Department of Biological Science and Engineering, Maulana Azad National Institute of Technology, Bhopal (India)

Corresponding author email: rashiraizada@gmail.com

Article Publishing History

INTRODUCTION

Chikungunya (CHIKV)  is an epidemic arbovirus that is often used to describe both the virus and the disease. The virus is transmitted mainly to humans through the bite of an infected mosquito of the genus Aedes ( Pialoux et al., 1953). The disease generally consists of such a severe infection that cause fever, rashes, and musculoskeletal pain (to walk bent over) is the hallmark of chikungunya that characterizes this dengue-like illness (Staples, Breiman and Powers, 2009; M Dubrulle et al – ‎2009; Caglioti et al., 2013; Lo Presti et al., 2014).

There have been several CHIKV outbreaks that have been contributed to describing chikungunya fever in detail and identified maculopapular rash predominantly on the thorax, facial edema a bullous rash with pronounced sloughing, and localized petechial rash. It intensely, affects main  extremities, large and  small joints eg: ankles, wrists, phalanges (Lo Presti et al., 2014). CHIKV is been carried by an infected female mosquito to the host the mosquito inoculates virus-containing saliva into the bloodstream of a new victim (Lo Presti et al., 2014) (Fig. 1).

Figure 1: Transmission cycle of CHIKV

CHIKV is an enveloped, spherical body of about 70nm in diameter. The virion genome consists of a Monopartite, linear single-stranded (ss), positive-sense RNA molecule of approximately 11.8 kb long, where the 5’ end is capped with a 7-methylguanosine while the 3’ end is poly-adenylate. The viral genome contains 2 polyproteins represent four non-structural proteins and five structural proteins (Fig. 2). The replication and propagation of the virus is regulated by nsp2 protein, therefore, it is hypothesized that a compound that inhibits the nsp2  will be a promising and potential drug molecule.  In the Era of drug reprofiling efforts can be made to identify a promising inhibitory molecule from the existing antiviral drugs for the treatment of CHIKV the identified potential inhibitors for CHIKV may serve as an inhibitory molecule for other viruses also.which may provide a clear potential path towards the identification of broad-spectrum drugs. (Singh et al., 2011) (Fig. 2).

Figure 2: Schematic description of both structural and nonstructural proteins within the polyprotein CHIKV. CHIKV RNA 11811 bases (top bar, purple color), translates into non-structural and structural precursor polyproteins of 2474 and 1244 residues, respectively, after maturation by protease cleavage, it gives 4 non-structural proteins (left bar, green color) and 5 structural proteins (right bar, red color).

MATERIAL AND METHODS

Retrieval of Target and Lead molecule: The nsp2 crystal structure was retrieved from the protein data bank (PDB) (www.rcsb.org). The retrieval of protein was followed by energy minimization using PYMOL (a user-sponsored molecular visualization system, version 2). The minimization process includes the removal of water molecules, sodium ion, l-peptide linking, and the gaps between amino acids. The lead compound for nsp2 protease was retrieved from PubChem and Drug bank database (Table1). The small molecules were optimized with AVOGADRO: open-source molecular builder and visualization tool( version 1). The optimization process was done with the false parameters that is the force field is off, steps per update is 4, the algorithm is the steepest descent.

Table 1. List of ligands involved in protein‐ligand interaction.

