flaviviridae family—emerges as the most prevalent arbovirus worldwide. DENV is the primary causative agent responsible for dengue fever, a mosquito-borne viral illness that affects millions of people worldwide. Aedes aegypti and Aedes albopictus mosquitoes are the main vectors for DENV transmission [4, 5]. DENV is grouped into four serotypes (1–4), which display limited cross-protection immunity due to antigenic and genetic differences. Infection with any of these serotypes can result in self-limiting dengue fever or progress to severe dengue hemorrhagic fever and dengue shock syndrome [5].

The structural characteristics of DENV particles have been elucidated through electron microscopy, revealing virions that consist of an electron-dense core enveloped by a lipid bilayer [6]. The DENV genome is comprised of a single-stranded, positive-polarity RNA, spanning approximately 11 kb in length. This RNA encodes a total of ten proteins: three structural proteins, namely core (or capsid), pre-membrane (prM), and envelope (E), responsible for forming the viral particle, and seven nonstructural (NS) proteins, denoted as NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, which play vital roles in viral genome replication and multiplication [6, 7].

Dengue has attained endemic status in various regions, including the Americas, the African continent, Eastern Mediterranean, South-East Asia, and the Western Pacific. Notably, over the past decade, cases have been reported in some European countries, showing its expanding geographical range [8]. Global records indicate a gradual increase in dengue incident cases, deaths, and disability-adjusted life years (DALYs) from 1990 to 2019, with larger epidemics occurring cyclically every 3 to 5 years [9, 10]. In 2019, the Americas reported the highest number of dengue cases, surpassing 3.1 million [5, 9]. The dengue fever burden is influenced by various factors, including socioeconomic development, climate warming, and population mobility. Research plays a pivotal role in reversing the escalating trend of DENV infection by improving methods, systems, and strategies for its treatment, prevention, and disease control.

To date, there are no antiviral agents or universal vaccines available for the treatment or prevention of dengue. Currently, the treatment for dengue primarily focuses on providing high-quality patient care to alleviate symptoms and minimize complications. Severe cases of dengue affect approximately 500,000 individuals per year and carry a mortality rate of up to 10% for hospitalized patients and 30% for non-hospitalized patients [11]. In cases where patients experience clinical manifestations, the use of paracetamol as an antipyretic and analgesic has been widely recommended [12, 13]. However, it is important to note that although it poses no significant risk at therapeutic doses, paracetamol can be hepatotoxic at high doses, and DENV infection often affects the liver and can lead to acute liver failure with fatal consequences. Then, considering these facts, when hepatocytes are already under stress due to DENV infection, the prolonged use of paracetamol can significantly impact the severity of the disease and exacerbate hepatic dysfunction [14, 15, 16].

Considering the lack of specific therapy, and the challenges associated with paracetamol use, there remains an urgent and imperative need to develop effective medications for dengue [11]. Extensive research in the field of antiviral therapies has explored both structural and nonstructural viral proteins as potential targets for inhibiting virus entry, viral genome replication, and virus maturation. Additionally, current research efforts have also focused on host factors as potential targets for the development of new therapies.

Investigating inhibitors of host processes involved in viral morphogenesis holds promise for the development of valuable therapeutics [17, 18]. However, traditional drug discovery entails a process that can take several years for development and approval. It involves significant time and resources, requiring meticulous research, rigorous testing, and regulatory approval before a new drug can be brought to market [19].

These challenges and issues can be addressed through the strategy of drug repositioning/repurposing, which involves exploring new indications for approved drugs or revitalizing failed drug candidates. This approach offers a promising solution by leveraging existing knowledge and resources to potentially identify alternative therapeutic applications for known drugs [20]. Presently, numerous studies are underway to identify drugs that have the potential to be repurposed for dengue treatment. One of the primary strategies employed involves identifying specific molecular targets associated with DENV multiplication and disease progression. A comprehensive understanding of the virus biology and its interactions with host cells is crucial for this approach [21]. Once these targets are identified, the studies focus on identifying existing drugs that can bind to these targets and regulate their activity, offering potential avenues for therapeutic intervention [22].

This chapter aims to provide a comprehensive overview of the current knowledge of the interaction of DENV with human host factors, as well as the modulation of these host factors during infection. The host factors discussed in this chapter play essential roles in lipid metabolism, the immune system pathways, and the autophagy and apoptosis processes. By identifying these elements, we can explore potential drug targets to develop effective therapeutics for dengue fever. Moreover, this review aims to identify existing molecules that interact with the host factors identified as potential drug targets such as antagonists, agonists, or inhibitors. These molecules could be explored in future studies to enhance our understanding of the role of host factors during infection and to investigate their potential as antiviral agents.

2. Dengue virus infection mechanisms and therapeutic implications

Dengue virus infection begins when the virus particle is released from the salivary gland of female mosquito vectors via saliva into the skin of a mammalian host [23]. DENV binds to a variety of host cell receptors, infecting a diverse cell type that includes but is not restricted to epithelial cells, fibroblast, monocytes, macrophages, dendritic cells, B cells, T cells, endothelial cells, and hepatocytes [23, 24]. Although there are several human host receptors that mediate DENV entry, such as dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), cluster of differentiation 4 (CD4), heat shock protein 90 (HSP90), heat shock protein 70 (HSP70), mannose receptors, T cell immunoglobin and mucin domain 1 (TIM-1), AXL, Claudin-1, it is known that the viral surface E protein is responsible for interacting with the cell membrane receptors and virus attachment to the cell [23, 25, 26].

The envelope (E) protein is a membrane glycoprotein that is differentially glycosylated according to the DENV serotype and the cell type in which the virus is propagated, being important for humoral immunity. The interaction between the E protein and its receptors initiates a cascade of events that affect both the viral particle and the cell membrane, as well as the cytoskeleton, facilitating the virus entry [24]. DENV entry can occur through two main mechanisms: direct fusion of the virus with the cell membrane or clathrin-dependent endocytosis. The specific mechanism depends on the tropism or infectivity of the virus, which is influenced by the DENV serotype and the host cell type. Once inside the endosomes, which have a mildly acidic pH range of 6.2–6.4, conformational changes occur in the E protein, leading to the release of the viral nucleocapsid into the cytoplasm. Following uncoating, the DENV genome becomes available for translation and replication through the host cell machinery [24, 27, 28].

Then, the DENV RNA genome is transported to the endoplasmic reticulum (ER), where it becomes accessible for two crucial processes: translation to produce a polyprotein and genome replication [23, 24]. It is important to note that the replication of the viral genome is coupled to the translation of the polyprotein, as viral proteins are necessary for RNA replication.

During translation, the viral positive-stranded genomic RNA is utilized as a template to synthesize a polyprotein. This polyprotein is subsequently cleaved by a combination of host cell enzymes and the viral NS3 protease complex, resulting in the production of three structural and seven nonstructural proteins [29, 30, 31]. While structural proteins are constituents of the virion, nonstructural proteins assemble into the replication complex and drive the invagination of the ER membrane forming specialized compartments known as viral replication complexes [23, 32]. The RNA-dependent RNA polymerase (RdRp) activity of the viral NS5 protein, along with the viral protease/helicase NS3 and other viral NS proteins collectively known as the replication complex, catalyzes the enzymatic reaction involved in RNA synthesis [33]. Thus, the replication complex replicates the viral RNA through a negative-stranded RNA intermediate, producing a positive-stranded RNA that is packaged into new nucleocapsids and envelopes, forming immature particles [30, 31] that undergo maturation through the Golgi and trans-Golgi membrane networks and are released into extracellular space from the infected cell [34, 35].

These intricate steps within the viral entry and replication processes offer invaluable insights into the underlying mechanisms of DENV infection, spotlighting the complex interplay between viral and cellular constituents. Likewise, throughout this course and within the context of infection, the virus adeptly modulates several host proteins to facilitate optimal replication, virion production, and successful infection. This comprehensive understanding presents a significant opportunity for the development of focused antiviral strategies aimed at disrupting critical stages of infection and replication. Ultimately, this holds the promise of effectively controlling and managing DENV infections.

In the subsequent sections, we highlighted the involvement of lipid metabolism, autophagy, apoptosis processes, the immune system, and signal transduction pathways in the context of DENV infection. We also explored the complex interplay of DENV with genes and proteins within the host, intricately linked with these pathways. Additionally, we investigated the modulation of genes and proteins within these pathways during infection, thereby enhancing the virus’s propagation. Considering these findings, we pointed out host factors operating within these pathways that warrant closer examination as potential antiviral drug targets. Furthermore, we present a comprehensive exploration of molecules, compounds, and drugs that interact with these host factors. These potential therapeutic agents could serve as valuable tools in future studies aimed at therapeutic development or a deeper investigation into the multifaceted role of host factors in the landscape of DENV infection.

