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Short Communication |
Novartis Institute for Tropical Diseases, 10 Biopolis Road, #05-01 Chromos, Singapore 138670
Correspondence
Siew Pheng Lim
siew_pheng.lim{at}novartis.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONREFERENCES |
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These authors contributed equally to this work. 
Supplementary figures and methods are available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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; Chambers et al., 1990a) with the following gene order: 5'-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3'. C, prM and E are structural proteins and NS1–NS5 are non-structural proteins involved in polyprotein processing and replication. Numerous studies have shown that the YFV NS3 protease, in complex with NS2B, is involved in co- and post-translational processing of the viral polyprotein and recognizes cleavage sequences (G/ARR
S/G) with consensus dibasic residues at the P1 and P2 positions to generate the N termini of NS2B, NS3, NS4A and NS5, and additional cleavage sites within core, NS2A and NS4A (Chambers et al., 1991, 1993, 1995, 2005; Amberg et al., 1994; Droll et al., 2000).
The YFV NS3 protease belongs to the trypsin superfamily and is relatively well conserved amongst members of the family Flaviviridae (Bazan & Fletterick, 1989; Gorbalenya et al., 1989). The essentiality of NS3 protease activity in viral replication, through at least its polyprotein-cleaving activity, has been demonstrated by mutational analysis of the residues predicted to form the canonical serine protease catalytic triad (H53, D77 and S138) (Chambers et al., 1990b, 1993, 1995, 2005; Droll et al., 2000).
An effective YFV vaccine was developed when the disease caused havoc in North America at the dawn of the 20th century. There still remains a chance that the disease can re-emerge in outbreak proportion because of its increased incidence in the past 25 years and risks of urban YFV in Africa and South America (Barrett & Higgs, 2007). This has led to calls for the development of chemotherapeutic agents for yellow fever so as not to be caught unprepared. Presently, there is no chemotherapy for any of the flaviviral infections.
The NS3 protease, being highly conserved amongst flaviviruses, would be an attractive potential target for a pan-flaviviral drug. Considerable efforts have been made recently to find such inhibitors to both dengue virus (DENV; Leung et al., 2001; Ganesh et al., 2005; Li et al., 2005; Yin et al., 2006a, b) and West Nile virus (WNV; Nall et al., 2004; Ganesh et al., 2005; Erbel et al., 2006; Knox et al., 2006) proteases. Similar undertakings have not been reported for the YFV protease. In this context, understanding the molecular interaction between flaviviral NS3 protease and inhibitors is a question of broad relevance and may facilitate the development of designed drugs. As DENV, WNV and YFV NS2B/NS3 proteases share similar cleavage recognition sites (Chambers et al., 1990a; Falgout et al., 1991), we reasoned that peptidic inhibitors generated against DENV (Yin et al., 2006a, b) and WNV (Erbel et al., 2006; Knox et al., 2006) proteases could potentially act on the YFV protease. To test this hypothesis, we studied the effects of these inhibitors on YFV NS3 protease activity by using a single-chain complex of YFV NS2B/NS3 protease (CF40-gly-NS3pro190) comprising 47 core amino acids of NS2B (Chambers et al., 1991; Falgout et al., 1993) linked via Gly4–Ser–Gly4 to the N-terminal 190 aa NS3 protease domain. Similar designs in DENV (Leung et al., 2001), WNV (Nall et al., 2004) and YFV (Bessaud et al., 2006) produced catalytically active proteases.