S.No.	Drugs	REFERENCE
1.	(R)‐Chloroquine	Andersag H et al., 1941
2.	Acarbose	S. P. Clissold et al 1988
3.	Acetaminophen	Kis B et al., 2005
4.	Amikacin Sulfate	Overington JP et al., 2006.
5.	Aspirin	Sneader W ., 2000
6.	Arbidol	Hui Peng et al., 2020
7.	Baicalein (Natural Compound)	Oliveira et al., 2017
8.	Bisdesethylchloroquine	Ajayi  FO et al., 1989
9.	Boceprevir	Jennifer J Kiser et al, 2013
10.	Boswellic acid	Arne Henkel et al, 2012
11.	Cefadroxil (Sumacef)	Leonardo Marsili., 1978
12.	Celecoxib	Yi Yu Ke et al., 2020
13.	Chloroquine	Vincent MJ et al., 2005
14.	Cletoquine	Dongre VG et al., 2009
15.	Curcumin	Fatemeh Zahedipour et al, 2020
16.	Desethylchloroquine	Frisk-Holmberg M et al., 1984
17.	Didesethylchloroquine Hydroxyacetamide	Abraham MJ et al., 2015
18.	Dihydrostreptomycin Sulfate	CURCI G ., 1951
19.	Diminazene Aceturate	R. Ghildiyal et al., 2019
20.	Docosanol	Hardman et al 2001
21.	Doxycycline	Dahl EL et al 2006
22.	E-64 (Zinc13493525)	Zheming Wang et al. 2008
23.	Etidronate (Etidronic Acid)	Rogovin et al 1968
24.	Fisetin  (Natural Compound)	Liu L et al 2019.
25.	Glucosamine Sulphate	Arvind Chopra et al, 2013
26.	Hesperetin	Samie A et al., 2018
27.	Hydroxychloroquine	Lim HS et al. 2009
28.	Ibandronate Sodium	Epstein S et al. 2005
29.	Ibuprofen	Casper D et al., 2000
30.	Imatinib	Deininger MW et al2003
31.	Iron Sucrose	Hörl WH 2007.
32.	Kanamycin Sulfate	Vetting MW et al. 2002.
33.	Ketotifen	Roy W. Bryant et al. 2011
34.	Leupeptin Hemisulfate	Pérez-Pérez et al 2019
35.	Mitoxantrone Hydrochloride (Novantrone)	Fox EJ 2006.
36.	N‐Acetyl (Mono) Desthylchloroquine	E. E. Essien et al1989
37.	Naproxen	Wongrakpanich S et al., 2018
38.	Nelfinavir	Kaldor SW et al 1997
39.	Niacin	Briggs gg, et al.,1998
40.	Officinalis acid	Mohammed Bourhia et al., 2019
41.	Pemetrexed Disodium Hemipentahydrate	Prateek Kumar et al.
42.	Pirodavir	Jef Peeters et al. 2007
43.	Pleconaril	Florea NR et al 2003
44.	Prednisolone	Maryam Daneshpazhooh ., 2020
45.	Quercetagetin  (Natural Compound)	Weiyou Wang et al 2016,
46.	Ribavirin	Sidwell RW et al. 2005
47.	Ribostamycin Sulfate	Zhou et al.1992
48.	Sennoside A	Esposito F et al 2016
49.	Sofosbuvir	Asselah T 2013
50.	Spectinomycin Hydrochloride Hydrate (Trobicin)	David R. White 1966
51.	Telaprevir	Kim JJ et al. 2012
52.	Zinc Acetate	Berni Canani R et al 2011

Molecular docking studies: Before performing molecular docking studies, we need to identify the binding pockets of the protein molecule. The Automated active site docking and scoring (AADS) is used in this analysis to identify binding pockets. The AADS (http://www.scfbio-iitd.res.in/dock/ActiveSite_new.jsp) utilizes the 3D structure of target molecules and identify top 10 possible binding sites with 100% precision in identifying the real (active) binding sites (Table 4). Once the protein binding pockets are identified, the Small Molecules Library (Table 1) is screened against these sites to identify the hit molecules using the software.  For this study, PyRx and AutoDockVina software were used to analyze the ligand-protein binding properties to the protein.

