Pharmacophore anchor models of flaviviral NS3 proteases lead to drug repurposing for DENV infection

Background Viruses of the flaviviridae family are responsible for some of the major infectious viral diseases around the world and there is an urgent need for drug development for these diseases. Most of the virtual screening methods in flaviviral drug discovery suffer from a low hit rate, strain-specific efficacy differences, and susceptibility to resistance. It is because they often fail to capture the key pharmacological features of the target active site critical for protein function inhibition. So in our current work, for the flaviviral NS3 protease, we summarized the pharmacophore features at the protease active site as anchors (subsite-moiety interactions). Results For each of the four flaviviral NS3 proteases (i.e., HCV, DENV, WNV, and JEV), the anchors were obtained and summarized into ‘Pharmacophore anchor (PA) models’. To capture the conserved pharmacophore anchors across these proteases, were merged the four PA models. We identified five consensus core anchors (CEH1, CH3, CH7, CV1, CV3) in all PA models, represented as the “Core pharmacophore anchor (CPA) model” and also identified specific anchors unique to the PA models. Our PA/CPA models complied with 89 known NS3 protease inhibitors. Furthermore, we proposed an integrated anchor-based screening method using the anchors from our models for discovering inhibitors. This method was applied on the DENV NS3 protease to screen FDA drugs discovering boceprevir, telaprevir and asunaprevir as promising anti-DENV candidates. Experimental testing against DV2-NGC virus by in-vitro plaque assays showed that asunaprevir and telaprevir inhibited viral replication with EC50 values of 10.4 μM & 24.5 μM respectively. The structure-anchor-activity relationships (SAAR) showed that our PA/CPA model anchors explained the observed in-vitro activities of the candidates. Also, we observed that the CEH1 anchor engagement was critical for the activities of telaprevir and asunaprevir while the extent of inhibitor anchor occupation guided their efficacies. Conclusion These results validate our NS3 protease PA/CPA models, anchors and the integrated anchor-based screening method to be useful in inhibitor discovery and lead optimization, thus accelerating flaviviral drug discovery. Electronic supplementary material The online version of this article (10.1186/s12859-017-1957-5) contains supplementary material, which is available to authorized users.

: Anchor residue mutation-activity data analysis Table S2: PA/CPA model anchor analysis by known inhibitors in accordance with its moiety preferences. Similarly, at S1 subsite we find a WHV8 anchor (D129, Y130, P131, Y161), which helps in anchorage of the 'Arg' side chain at P1 region of the inhibitor [2]. Adjacent to WHV8 and deeply located is a WH5 anchor (Y130, T134, S135) available for H-bonding (analogous to DH2). The specific anchor, WH2 (Y161, G151, N152, G153) engages the amino acid backbone carbonyl groups of the peptide substrate by H-bonding. The anchor WV5 forms interaction with compound hydrophobic moieties (like with phenyl group of inhibitor in 2FP7) by anchor residues Y151, G153, V154 and I155. The two anchors WH6 and WV6, engage the substrate/inhibitor functional groups at the S3 subsite of the WNV protease.
In the JEV NS3 protease PA model, we observed 13 anchors (5 core and 8 specific) (Fig. S2D). Among the specific anchors, JHV3, JHV4, JHV7 and JEH4 anchors belonged to the dual interaction type; JH2 and JH6 were H-type; JV1 and JV10 were V-type. In the vicinity of the S1 sub-pocket, we find the three mixed-type anchors and a JH2 anchor. The JHV3 and JHV7 anchors supported H-bonding (with polar amide, ketone, alcohol groups) and van der Waals interactions (with aromatic, alkyl and aliphatic moieties). The JEH4 anchor preferred electrostatic bonding of D129 residue with charged groups like -SO2-of compounds, while residues like Y130 helped in forming H-bond with polar carbonyl moieties. The JH2 anchor located deep in the S1 sub-pocket is analogous to DH2 and WH5 anchors sharing similar binding features. The JH6 (near the core anchor CV3) aids to stabilize the main chains of substrate or peptide inhibitor by H-bonding. At the S3 site, there exists the JHV4 anchor (A125, G151, N152 and G153) H-bonding with polar groups (amide, tertiary amine and alcohol moieties) and forming van der Waals interactions with hydrophobic groups (aromatic, alkyl and phenolic moieties). Finally, JV1 at S3 site and JV10 at S2 site, hold the substrate/inhibitor hydrophobic functional groups in position by van der Waals interactions.

