Therapeutic potential of marine natural products against dengue virus

Abstract

The causative agent for dengue is dengue virus (DENV), with humans and mosquitoes serving as vectors. The DENV (family = Flaviviridae) is a positive-stranded RNA virus with four serotypes, DENV-1, DENV-2, DENV-3, and DENV-4. As of 2023, the disease had been reported in 80 countries with over 6.5 million cases and 7300 mortalities since then. Despite the concerning incidence and prevalence of this disease, less attention is given to the development of therapeutic remedies and preventive alternatives. The search for new antiviral bioactive compounds is therefore important and urgent. Marine organisms have been a source of diverse bioactive natural products. The large marine biodiversity and the structural diversity of their specialized metabolites provide an explorative opportunity for the discovery of novel antiviral specialized metabolites. Reported marine natural products with anti-DENV activities include fucoidan, scequinadoline A, indole derivatives, and others. Nevertheless, marine organisms are still largely underexplored as sources of antiviral drugs. With advances in metabolomics and computational screening, we may discover additional marine natural products to help with dengue treatment.

Key Words: Marine natural products; Dengue; Therapeutic; Antiviral; Virus

Core Tip: The dengue disease, caused by the dengue virus, is mosquito-borne. Recent epidemiological studies of this highly contagious disease revealed its increasing incidence and mortality rates. Despite these concerns, approved therapeutic and prophylactic measures are still lacking. The ocean houses organisms that are prolific producers of specialized metabolites with pharmacological importance. Marine natural products have potential as drug molecules with anti-dengue virus activity.

INTRODUCTION

The dengue virus (family = Flaviviridae), the causative agent of dengue, is a positive-stranded RNA virus with four serotypes, DENV-1, DENV-2, DENV-3, and DENV-4[1]. The dengue disease is currently the most contagious mosquito-borne disease worldwide, transmitted by Aedes aegypti and Aedes albopictus[2]. Following infection, the virus incubates for four to seven days, after which the full-blown disease takes a triad phase – febrile, critical, and recovery phases[3]. Monophasic courses also occur[3]. About 75% of the disease cases are asymptomatic while other clinical presentations could be mild or severe. Severe presentations include hemorrhage, organ impairment-associated shock, and death[3]. As of 2023, over 80 countries were reported to have been affected and since then, over 6.5 million cases and 7300 deaths associated with dengue have been reported[4]. Stanaway et al[5] estimated 9221 mortalities per year between 1990 and 2013, ranging from a low of 8227 in 1992 and peaking in 2010 with 11302 deaths. The increasing trend in dengue incidence and consequent mortality can be linked to globalization and urbanization, causing the emergence of breeding sites for mosquitoes. These factors and a rising global population increase the accumulation of stagnant water and the deposition of waste that either house the mosquitoes or foster their development[6]. Increasing temperatures due to climate change may further worsen the situation, especially in the currently low-risk regions of the world. All these contribute to the predicted global risk population of 63% in 2080[7].

DENGUE VIRAL DRUG TARGETS

Specific treatment options for dengue are currently lacking, as the disease is only managed by either improving the patient’s immunity or controlling the symptoms[3]. Encoded in the viral genome are three structural proteins and seven non-structural proteins, some of which may serve as potential anti-dengue drug targets[8]. The capsid, a structural protein, has been targeted as a retroviral inhibition approach, where the viral capsid protein is fused with a ligand that inhibits the virus[9]. This capsid-targeted viral inactivation approach elicited moderate anti-dengue effect in vitro, and the prophylactic model also decreased the viral infectious titers by 10³ to 10⁴-fold[10]. Viral growth involves steps like viral attachment, viral cell fusion, reverse transcription, integration, and translation, which are potential targets for antiviral drug molecules[11]. Crucial enzymes involved in the synthesis of DNA, RNA, and glycoproteins may also be targeted[12]. The knowledge of the structure and replication cycle of the DENV is a vital tool in the development of new anti-dengue drug molecules. While macromolecules like fusion proteins have been developed, small molecules also have potential as antivirals.

ANTI-DENV MARINE NATURAL PRODUCTS

Lately, the marine environment has been a hub for the discovery of specialized metabolites with diverse bioactivities[12]. The MarinLit database, a collection of marine natural products contains 42237 compounds from marine organisms, especially invertebrates and microorganisms. A lot of these compounds have made it to the clinical trial phase while several of them have obtained approval for commercial distribution. The anti-HSV drug, vidarabine, was developed from the lead, spongouridine, which was first isolated from marine sponges[13]. The microbial diversity in the seawater presents great potential for drug development. Marine cyanobacteria, actinomycetes, bacteria, and fungi are prolific producers of alkaloids, terpenes, peptides, polyketides, shikimates, etc, with antitumor, antibacterial, and antiviral activities[12]. While the cyanobacterial-derived anti-HIV cyanovirin N was involved in preclinical studies, the marine microbiomes still lack extensive exploration as sources of anti-viral agents for clinical studies[14,15]. Algae have also been a reservoir for antiviral specialized metabolites. Algal-derived compounds such as sulfated polysaccharides, carrageenan, lectin, fucoidan, and sulfated galactan have been reported to inhibit different stages of viral replication[16]. Their impressive potential as sources of bioactive molecules can be utilized for anti-dengue drug discovery.