The blind dockings were performed in which the grid boxes’ size was adjusted to cover the binding site. Once the docking is complete the resulting PDBQT output file was opened in the PyMOL software for converting all protein conformations into one file analysis on further studies. Afterward, each conformation was examined using Discovery Studio  2.5  software, using information like binding affinities, interaction energies, van der Waals energies, electrostatic energies, hydrogen bonding,pi-pi interactions, pi-cation interactions and close contacting residues were obtained and recorded. The compounds were screened against nsp2 using the PyRx tool to identify the ligands with the best conformers to the target protein.

Table 2. Parameters used for molecular docking of top ten ligands with the protein of interest. All grid boxes with a spacing size of 1.000 A˚ have sufficient sizes to cover the entire protein structures during molecular docking.

S.No.	Ligands with Protein	Center-X	Center-Y	Center-Z	Size-X	Size-Y	Size-Z
1.	3trk_Acarbose	12.8155666274	26.2634859746	21.5992382951	82.0109838983	84.344657921	61.1584777994
2.	3trk_Baicalin	11.697598268	23.47479489	28.5074738773	67.5700208963	85.8025203587	98.2122134629
3.	3trk_Doxycycline	11.37419874	23.0596327058	21.6856001381	71.6738245391	86.7873081266	53.1784838552
4.	3trk_Fisetin	28.9252280574	24.6848042594	19.2251198554	115.892612756	88.4610580017	86.8902924293
5.	3trk_Ibandronate sodium	28.9252280574	24.6848042594	19.2251198554	115.892612756	88.4610580017	86.8902924293
6.	3trk_Mitoxantrone hydrochloride	28.6614606151	20.2939940445	19.342323373	104.001803984	95.7958489908	74.5569102783
7.	3trk_Quercetagetin	12.2522153681	25.5314252099	22.5563495924	70.093582201	93.8091943285	81.4775121373
8.	3trk_Sennoside A	12.2522153681	25.5314252099	22.5563495924	70.093582201	93.8091943285	81.4775121373
9.	3trk_spectinomycinhydrochloride	16.1736849469	22.7882300823	17.3275975767	88.0971554836	88.3232612193	73.2683286745
10.	3trk_Telaprevir	12.2522153681	25.5314252099	22.5563495924	70.093582201	93.8091943285	81.4775121373

RESULTS AND DISCUSSION

During Retrieval of the target molecule, the nsP2 with the PDB ID – 3TRK was retrieved and 52 lead compounds were listed (Table1) these lead compounds were screened for potential inhibitory activities against the top 10 binding sites (Table 4)  CHIKV’s non-structural protein nsp2. The docking studies for the top 10 binding sites of nsp2 (Table3)  the docking studies of all 52 lead compounds with 10 binding sites. The study suggests out of 52 lead compounds the four compound Telaprevir, Doxycycline, Acarbose, Sennoside A showed significant binding affinity whereas spectinomycin hydrochloride (trobicin), Baicalin, Ibandronate sodium, Quercetagetin, Mitoxantrone hydrochloride, and Fisetin showed promising binding affinity ( Table.4 and5.).  Telaprevir showed the strongest binding affinity (-12.3kcal/mol), is a member of protease blockers (a group of antiviral medicine). These affinities and energies are due to interaction and bond formation between lead molecules and binding site amino acid of nsp2.

The result shows the amino acid residue found in the binding pocket between Telaprevir and nsP2, are SER1048, GLN1241, TRP1084, TYR1047, ASN1082, TYR1079, ALA1046, CYS1013, LYS1091, GLU1048, VAL1051, ARG1271, THR1268, ARG1267, TRP1014, HIS1083, LEU1205, and GLU1204 a Fig. 3. The hydrogen bonds between Telaprevir, Doxycycline, Acarbose, Sennoside A, spectinomycin hydrochloride (trobicin), Baicalin,  Ibandronate sodium, Quercetagetin,  Mitoxantrone hydrochloride, Fisetin, and 3TRK are also as shown in (Table 4). between Telaprevir and nsP2, are SER1048, GLN1241, TRP1084, TYR1047, ASN1082, TYR1079, ALA1046, CYS1013, LYS1091, GLU1048, VAL1051, ARG1271, THR1268, ARG1267, TRP1014, HIS1083, LEU1205, and GLU1204 a Fig. 3. The hydrogen bonds between Telaprevir, Doxycycline, Acarbose, Sennoside A, spectinomycin hydrochloride (trobicin), Baicalin,  Ibandronate sodium, Quercetagetin,  Mitoxantrone hydrochloride, Fisetin, and 3TRK are also as shown in (Table 4).