3. The intimate relationship between lipid metabolism and Dengue virus infection

Viruses do not possess the machinery for lipid synthesis, so most of them, such as flaviviruses, depend on and influence the host’s lipid metabolism pathways to obtain energy to complete their multiplication cycle [36, 37]. Indeed, lipid metabolism plays a crucial role in the cellular entry, replication, and assembly of the Dengue virus. Several key molecules and pathways involved in de novo fatty acid (FA) synthesis, lipid droplet formation, cholesterol biosynthesis, and lipoprotein metabolism have been identified as important factors in DENV infection. Understanding the interplay between DENV and lipid metabolism provides valuable insights into potential therapeutic targets and strategies for combating dengue fever. Further research in this field will contribute to unraveling the mechanisms underlying lipid-mediated processes and may lead to the development of novel antiviral interventions.

3.1 Fatty acid synthesis

Multiple studies have provided evidence demonstrating the involvement of molecules associated with de novo fatty acid synthesis in the successful infection of DENV.

De novo fatty acid synthesis occurs via acetyl coenzyme A (acetyl-CoA) carboxylation using adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). Acetyl-CoA carboxylase (ACC) catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA [37]. The enzyme fatty acid synthase (FAS) utilizes acetyl-CoA, malonyl-CoA, and NADPH as substrates to catalyze the synthesis of the saturated fatty acid palmitate. FAS can also produce shorter fatty acids such as stearate. Furthermore, the lipids synthesized by FAS are stored in lipid droplets [38].

The inhibition of ACC caused a notable reduction in DENV-2 proliferation both in vitro and in vivo [38, 39]. The NS3 protein interacts with FAS, facilitating the recruitment of this enzyme to sites where cytosolic replication complexes are formed, thereby enabling fatty acid generation. Moreover, DENV-2 infection leads to an upregulation of FAS activity [40]. In addition, Tongluan et al. described that the knockdown of FAS led to a decrease in DENV-2 and DENV-4 production, indicating that DENV exploits the fatty acid biosynthetic pathway to establish its replication complexes [41].

These reports underscore the crucial role of de novo fatty acid synthesis in DENV infection, revealing potential targets for antiviral strategies.

3.2 Lipid droplets

It is known that lipid droplets (LDs) have an important role in the process of de novo fatty acid synthesis. LDs are organelles that originate from the endoplasmic reticulum (ER) through the accumulation of neutral lipids such as triacylglycerols (TAGs) and sterol esters, which are synthesized by diacylglycerol acyltransferases (DGATs) and cholesterol acyltransferases (ACATs/SOATS), respectively [42, 43].

Studies already demonstrated that DENV infection activates lipophagy, leading to an increase in the release of free fatty acids (FFAs) from LDs. Concurrently, the virus induces β-oxidation, thus promoting energy production [44, 45]. Moreover, transcriptomic analysis showed a downregulation of acat2 in hepatocytes infected with DENV-2 [46].

In addition to lipids, LDs are composed of several proteins, such as seipin, perilipins, fat storage-inducing transmembrane proteins, and ER-shaping proteins [47]. Among these, the perilipins play a critical role in mediating the binding of DENV capsid to LDs, with particular significance attributed to perilipin 3 [48].

Transmembrane Protein 41B (TMEM41B) and Vacuole Membrane Protein 1 (VMP1) are ER-associated proteins that play a crucial role in the mobilization of LDs [49, 50]. TMEM41B- and VMP1-deficient human embryonal kidney cells resulted in the accumulation of lipids, FA and triglyceride decrease, impaired β-oxidation, and a significant reduction in DENV infection across all serotypes. These findings highlight the involvement of TMEM41B and VMP1 in lipid mobilization, β-oxidation, and consequently, in DENV replication [50].

The inhibition of subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), a modulator of lipid homeostasis, resulted in decreased formation of LDs and attenuated DENV infection [51]. Likewise, the inhibition of FAS leads to a decrease in LD levels and impairs DENV-2 assembly [52].

Ancient ubiquitous protein 1 (AUP1), a type-III membrane protein, and regulator of LDs serves as a crucial host cofactor involved in DENV assembly and production [45, 53]. AUP1 interacts with NS4A and NS4B, stimulating lipophagy to generate the energy necessary for DENV replication. Notably, the knockdown of aup1 decreases viral replication and significantly impacts virus assembly [45].

Studies documenting the interaction between DENV-2 capsid protein and LDs have demonstrated the pivotal role of this interaction in manipulating specific intracellular ion concentrations, thereby establishing an advantageous environment for viral multiplication. Consequently, this interaction decreases the production of infectious virions [48, 54]. Additionally, individuals infected with DENV exhibit altered levels of LDs [55].

Hence, DENV exploits LDs and their modulators to promote viral infection and replication. Indeed, further studies to investigate the roles of ACATs and DGATs in DENV pathogenesis would contribute to unraveling the mechanisms underlying LD-mediated processes. These investigations can potentially provide valuable insights into additional targets within LD biogenesis.

3.3 Cholesterol biosynthesis

The process of cholesterol biosynthesis not only influences cellular cholesterol levels but also plays a significant role in DENV infection. The connection between cholesterol metabolism and DENV infection is multifaceted, with various key enzymes and pathways serving as potential targets for antiviral strategies. Understanding and manipulating these pathways offer promising avenues for combating dengue and mitigating its severity.

The biosynthesis of cholesterol begins with the conversion of two molecules of acetyl-CoA into acetoacetyl-CoA by ACATs. Subsequently, the condensation of acetoacetyl-CoA and acetyl-CoA, catalyzed by HMG-CoA synthase (HMGCS), forms 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). HMG-CoA reductase (HMGCR) then synthesizes mevalonate from HMG-CoA. Mevalonate is further converted into activated isoprenes that undergo condensation reactions leading to the formation of squalene. Sequential reactions then transform squalene into cholesterol [56, 57].

It has been reported that hepatocytes infected with DENV-2 exhibit elevated cholesterol levels. The inhibition of the Niemann-Pick C1-Like 1 (NPC1L1) receptor, which is essential for cholesterol uptake, leads to a significant reduction in infection and viral RNA synthesis [58, 59]. Patients diagnosed with dengue fever exhibit lower levels of cholesterol in their blood, which has been associated with an increased risk of developing severe dengue [60].

Blocking cholesterol trafficking in DENV-2-infected cells resulted in the inhibition of cholesterol biosynthesis and a decrease in virus multiplication [61]. The inhibition of sterol regulatory element-binding protein 2 (SREBP2), a regulator of cholesterol biosynthesis, resulted in the downregulation of genes coding for other key enzymes involved in cholesterol biosynthesis, including HMGCS, HMGCR, and low-density lipoprotein receptor (LDLR). This downregulation led to a decrease in cholesterol biosynthesis, which impaired virus yield, genome replication, and viral protein synthesis [62].

The enzyme diphosphomevalonate decarboxylase (MVD) plays a crucial role in cholesterol biosynthesis, and mvd knockdown effectively inhibited endogenous cholesterol production as well as virus multiplication. Additionally, inhibiting key enzymes involved in the cholesterol syntheses pathway, such as HMGCR and squalene synthetase, had a significant impact on virus replication [63].

Furthermore, the inhibition of 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12), an enzyme involved in the steroid metabolism pathway, resulted in decreased DENV replication and reduced production of infectious viral particles [64].

3.4 Lipoproteins and apolipoproteins

Triglycerides and cholesterol are transported through the bloodstream in association with proteins known as lipoproteins [65]. Lipoproteins are macromolecular complexes composed of lipids and specific proteins. They consist of a core of neutral lipids, including triglycerides and cholesterol esters, enclosed by phospholipids, free cholesterol, and apolipoproteins [66, 67]. The classes of lipoproteins include chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), intermediate-density lipoprotein (IDL), high-density lipoproteins (HDL), and lipoprotein (a) (Lp(a)) [66, 68, 69, 70, 71].

Plasma analysis of patients infected with DENV, regardless of the severity of the infection, revealed decreased levels of VLDL [72]. However, contrasting these findings, another study observed a notable increase in VLDL levels in the serum of patients with severe dengue [73]. Previous reports have documented the interaction between the capsid protein of DENV-2 and VLDL [74]. Moreover, patients diagnosed with dengue fever exhibit elevated levels of high-density lipoprotein (HDL) in their serum and lower concentrations of LDL in their plasma [72, 73]. Additionally, the interaction between NS1 and HDL has been documented in the plasma of individuals with dengue fever [75]. Interestingly, fluorescence microscopy analysis has provided evidence that synthetic peptides derived from the DENV envelope colocalize with LDL on the cell surface, indicating that LDL may serve as a mechanism for viral entry [76]. These findings provide valuable insights into the potential role of lipoproteins in the pathogenesis of dengue, highlighting their potential involvement in the early stages of DENV infection and the virus replication cycle.

