INTRODUCTION:

Dengue is an arthropod borne disease affecting millions of people globally, caused by the bite of infected Aedes mosquito transmitting the dengue virus (DENV) belonging to family flaviviridae. The incidence of the disease has grown dramatically in last few decades affecting over hundred countries globally.^1-
6

Dengue leads to flu-like symptoms and lasts for 2-7 days. Dengue fever (DF) generally occurs after an incubation period of 4-10 days following the bite of the infected mosquito. The symptoms of dengue include high fever (40°C/ 104°F) which is usually accompanied with at least 2 symptoms from headache, malaise, nausea/vomiting, abdominal pain and sometimes rash, retro-orbital pain, myalgia and arthralgia. The other symptoms include bleeding manifestations: petechiae, epistaxis, gum bleeding, hematemesis, melena, or positive tourniquet test, leucopenia, thrombocytopenia and risen haematocrit.^7-9

DENV is a positive-stranded encapsulated RNA virus existing in 4 serotypes- DENV 1, 2, 3 and 4.

The virus has 3 structural protein genes, which encode the nucleocapsid (C) protein, a membrane-associated (M) protein, an enveloped (E) glycoprotein and 7 non-structural (NS) proteins- NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS5. These proteins play an important role in viral fusion, entry, replication, maturation and release, making them a potential antiviral target.¹⁰

Currently there are no approved antiviral drugs for dengue, however one dengue vaccine Dengvaxia by Sanofi Pasteur has been approved but with major restrictions due to its potential safety concerns.^11,12Thus a number of synthetic and herbal interventions have been explored to treat dengue and its symptoms.^13-17 Various phytoconstituents like piperine, quinine, artemisinin, berberine, andrographolide, carpaine have been reported to be active against dengue virus and some have also been taken up to clinical trials levels.¹⁸

Further, the use of structure based drug design technique for the development of dengue antivirals may be a useful starting point before wet lab experiments. Various studies have been done using several viral drug targets with phytoconstituents.^19-29

Cynodon dactylon (CD), family Poacea, is a perennial grass with its presence worldwide, rich in metabolites like proteins, carbohydrates, minerals, alkaloids, flavonoids, carotenoids, glycosides and triterpenoids. It consists of flavonoids like apigenin, luteolin, myricetin, orientin, vitexin.^30,31 Flavonoids have been extensively studied for their anti-viral potential.^32-35CD is also reported to have hypolipidemic³⁶, anticancer³⁷, antidiabetic, antioxidant³⁸, anti-arthritic³⁹, immunostimulant and DNA protective^40,41, CNS depressant activity⁴². The extracts of CD have been found to exhibit potent antiviral activity against white spot syndrome virus (WSSV), Vaccinia virus and chikungunya virus.^43,44 Molecular docking analysis of selected phytoconstituents from CD against WSSV structural protein VP26 has also been reported.⁴⁵ In the present study, CD owing to its rich phytochemical profile was explored for potential against NS2B –NS3 dengue viral target by molecular docking studies.The molecules with lowest docking energy and interaction with essential amino acids have been identified.

MATERIALS AND METHODS:

Retreival of target proteins:

The crystal structure of Dengue virus NS2B-NS3 protease non structural protein was retrieved from protein data bank. (PDB ID-3U1I, PDB database, www.rcsb.org). The downloaded protein structure was prepared prior to docking using Schrödinger Maestro release 2016-4. Briefly the protein preparation was done by preprocessing the structures for assignment of bonds and bond orders, addition of hydrogens, filling in missing loops or side chains, capping uncapped termini, adjusting bonds and formal charges for metals, and correcting mislabeled elements, removing water molecules, removing unwanted chains and optimization of hydrogen bonded structures followed by refinement.

