Flavonoid from Carica papaya inhibits NS2B-NS3 protease and prevents Dengue 2 viral assembly

Background

Dengue is a serious public health problem worldwide [1]. There are currently no vaccines available for the prevention and treatment of DENV infection [2]. DENV is a positive – stranded encapsulated RNA virus and is composed of three structural protein genes, which encode the nucleocapsid or core (C) protein, a membrane-associated (M) protein, an enveloped (E) glycoprotein and seven non-structural (NS) proteins [3]. DENV is a member of the genus Flavivirus and belongs to the Flaviviridae family [4]. DENV causes a wide spectrum of clinical illnesses ranging from relatively mild self limiting disease associated with fever, malaise and other non specific symptoms to the more severe, potential lethal disease manifestations that leads to dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) [5]. There are four antigenically distinct, but closely related serotypes of the dengue virus (DENV -1, DENV -2, DENV -3 and DENV -4), that exhibits a 65 – 70% sequence homology [6]. Dengue is transmitted mainly by Aedes aegypti mosquito and to some extent by Aedes albopictus [7]. The DENV-2 is the most widespread of the four serotypes [8]. In India, the earliest isolation of DENV-2 is from Vellore, South India [9]. The two component of viral serine protease, NS2B and NS3,play a crucial role in viral replication as it is required for the synthesis of the polyprotein precursor prior to the assembly of the viral complex [10]. Thus NS2B-NS3 is considered to be a significant target for development of anti-dengue drugs.

Plant derived compounds remain a significant source for the development of new antiviral agents. Studies reveal the importance of phytochemical agents against DENV [11–13]. Carica papaya, commonly known as papaya is a tree-like herbaceous plant. Carica papaya is a member of the plant family Caricaceae. Carica papaya leaf extracts are prescribed as a tonic for heart and also for the treatment of fever, pyrexia, diabetes, gonorrhea, syphilis, inflammation and for dressing foul wounds [14–16]. Previous phytochemical analysis [17] reveals the presence of flavonoids, alkaloids, carbohydrates, saponins, glycosides, phytosterols, phenolics, terpenoids and tannins in the leaves of Carica papaya. Previous report [18] states that the administration of aqueous leaf extracts of Carica papaya exhibits potential anti-dengue activity as indicated by the increased platelet count from 55×103/µL to 168×103/µL, White blood cells from 3.7×103/µL to 7.7×103/µL and neutrophils from 46% to78% in patients with dengue fever.

GCMS analysis of previous report [19] states that the leaves of Carica papaya mainly consists of seven phenolic compounds namely quercetin, protocatechuic acid, p-coumaric acid, caffeic acid, chlorogenic acid, kaempferol, and 5,7-dimethoxycoumarin. However, these compounds are not analyzed for their anti-dengue properties. Hence we attempted this research, and our results suggest that, the flavonoid quercetin has inhibitory activity on NS2B-NS3 protease which is very essential for the viral assembly. We believe our study might be a good starting point for the development of anti-dengue molecules from Carica papaya.