YFV CF40-gly-NS3pro190 was expressed as an N-terminally His-tagged fusion protein in Escherichia coli and purified from an Ni2+ affinity column from clarified bacterial lysate [Supplementary Figure S1(a), available in JGV Online]. The total amount of active protein in the purified fraction was 100 %, as determined after active-site titration with the competitive, tight-binding inhibitor aprotinin (Ki=17.61±0.56 nM; Supplementary Figure S2, available in JGV Online) and curve fitting according to the Morrison equation (Copeland, 2000; Supplementary Figure S2). The proteolytic activity of YFV CF40-gly-NS3pro190 was first assessed with two different fluorogenic substrates: the tripeptide Boc-GRR-AMC (Yusof et al., 2000; Bessaud et al., 2006) and the tetrapeptide Bz-nKRR-AMC (Li et al., 2005). We observed that its catalytic efficiency for Boc-GRR-AMC (kcat/Km=239.05±7.7 M–1 s–1; Table 1) was comparable to that reported by Bessaud et al. (2006) (kcat/Km=105 M–1 s–1). However, the enzyme exhibited an almost 10-fold higher affinity for the tetrapeptide substrate and also processed it with 34-fold greater efficiency (Table 1). This finding may in part be due to the additional P4 residue (norleucine) in the tetrapeptide sequence. In DENV, tetrapeptides were also shown to give improved substrate binding (giving rise to a better Km value) and turnover (better kcat value) compared with tripeptides (Niyomrattanakit et al., 2006). The tetrapeptide substrate was thus employed for subsequent enzyme characterization and inhibitory studies with peptidic inhibitors. No activity was obtained with an S138A mutant construct of YFV CF40-gly-NS3pro190 when tested against Bz-nKRR-AMC at enzyme concentrations ranging from 0 to 1000 nM and even after incubation with the substrate for 6 h (Supplementary Figure S3, available in JGV Online). This indicates that the enzymic activity of wild-type YFV CF40-gly-NS3pro190 was not due to contamination by bacterial proteases.
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), WNV (Nall et al., 2004) and YFV (Bessaud et al., 2006). Furthermore, similar to reports on DENV (Leung et al., 2001) and WNV (Nall et al., 2004), YFV activity was also enhanced by addition of glycerol (Supplementary Figure S4, available in JGV Online). Based on these results, we used these buffer conditions (50 mM Tris/HCl, pH 8.5; 1 mM CHAPS; 20 % glycerol) to assay the properties of the peptidic inhibitors.
We first employed the aldehyde derivative of the substrate-based peptide Bz-nKRR-H (1; Table 2), which has Ki values of 5.8 and 2.1 µM for DENV (Yin et al., 2006a, b) and WNV (Erbel et al., 2006; Knox et al., 2006), respectively. Against YFV, it was almost 15-fold more potent than DENV and 5-fold better than WNV (Ki=0.4 µM; Table 2). Its potency could be improved by 8-fold by substituting the aldehyde warhead with the more electrophilic group boronic acid (14; Table 2). Replacement of P1 (Arg) in Bz-nKRR-H with Ala (2; Table 2) reduced its potency dramatically by 83-fold. Replacement with Phe (6; Table 2) had an even more pronounced effect (155-fold decrease). Interestingly, the P1 position could not be substituted with positively charged Lys, as this reduced potency by 28-fold (10; Table 2). Substitution of P2 Arg with Ala resulted in a complete loss of inhibition (3; Table 2), whilst replacement with Phe caused a 39-fold decrease (7; Table 2). Unlike in the P1 site, substitution with Lys in the P2 site was tolerated, as enzyme activity was reduced marginally by about 3-fold (11; Table 2). Substitutions of P3-Lys and P4-Nle with either Ala or Phe did not affect inhibitory activity significantly (4, 5, 8, 9; Table 2). Likewise, when the peptide-inhibitor sequence was shortened to tri- and dipeptides, Ki values also did not change significantly (12, 13; Table 2). Taken together, these data show that S1 and S2 are the major determinants for the binding of the peptidic inhibitor.
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) based on the crystal structure of WNV NS2B/NS3 in complex with Bz-nKRR-H (2FP7; Erbel et al., 2006) and replaced the latter with individual peptidic aldehydes by using SYBYL 6.9 followed by optimization with MacroModel 9.0, to study their effects on interactions with amino acids in the YFV active site (Supplementary Methods, available in JGV Online).