Table 3. Cavity details of Nsp2 Protein

S.No.	Cavity Points			V	A	D	R	h
1.	124.023	55.309	63.320	0.94	0.39	0.42	1.00	0.68	0.6857
2.	100.718	64.861	70.832	0.94	0.56	0.50	0.75	0.63	0.6755
3.	109.046	35.144	85.035	1.00	0.17	0.77	0.50	0.80	0.6465
4.	86.588	66.635	79.871	0.71	0.39	0.46	0.62	1.00	0.6371
5.	92.209	49.198	90.901	0.78	1.00	0.62	0.25	0.41	0.6107
6.	-16.257	-22.551	-4.530	0.98	1.00	1.00	0.83	0.35	0.8326
7.	-23.802	-30.007	2.100	1.00	0.22	0.57	1.00	0.61	0.6804
8.	-7.096	-43.641	-3.465	0.98	0.72	0.53	0.67	0.40	0.6595
9.	-5.740	-45.699	-23.319	0.62	0.72	0.77	0.50	0.48	0.6173
10.	-24.954	-46.789	4.600	0.30	0.67	0.47	1.00	0.53	0.5934

Table 4. Hydrogen Bonding Between the top hit compounds from the blind docking and CHIKV Nsp2. This table documents the Residues involved in the Discovery Studio 2.5. The binding affinities as ranked by the PyRx 8.0 and Auto Dockvinal 1.5.6 are recorded in the final column of the table.

Ligands with Protein	Hydrogen bonds	AngleDHA(○)	Distance(A˚)	Binding affinity (kcal/mol)
3trk andTelaprevir	:UNL1:HN – A:ASN1082:O :UNL1:HN – :UNL1:O :UNL1: HN – A: TYR1047:O	119.232 133.404 151.652	2.43416 2.79452 2.0751	-12.3
3trk and doxycycline	A: TYR1047: HN – :UNK0: O A: TRP1084: HE1 – :UNK0: O	146.817 155.005	2.86321 1.69013	-11.8
3trk andAcarbose	A: TYR1047: HN – :UNK0: O A: SER1048: HG – :UNK0: O A: TRP1084: HE1 – :UNK0: O	148.924 107.507 147.087	2.3149 2.42619 2.46832	-10.9
3trk andSennoside A	A: TYR1079: HH – :UNK0: O A: TRP1084:HE1 – :UNK0: O A: GLN1241: HE22 – :UNK0: O	152.837 135.362 108.266	1.82834 2.37549 2.53045	-10.9
3trk and spectinomycin hydrochloride(trobicin)	A: TRP1084: HE1 – :UNK0: O A: TRP1084: HE1 – :UNK0: O :UNK0: H – A:TYR1079: OH	160.856 142.698 94.399	2.14139 2.27101 2.72027	-8.9
3trk and baicalin	A: TRP1084: HE1 – :UNK0: O A: GLN1241: HE22 – :UNK0: O :UNK0: H – A:TYR1079: OH :UNK0: H – A:ASN1082: OD1	150.648 99.059 138.871 150.896	2.03652 2.87101 2.70173 2.76228	-8.1
3trk and Ibandronate sodium	A: TYR1047: HN – :UNK0: O A: TRP1084: HE1 – :UNK0: O :UNK0: H – A:TYR1079: OH : UNK0: H – A: TYR1047: O : UNK0: H – A: TYR1047: O	162.318 132.257 102.006 137.74 147.153	2.22615 2.66686 2.77491 2.21746 2.12432	-8
3trk andQuercetagetin	A: TYR1047: HN – :UNK0: O A: TYR1047: HN – :UNK0: O A: TRP1084: HE1 – :UNK0: O	149.998 165.015 135.434	2.84355 2.2247 2.27803	-7.9
3trk and Mitoxantrone hydrochloride	A: TYR1047: HN – :UNK0: O A: TRP1084: HE1 – :UNK0: O	157.204 173.755	2.30123 1.82032	-7.8
3trk andFisetin	A: SER1048: HG – :UNK0: O : UNK0: H – A: TYR1047: O :UNK0: H – A:ASP1246: OD2	154.341 140.152 150.743	2.30796 2.06479 2.84791	-7.7

