Enzymatic characterization and homology model of a catalytically active recombinant West Nile virus NS3 protease

West Nile Virus (WNV) is a mosquito-borne flavivirus with a rapidly expanding global distribution. Infection causes severe neurological disease and fatalities in both human and animal hosts. The West Nile viral protease (NS2B-NS3) is essential for post-translational processing in host-infected cells of a viral polypeptide precursor into structural and functional viral proteins, and its inhibition could represent a potential treatment for viral infections. This article describes the design, expression, and enzymatic characterization of a catalytically active recombinant WNV protease, consisting of a 40-residue component of cofactor NS2B tethered via a noncleavable nonapeptide (G4SG4) to the N-terminal 184 residues of NS3. A chromogenic assay using synthetic para-nitroanilide (pNA) hexapeptide substrates was used to identify optimal enzyme-processing conditions (pH 9.5, I <0.1 m, 30% glycerol, 1 mm CHAPS), preferred substrate cleavage sites, and the first competitive inhibitor (Ac-FASGKR-H, IC50 approximately 1 microm). A putative three-dimensional structure of WNV protease, created through homology modeling based on the crystal structures of Dengue-2 and Hepatitis C NS3 viral proteases, provides some valuable insights for structure-based design of potent and selective inhibitors of WNV protease.     

黄病毒NS2B-NS3pro蛋白酶的结构研究进展

黄病毒能引起严重的人类疾病,但是并无特定药物来治疗病毒感染。黄病毒非结构蛋白NS3的N端区域及其辅因子NS2B构成蛋白酶,该酶切割病毒的多聚蛋白形成成熟的结构蛋白和非结构蛋白来帮助病毒完成增殖过程。NS2B-NS3pro蛋白酶在黄病毒生命周期中起关键的作用,使之成为抗病毒药物研发的重要靶标。本文综述了黄病毒属中寨卡病毒、登革热病毒、西尼罗病毒的NS2B-NS3pro蛋白酶结构的研究进展,并介绍了相关抑制剂与蛋白酶形成的复合物结构,以期为研发抗黄病毒药物提供必要的参考。     

NMR Analysis of the Dynamic Exchange of the NS2B Cofactor between Open and Closed Conformations of the West Nile Virus NS2B-NS3 Protease

Background

The two-component NS2B-NS3 proteases of West Nile and dengue viruses are essential for viral replication and established targets for drug development. In all crystal structures of the proteases to date, the NS2B cofactor is located far from the substrate binding site (open conformation) in the absence of inhibitor and lining the substrate binding site (closed conformation) in the presence of an inhibitor.

Methods

In this work, nuclear magnetic resonance (NMR) spectroscopy of isotope and spin-labeled samples of the West Nile virus protease was used to investigate the occurrence of equilibria between open and closed conformations in solution.

Findings

In solution, the closed form of the West Nile virus protease is the predominant conformation irrespective of the presence or absence of inhibitors. Nonetheless, dissociation of the C-terminal part of the NS2B cofactor from the NS3 protease (open conformation) occurs in both the presence and the absence of inhibitors. Low-molecular-weight inhibitors can shift the conformational exchange equilibria so that over 90% of the West Nile virus protease molecules assume the closed conformation. The West Nile virus protease differs from the dengue virus protease, where the open conformation is the predominant form in the absence of inhibitors.

Conclusion

Partial dissociation of NS2B from NS3 has implications for the way in which the NS3 protease can be positioned with respect to the host cell membrane when NS2B is membrane associated via N- and C-terminal segments present in the polyprotein. In the case of the West Nile virus protease, discovery of low-molecular-weight inhibitors that act by breaking the association of the NS2B cofactor with the NS3 protease is impeded by the natural affinity of the cofactor to the NS3 protease. The same strategy can be more successful in the case of the dengue virus NS2B-NS3 protease.     

Structural evidence for regulation and specificity of flaviviral proteases and evolution of the Flaviviridae fold

Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an "induced fit" mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.     

Characterization of the West Nile virus protease substrate specificity and inhibitors

West Nile virus (WNV), a mosquito-borne member of Flaviviridae, is a human pathogen causing widespread disease for which there is no vaccine or chemotherapy. The two-component viral serine protease consists of a heterodimeric complex between the hydrophilic domain of the cofactor, NS2B (NS2BH) and the protease domain (NS3-pro). The protease is essential for polyprotein processing followed by assembly of viral replicase and genome replication. Therefore, the protease is an excellent target for development of antiviral therapeutics. Here, we report the expression in Escherichia coli, purification, and characterization of biochemical and kinetic properties of the WNV protease. Furthermore, we show that the WNV and the dengue virus type 2 (DENV-2) proteases are inhibited by aprotinin with inhibitor constants of 0.16 and 0.026 microM, respectively. Molecular modeling of the WNV protease/aprotinin complex, based on the known crystal structures of the WNV NS2BH-N3pro and aprotinin, suggest a potentially strong interaction between the P2 Lys and the protease activator peptide, NS2BH. This conclusion based on molecular modeling is in agreement with our data of a higher k(cat)/Km value with the substrate, Boc-Gly-Lys-Arg-MCA than the Boc-Gly-Arg-Arg-MCA and is also consistent with the results of an earlier study that were based on substrate-based inhibitor peptides.     