Apolipoprotein AI (ApoAI) is the primary protein constituent of HDL and plays a crucial role in cellular cholesterol transport [77]. ApoAI is essential for HDL assembly by functioning as the receptor for cholesterol uptake from cells [77, 78]. Several studies have unveiled the involvement of ApoAI, as well as human proteins that interact with ApoAI, in DENV infection. A yeast two-hybrid assay study revealed the interaction between ApoAI and the protein NS2A of DENV-2 [79]. It has been suggested that ApoAI interacts with DENV-2, facilitating the virus’s attachment to host cells and increasing its infectivity in pro-monocytic cells. Coelho et al. demonstrated that NS1 interacts with ApoAI, and infected individuals exhibit decreased levels of this protein, implying that DENV infection reduces ApoAI levels and consequently increases inflammation [80]. Furthermore, the levels of ApoAI proteins are significantly increased in individuals infected with DENV [81]. Conversely, the transcript levels of apoaI and other apolipoproteins have been found to be downregulated in hepatocytes infected with DENV-2 [46]. The differential regulation of apoaI suggests a potential role for these molecules in the host response to DENV infection and may indicate a disruption in lipid metabolism and transport processes. Further investigation into the functional implications of these changes could provide valuable insights into the interplay between DENV and host lipid homeostasis. ApoAI interacts with membrane lipid transporters, including those of the ATP-binding cassette (ABC) family and scavenger receptor class B type 1 (SR-BI), in the extracellular environment to accumulate lipids and facilitate the formation of fully developed HDL particles [82]. Proteins from the ABC family and SR-BI have been implicated in the entry and cell infection of various viruses, including the human immunodeficiency virus (HIV), and hepatitis C virus (HCV) [83, 84, 85, 86]. Additionally, SR-BI has been identified as a cell surface binding receptor for NS1 and appears to mediate viral entry and enhance infectivity in hepatocytes [87, 88].

Regarding other apolipoproteins, interactions have been observed between DENV-2 PrM and the nonstructural proteins NS2A and NS5 with Apolipoprotein AII (ApoAII) and Apolipoprotein B (ApoB), respectively [79, 89]. Furthermore, Apolipoprotein E (ApoE) interacts with DENV protein C [90]. Interestingly, the knockdown of apoe did not lead to a decrease in DENV-infected particle production; however, it did have an impact on the assembly and maturation of HCV [91].

Lipoproteins’ and apolipoproteins’ interactions with DENV provide valued insights into their significant roles in various stages of the infection process, suggesting a multifaceted connection between lipid homeostasis and the virus. Further investigations are required to uncover potential targets for antiviral interventions.

3.5 Lipid rafts

Lipid rafts are microdomains on the cell membrane characterized by the association of cholesterol, sphingolipids, and protein receptors [92, 93].

Analysis of endothelial cells infected with DENV-2 revealed the colocalization of Caveolin 1 (Cav-1) and flotillin 1 (Flt-1), which are lipid raft markers, with NS2B and NS3 proteins. However, this association was found to be stronger with Cav-1 [94].

The protein disulfide isomerase (PDI), which is localized on lipid rafts, interacts with the NS1 protein, and, upon cholesterol inhibition, PDI and lipid raft expression are increased during DENV infection. These data provide evidence for the role of PDI and lipid rafts in DENV cell entry and potentially in its replication [95].

Following the use of lipid raft disruptors, Soto-Acosta et al. reported a decrease in DENV-2 and DENV-4 yield, as well as NS1 secretion, providing further evidence for the involvement of lipid rafts in virus cell entry. An increase in lipid raft concentration was also observed following DENV-4 infection [96]. Additionally, Coelho et al. also demonstrated that DENV infection triggers the accumulation of lipid rafts by activating inflammation through NS1, which facilitates virus attachment. Nevertheless, the induction of lipid rafts can be impeded by the presence of ApoAI [80].

In summary, lipid rafts emerge as crucial microdomains for DENV infection, with their characteristic components, such as cholesterol and sphingolipids, playing pivotal roles.

3.6 Acyl-CoA thioesterases and other lipids

Acyl-CoA thioesterases (ACOTs) are enzymes involved in the regulation of β-oxidation of fatty acids by hydrolyzing acyl-CoA into free fatty acids (FFAs) and coenzyme A [97, 98]. Acyl-CoA thioesterase 2 (ACOT2) and Acyl-CoA thioesterase 7 (ACOT7) have been implicated in DENV-2 replication and infectivity, as acot2 and acot7 knockdown resulted in reduced viral particle number and infectivity. However, the combined knockdown of acot1 and acot2 had the opposite effect, causing an increase in viral replication [99].

High expression of stearoyl-CoA-desaturase (SCD), an enzyme involved in the conversion of saturated fatty acids to monounsaturated fatty acids, has been observed during the early stages of DENV-2 infection. Interestingly, suppression of SCD significantly reduced DENV-2 replication [100, 101].

Inhibition of neutral sphingomyelinase 2 (nSMase2), an enzyme involved in the hydrolysis of sphingomyelin, a key phospholipid, has demonstrated potent antiviral effects against West Nile virus (WNV) and Zika virus (ZIKV) infections, as it led to a reduction in viral yield. However, the involvement of nSMase2 in DENV infection remains to be explored [102].

3.7 Molecules targeting host factors from lipid metabolism

Several host factors involved in lipid metabolism have been identified to interact with specific drugs and compounds such as statins, soraphen-A, orlistat, and hesperetin [103]. Additionally, extensive research has been carried out to investigate the effectiveness of these molecules against DENV.

Statins, a class of HMGCR inhibitors, have been extensively studied to explore their potential in treating dengue fever (Table 1). Soraphen-A, a compound that targets ACC2, demonstrated remarkable efficacy in reducing DENV-2 viremia in infected mice [119]. Orlistat, a FAS inhibitor, exhibited potent antiviral effects by suppressing replication and infection of all DENV serotypes [107]. Hesperetin, an Acyl CoA: cholesterol acyltransferase 1 (ACAT1) inhibitor, effectively interacted with and inhibited the activity of DENV-2 NS2B/NS3 [104].

Host target	Molecule	Action	Drugbank ID	Studies involving DENV
ABC subfamily A member 1	Glyburide	Inhibitor	DB01016	—
	Probucol	Inhibitor	DB01599	—
ABC subfamily B member 5	Biricodar	—	DB04851	—
	Dofequidar	Inhibitor	DB14067	—
ABC sub-family C member 8	Gliclazide	Binder	DB01120	—
	Nateglinide	Inhibitor	DB00731	—
	Repaglinide	Inhibitor	DB00912	—
	Mitiglinide	Inhibitor	DB01252	—
	Glipizide	Inhibitor	DB01067	—
	Tolbutamide	Inhibitor	DB01124	—
	Chlorpropamide	Inhibitor	DB00672	—
	Gliquidone	Inhibitor	DB01251	—
	Glimepiride	Inducer	DB00222	—
	Glymidine	Inducer	DB01382	—
ABC sub-family C member 9	Glyburide	Modulator	DB01016	—
	Nicorandil	Activator	DB09220	—
ABC sub-family G member 2	Biricodar	—	DB04851	—
ACAT1	Hesperetin	Inhibitor	DB01094	[104, 105, 106]
	Ezetimibe	Inhibitor	DB00973	[60]
ACAT2	Hesperetin	Inhibitor	DB01094	[104, 105, 106]
ACC1	Biotin	Cofactor	DB00121	—
ACC2	Biotin	Cofactor	DB00122	—
	Soraphen A	—	DB02859	[107]
	Haloxyfop-P	—	DB07870	—
ACOT	2-Undecanone	—	DB08688	—
ApoAI	Copper	—	DB09130	[108, 109]
	Zinc	—	DB01593	[110, 111, 112]
	Zinc acetate	—	DB14487	—
	Zinc chloride	Inducer; Ligand	DB14533	—
	Zinc sulfate	Inducer; Ligand	DB14548	—
ApoB and ApoE	Zinc acetate	—	DB14487	—
	Zinc chloride	Antagonist	DB14533	—
	Zinc sulfate	Antagonist	DB14548	—
FAS	Cerulenin	Inhibitor	DB01034	[45]
	Orlistat	Inhibitor	DB01083	[42, 107]
HMGCR	Lovastatin	Inhibitor	DB00227	[113, 114, 115, 116]
	Cerivastatin	Inhibitor	DB00439	—
	Simvastatin	Inhibitor	DB00641	[115, 117]
	Atorvastatin	Inhibitor	DB01076	[115, 117]
	Rosuvastatin	Inhibitor	DB01098	—
	Fluvastatin	Inhibitor	DB01095	[64, 117, 118]
	Pravastatin	Inhibitor	DB00175	[117]
	Mevastatin	Inhibitor	DB06693	[64, 118]
	Pitavastatin	Inhibitor	DB08860	—
	Meglutol	—	DB04377	—
	TAK-475	—	DB05317	—
	Nabiximols	Stimulator	DB14011	—
HMGCS	(7R,12R,13R)-13-formyl-12,14-dihydroxy-3,5,7-trimethyltetradeca-2,4-dienoic acid	—	DB07740	—
NPC1L1	Ezetimibe	Inhibitor	DB00973	[60]
PDI	Ribostamycin	—	DB03615	—
	Artenimol	Ligand	DB11638	—
	Copper	—	DB09130	[110, 111, 112]
	Zinc	—	DB01593	[110, 111, 112]
	Zinc acetate	—	DB14487	—
	Zinc chloride	Binder	DB14533	—
	Zinc sulfate	Binder	DB14548	—
Perilipin-3	Idursulfase	—	DB01271	—
	Galsulfase	—	DB01279	—
SR-BI	Phosphatidylserine	—	DB00144	—
SREBP2	Omega-3-acid ethyl esters	Inhibitor	DB09539	—
	Doconexent	Inhibitor	DB03756	—
	Omega-3 fatty acids	Inhibitor	DB11133	—

Table 1.