Figure 3: 2D diagram of the interaction between telaprevir and nsP2. The diagram shows the ligand-receptor interactions and close amino acid residues found in the binding pocket.

The result of computational studies recommends that Telaprevir, Doxycycline, Acarbose, Sennoside A can be used as nsp2 inhibitors for chikungunya. These lead compounds already exist and were listed in antiviral medicines especially protease blocker so no harm in exploring these drugs for CHIKV inhibition. This significant outcome is for country path in drug reprofiling studies and here we are proposing molecular docking as a tool for exploring new drug prospects from old drugs.

Table 5. Analysis of ligand-receptor interactions

S.No.	Ligand	Binding Affinity	Rmsd/Ub	Rmsd/Lb
1.	3trk_Telaprevir	-12.3	2.456	1.087
2.	3trk_Doxycycline	-11.8	5.49	1.561
3.	3trk_Acarbose	-10.9	5.22	2.49
4.	3trk_Sennoside A	-10.9	8.368	0.016
5.	3trk_Spectinomycin hydrochloride (Trobicin)	-8.9	4.485	1.768
6.	3trk_Baicalin	-8.1	8.143	5.091
7.	3trk_Ibandronate sodium	-8	10.415	9.23
8.	3trk_Quercetagetin	-7.9	31.104	30.446
9.	3trk_Mitoxantrone hydrochloride	-7.8	5.817	0.058
10.	3trk_Fisetin	-7.7	6.404	2.937
11.	3trk_Imatinib	-7.7	21.294	19.022
12.	3trk_Proteinase inhibitor E64	-7.7	12.243	10.903
13.	3trk_N acetyl Desethylchloroquine	-7.6	13.291	11.618
14.	3trk_Nelfinavir	-7.5	28.101	24.91
15.	3trk_Beta-Boswellic acid	-7.5	26.375	23.199
16.	3trk_Etidronic acid	-7.4	2.29	0.784
17.	3trk_Celecoxib	-7.4	5.297	3.179
18.	3trk_Officinalic acid	-7.4	13.228	9.67
19.	3trk_Pleconaril	-7.2	19.127	14.524
20.	3trk_Hesperetin	-7.1	8.237	2.364

Figure 4: The receptor-ligand interactions, and bonds between them with the highest binding affinities of Acarbose, Baicalin. Doxycycline, Fisetin, Ibandronate sodium, Mitoxantrone hydrochloride, Quercetagetin, Sennoside A, spectinomycin hydrochloride, and Telaprevir (Grey, Red, and Blue stick structure) when docked against Nsp2 protein (dark green colored ball and stick structure).

• 3trk_Telaprevir with the binding affinity of -12.3 kcal/mol
• 3trk_Doxycycline with the binding affinity of -11.8 kcal/mol
• 3trk_Acarbose with the binding affinity of -10.9 kcal/mol
• 3trk_Sennoside A with the binding affinity of -10.9 kcal/mol
• 3trk_Spectinomycin hydrochloride with the binding affinity of -8.9 kcal/mol
• 3trk_Baicalin with the binding affinity of -8.1 kcal/mol
• 3trk_Ibandronate sodium with the binding affinity of -8 kcal/mol
• 3trk_Quercetagetin with the binding affinity of -7.9 kcal/mol
• 3trk_Mitoxantrone hydrochloride with the binding affinity of -7.8 kcal/mol
• 3trk_Fisetin with the binding affinity of -7.7 kcal/mol.