Ligand preparation and molecular docking:

The structures of the selected 24 major phytoconstituents of CD (Table 1) were downloaded from pubchem (https://pubchem.ncbi.nlm.nih.gov/). The energy minimisation was done using Avogadro software and structures were saved in sdf format. The minimised structures were docked on the prepared protein targets after converting to 3d structures and refinement, using FlexX Lead IT 2.3.2 software. The best phytoconstituent was identified on the basis of binding energy and interaction with amino acid residues important for viral replication. Application of Lipinski rule of five was done using SWISS ADME software.

Table 1: List of phytoconstituents

Sr. No.	Phytoconstituent	Sr. No.	Phytoconstituent
1	Apigenin	13	Isovitexin
2	Arundoin	14	Luteolin
3	Betaionone	15	o-Hydroxyphenylacetic acid
4	β-sitosterol	16	Orientin
5	Ergonovine	17	p-Coumaricacid
6	Ergonovinine	18	Phenylacetaldehyde
7	Ferulicacid	19	p-hydroxy benzoic acid
8	Furfural	20	Syringicacid
9	Furfuryl Alcohol	21	Triglochinin
10	Hexadecanal	22	Tritriacontane
11	Hexadecanoic Acid	23	Vanilic acid
12	Isoorientin	24	Vitexin

RESULTS AND DISCUSSION:

The present study revealed the interaction of selected phytoconstituents of CD with DENV NS2B-NS3 protease.

The 3D structure of the protein was obtained from protein data bank which was in complex with the aldehyde inhibitor Bz-nKRR-H (PDB ID- 3U1I).⁴⁶The binding site of the inhibitor was used to study the remaining phytoconstituents.

The DENV NS2B- NS3 protease is important for viral replication with conserved and significant motifs including- the catalytic triad (H51, D75, and S135) and the oxanion hole (G133, T134, S135) which may be well explored in antiviral research.⁴⁶

The lowest binding energy solutions included isoorientin, luteolin, triglochinin, and apigenin with binding energy of -30.4214, -29.4214, -29.0361, -26.2859 Kcal/mol respectively. Isovitexin, vitexin and orientin also showed good binding interaction with the target molecule. All these molecules formed multiple hydrogen bonding interactions with the target protein.

The phytoconstituents of CD- iso-orientin and apigenin bound to S135 through hydrogen bond interactions with bond distance of less than 2A^O units stating their interaction with the catalytic traid of the serine protease NS2B- NS3. Apigenin also forms a pi pi stacking interaction with His 51 and Tyr 150. Apigenin has already been reported to have inhibitory activity against Zika virus NS2B-NS3 protease.⁴⁷ Also, apigenin and luteolin have been reported to have anti- chikungunya activity.⁴⁴ Thus, these flavones and flavonoids may be potential leads for NS2B-NS3 protease dengue virus inhibitors.

A no. of other molecules have been studied and reported to have NS2B-NS3 protease inhibitory activity like derivatives of anthracene, quinoline containing compounds, phthalazine-based compounds, α-ketoamides and flavonoids.^48-50 In the present study, flavones and flavonoid glycosides were found to show good interaction with target protein with low dock score. Also cyanogenic glycoside triglochinin was found to have low binding energy. A low binding energy is indicative of a more stable complex. Further, apigenin and luteolin were found to show good drug likeliness and follow majority of rules unlike triglochinin and iso-orientin. However, these can be used as leads to modify and develop other NS2B-NS3 protease inhibitors.

Three phytoconstituents phenylacetaldehyde, hexadecanal and tritriacontane showed no interaction with the target.

The details of the phytoconstituents binding energy, interacting residues, bond type and bond distance to the target are given in Table 2. The details of Lipinski rule of 5 applied to the top molecules is given in Table 3. The 2d and 3d interaction images of the top 4 phytoconstituents, iso-orientin, luteolin, triglochinin and apigenin with NS2B –NS3 is given in Figure 1, 2, 3 and 4 respectively.