Molecules targeting host lipid metabolism.

The table represents existing molecules that target the described host factors from the lipid metabolism, as identified through a search in the DrugBank Database [103]. The table also displays the specific action of each molecule on the targeted host factor, the corresponding DrugBank ID, and references to studies related to the molecule’s interaction with the Dengue virus or their role during the infection.

Nonetheless, numerous ligands associated with other host factors involved in lipid metabolism remain unexplored or require additional investigation. These include SREBP2 inhibitors, apolipoprotein antagonists, and ATP-binding cassette protein inhibitors (Table 1).

Further investigation into these molecules would contribute to a better understanding of their target role during DENV infection and could potentially establish them as promising candidates for dengue fever treatment.

4. Autophagy and apoptosis processes and their potential influence upon infection with Dengue virus

4.1 Autophagy

Autophagy acts as a defense mechanism against viral infections. However, certain viruses, such as DENV, are capable of modulating autophagy-related genes (ATGs) to enhance their own multiplication and facilitate successful infection [105].

Autophagy is a catabolic process that involves the formation of the autophagosome, which sequesters damaged proteins, organelles, and other cellular components. Subsequently, the autophagosome fuses with a lysosome, forming an autolysosome. Within the autolysosome, the captured material is degraded and broken down into smaller molecules and components that can be recycled and reused by the cell [106].

This process is regulated by a set of key proteins coded by ATGs including serine/threonine-protein kinase Unc-51 like autophagy activating kinase 1 (ULK1) Beclin-1, and microtubule-associated protein light-chain 3 (LC3). ULK1 is involved in autophagy initiation, while Beclin-1 plays critical roles in the generation of phosphatidylinositol 3-phosphate (PI3P) and autophagosome formation. LC3 is essential for autophagosome elongation and cargo recognition [108, 109]. For instance, infection with all DENV serotypes stimulates the expression and secretion of Macrophage Migration Inhibitory Factor (MIF) in hepatoma cells, thereby triggering autophagy and subsequently leading to increased replication and infection of DENV [110]. Depletion of atg5 in hepatomas resulted in a significant decrease in the DENV infection rate and the release of viral particles. These particles were found to be associated with LC3 [111].

The employment of mycophenolic acid, a DENV replication inhibitor, which effectively inhibits viral replication by targeting inosine monophosphate dehydrogenase, resulted in the downregulation of several crucial ATGs, including those coding for Unc-51 like autophagy activating kinase 2 (ULK2), Cathepsin D, transmembrane protein 74 (TMEM74), and estrogen receptor protein 1 (ESR1). Additionally, a significant reduction was observed in the expression levels of lipid-conjugated LC3 (an LC3-phospholipid conjugate (LC3-II)) and p62 proteins [112]. The p62 protein serves as an autophagy adaptor protein and is known to associate with LC3 [113, 114]. These findings indicate a direct correlation between the downregulation of autophagy genes and the decrease in viral replication. Furthermore, p62 specifically targets the capsid of DENV-2, facilitating autophagy. Interestingly, the absence of p62 resulted in heightened susceptibility to DENV-2 infection [115].

The serine/threonine-protein kinase known as the mammalian target of rapamycin (mTOR) comprises two distinct complexes called mTOR complex 1 (mTORCl) and mTOR complex 2 (mTORC2). This signaling pathway holds significant importance in various cellular processes, including the modulation of autophagy, which can be suppressed upon its activation [116]. Endothelial cells infected with DENV-2 exhibited reduced levels of mTOR phosphorylation, along with ATG13 and ULK1, while presenting elevated levels of LC3-II [117]. During DENV infection in vitro, mTORC1 becomes dysregulated, and its inhibition leads to an increase in viral replication through the activation of lipophagy [118, 120]. Inhibition of mTOR resulted in a reduction in the levels of the C protein of DENV-2 [115]. Moreover, Carter et al. observed that DENV infection stimulated mTORC2 signaling and that NS5 interacts with both mTORC1 and mTORC2 [121]. These findings imply that the modulation of the mTOR pathway is one potential mechanism through which DENV infection triggers autophagy.

Recently, high-mobility group box 1 (HMGB1) has emerged as a significant host factor involved in mediating autophagy [122]. The knockdown of hmgb1 in lung cells infected with DENV-2 led to decreased expression of NS5 and C proteins, as well as reduced levels of autophagy markers LC3-II, p62, autophagy-related protein 5 (ATG5), and autophagy-related protein 7 (ATG7). Conversely, stimulating HMGB1 resulted in increased LC3-II levels, decreased p62 levels, and enhanced viral production. Furthermore, when hmgb1 was knocked down, Beclin-1 levels exhibited a reduction, whereas Beclin-1 levels increased upon stimulation, indicating that HMGB1 mediates autophagy modulation during DENV infection through the Beclin-1 pathway [123].

In summary, autophagy initially acts as a cellular defense mechanism against viral replication, but the Dengue virus has evolved strategies to manipulate ATGs and exploit this process for its own benefit. The depletion of ATG proteins, downregulation of autophagy genes, dysregulation of the mTOR signaling pathway, and involvement of other host factors like HMGB1 all contribute to the modulation of autophagy during dengue infection.

4.2 Apoptosis

Apoptosis serves as a cellular process responsible for programmed cell death, predominantly executed by caspases [124]. This cellular death pathway can also serve as an antiviral response.

Apoptosis can be induced through either the extrinsic or intrinsic apoptotic pathway. The extrinsic pathway involves the activation of tumor necrosis factor receptor (TNFR) and FAS-ligand (FASL) receptor, leading to the formation of death receptor adaptor proteins that trigger the apoptotic cascade by activating caspase-8, −3, and − 7 [125, 126]. The intrinsic apoptotic pathway, which is regulated by B-cell lymphoma 2 (BCL-2) family proteins, involves the disruption of mitochondrial outer membrane permeabilization (MOMP) and the activation of caspase-9 [127, 128]. Indeed, viruses employ both pro-apoptotic and anti-apoptotic mechanisms to facilitate their replication and dissemination. Studies have already demonstrated the regulatory role of various DENV proteins.

Dengue virus proteins, C, M, E, NS2B-NS3, and NS5, interact with and modulate apoptosis factors such as death-associated protein 6 (Daxx), receptor-interacting serine/threonine-protein kinase 2 (RIPK2), and nuclear factor kappa B (NF-κB). C protein has been identified as a pro-survival protein, PrM, M, E, NS2A, NS2B, and NS3 have been found to possess pro-apoptotic properties [129, 130].

Apoptosis-linked gene-2-interacting protein X (ALIX) plays a role in vesicular trafficking associated with the initiation of apoptosis [131]. ALX interacts with and colocalizes with the NS3, and when alx is overexpressed, there is a significant increase in viral titer, indicating enhanced viral replication. Conversely, in the absence of alx expression, viral production is significantly reduced, suggesting that ALX plays a critical role in facilitating DENV multiplication [132].

The proteins, such as BCL2-like 1 (BCLX_L/BCL2L1), BCL2-like 2 (BCLW/BCL2L2), and Myeloid cell leukemia 1 (MCL1) of the BCL-2 family, play a crucial role in preventing or delaying cell death as anti-apoptotic proteins. They inhibit the activation of pro-apoptotic proteins, thereby preserving cell survival [133]. MCL1 expression undergoes a reduction upon DENV-4 infection. Additionally, bcl2l1-deficient cells demonstrate higher levels of apoptosis, while mcl1-deficient cells do not exhibit such effects. Furthermore, the inhibition of BCL-2, BCL2L2, and BCL2L1 in DENV-4-infected cells results in diminished viral production alongside augmented cell death [134].