Methodology

Results

Discussion

Dengue infection has been re-emerging as a serious life threat with increase in the infection rates every year. The number of cases of severe dengue disease continues to grow in endemic areas of Southeast Asia, Central and South America, and other subtropical regions [30]. Therefore, there is an urgent need to develop effective anti-dengue compounds to combat this epidemic infection. In the present study we have analysed the activity of compounds in Carica papaya leaf extracts against NS2B-NS3 serine protease of DENV2 virus. DENV2 is a single stranded RNA with type I cap structure at the 5' end and codes for single polyprotein precursor arranged in order NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH [31]. It is well known that DENV NS3 is a multifunctional protein with an N-terminal protease domain (NS3pro) [32]. The viral protease is comprised of two viral proteins NS2B and NS3 that are associated with each other to form a heterocomplex. The N-terminal region of the non-structural 3 (NS3) protease forms complex with NS2B cofactor which is essential for viral replication [33]. Prohibiting the processing and release of the viral proteins from the polyprotein precursor would inhibit viral genome replication, thus reducing the number of virion progency produced. Since NS2B-NS3 protease plays a central role in the viral life cycle, it acts as an attractive therapeutic target for antiviral compound. Several studies provide considerable evidence for the importance of NS2B-NS3 protease as potential drug target [34,35]. Receptor-ligand bindinginteraction studies carried out by performing docking of the ligands that are found to be competitively inhibiting the activities of the DENV-2 NS2B-NS3 serine protease. From our analysis we found quercetin as best inhibitors as they show better ligand-enzyme interactions and stability. Our results correlate with the experimental work done by Zandi et al. (2011) [36], which show significant antiviral activity against DENV-2 in Vero cells. Quercetin has also been reported to have inhibitory effects on several viruses [37]. Even though p-Coumaric acid shows best binding affinity in terms of hydrogen bond it shows poor values in case of ligand-receptor atom pair interaction. Considering protocatechuic acid the D score which reflects electrostatic and hydrophobic energy showed the best score whereas all the other parameters gave poor results. Chlorogenic acid, 5, 7-dimethoxycoumarin and kaempferol forms five hydrogen bonds with the active site residues of NS2B-NS3 protease but shows low binding affinity comparing to quercetin. Caffeic acid shows only four hydrogen bond interactions which are the least among the seven compounds analysed. Evaluation of Absorption, Distribution, Metabolism, and Excretion (ADME) properties of lead compounds is a major challenge in the process of drug development [38]. Most drug fails in the drug development process are due to poor pharmacokinetic properties and toxicity [39]. Development of high throughput and fast ADMET profiling assays facilitate the identification of active lead compounds at early drug discovery. ADME profiling into the earliest phase of the discovery process, leads to the development of effective lead compounds in the process of drug design [40]. ADME profiling of active compounds proves that all the compounds except chlorogenic acid have no side effects on absorption. Presence of high-resistance tight junctions between endothelial cells of brain capillaries forms the barrier and prevents the brain uptake of most pharmaceuticals [41]. Experimentally, Blood brain barrier (BBB) is measured as the ratio of the compound concentration in the brain to that in the blood. Knowledge of the penetration of drugs through BBB is one of the key parameters to be optimized in drug discovery [42]. Oral bioavailability often considered as an important parameter to determine the drug likeness of active compounds as therapeutic agents [43]. Oral drug bioavailability can also be markedly influenced by physiological, physiochemical and certain biopharmaceutical factors [44]. High penetration of BBB is required for central nervous system (CNS)-active drugs, whereas for non- CNS low penetration is desirable to minimize CNS-related side effects. Incidence of CNS involvement in patients with dengue infection has been reported in the many studies [45]. Recently two case of meningitis caused by oligosymptomatic dengue has been reported in the city of Kolkata, West Bengal, India [46]. The BBB permeability of compound depends on several factors such as lipophilicity, hydrogen-bond desolvation potential, molecular size and pKa/charge [47,47]. ADMET-associated properties of the active compounds for different types of models such as BBB penetration, P-glycoprotein substrate, renal organic cation transporter, human intestinal absorption and Caco2 permeability showed positive results which strongly supports the ability of compounds to act as drug. Cytochrome P450 (CYP) is group of isozymes involves in the metabolism of drugs, fatty acids, steroids, bile acids and carcinogens. Human genome encodes nearly fifty-seven CYP of which fifteen are involved in the metabolism of drugs and other xenobiotic chemicals [49]. Nearly 75% of phase I drug metabolism depends on the involvement of CYP enzymes [50]. In the case of metabolism, various CYP substrate and inhibitor models are calculated and the result shows that these active compounds are Non-substrate and Non-inhibitor of CYP enzymes. In terms of toxicity, all the compounds are found to be non toxic. Our findings reveal that among the seven compounds screened, quercetin has potential inhibitory activity against NS2B-NS3 serine protease. Further ADME and toxicity risk assessment strongly suggest that quercetin have marked antiviral activity against DENV2 virus. Based on our observations, we suggest that, the flavonoid quercetin in Carica papaya might exert its antiviral activity by blocking the viral assembly mechanism of DENV2 virus. Our results strongly suggest that quercetin is a good candidate for the development of effective anti-dengue compounds. On the whole, we conclude that the flavonoid quercetin could be further investigated and can possibly be developed as an effective anti-dengue compound.

Supplementary material