CONCLUSION

In our current study, we conclude briefly that Telaprevir, Doxycycline, Acarbose, Sennoside A possesses interactions with CHIKV non-structural protein to (NSP2)  which plays a role in the virus replication cycle. These findings enhance our understandings of the possibility of an existing antimicrobial drug molecule to be used for treatment against chikungunya fever. The repurposing of these old drugs to treat chikungunya will become an attractive proposition because it involves the use of no risk compounds with considerably lower development cost and minimal discovery timeline hence further studies on this target protein and ligands will enhance the development of a novel anti-CHIKV drug.

ACKNOWLEDGEMENTS

The authors are thankful to the Department of Biological Science and Engineering, Maulana Azad National Institute of Technology, Bhopal (India).

Conflict of Interests: We, the authors of the submitted manuscript declare that the work and data present in the manuscript entitled – Chikungunya virus: new drug prospects emerging from molecular docking studies for medicinal biotechnology is genuine research carried out by us. The work finally belongs to the institute. We have not misused the data previously published and have not manipulated the original work.

REFERENCES

Agarwal, T., Asthana, S., and Bissoyi, A. (2015). Molecular Modeling and Docking Study to Elucidate Novel Chikungunya Virus nsP2 Protease Inhibitors. Indian journal of pharmaceutical sciences, 77(4), 453–460. https://doi.org/10.4103/0250-474x.164769

Bora L (2012) Homology Modeling and Docking to Potential Novel Inhibitor for Chikungunya (37997) Protein nsP2 Protease. J Proteomics Bioinform 5: 054-059. doi:10.4172/jpb.1000213

Choi, H. K., Tong, L., Minor, W., Dumas, P., Boege, U., Rossmann, M. G., and Wengler, G. (1991). Structure of Sindbis virus core protein reveals a chymotrypsin-like serine proteinase and the organization of the virion. Nature, 354(6348), 37–43. https://doi.org/10.1038/354037a0

Clissold, S. P., and Edwards, C. (1988). Acarbose. A preliminary review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential. Drugs, 35(3), 214–243. https://doi.org/10.2165/00003495-198835030-00003

CURCI G. (1951). La idrossistreptomicina [Dihydrostreptomycin]. Archivio di tisiologia e delle malattie dell’apparato respiratorio, 6(3), 104-6.

Delogu, I., Pastorino, B., Baronti, C., Nougairède, A., Bonnet, E., and de Lamballerie, X. (2011). In vitro antiviral activity of arbidol against Chikungunya virus and characteristics of a selected resistant mutant. Antiviral Research, 90(3), 99–107. https://doi.org/10.1016/j.antiviral.2011.03.182

Dongre, V. G., Ghugare, P. D., Karmuse, P., Singh, D., Jadhav, A., and Kumar, A. (2009). Identification and characterization of process-related impurities in chloroquine and hydroxychloroquine by LC/IT/MS, LC/TOF/MS, and NMR. Journal of pharmaceutical and biomedical analysis, 49(4), 873–879. https://doi.org/10.1016/j.jpba.2009.01.013

Dubrulle, M., Mousson, L., Moutailler, S., Vazeille, M., and Failloux, A. B. (2009). Chikungunya virus and Aedes mosquitoes: saliva is infectious as soon as two days after oral infection. PloS one, 4(6), e5895. https://doi.org/10.1371/journal.pone.0005895.