Thus, apoptosis serves as an intrinsic antiviral response. However, in the case of dengue infection, the virus employs both pro-apoptotic and anti-apoptotic mechanisms to support its replication and spread. Various DENV proteins interact with apoptosis factors and modulate their activity, promoting either cell survival or cell death. Proteins like ALIX and MCL1 have been implicated in facilitating DENV propagation or preventing apoptosis, respectively. Inhibiting the anti-apoptotic proteins BCL-2, BCLW, and BCLXL has shown promising results in reducing viral production and increasing cell death in DENV-infected cells.

4.3 Autophagy and apoptosis association

Several studies have revealed a compelling and well-established association between autophagy and apoptosis during DENV infection. Inhibition of autophagy in endothelial cells infected with DENV-2 resulted in an increase in apoptosis, while inhibition of apoptosis led to an elevation in autophagy characterized by elevated LC3 levels [135].

Autophagy is induced during early DENV-2 infection, while apoptosis is suppressed. In the late phase, an augmented cleavage of Beclin-1 coincided with an increase in apoptosis. Interestingly, the knockdown of becn1 led to a reduction in LC3-II levels, indicating a decrease in autophagy. This was accompanied by an increase in apoptosis and a subsequent decrease in DENV-2 replication. Notably, the interaction between NS1 and Beclin-1 was observed, which led to a decrease in Beclin-1 cleavage and subsequently increased autophagy. Additionally, inhibiting autophagy using 3-methyladenine (3-MA) in DENV-infected cells resulted in the accumulation of LC3-II and degradation of p62 during early infection and a further elevation in apoptosis in late infection [136]. Moreover, inactivation of mTORC2 signaling results in increased apoptosis and reduced viral replication. These findings indicate that the virus utilizes mTORC2 activation to suppress apoptosis and promote viral replication [121].

X-linked inhibitor of apoptosis (XIAP)-associated factor 1 (XAF1) was found to be involved in apoptosis induction during DENV-2 infection, whereas Interferon-alpha-inducible protein 6 (IFI6) is known to regulate apoptosis [135, 137]. DENV-2-infected cells that overexpressed xaf1 presented enhanced LC3 protein expression, indicating heightened autophagy. Conversely, DENV-2-infected cells overexpressing ifi6 exhibited lower levels of LC3, suggesting a correlation between autophagy and apoptosis during the infection [135].

Transcriptomic analysis revealed that upregulated genes involved in autophagy and apoptosis were expressed in hepatocytes infected with DENV-2. Among them, the genes encoding the proteins, including caspase-1, −2, and −4, as well as Daxx are already described. In addition, several other genes encoding proteins involved in these processes were also upregulated, for example, immunity-related GTPase family M member 1 (IRGM1), ring finger protein 152 (RNF152), La ribonucleoprotein 1 (LARP1), Endophilin-B1, TANK (TRAF-associated NF-kB)-binding kinase 1 (TBK1), lysosomal-associated membrane protein 2 (LAMP2), and Optineurin (OPTN) [46]. IRGM1 acts as an AMP-activated protein kinase (AMPK) activator, while RNF152 and LARP1 function as downstream regulators of mTORC1 [138, 139]. Endophilin-B1 is involved in the activation of BAX/BAK1 [140]. TBK1 mediates ATG8 and ATG7 phosphorylation and is inactivated by NS2A and NS4B of DENV-1, −2, and −4 [141, 142]. Furthermore, LAMP2 is required for the fusion of autophagosomes with lysosomes and OPTN plays a role in modulating proteins from the LC3 family [142, 143].

Therefore, a dynamic interplay between autophagy and apoptosis is a well-established aspect of DENV infection. Autophagy and apoptosis are intricately linked, with the inhibition of one pathway often resulting in the upregulation of the other. The timing of their activation during DENV-2 infection is distinct, with early autophagy induction and late apoptosis, highlighting the complex temporal regulation of these processes. This network of interactions offers merits further research into the mechanisms of DENV infection and the potential development of antiviral strategies.

4.4 Molecules targeting host factors involved in autophagy and apoptosis processes

Numerous molecules exhibit affinity for host factors involved in autophagy and apoptosis [103] (Table 2). For instance, minocycline, a negative modulator of caspase-1 and -3, demonstrated inhibitory effects on DENV infection and replication [110, 158]. In vitro studies revealed that minocycline effectively inhibits DENV infection by suppressing autophagy induced by MIF. Furthermore, minocycline reduced the secretion of MIF, inhibited the formation of autophagy, and diminished viremia induced by DENV in mice [110].

Host target	Molecule	Action	Drugbank ID	Studies involving DENV
BCLW/BCL2L2	Navitoclax	—	DB12340	—
BCLXL-/BCL2L1	Fostamatinib	Inhibitor	DB12010	—
Beclin-1/BECN1	Estradiol	Binder	DB00783	—
	Estradiol acetate	—	DB13952	—
	Estradiol benzoate	—	DB13953	—
	Estradiol cypionate	—	DB13954	—
	Estradiol dienanthate	—	DB13955	—
	Estradiol valerate	—	DB13956	—
Caspase-1	Minocycline	Negative modulator	DB01017	[125, 144]
	Pralnacasan	—	DB04875	—
	Emricasan	—	DB05408	—
	VX-765	—	DB05507	—
	1-methyl-3-trifluoromethyl-1H-thieno[2,3-C] pyrazole-5-carboxylic acid (2-mercapto-ethyl)-amide	—	DB07733	—
	Z-Val-Ala-Asp fluoromethyl ketone	—	DB07744	—
	3-{6-[(8-hydroxy-quinoline-2-carbonyl)-amino]-2-thiophen-2-YL-hexanoylamino}-4-oxo-butyric acid	—	DB07916	—
	Acetylsalicylic acid	Inhibitor/ Downregulator	DB00945	—
Caspase-3	Minocycline	Negative modulator	DB01017	[125, 144]
	5-[4-(1-Carboxymethyl-2-Oxo-Propylcarbamoyl)-Benzylsulfamoyl]-2-Hydroxy-Benzoic Acid	—	DB03124	—
	Emricasan	—	DB05408	—
	2-hydroxy-5-(2-mercapto-ethylsulfamoyl)-benzoic acid	—	DB06862	—
	methyl (3S)-3-[(tert-butoxycarbonyl)amino]-4-oxopentanoate	—	DB07696	—
	[N-(3-dibenzylcarbamoyl-oxiranecarbonyl)-hydrazino]-acetic acid	—	DB08229	—
	4-[5-(2-carboxy-1-formyl-ethylcarbamoyl)-pyridin-3-YL]-benzoic acid	—	DB08251	—
	(1S)-2-oxo-1-phenyl-2-[(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-5-yl)amino]ethyl acetate	—	DB08497	—
	(1S)-1-(3-chlorophenyl)-2-oxo-2-[(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-5-yl)amino]ethyl acetate	—	DB08498	—
	N-[3-(2-fluoroethoxy)phenyl]-N′-(1,3,4-trioxo-1,2,3,4-tetrahydroisoquinolin-6-yl) butanediamide	—	DB08499	—
	Glycyrrhizic acid	Antagonist	DB13751	[145, 146, 147]
	Acetylsalicylic acid	Inhibitor/Downregulator	DB00945	—
	Incadronic acid	Activator	DB06255	—
	Pamidronic acid	Activator	DB00282	—
	PAC-1	Activator	DB13048	—
	Tributyrin	Activator	DB12709	—
	Oleandrin	Regulator	DB12843	—
Caspase-4	Incadronic acid	Activator	DB06255	—
Caspase-7	Fica	—	DB03384	—
	Emricasan	—	DB05408	—
	Incadronic acid	Activator	DB06255	—
Caspase-8	Bardoxolone	—	DB12651	[148]
	Bryostatin 1	Inhibitor	DB11752	—
	AN-9	Regulator	DB05103	—
	Trichostatin A	Activator	DB04297	—
	Oleandrin	Regulator	DB12843	—
Caspase-9	Pamidronic acid	Activator	DB00282	—
Cathepsin D	S-Methylcysteine	—	DB02216	—
	1H-Benzimidazole-2-carboxylic acid	—	DB03028	—
	2-Morpholinoethylamine	—	DB03096	—
	5-Amino-6-cyclohexyl-4-hydroxy-2-isobutyl-hexanoic acid	—	DB07542	—
	Cyclohexylmethyl-2,3-Dihydroxy-5-Methyl-Hexylamide	—	DB08740	—
Daxx	Calcium citrate	Agonist	DB11093	—
	Calcium phosphate	Agonist	DB11348	—
	Zinc	—	DB01593	[110, 111, 112]
HMGB1	Ethyl pyruvate	—	DB05869	[149, 150]
	Chloroquine	Inhibitor	DB00608	[151, 152, 153, 154]
MIF	7-Hydroxy-2-Oxo-Chromene-3-Carboxylic Acid Ethyl Ester	—	DB02728	—
	3,4-Dihydroxycinnamic acid (caffeic acid)	—	DB01880	[155, 156]
	4-Hydroxyphenylpyruvic acid	—	DB07718	—
	3-(4-Hydroxyphenyl)-4,5-Dihydro-5-Isoxazole-acetic acid methyl ester	—	DB07888	—
	4-Hydroxybenzaldehyde O-(cyclohexylcarbonyl)oxime	—	DB08333	—
	3-Fluoro-4-hydroxybenzaldehyde O-(cyclohexylcarbonyl)oxime	—	DB08334	—
	4-Hydroxybenzaldehyde O-(3,3-dimethylbutanoyl) oxime	—	DB08335	—
	6-Hydroxy-1,3-benzothiazole-2-sulfonamide	—	DB08765	—
mTORC	SF1126	—	DB05210	—
	Rimiducid	Ligand	DB04974	—
	XL765	—	DB05241	—
	Everolimus	Inhibitor	DB01590	—
	Ridaforolimus	Inhibitor	DB06233	—
	Temsirolimus	Inhibitor	DB06287	—
	Sirolimus	Inhibitor	DB00877	[157]
	Pimecrolimus	Potentiator	DB00337	—
	Fostamatinib	Inhibitor	DB12010	—
	GSK-1059615	Inhibitor	DB11962	—
	Oleandrin	Downregulator	DB12843	—
RIPK2	Fostamatinib	Inhibitor	DB12010	—
SPHK2	Opaganib	Inhibitor	DB12764	—
TBK1	Fostamatinib	Inhibitor	DB12010	—
TNFR	Urelumab	—	DB12077	—
	Tasonermin	Agonist	DB11626	—
	Brentuximab vedotin	Binder; Antibody; Regulator	DB08870	—
	Iratumumab	Antibody; Regulator	DB05550	—
	Belantamab mafodotin	Binder	DB15719	—
	Idecabtagene vicleucel	Binder	DB16665	—
	Ciltacabtagene autoleucel	Binder	DB16738	—
	Teclistamab	Antibody; Regulator	DB16655	—
	ENMD-1198	—	DB05959	—
ULK1/2	Fostamatinib	Inhibitor	DB12010	—

