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. 2016 Nov 21;60(12):7382-7395.
doi: 10.1128/AAC.01421-16. Print 2016 Dec.

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

Pratyush Kumar Das  1 , Laura Puusepp  2 , Finny S Varghese  3 , Age Utt  1 , Tero Ahola  3 , Dzmitry G Kananovich  4 , Margus Lopp  4 , Andres Merits  5 , Mati Karelson  6
Affiliations

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

Pratyush Kumar Das et al. Antimicrob Agents Chemother. .

Abstract

Chikungunya virus (CHIKV; genus Alphavirus) is the causative agent of chikungunya fever. CHIKV replication can be inhibited by some broad-spectrum antiviral compounds; in contrast, there is very little information about compounds specifically inhibiting the enzymatic activities of CHIKV replication proteins. These proteins are translated in the form of a nonstructural (ns) P1234 polyprotein precursor from the CHIKV positive-strand RNA genome. Active forms of replicase enzymes are generated using the autoproteolytic activity of nsP2. The available three-dimensional (3D) structure of nsP2 protease has made it a target for in silico drug design; however, there is thus far little evidence that the designed compounds indeed inhibit the protease activity of nsP2 and/or suppress CHIKV replication. In this study, a set of 12 compounds, predicted to interact with the active center of nsP2 protease, was designed using target-based modeling. The majority of these compounds were shown to inhibit the ability of nsP2 to process recombinant protein and synthetic peptide substrates. Furthermore, all compounds found to be active in these cell-free assays also suppressed CHIKV replication in cell culture, the 50% effective concentration (EC50) of the most potent inhibitor being ∼1.5 μM. Analysis of stereoisomers of one compound revealed that inhibition of both the nsP2 protease activity and CHIKV replication depended on the conformation of the inhibitor. Combining the data obtained from different assays also indicates that some of the analyzed compounds may suppress CHIKV replication using more than one mechanism.

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Figures

FIG 1
FIG 1
Pharmacophoric characterization of the starting compound, B1. The compound has two terminal aromatic/hydrophobic groups and hydrogen bonding donor and acceptor centers in the bridge structure.
FIG 2
FIG 2
Structures of compounds 1 to 12.
FIG 3
FIG 3
Rationally selected compounds inhibit the ability of CHIKV nsP2 to process a recombinant protein substrate. The ratio of enzyme to substrate was ∼1:17. Reactions were carried out at 30°C for 60 min; products were separated by SDS-PAGE and visualized using Coomassie blue staining. (A) Determination of maximally tolerated solvent (DMSO) concentration. (B) Effects of 1 mM compounds 1 to 12 (indicated at the top) on the protease activity of CHIKV nsP2. The image combines three gels that have been rearranged to support the final numbering of compounds; the dotted lines show where different lanes have been merged. (C) Effects of 1 mM compounds B1, B2, B8, B10, and B11 (indicated at the top) on the protease activity of CHIKV nsP2. The dotted lines show where different lanes have been merged. The experiments for each panel were repeated at least three times, with highly similar results.
FIG 4
FIG 4
Compounds 1 to 12 inhibit the ability of CHIKV nsP2 to process a peptide substrate. In all assays the enzyme concentration was 78 nM, while the concentration of the substrate was 15 μM. Reactions were carried out at 30°C for 60 min. Vertical axes show EDANS fluorescence in relative fluorescence units (RFU). Horizontal axes show reaction time. (A) Effects of compounds 1, 2, 5, 6, 7, 8, and 9 at 200 μM. (B) Effects of compounds 3, 4, 10, 11, and 12 at 200 μM. (C to F) Concentration-dependent inhibition of protease activity by compounds 1 (C), 3 (D), 8 (E), and 11 (F) is shown. Each graph represents averages from at least two independent experiments.
FIG 5
FIG 5
Structures of isomers of compound 1.
FIG 6
FIG 6
Schema for the synthesis of isomers 1a to 1d of compound 1. Reagents and conditions: (a) Ph3PMe+Br−, t-BuOK, tetrahydrofuran (THF); (b) N2CHCO2Et, Rh2(OAc)4 (0.5 mol%), dichloromethane (DCM); (c) 2 M aqueous NaOH (0.65 eq), THF-EtOH, room temperature, and then extraction and chromatography; (d) 2 M aqueous NaOH (5 eq), THF-EtOH, 65°C; (e) (COCl)2, THF; (f) d-(−)-phenylglycinamide, N-ethylmorpholine, 4-(dimethylamino)pyridine (DMAP), THF.
FIG 7
FIG 7
Inhibitory properties of different stereoisomers of compound 1. (A) Effects of compounds 1a to 1d and 1aL to 1dL on the ability of CHIKV nsP2 to cleave a recombinant protein substrate. The assay was performed and data are presented as described for Fig. 3. (B) Effects of compounds 1a to 1d and 1aL to 1dL on the ability of CHIKV nsP2 to cleave a peptide substrate. The assay was performed and data are presented as described for Fig. 4A. (C) Concentration-dependent inhibition of protease activity by compound 1c. (D) Molecular docking of compound 1c to the active site of CHIKV nsP2 protease. Hydrogen bonding between either of the amide N-H hydrogens of the inhibitor molecule and the oxygen atom of Asn1082 is shown with a dotted line.
FIG 8
FIG 8
Compounds 3, 8, and 11 inhibit CHIKV positive-strand RNA synthesis and release of infectious virions. (A) Demonstration of concentration-dependent inhibition. Compounds (names and concentrations are shown above the panels) were added to the cells together with virus and present throughout experiment. Control cells were treated with an appropriate amount of a solvent control (DMSO). Total RNAs were extracted and separated; CHIKV positive-stand RNAs were detected using a probe complementary to the 3′ untranslated region of the virus genome. Arrows point out the positions of CHIKV genomic and subgenomic RNAs. (B) Time-of-addition assay (inhibition of positive-strand RNA synthesis). The illustration at the top represents the schema of the experiment. All compounds were used at 100 μM; samples were analyzed and results are presented as described for panel A. (C) Time-of-addition assay (inhibition of infectious virus release). The illustration at the top represents the schema of the experiment. All compounds were used at 100 μM; amounts of released virions were determined using plaque titration. Results were normalized to those from DMSO-treated control cells and expressed as log10 fold reduction of infectious virus titers. Each column represents an average from two experiments performed in triplicate; error bars represent standard deviations.
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