Esposito, F., Carli, I., Del Vecchio, C., Xu, L., Corona, A., Grandi, N., Piano, D., Maccioni, E., Distinto, S., Parolin, C., and Tramontano, E. (2016). Sennoside A, derived from the traditional Chinese medicine plant Rheum L., is a new dual HIV-1 inhibitor effective on HIV-1 replication. Phytomedicine: international journal of phytotherapy and phytopharmacology, 23(12), 1383–1391. https://doi.org/10.1016/j.phymed.2016.08.001

Felix, R. A., 2nd, Kadner, A., and Berrebi, A. S. (2012). Effects of ketamine on response properties of neurons in the superior para olivary nucleus of the mouse. Neuroscience, 201, 307–319. https://doi.org/10.1016/j.neuroscience.2011.11.027

Hahn, C. S., and Strauss, J. H. (1990). Site-directed mutagenesis of the proposed catalytic amino acids of the Sindbis virus capsid protein auto protease. Journal of virology, 64(6), 3069–3073.

Hörl W. H. (2007). Clinical aspects of iron used in the anemia of kidney disease. Journal of the American Society of Nephrology: JASN, 18(2), 382–393. https://doi.org/10.1681/ASN.2006080856

Kaur, P., Thiruchelvan, M., Lee, R. C., Chen, H., Chen, K. C., Ng, M. L., and Chu, J. J. (2013). Inhibition of chikungunya virus replication by harringtonine, a novel antiviral that suppresses viral protein expression. Antimicrobial agents and chemotherapy, 57(1), 155–167. https://doi.org/10.1128/AAC.01467-12

Kawatkar, S., Wang, H., Czerminski, R., and Joseph-McCarthy, D. (2009). Virtual fragment screening: an exploration of various docking and scoring protocols for fragments using Glide. Journal of computer-aided molecular design, 23(8), 527–539. https://doi.org/10.1007/s10822-009-9281-4

Lani, R., Hassandarvish, P., Chiam, C. W., Moghaddam, E., Chu, J. J., Rausalu, K., Merits, A., Higgs, S., Vanlandingham, D., Abu Bakar, S., and Zandi, K. (2015). Antiviral activity of silymarin against the chikungunya virus. Scientific reports, 5, 11421. https://doi.org/10.1038/srep11421

Lee, N., Wong, C. K., Lam, W. Y., Wong, A., Lim, W., Lam, C. W., Cockram, C. S., Sung, J. J., Chan, P. K., and Tang, J. W. (2006). Chikungunya fever, Hong Kong. Emerging infectious diseases, 12(11), 1790–1792. https://doi.org/10.3201/eid1211.060574

Lopresti, A. L., Maes, M., Maker, G. L., Hood, S. D., and Drummond, P. D. (2014). Curcumin for the treatment of major depression: a randomized, double-blind, placebo-controlled study. Journal of affective disorders, 167, 368–375. https://doi.org/10.1016/j.jad.2014.06.001
National Center for Biotechnology Information (2020). PubChem Database. Etidronic acid, CID=3305, https://pubchem.ncbi.nlm.nih.gov/compound/Etidronic-acid (accessed on January 9, 2020)

Nguyen, P. T., Yu, H., and Keller, P. A. (2014). Discovery of in silico hits targeting the nsP3 macro domain of the chikungunya virus. Journal of molecular modeling, 20(5), 2216. https://doi.org/10.1007/s00894-014-2216-6

Overington, J. P., Al-Lazikani, B., and Hopkins, A. L. (2006). How many drug targets are there?. Nature reviews. Drug discovery, 5(12), 993–996. https://doi.org/10.1038/nrd2199

Perera, R., Owen, K. E., Tellinghuisen, T. L., Gorbalenya, A. E., and Kuhn, R. J. (2001). Alphavirus nucleocapsid protein contains a putative coiled-coil alpha-helix important for core assembly. Journal of Virology, 75(1), 1–10. https://doi.org/10.1128/JVI.75.1.1-10.2001