Table 2.

Molecules targeting host proteins involved in autophagy or apoptosis process.

The table represents existing molecules that target the described host factors involved in the autophagy or apoptosis process, as identified through a search in the DrugBank Database [103]. The table also displays the specific action of each molecule on the targeted host factor, the corresponding DrugBank ID, and references to studies related to the molecule’s interaction with the Dengue virus or their role during the infection.

Glycyrrhizic acid also suppresses autophagy in cells infected with DENV-2. This suppression is attributed to the inhibition of HMGB1 and leads to a reduction in DENV-2 replication [123, 159]. Furthermore, glycyrrhizic acid derivatives exhibit antiviral activity against both DENV-1 and DENV-2, decreasing virus yield [160]. Consequently, glycyrrhizic acid has garnered attention as a potent inhibitor of dengue fever, holding promise for potential therapeutic applications. Bardoxolone has demonstrated a decrease in virus infectivity in cells infected with the DENV [160]. Conversely, inhibiting HMGB1 with ethyl pyruvate has been shown to enhance DENV-2 propagation [161, 162].

Additionally, caffeic acid has displayed inhibitory effects on DENV-1 infection [163, 164]. Sirolimus has been proposed as a potential candidate for repurposing in the treatment of dengue [20].

Although tested for their effects on dengue fever, a significant portion of these molecules that target host factors involved in autophagy and apoptosis have not been adequately studied regarding their specific impact on dengue infection. This highlights the necessity for further research to comprehensively evaluate their potential antiviral effects in the context of DENV infection.

5. The interaction between Dengue virus and the human immune system: exploring old and new putative targets

The immune system assumes the initial responsibility for viral clearance. Upon DENV inoculation in the skin by the mosquito vector, Langerhans cells and skin macrophages are the primary targets of viral infection. These infected cells subsequently undergo migration to lymph nodes, facilitating the dissemination of the virus throughout the bloodstream and to various tissues when more cells like monocytes, macrophages, and dendritic cells can also be infected [165, 166].

5.1 Pattern-recognition receptors (PRRs)

The primarily DENV-infected cells express specific pattern-recognition receptors (PRRs) including the antiviral innate immune response receptor RIG-I (retinoic acid-inducible gene I), melanoma differentiation-associated protein 5 (MDA5), and endosomal Toll-like receptor 3 (TLR3). These PRRs play a role in recognizing pathogen-associated molecular patterns (PAMPs) and initiating an immune response against the viral infection. The recognition of viral factors triggers a cascade of events, starting with the activation of mitochondrial antiviral signaling (MAVS), followed by the activation of TANK-binding kinase 1 (TBK1) and IκB kinase-ϵ (IKKϵ). These kinases phosphorylate interferon regulatory factor 3 (IRF3) and interferon regulatory factor 7 (IRF7), leading to the production of type I/III interferons (IFNs), proteins with known antiviral properties, during the initial stages of infection [167, 168]. TLR3 and Toll-like receptor 7 (TLR7) activate the recruitment of TIR-domain-containing adapter-inducing IFNβ (TRIF) and myeloid differentiation primary response gene 88 (MyD88). This recruitment leads to the activation of nuclear factor-kappa B (NF-κB) and IRF3 and IRF7, ultimately resulting in the production of IFNs. Elevated levels of IFNs stimulate the complement system, activate the Janus kinase (Jak)/signal transducer and activator of the transcription (STAT) pathway, and elicit an adaptive immune response, all working in synergy to eliminate the viral infection [168, 169].

The PRRs RIG-I and MDA5 belong to the RIG-I-like receptor (RLR) family, which constitutes a subset of DEAD/H-box helicases [170]. DEAD/H-box helicases serve as viral RNA sensors or co-sensors and play a significant role in immune response modulation. The RLR family consists of multiple members that can act as either pro-viral or antiviral factors, highlighting their diverse functions in viral infections [144, 145].

The knockdown of rig-i and mda5 resulted in a decrease in the production of IFNβ, interferon lambda 1 (IFNL1), interferon lambda 2 (IFNL2), interleukin-27 (IL-27), interleukin 1 beta (IL-1β), interleukin 6 (IL-6), and tumor necrosis factor (TNF) during DENV-2 infection [146, 147]. Additionally, the knockdown of rig-i, mda5, and mavs inhibited the expression of CCL2 (macrophage inflammatory protein-1, MCP-1), CCL3 (macrophage inflammatory protein-1α, MIP-1α), and CCL4 (macrophage inflammatory protein-1α, MIP-1β) [147]. Cells infected with DENV-2 and exposed to a RIG-I agonist showed impaired viral infection, while depletion of rig-i increased the infection [148]. Moreover, activation of RIG-I by designed immune-modulating RNAs inhibited DENV-2 infection [149]. Both structural and nonstructural proteins of DENV are recognized for their role in modulating viral infection through the regulation of RLRs. The NS2A and NS2B proteins of DENV-4 were found to negatively regulate rig-i, mda5, mavs, and tbk1, resulting in reduced transcription of ifnβ [150]. Besides, the expression of DENV-2 NS2A led to a decrease in interferon beta (IFN-β) levels by inhibiting RIG-I, whereas the presence of DENV-2 NS2B, NS4A, and NS4B resulted in a reduction of IFN-β expression through inhibition of both RIG-I and MAVS [155]. Regarding structural proteins, studies have demonstrated that DENV-2 prM interacts with MDA5, resulting in the inhibition of IFN-β production [156].

Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) represents another crucial PRR and holds promise as a potential target for dengue therapeutics. It serves as both an attachment factor for DENV and facilitates viral cell entry [157]. The use of DC-SIGN antagonists inhibited viral infection in DENV-2-infected cells [151]. Additionally, blocking DC-SIGN and LDLR has been shown to reduce DENV-2 infection and protect mice from developing illness [152]. Therefore, targeting DC-SIGN holds promise as an effective approach for inhibiting viral infection in dengue therapeutics.