Table 2: Phytoconstituents binding to NS2B- NS3 protease (PDB ID: 3U1I)

Sr. No.	Ligand	Free Binding Energy (Kcal mol^-1)	Interacting Residue	Bond Type	Bond distance ()
1.	Iso-orientin	-30.4214	Ser B:135	H-Bond	2.206
			Phe B:130	H-Bond	1.85
			Tyr B:150	H-Bond	2.00
			Gly B:153	H-Bond	2.198
			Thr A:83	H-Bond	1.67
2.	Luteolin	-29.4214	Lys B:131	H-Bond	2.25
			TyrB:150	H-Bond	2.28
			Gly B:153	H-Bond	1.62
3.	Triglochinin	-29.0361	Gly B:153	2 H-Bond	2.23, 2.27
			Gly B:133	H-Bond	1.38
			Gly B:151	H-Bond	2.278
4.	Apigenin	-26.2859	His B: 51	Pi-Pi stacking	5.43
			Phe B:130	H-Bond	1.544
			Tyr B:150	Pi-Pi stacking	5.394
			SerB:135	H-Bond	1.84
5.	o-hydroxyphenyl acetic acid	-24.5667	Tyr 161	Pi-Pi stacking	5.28
			Gly 133	H-Bond	1.98
			Lys 131	H-Bond	1.91
6.	Isovitexin	-24.161	Gly B:153	H-Bond	1.898
			Tyr B:161	H-Bond	2.731
			Phe B:130	2 H-Bond	1.79,2.02
			Tyr B:150	H-Bond	2.33
			Ser B:135	H-Bond	1.92
7.	Ferulic acid	-23.8875	Gly 153	H-Bond	2.22
			Tyr 161	H-Bond	2.09
			Lys 131	H-Bond	1.63
			Gly 133	H-Bond	2.09
8.	p-coumaric acid	-23.5296	Asp 129	H-Bond	1.847
8.	p-coumaric acid	-23.5296	Gly 133	H-Bond	2.098
9.	o-hydroxybenzoic acid	22.3949	Phe 130	H-Bond	2.01
9.	o-hydroxybenzoic acid	22.3949	Gly 133	H-Bond	1.837
10.	Orientin	-21.9439	Asn B:152	H-Bond	1.628
			Tyr B:161	H-Bond	1.529
			Gly B:151	H-Bond	2.124
			Gly B:153	H-Bond	1.967
			Phe B:130	H-Bond	2.183
			Lys B:131	H-Bond	2.027
			Arg B:54	H-Bond	1.988
			His B:51	H-Bond	2.272
11.	Vitexin	-20.1573	Asp 129	H-Bond	2.174
			Tyr 150	Pi-Pi Stacking	5.479
			Lys 131	H-Bond	1.84
			Ser 135	H-Bond	2.167
			Thr 134	H-Bond	2.48
12.	Ergonovine	-19.0809	Asp 129	Salt bridge	3.044
			Lys 131	H-Bond	1.63
			Ser 135	H-Bond	2.495
			Thr 134	H-Bond	2.709
13.	Ergonovinine	-18.4192	Asp B: 129	H-Bond	1.672
			Tyr B:161	Pi-Pi Stacking	4.213
			Tyr B:161	H-Bond	2.103
			Gly B:153	H-Bond	2.039
			Gly B:153	H-Bond	1.607
14.	Vanillic acid	-18.1843	Phe 130	H-Bond	1.807
15.	Syringic acid	-17.5216	Val 36	H-Bond	2.19
			Arg 54	H-Bond	1.98
			Arg 54	H-Bond	1.92
16.	β-ionone	-14.9432	Gly 153	H-Bond	2.167
16.	β-ionone	-14.9432	Tyr 161	H-Bond	2.109
17.	Phenylacetaldehyde	-13.998	No interaction	--	--
18.	Furfural	-13.5688	Thr 134	H-Bond	2.066
18.	Furfural	-13.5688	Gly 133	H-Bond	1.828
19.	Furfuryl alcohol	-12.4083	Ser 135	H-Bond	2.118
19.	Furfuryl alcohol	-12.4083	Thr 134	H-Bond	1.896
20.	β -sitosterol	-8.1567	Ser 135	H-Bond	1.81
21.	Hexadecanoic acid	-7.9659	ArgB:54	H-Bond	1.789
			ArgB:54	Salt bridge	4.158
			Trp B:50	H-Bond	1.786
22.	Arundoin	-6.1574	Gly 133	H-Bond	2.14
23.	Hexadecanal	8.8473	No interaction	--	--
24.	Tritriacontane	24.5906	No interaction	--	--