Pialoux, G., Gaüzère, B. A., Jauréguiberry, S., and Strobel, M. (2007). Chikungunya, an epidemic arbovirosis. The Lancet. Infectious diseases, 7(5), 319–327. https://doi.org/10.1016/S1473-3099(07)70107-X

Schuster, R. K., Wibbelt, G., and Kinne, J. (2014). On the life cycle and morphology of development stages of Paraspiralatus sakeri Gibbons et al., 2004 (Nematoda: Spiroidea, Spirocercidae), a heterogenic stomach parasite of falcons. Parasitology Research, 113(6), 2047–2051. https://doi.org/10.1007/s00436-014-3852-6

Singh KhD, Kirubakaran P, and Nagarajan S (2012) Homology modeling, molecular dynamics, e-pharmacophore mapping, and docking study of Chikungunya virus nsP2 protease. J Mol Model. 2012; 18(1):39-51. DOI: 10.1007/s00894-011-1018-3

Soni, A., Pandey, K. M., Ray, P., and Jayaram, B. (2013). Genomes to hits in silico – a country path today, a highway tomorrow: a case study of chikungunya. Current pharmaceutical design, 19(26), 4687–4700. https://doi.org/10.2174/13816128113199990379

Staples, J. E., Breiman, R. F., and Powers, A. M. (2009). Chikungunya fever: an epidemiological review of a re-emerging infectious disease. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 49(6), 942–948. https://doi.org/10.1086/605496

Sun, L., Wu, J., Du, F., Chen, X., and Chen, Z. J. (2013). Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science (New York, N.Y.), 339(6121), 786–791. https://doi.org/10.1126/science.1232458

Taubitz, W., Cramer, J. P., Kapaun, A., Pfeffer, M., Drosten, C., Dobler, G., Burchard, G. D., and Löscher, T. (2007). Chikungunya fever in travelers: clinical presentation and course. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America, 45(1), e1–e4. https://doi.org/10.1086/518701

Vetting, M. W., Hegde, S. S., Javid-Majd, F., Blanchard, J. S., and Roderick, S. L. (2002). Aminoglycoside 2′-n-acetyltransferase from mycobacterium tuberculosis in complex with coenzyme a and aminoglycoside substrates. Nature structural biology, 9(9), 653-658. https://doi.org/10.1038/nsb830

Vincent, M. J., Bergeron, E., Benjannet, S., Erickson, B. R., Rollin, P. E., Ksiazek, T. G., Seidah, N. G., and Nichol, S. T. (2005). Chloroquine is a potent inhibitor of SARS coronavirus infection and spread. Virology journal, 2, 69. https://doi.org/10.1186/1743-422X-2-69

Wang, Z., Gu, C., and Colby, T. (2008). β-Lactone probes identify a papain-like peptide ligase in Arabidopsis thaliana. Nat Chem Biol 4, 557–563 https://doi.org/10.1038/nchembio.104

Wu, D., Wu, J., Zhang, Q., Zhong, H., Ke, C., Deng, X., Guan, D., Li, H., Zhang, Y., Zhou, H., He, J., Li, L., and Yang, X. (2012). Chikungunya outbreak in Guangdong Province, China, 2010. Emerging infectious diseases, 18(3), 493–495. https://doi.org/10.3201/eid1803.110034

Zhang, W., Fisher, B. R., Olson, N. H., Strauss, J. H., Kuhn, R. J., and Baker, T. S. (2002). Aura virus structure suggests that the T=4 organization is a fundamental property of viral structural proteins. Journal of virology, 76(14), 7239–7246. https://doi.org/10.1128/jvi.76.14.7239-7246.2002

Zhou, Q. S., Zhao, Y. M., Bai, X., Li, P. X., and Ruan, C. G. (1992). Effect of new-breviscapine on fibrinolysis and anticoagulation of human vascular endothelial cells. Acta pharmacologica Sinica, 13(3), 239–242.