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). A large portion of the S1 pocket comprised the aromatic side chains of Y153 and F164 (Fig. 1c). As was observed in the WNV protease, a cation–
stacking occurred, with the guanidine group from P1-Arg sandwiched between the phenyl rings of the Bz cap of the inhibitor and F164. Two additional residues, D132 and G154, formed key interactions with P1-Arg via its side chain and backbone, respectively (Fig. 1c). The S2 pocket was narrower and shallower than S1 and was lined with hydrogen-bond donors and acceptors from NS2B and the NS3 domain, with three hydrogen bonds formed between S2 and P2-Arg: two with NS3 (D77 and N155) and one with NS2B (G82; Fig. 1c). The S3 pocket was larger, but less well defined, than S1 and S2, again comprising residues from both NS2B and the NS3 domain. Interestingly this pocket possessed a dual character in which half of the pocket was hydrophobic (F84 from NS2B; I157, L158 from NS3), whilst the other half comprised hydrogen-bond donor/acceptor atoms from the backbone of E83, K85 (NS2B) and G156 (NS3). P3-Lys also formed three hydrogen bonds; the first was a backbone hydrogen bond to G156 in NS3, whilst the remaining two were side-chain hydrogen bonds to E83 and F84 in NS2B (Fig. 1c). The S4 pocket was the least well defined and was made up entirely of L158, yielding a hydrophobic, solvent-exposed patch on the surface (Fig. 1c). No hydrogen bonds were found between the side chain of L158 and P4-Nle. Overall, the YFV homology model resembled the WNV structure closely, in that the S1 and S2 pockets were the dominant recognition sites for the peptidic inhibitor (Erbel et al., 2006).
We next replaced Bz-nKRR-H in the YFV homology model with the other peptidic inhibitors tested. We observed that side-chain interactions with charged and aromatic residues lining the S1 and S2 pockets were lost when P1 and P2 Arg were replaced with Ala or Phe in peptidic inhibitors 2, 3, 6 and 7, leading to dramatic drops in the inhibitory activity (>40-fold, Table 2; D132 for 2 and 6; G82 and N155, D77 for 3 and 7). Furthermore, these substitutions disrupted the -stacking of P1-Arg, the Bz cap and F164. Although P1-Ala substitution (2) resulted in loss of the same hydrogen bonds as P1-Phe (6), there was a nearly 2-fold difference in their inhibitory activities (Table 2). One explanation for this difference may be that the smaller size of Ala allowed the hydrophobic Bz cap to bury more deeply into the S1 pocket, giving rise to a more stable complex than Bz-nKRF-H.
In contrast, the inhibitory activity of Bz-nKKR-H (11) was comparable to that of 1. Bz-nKKR-H retained the key hydrogen bonding with D77, and its P2-Lys terminal 
interacted similarly with G82 (NS2B domain) as P2-Arg in Bz-nKRR-H (1). In the case of Bz-nKRK-H (10), the P1-Lys 
group failed to form the optimal cation– interaction found in Bz-nKRR-H (1). Nevertheless, it retained the hydrogen bond with the side chain of D132; this probably explains why it is less potent than 1, but more potent than the corresponding Ala (2) and Phe (6) substitutions in P1 (Table 2). The WNV protease also shows the same Lys tolerance in P2, but not in P1 (Erbel et al., 2006; Knox et al., 2006). However, for the DENV protease, Lys substitutions in both P1 and P2 are not tolerated (Yin et al., 2006b), suggesting that there may be differences in the DENV S1 pocket. This was supported by recent findings reported by Chappell et al. (2006), showing that the WNV protease was twice as active against substrates with Lys rather than Arg in P2, whilst DENV protease behaved in the opposite manner.
Side-chain interactions of S3 residues E83 and F84 with P3-Lys were lost with Ala or Phe substitutions (4, 8), whilst backbone hydrogen-bonding interaction with G156 was retained. As peptidic inhibitors 4 and 8 and the tripeptidic inhibitor (12) did not exhibit reduced potency, these side-chain interactions are probably not important. Finally, studies with both the dipeptide inhibitor and P4 replacements with Ala and Phe (5, 9) also showed that norleucine did not play a significant role in inhibitor binding and are consistent with the homology model, where the P4 residue does not form any hydrogen bonds with S4 residues.
In summary, the YFV protease shares similar characteristics with the DENV and WNV proteases and can be inhibited by the same peptidic inhibitor, Bz-nKRR-H. In all three cases, inhibitor potency is driven predominantly by interactions with amino acid residues in the S1 and S2 pockets, as substitutions in the P1 and P2 sites have the greatest impact on inhibitor potency. The information from these studies suggests that the design of pan-flavivirus drugs against NS2B/3 protease may be possible and that the peptidic inhibitors can serve as useful starting points for rational drug design.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONREFERENCES |
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Received 24 November 2006;
accepted 11 April 2007.
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