Table 3: ADME properties of top phytoconstituents

Molecule name	Apigenin	Isoorientin	Luteolin	Triglochinin
Formula	C₁₅H₁₀O₅	C₂₁H₂₀O₁₁	C₁₅H₁₀O₆	C₁₄H₁₇NO₁₀
MW	270.24	448.38	286.24	359.29
H-bond acceptors	5	11	6	11
H-bond donors	3	8	4	6
iLOGP	1.89	2.12	1.86	0.46
GI absorption	High	Low	High	Low
BBB permeant	No	No	No	No
Lipinski violations	0	2	0	2
Ghose violations	0	1	0	1
Veber violations	0	1	0	1
Egan violations	0	1	0	1
Muegge violations	0	3	0	3
Bioavailability Score	0.55	0.17	0.55	0.11

Fig 1a) Fig 1b)

Figure 1 a) 2D image and b) 3D image of isoorientin interacting with NS2B-NS3 protease (PDB ID- 3U1I) of dengue virus. Interactions- H-Bond ( 1.67 Å)with Thr 83, H-Bond ( 1.85 Å)with Phe 130, H-Bond ( 2.206 Å)with Ser135, H-Bond ( 2.00 Å)with Tyr 150 and H-Bond

(2.198 Å)with Gly 153

Fig 2a) Fig 2b)

Figure 2 a) 2D image and b) 3D image of luteolin interacting with NS2B-NS3 protease (PDB ID- 3U1I) of dengue virus. Interactions- H-Bond (2.25 Å) with Lys 131, H-Bond (2.28 Å)with Tyr 150, H-Bond ( 1.62 Å) with Gly 153

Fig 3a) Fig3b)

Figure 3 a) 2D image and b) 3D image of triglochinin interacting with NS2B-NS3 protease (PDB ID- 3U1I) of dengue virus. Interactions- H-Bond (1.38 Å) with Gly 133, H-Bond (2.28 Å )with Gly 151, 2 H-Bonds (2.23 and 2.27 Å) with Gly 153

Fig 4a) Fig4b)

Figure 4 a) 2D image and b) 3D image of apigenin interacting with NS2B-NS3 protease (PDB ID- 3U1I) of dengue virus. Interactions- Pi pi stacking interaction (5.43 Å) with His 51, H-Bond (1.54 Å)with Phe 130 , H-Bond (1.84 Å)with Ser 135, Pi pi stacking interaction (5.39 Å) with Tyr 150

CONCLUSION:

Dengue is still a much explored but unanswered question, as no medicine is available till date when it comes to the treatment. A no. of studies have been performed to identify synthetic as well as herbal molecules that may be potential inhibitors of dengue virus. In the current study it was found that various phtyoconstituents of Cynodon dactylon bound well with the dengue NS2B-NS3 protease. Iso-orienitn, luteolin, triglochinin and apigenin showed the least binding energy with promising interactions with catalytic triad essential for viral replication and progression. These flavonoids and glycosides may serve as potential leads for developing NS2B- NS3 protease inhibitors of dengue virus.

ACKNOWLEDGEMENT:

The work was supported by Bioinformatics Research Laboratory, Dr. D. Y. Patil Vidyapeeth, Dr. D. Y. Patil Biotechnology and Bioinformatics Institute, Tathawade, Pune.

CONFLICT OF INTEREST:

None.

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Received on 09.06.2019 Modified on 14.07.2019

Research J. Pharm. and Tech. 2019; 12(12): 5865-5870.

DOI: 10.5958/0974-360X.2019.01017.5