Tripartite motif (TRIM) proteins act as crucial regulators of PRR signaling pathways, modulating RLRs, toll-like receptors (TLRs), DNA sensor cGAS (cyclic guanosine monophosphate–adenosine monophosphate (cGAMP) synthase), and the interferon alpha/beta (IFNα/β) receptors. Consequently, they play a vital role in the host defense against viral infections. TRIM proteins can form complexes with TRIF and stimulator of interferon genes (STING) to potentiate TLR3 and STING antiviral signaling pathways [153]. Notably, overexpression of trim56 and trim69 suppresses DENV-2 propagation in vitro by impairing genome replication, while their knockdown increases viral load [154, 171]. In vivo suppression of trim69 also elevates virus RNA levels and virus titer. Furthermore, TRIM69 colocalizes with DENV-2 NS3 in both in vitro and in vivo assays and with NS2B [171, 172]. Additionally, the knockdown of trim25 increases DENV-2 replication [173, 174]. Therefore, TRIM proteins represent potential antiviral targets for treating dengue fever. Exploring agonists, activators, or their development could be a promising strategy to discover potential drugs. Additionally, the characterization of other proteins from the TRIM family should be conducted to expand our knowledge about these proteins during DENV infections.

The immune response against DENV infection is orchestrated through PRRs, such as RIG-I, MDA5, TLR3, TLR7, DC-SIGN, TRIM, and others. These receptors recognize viral factors and trigger a sequence of events, ultimately leading to the production of interferons and the activation of various immune pathways. The modulation of RLRs by DENV structural and nonstructural proteins further highlights the virus’s strategies to evade host defenses. These multifaceted immune responses to DENV infection present potential targets for antiviral interventions.

5.2 RNA helicases of the DEAD box and related DExD/H proteins

Many studies have elucidated the role of other DEAD/H-box RNA helicases for DENV replication. During DENV-2 infection, ddx3x is upregulated, and its knockdown results in increased virus replication and a decrease in IFN-β transcript levels during initial infection, followed by an increase in late infection. When ddx3x is overexpressed, DENV production is decreased, and the IRF3-responsive PRD (III–I) promoter is activated. Conversely, the suppression of ddx50 and Integrator complex subunit 6 (ints6) leads to a decrease in viral production. These findings suggest that DEAD (Asp-Glu-Ala-Asp) box helicase 3, X-linked (DDX3X) could inhibit DENV replication, while DEAD (Asp-Glu-Ala-Asp) box polypeptide 50 (DDX50) and integrator complex subunit 6 (INTS6) act as pro-viral factors [175].

Like DDX3X, another DEAD-box RNA helicase, DDX25, is also upregulated during DENV-2 infection. The knockdown of ddx25 resulted in impaired DENV replication and an increase in IFN-β transcript levels after DENV-2 infection. As expected, the overexpression of ddx25 led to an increase in viral load and a decrease in ifn-β transcript levels after DENV-2 infection during initial infection, followed by an increase in ifn-β transcript levels in late infection [176]. Therefore, DDX25 appears to have a similar role as DDX3X during DENV-2 infection, acting as an antiviral molecule and playing a crucial role in the regulation of IFN-β pathways during the infection.

Considering the exposed, investigations using agonists of DDX3X and DDX25, as well as antagonists or inhibitors of DDX50 and INTS6, would be useful in identifying potential drug candidates.

5.3 Interferon type I pathways and ubiquitination

Interferon type I pathways are also regulated by ubiquitination, a process catalyzed by ubiquitin-related enzymes such as ubiquitin-conjugating enzyme E2 (UBE2). When the transcript levels of ube2b and ube2j1 are diminished, DENV-2 replication also exhibits a decline. On the other hand, the knockdown of ube2s, ube2u, ube2v1, and ube2i resulted in an increase in DENV-2 replication. During a DENV-2 in vitro infection, an upregulation of ube2j1 expression was observed, and its overexpression resulted in an augmentation of viral replication. Additionally, suppression of ube2j1 resulted in a reduction in ifn-β expression induced by RIG-I-N, tumor necrosis factor receptor-associated factor 3 (TRAF3), and IRF3-5D in DENV-2-infected cells [177]. Collectively, these data suggest that UBE2B and particularly UBE2J1 play a pro-viral role, while ubiquitin-conjugating enzyme 2S (UBE2S), ubiquitin-conjugating enzyme E2U (UBE2U), ubiquitin-conjugating enzyme E2 V1 (UBE2V1), and ubiquitin-conjugating enzyme E2 I (UBE2I) seem to have antiviral roles. However, further studies are required to substantiate these findings.

Throughout DENV-2 infection in vitro, the expression of E3 ubiquitin ligases seven in absentia homolog (SIAH1), SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1), and SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2) is increased, and the knockdown of siah1 and smurf2 resulted in a decrease in DENV-2 titer. Furthermore, during DENV-2 infection in vitro, myd88 expression was diminished, while the knockdown of siah1 induced upregulation of myd88 expression, showing that SIAH1 negatively regulated this TLR signaling adaptor during the infection. Also, the depletion of myd88 did not lead to a reduction in viral titer [178]. So, the inhibition of SIAH1 could maintain or increase MyD88 levels, resulting in a decrease in infection. However, a separate study demonstrated that inhibiting MyD88 using a small molecule resulted in impaired DENV replication [179]. Therefore, investigating whether MyD88 and its modulators, such as SIAH1, act as pro-viral or antiviral factors and validating them as potential targets for antiviral strategies aimed at treating dengue is necessary.

Interferon-regulatory factors (IRFs) also hold promise as potential antiviral targets. In DENV-infected mice, the absence of irf3, irf5, and irf7 prevented severe dengue and promoted survival, whereas irf1-deficient mice succumbed to the infection. Furthermore, the simultaneous depletion of these four genes led to the development of severe disease [180]. These findings suggest that IRF-3, -5, and -7 may contribute to dengue severity. Thus, investigations focused on reducing these IRFs and increasing IRF-1 expressions could offer a promising approach to preventing disease progression to severe cases. Besides, IF16 has been reported to colocalize with DENV-2 NS4B, and the reduction of IF16 activity led to the suppression of genome replication, highlighting its critical role in this process [181].

Individuals with DENV infection exhibit heightened levels of IFN-λ. Additionally, epithelial cells infected with DENV-2 showed increased expression of interferon lambda 1 (IFN-λ1) and interferon lambda (IFN-λ2). The administration of IFN-λ1 and IFN-λ2 induces the inhibition of DENV-2 replication. Notably, this reduction in viral replication is further amplified in the presence of interferon alpha (IFN-α) [182]. Investigations using the bioactive component celastrol have revealed that hepatoma cells infected with all DENV serotypes and exposed to celastrol exhibited an increase in IFN-α production through the JAK-STAT pathway. This induction of the antiviral response was evidenced by the reduction of DENV replication and protein synthesis in hepatoma cells. Celastrol also induced IFN-α production in mice infected with DENV-2, resulting in reduced disease outcome and lethality in these animals [183]. Hence, upregulating interferon alpha (IFN-α) and interferon lambda (IFN-λ) could be a promising strategy to both reduce viral loads and hamper disease progression.

5.4 Complement system and other immune system factors

The complement system comprises diverse proteins that constitute the innate immune response, including the classic, lectin, and alternative pathways. The classical and lectin pathways are activated by pathogen recognition, while the alternative pathway is constitutively active but regulated by the protein factor H (complement factor H (CFH)). Once activated, the complement system initiates an enzymatic cascade that facilitates phagocytosis, lysis of infected cells, and regulation of the inflammatory response. Notably, during viral infections, the complement system plays a significant role in viral clearance, rendering it a promising target for therapy strategies [184, 185].

In individuals infected with DENV, monocytes present in peripheral blood mononuclear cells (PBMCs) exhibit downregulation of complement receptors (CRs) such as cr3 (cd11b) and cr4 (cd11c). Additionally, cd59 is downregulated in monocytes from DENV-infected patients, with cr4 (cd11c) showing more pronounced downregulation in patients with severe disease compared to non-severe cases. In vitro assays have demonstrated that suppressing cr3 in monocytes infected with DENV-2 reduces virus infection, a process mediated by IFN-α and TNF-α [186]. In addition, studies indicated that DENV-2 infection induces an increase in transcript levels of cfh in endothelial cells and monocytes [187].

Both DENV nonstructural and structural proteins also engage with complement system factors. C1q, a pattern-recognition molecule of the classical pathway, binds to recombinant DENV-1 and -2 NS1 and E proteins from all serotypes. Monocytes pre-exposed to C1q exhibited reduced DENV-2 infection, possibly impeding cell entry, as DC-SIGN transcript levels were also decreased [188, 189]. Moreover, NS1 from DENV-2 binds to mannose-binding lectin (MBL), a pattern-recognition molecule of the lectin pathway, and inhibits DENV-2 neutralization mediated by MBL. This suggests that MBL plays a role in impairing DENV virus entry and inhibiting infection, while NS1 binds to MBL to facilitate cell entry [190]. Considering these findings, investigations into compounds that suppress CRs and induce C1q and MBL should be conducted to explore their potential as antiviral drugs.

Regarding the cytokine TGF-β, its levels are elevated during DENV infection. Inhibition of transforming growth factor beta 1 (TGF-β1) in macrophages infected with DENV-2 has been shown to reduce viral load [191, 192]. Accordingly, exploring TGF-β inhibition could provide valuable insights to validate this cytokine as a potential drug target for decreasing viral load and/or assisting in the treatment of the pathogenesis of the disease.