Despite the potential of the ocean and drug source, only a few marine-sourced anti-dengue specialized metabolites, including fucoidan, scequinadoline A, and indole derivatives, have been reported (Figure 1). Fucoidan, found in Sargassum spp. polysaccharide extract elicited anti-dengue activity by preventing viral attachment to the host cell[17]. Fucoidan is a sulfated polysaccharide composed of two main monosaccharides, fucose and glucuronic acid. Structure diversity is a major strength of sulfated polysaccharides as antiviral agents[17]. They elicit antiviral activity by blocking viral attachment, adsorption, and reproduction. A rich source of sulfated polysaccharides is marine algae[17]. These polysaccharides have been studied for activity against influenza A, mumps, measles, HSV-1, HIV-1, and human cytomegalovirus[18]. Carrageenan is another polysaccharide from red algae, which moderately reduced viral replication to varying extents in the four dengue serotypes[19]. Scequinadoline A, a specialized metabolite from the marine-derived fungus Dichotomomyces cejpii, inhibited DENV serotype 2 viral production by plaque assay[20]. Scequinadoline A is an alkaloid made up of the pyrazino[2,1-b]quinazoline-3,6-dione and 3,3a-dihydroimidazo[1,2-a]indol-1-one moieties. The two chiral centers on the pyrazino[2,1-b]quinazoline-3,6-dione moiety may be important for activity[20]. Indole alkaloids have also been studied for activity against DENV. Bardiot et al[21] performed a medium-throughput cytopathic effect reduction assay using the CD3 (Center for Drug Design and Discovery, KU Leuven) compound library. This led to the discovery of 2-[(3,4-dimethoxyphenyl)amino]-1-(1H-indol-3-yl)-2-phenylethan-1-one, a novel anti-dengue molecule. Other indole derivatives with significant viral inhibitory activity were also identified[21]. These compounds prevented viral replication by blocking the interaction between NS3 and NS4B viral proteins in vitro[22,23]. A follow-up lead optimization effort yielded two compounds with improved spectrum of activity, ADME, and pharmacokinetic properties[24].

Figure 1 Known marine natural products with anti-dengue virus activity. A: Fucoidan; B: Indole derivative; C: Scequinadoline A.

Advances in bioinformatic tools and the application of molecular biology to natural product drug discovery give room for rational natural product discovery endeavors. Unlike the traditional trial-and-error and bioassay-guided fractionation approaches, the genomes of marine microbes can be mined for biosynthetic gene clusters responsible for specialized metabolite production. Molecular network generation has also been used to identify metabolites with important pharmacological activities from extracts of marine microbial cultures[25]. Computational methods can also be applied to anti-DENV small molecules from the ocean.

COMPUTATIONAL PERSPECTIVES ON TARGETING THE DENV

Computational methods are increasingly important for identifying potential treatment options for the DENV. By leveraging computational tools, medicinal chemists can identify potential drug targets, perform virtual screening of large chemical libraries, and evaluate the binding and activity of potential lead compounds, thus accelerating the discovery of newer and more effective therapeutic options for the DENV.

The highly conserved serine NS2B/NS3 protease, which functions in both the viral replication process and immune system invasion, is considered the major therapeutic target for anti-flavivirus agents[26-28]. Crystal structures of the DENV NS2B/NS3 protease reveal a negatively charged active site, which favors molecular interactions with positively charged residues such as arginine and lysine. Its flat surface and preference for binding positively charged residues present a significant challenge for developing effective inhibitors[29]. Hence, allosteric sites within the protease are being explored for inhibition. A diverse array of synthetic compounds has been identified as orthosteric and allosteric inhibitors of the DENV NS2B/NS3 protease[28]. Moreover, the synthetic compound NITD-88 is currently being developed in clinical trials as a drug candidate for dengue. NITD-88 acts as an NS4B/NS3 inhibitor, disrupting critical interactions between these two protein counterparts[30].

The potential of plant-based natural products as inhibitors of the DENV protease has been elucidated through molecular docking and molecular dynamics simulation studies. Compounds including rosmarinic acid, niloticin, luteolin, methoxyflavones, diterpenes, diterpenoids, and their derivatives have been shown to inhibit DENV’s non-structural proteins[31-35]. Databases of phytochemicals have also been screened against the dengue viral proteases NS1, NS3/NS2B, and NS5, generating novel scaffolds for drug development against the DENV[36].

A virtual screening of the Comprehensive Marine Natural Products Database, containing 31561 marine compounds, against the DENV NS1 generated hit compounds with good binding affinities, some of which can be further optimized to serve as lead molecules for the development of potential drug candidates for DENV[37]. Based on the conservation of the NS2B/NS3 protease in the flavivirus genus, a multi-target drug development approach is more commonly explored. However, as part of the drug discovery and development process, a more focused approach for the selective targeting of dengue viral protease should also be considered. This can be achieved by investigating other less conserved proteases or exploring novel allosteric sites with less conservation among the flaviviruses.

CONCLUSION

The currently high incidence and mortality rates of the DENV require urgent public health attention. There is yet no approved drug for the treatment and prophylaxis of the viral infection. Therefore, endeavors to discover new anti-DENV drugs need to be heightened. With the ocean being a known source of bioactive specialized metabolites, marine natural products have high potential as sources of these novel drug molecules. Bioinformatic and computational advancements may aid and accelerate these discoveries.