Antiviral phytochemicals identification from Azadirachta indica leaves against HCV NS3 protease: an in silico approach

Abstract Hepatitis C virus (HCV) is a major health problem across the world affecting the people of all age groups. It is the main cause of hepatitis and at chronic stage causes liver cirrhosis and hepatocellular carcinoma. Various therapeutics are made against HCV but still there is a need to find out potential therapeutics to combat the virus. The goal of this study is to identify the phytochemicals of Azadirachta indica leaves having antiviral activity against HCV NS3 protease through molecular docking and simulation approach. Results show that the compound 3-Deacetyl-3-cinnamoyl-azadirachtin possesses good binding properties with HCV NS3/4A protease. It can be concluded from this study that Deacetyl-3-cinnamoyl-azadirachtin may serve as a potential inhibitor against NS3/4A protease. Graphical abstract


Introduction
HCV is characterised as a worldwide health distress which affects about 170 million people worldwide, and 10 million people in Pakistan are affected due to this appalling disease (Ashfaq et al. 2011). HCV is a single-stranded RNA virus with a positive sense strand.

ABSTRACT
Hepatitis C virus (HCV) is a major health problem across the world affecting the people of all age groups. It is the main cause of hepatitis and at chronic stage causes liver cirrhosis and hepatocellular carcinoma. Various therapeutics are made against HCV but still there is a need to find out potential therapeutics to combat the virus. The goal of this study is to identify the phytochemicals of Azadirachta indica leaves having antiviral activity against HCV NS3 protease through molecular docking and simulation approach. Results show that the compound 3-Deacetyl-3-cinnamoyl-azadirachtin possesses good binding properties with HCV NS3/4A protease. It can be concluded from this study that Deacetyl-3-cinnamoyl-azadirachtin may serve as a potential inhibitor against NS3/4A protease. The genome length of HCV is 9.6 kb approximately. This positive sense RNA of HCV has a 5′ non-coding region and a long open reading frame (ORF) followed by a 3′ non-coding region. The ORF encodes a single polyprotein of length 3010 amino acid. This polyprotein further cleaved into nine proteins, i.e. (NH (2)-Core-e1-e2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS 5B-COOH) (Idrees et al. 2013). From all of these HCV proteins, NS3/4A serine protease can serve as an important target in the effort to combat HCV because it is involved in HCV replication. NS3/4A catalytic activity is based on three amino acid residues: His 57, Asp 81 and Ser 139 (Randall et al. 2003). Rapid increase in HCV infection cases and absence of effective treatment required the preparation of particular drug compounds which can target significantly the life cycle of HCV (Ashfaq et al. 2011). In recent years, Azadirachta indica (neem) has gained global significance due to its wide-ranging medicinal activities (Del Serrone et al. 2015). It has considerable antinociceptive, emollient, antiviral, anti-inflammatory, antiseptic, antifungal, astringent, insecticidal, anthelmintic and antibacterial properties (Ilango et al. 2013). More than 140 active phytochemicals were found in neem, including leaf, flower, fruit, seed, root and bark, which have been used for therapeutic purposes (Subapriya & Nagini 2005). It has been reported that phytochemicals of A. indica can assist as a potential drug candidate against HCV NS3/4A by doing few chemical structure modifications (Ashfaq & Idrees 2014). Keeping the above discussion in view, this study was designed to screen the phytochemicals of A. indica leaves against NS3/4A protease using in silico techniques.

Results and discussion
HCV is a serious health issue worldwide. HCV genome encodes three structural and six non-structural proteins. Among non-structural proteins, NS3/4A serine protease and helicase are proved to be effective drug targets to develop anti-HCV drug candidates. Different vaccines and drugs are reported against HCV, but still there is a strong need to develop and identify specific drug compounds which can target HCV life cycle and inhibit its viral activity because present treatment methods and drugs are less effective, have side effects and expensive (Ashfaq et al. 2011). Medicinal plants are used as a source of medicine by 75% of the world. Phytochemicals are chemical compounds found in medicinal plants which can be used as drug candidates. Innovations in the field of computational biology have broadened the scope of research in the area of drug designing. Molecular docking has become a key technique in drug designing and screening antiviral compounds against different diseases (Ul Qamar et al. 2014).

Docking analysis
Neem leaves' active phytochemicals were docked against HCV NS3/4A protease. Docking study was conducted using MOe by selecting pocket. All the docked output conformations were analysed by ligX option of MOe. The conformations having top minimum S-score were saved in a separate database file. Docking study indicated that the compound-01 has a minimum S-score in the output database, which shows that it can be a potent inhibitor against NS3/4A protease. The other docked compounds also have adjacent minimum S-score values. The docked ligands were analysed and their interactions with active site were also illustrated. Interaction images were taken through MOe-ligand interaction analysis option. Interaction images of top four phytochemicals with HCV NS3/4A protein are shown in Figure 1.

Binding interactions of ligands and protein
Molecular docking study has revealed that phytochemicals of A. indica bind to the active sites of the target protein. Compound-01 has a minimum S-score value and was ranked at top followed by compound-02, compound-03 and compound-04. Compound-01 made hydrogen bonding with Gln526, compound-02 made bonding with Arg155, Arg123 and Ala157, compound-03 made bonding with His57, Arg155, Ala157, Arg123, His528 and Cys159, and compound-04 made bonding with Ser139 and His57 residues of pocket molecules as shown in Figure 1. All the ligands have a strong potential to bind with the active site of NS3/4A and the ligands with top minimum values are shown in Table 1.

Conclusion
Current molecular docking study has shown the important interactions of neem leaves' active phytochemicals, especially 3-Deacetyl-3-cinnamoyl-azadirachtin with NS3/4A protease. Thus, it is concluded that the neem leaves' phytochemicals could be the potential inhibitor against NS3/4A protease of HCV and it could be helpful in designing the inhibitory drugs with simple structural modifications against HCV.

Disclosure statement
No potential conflict of interest was reported by the authors.