5.5 Molecules targeting host factors from the immune system

As depicted, there are numerous functional investigation studies concerning immune system host factors and DENV infection, but only a limited few explore molecules with affinity for these host factors.

Notably, zinc has been recognized to interact with factor H, and research has indicated that zinc chelation and zinc nanoparticles exhibit anti-DENV-2 activity [193, 194].

Investigations have demonstrated the anti-DENV-2 effects of IFN-λ inhibitors in vitro [195]. Promisingly, there exist inhibitors of TBK1 and TGF-β1 that warrant exploration as potential anti-DENV candidates (Table 3). Additionally, agonists of IFN-λ, TRIM13, and TGF-β1 hold the potential to enrich our understanding of the interactions between these proteins and DENV infection (Table 3).

Host target	Molecule	Action	Drugbank ID	Studies involving DENV
C1q	Etanercept	Ligand	DB00005	—
Factor H (CFH)	Copper	—	DB09130	—
	Zinc	—	DB01593	[125, 144]
	Zinc chloride	Ligand	DB14533	—
	Zinc sulfate	—	DB14548	—
IFN-α	Sifalimumab	—	DB12773	—
IFN-λ	Glucosamine	Inhibitor	DB01296	[195]
	VIR201	—	DB05110	—
	Fontolizumab	—	DB05111	—
	Olsalazine	—	DB01250	—
	Foreskin fibroblast	Agonist	DB10770	—
	Foreskin keratinocyte	Agonist	DB10772	—
	Emapalumab	Neutralizer	DB14724	—
MBL	O3-sulfonylgalactose	—	DB01818	—
	Methyl alpha-D-mannoside	—	DB01979	—
	4-(hydrogen sulfate)-beta-D-galactopyranose	—	DB02837	—
	Methyl beta-L-fucopyranoside	—	DB03194	—
	N-acetyl-alpha-neuraminic acid	—	DB03721	—
	Alpha-L-methyl-fucose	—	DB03879	—
	Alpha-Methyl-N-Acetyl-D-Glucosamine	—	DB04426	—
TBK1	Fostamatinib	Inhibitor	DB12010	—
TGF-β1	Hyaluronidase	Inhibitor	DB14740	—
	Foreskin fibroblast (neonatal)	Agonist	DB10770	—
	Foreskin keratinocyte (neonatal)	Agonist	DB10772	—
	Terazosin	Inducer	DB01162	—
TRIM13	Etorphine	Agonist	DB01497	—
	Diprenorphine	—	DB01548	—
	Dihydromorphine	Agonist	DB01565	—
UBE2	Artenimol	Ligand	DB11638	—

Table 3.

Molecules targeting the human immune system.

The table represents existing molecules that target the described host factors from the immune system, as identified through a search in the DrugBank Database [103]. The table also displays the specific action of each molecule on the targeted host factor, the corresponding DrugBank ID, and references to studies related to the molecule’s interaction with the Dengue virus or their role during the infection.

These prospects, along with other molecules that engage with distinct host factors within immune pathways, suggest promising avenues for further exploration. Therefore, in-depth studies employing these molecules should be actively pursued.

6. Final remarks

It is well established that the Dengue virus interacts with and modulates a wide range of host factors involved in several metabolic processes. This chapter explores human factors associated with autophagy, apoptosis, lipid metabolism, and the immune system. We have identified 99 host factors that, based on various studies, show promise as potential therapeutic targets for dengue treatment and warrant further exploration within this context (see Figure 1).

Figure 1.

Schematic representation of potential host factors as drug targets for dengue fever treatment. The host factors represented in the scheme are associated with various metabolic processes, including autophagy, apoptosis, lipid metabolism, and the immune system. In gray: Host factors with evidence of influencing DENV infection, with unclear mechanisms of interaction and infection suppression. In green: Host factors for which there is evidence suggesting that their activation suppresses DENV infection. In red: Host factors for which there is evidence suggesting that their inhibition suppresses DENV infection. Above is a brief mention of other processes and pathways containing host factors with potential as drug targets for dengue fever treatment, although these were not addressed in this chapter.

Some of these potential targets are activated or upregulated during the DENV infection process. Blocking or inhibiting these factors would block the virus at different stages, such as entry, genome replication, assembly, or production. Conversely, some other host factors are negatively regulated or inhibited during infection, suggesting that their activation may help suppress the infection (see Figure 1).

Lipid metabolism, key molecules, and pathways, such as de novo fatty acid synthesis, lipid droplet formation, cholesterol biosynthesis, and lipoprotein metabolism, play crucial roles in DENV infection, offering potential therapeutic targets for combating dengue fever. Autophagy and apoptosis also play unique roles in DENV infection. The virus modulates autophagy, enhancing its replication through ATGs and the mTOR signaling pathway modulation. Indeed, this process intersects with apoptosis during DENV infection, where the virus initially induces autophagy but later promotes apoptosis, involving various host factors such as ALX and BCL-2 family proteins in this complex interaction. The immune system serves as an antiviral defense but becomes a target during DENV infection. Receptors like RIG-I, MDA5, and DEAD/H-box RNA helicases, including DDX50 and DDX25, modulate DENV replication, while ubiquitination can have pro-viral and antiviral roles. TRIM proteins are pivotal regulators of PRR signaling pathways, with some, such as TRIM56 and TRIM69, capable of suppressing DENV propagation.

In Figure 1, it is possible to note that—focusing on the autophagy/apoptosis processes but mainly the lipid metabolism—most host factors are positively regulated (red) during dengue and act as pro-viral agents (red) during DENV infection and act as pro-viral agents. This is not a surprise since autophagy and apoptosis share certain host factors, as these processes are interconnected during infection. Moreover, autophagy is intricately linked to lipid metabolism and is modulated during DENV infection, primarily to facilitate ATP production that is essential for virus replication. Hence, a positive regulation of host factors involved in these processes is expected. On the other hand, major host factors associated with the immune system are typically downregulated or inhibited (green) during DENV infection. This aligns with the immune system’s role in viral clearance.

Noteworthy, there are other metabolic pathways such as signal transduction pathways, energy and carbohydrate metabolism, and genetic information processing, where studies have identified host factors as potential therapy targets for dengue treatment. Therefore, it is essential to explore these processes in future studies with the aim of discovering and validating host factors as drug targets for dengue therapy.

Despite the multitude of studies exploring host factors and their essential role in successful DENV infection, there is a lack of research dedicated to developing and screening ligands for these host factors to find drugs for dengue therapy. Given this, the chapter provides a comprehensive list of various ligands for these host factors to support the search for specific ligands in future studies (see Tables 1–3). Various molecules targeting host factors within lipid metabolism, autophagy, apoptosis, and the immune system have undergone extensive investigation as potential antiviral agents against DENV. These include statins, soraphen-A, hesperetin, minocycline, glycyrrhizic acid, orlistat, bardoxolone, sirolimus, caffeic acid, zinc, interferon lambda (IFN-λ) inhibitors, IFN-λ agonists, TBK1 inhibitors, and TGF-β1 inhibitors, representing potential alternatives for antiviral intervention.

In conclusion, this chapter emphasizes the need for continued research into host factors as drug targets to develop therapeutic strategies to combat DENV infection.

7. Future directions

Efforts directed at identifying potential drug targets, comprehending their interactions, and investigating existing molecules for therapeutic applications can represent a viable approach to discovering effective antiviral strategies against dengue. Some approved drugs are already designed to impact many of the host factors mentioned in this chapter and warrant exploration in studies seeking potential candidates for drug repurposing. Nevertheless, challenges like clinical validation, safety assessments, and optimization of drug dosages must be addressed for successful drug repurposing.

Additionally, this comprehensive chapter underscores the importance of host-virus interactions in DENV infection and emphasizes the potential of host factors as a strategy for dengue fever therapy. Furthermore, exploring existing drugs or compounds that interact with molecules identified as potential targets for dengue therapy can have broader applications in the context of flaviviruses and other arboviruses. For instance, a validated target for treating dengue fever may also prove effective in treating Zika fever. Similarly, a drug repurposing candidate initially intended for dengue fever treatment can also be considered for repurposing in the context of yellow fever therapy. The drug targets and molecules described here would provide ample prospects for further exploration and investigation.

Acknowledgments

The authors thank the René Rachou Institute - Fiocruz Minas, and the Vice Presidency of Research and Biological Collections (VPPCB) of Fiocruz for their support. We also extend our gratitude to Minas Gerais State Agency for Research and Development (FAPEMIG) for funding (PPE-00038-22, BPD-00023-22, and APQ-01688-23).

Conflict of interest

The authors declare no conflict of interest.