Flaviviral NS3 protease sequence and structure analysis:
We analyzed the four flaviviral NS3 protease sequences by CLUSTALW, a multiple sequence alignment (MSA) tool [3] and structures by CEalign, a structure alignment tool [4]. We observed a significant sequence similarity in aligned NS3 protease (Fig. S1A), except for HCV NS3/4A protease which differs distinctly by the use of NS4A as a cofactor unlike others which used the cofactor NS2B. In addition, the phylogenetic evolutionary tree derived from MSA for NS3 and the co-factors showed that the HCV protease is branched farther away from other viruses denoting its distant evolution (Fig. S1B). The structural alignment revealed a conserved chymotrypsin-like fold with aligned catalytic triad residues His-Ser-Asp (Fig. S1C). In DENV, WNV and JEV NS3 proteases, the cofactor NS2B extends to the substrate binding site for substrate stabilization making a deeper active site, but the cofactor 4A in HCV NS3 protease does not extend to the substrate binding pockets resulting in a much flat and wider active site. These subtle similarities and differences in proteases are of great importance in inhibitor design and discovery.

Evolutionary conservation and mutational analysis of anchor residues:
To verify our PA/CPA models, we primarily evaluated anchor-residue conservation by ConsurfDB residue conservation scores [5]. The NS3 protease residues were grouped into four categories as core anchor residues, specific anchor residues, binding site residues (active site residues (<8Å) but not anchor residues) and other residues (not in above groups). Based on the residue conservation scores for four proteases (Fig. S4A), we observed that the >60% of core anchor residues and >40% of specific anchor residues had the highest conservation score of 9. This high conservation of core anchor residues confirms their critical role in the protease structure and function. The specific anchor residues were less conserved compared to core anchors as they tend to have subtle differences among species. Moreover, when comparing the anchor conservation scores (an average of conservation scores of anchor residues) the highest scores 8-9 were attained by core (>60%) and specific (>40%) anchors in all the four proteases (Fig. S4B). This shows that core anchors are critically conserved across the protein family during evolution followed by specific anchors, and points out their key role.
The PA/CPA models were further verified by analyzing the effect of the anchor residue mutations on the overall protease enzymatic activity. For this, we collected the mutation-activity effect data of HCV, DENV, and WNV NS3 protease residues from literature (Table. S2). In general, we observed that anchor residue led to abrogation of enzyme activity depicting their functional role pointing out their targeting for function inhibition.
In the HCV NS3 protease (Table. S2A), when His1057 (a catalytic residue) involved in four core and two specific anchors was mutated to Ala, the positively charged His side chain was lost resulting in disruption of the protease activity evident by the inactive mutant. Also, another anchor residue D1081 of CH7 and HHV4 anchors when mutated to Gly, lost it negative charged aspartate side chain leaving the mutant enzyme inactive. But when residue R1123 of specific anchor HV6 mutates to threonine the enzyme retains its biological activity similar to wild type, as both Arg and Thr have similar hydrophobic interactions at the anchor. In the DENV NS3 protease (Table. 2B), the CEH1 anchor residue G133 forming the catalytic oxyanion hole, when mutated to Ala can no longer accommodate oxyanion during catalysis resulting in its undetectable protease activity. Similarly, mutations in residues S135, Y150, G151, N152 and G153 all involved in core and specific anchors resulted in mutant enzymes with undetectable or very low activities. We see similar observations in the residue mutations occurring in the WNV NS3 protease (Table. 2C). Catalytic D75 involved in CH7, WHV4 and WV6 anchors if mutated to Ala lost the ability to stabilize substrate, resulting in an enzymatically inactive enzyme. Interactions of WHV4 and WV6 anchor residues D82 and G83 with substrates are disrupted due to their mutations causing enzyme inactivity.
From this analysis, we inferred that enzymatic activity is preserved in mutant when the anchor interaction is preserved in spite of the anchor residue change; while activity is lost when anchor interaction is lost due to residue mutation. Hence, the role of anchors (consensus interactions) in enzymatic activity as well as inhibition is justified.        - [30]