Homology modeling and molecular dynamics study of West Nile virus NS3 protease: a molecular basis for the catalytic activity increased by the NS2B cofactor

The West Nile virus (WNV) NS3 serine protease, which plays an important role in assembly of infective virion, is an attractive target for anti-WNV drug development. Cofactors NS2B and NS4A increase the catalytic activity of NS3 in dengue virus and Hepatitis C virus, respectively. Recent studies on the WNV-NS3 characterize the catalytically active form of NS3 by tethering the 40-residue cofactor NS2B. It is suggested that NS2B is essential for the NS3 activity in WNV, while there is no information of the WNV-NS3-related crystal structure. To understand the role of NS2B/substrate in the NS3 catalytic activity, we built a series of models: WNV-NS3 and WNV-NS3-NS2B and WNV-NS3-NS2B-substrate using homology modeling and molecular modeling techniques. Molecular dynamics (MD) simulations were performed for 2.75 ns on each model, to investigate the structural stabilization and catalytic triad motion of the WNV NS3 protease with and without NS2B/substrate. The simulations show that the NS3 rearrangement occurs upon the NS2B binding, resulting in the stable D75-OD1...H51-NH hydrogen bonding. After the substrate binds to the NS3-NS2B active site, the NS3 protease becomes more stable, and the catalytic triad is formed. These results provide a structural basis for the activation and stabilization of the enzyme by its cofactor and substrate.     

Substrate inhibition kinetic model for West Nile virus NS2B-NS3 protease

West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested that peptide substrates could bind to two binding sites on the protease. Reaction product analogues also showed inhibition of the protease, demonstrating product inhibition in addition to and distinct from substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1'-P3' interaction surface (i.e., the S1'-S3' sites). Optimization of substrate analogue inhibitors that target these two independent sites may lead to novel anti-WNV drugs.     

Activation of the West Nile virus NS3 protease: Molecular dynamics evidence for a conformational selection mechanism

The flaviviral nonstructural 3 protease (NS3pro) is essential for virus replication and is therefore a pharmaceutically relevant target to fight Dengue and West Nile virus (WNV). NS3pro is a chymotrypsin‐like serine protease which requires a polypeptide cofactor (NS2B) for activation. Recent X‐ray crystallography studies have led to the suggestion that the substrate binds to the two‐component NS2B‐NS3pro enzyme by an induced‐fit mechanism. Here, multiple explicit water molecular dynamics simulations of the WNV NS2B‐NS3pro enzyme show that the active conformation of the NS2B cofactor (in which its β‐loop is part of the substrate binding site) is stable over a 50‐ns time scale even in the absence of the inhibitor. The partial and reversible opening of the NSB2 β‐loop and its correlated motion with an adjacent NS3pro loop, both observed in the simulations started from the active conformation, are likely to facilitate substrate binding and product release. Moreover, in five of eight simulations without inhibitor (started from two X‐ray structures both with improperly formed oxyanion hole) the Thr132‐Gly133 peptide bond flips spontaneously thereby promoting the formation of the catalytically competent oxyanion hole, which then stays stable until the end of the runs. The simulation results provide evidence at atomic level of detail that the substrate binds to the NS2B‐NS3pro enzyme by conformational selection, rather than induced‐fit mechanism.     

Functional characterization of cis and trans activity of the Flavivirus NS2B-NS3 protease

Flaviviruses are serious human pathogens for which treatments are generally lacking. The proteolytic maturation of the 375-kDa viral polyprotein is one target for antiviral development. The flavivirus serine protease consists of the N-terminal domain of the multifunctional nonstructural protein 3 (NS3) and an essential 40-residue cofactor (NS2B(40)) within viral protein NS2B. The NS2B-NS3 protease is responsible for all cytoplasmic cleavage events in viral polyprotein maturation. This study describes the first biochemical characterization of flavivirus protease activity using full-length NS3. Recombinant proteases were created by fusion of West Nile virus (WNV) NS2B(40) to full-length WNV NS3. The protease catalyzed two autolytic cleavages. The NS2B/NS3 junction was cleaved before protein purification. A second site at Arg(459) decreasing Gly(460) within the C-terminal helicase region of NS3 was cleaved more slowly. Autolytic cleavage reactions also occurred in NS2B-NS3 recombinant proteins from yellow fever virus, dengue virus types 2 and 4, and Japanese encephalitis virus. Cis and trans cleavages were distinguished using a noncleavable WNV protease variant and two types of substrates as follows: an inactive variant of recombinant WNV NS2B-NS3, and cyan and yellow fluorescent proteins fused by a dodecamer peptide encompassing a natural cleavage site. With these materials, the autolytic cleavages were found to be intramolecular only. Autolytic cleavage of the helicase site was insensitive to protein dilution, confirming that autolysis is intramolecular. Formation of an active protease was found to require neither cleavage of NS2B from NS3 nor a free NS3 N terminus. Evidence was also obtained for product inhibition of the protease by the cleaved C terminus of NS2B.     

Site-directed mutagenesis and kinetic studies of the West Nile Virus NS3 protease identify key enzyme-substrate interactions

The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ac-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in WNV NS3 protease at S1 (V115A/F, D129A/E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases.     

Mutagenesis of D80-82 and G83 residues in West Nile Virus NS2B: Effects on NS2B-NS3 activity and viral replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D⁸⁰DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D⁸⁰DDG in NS2B on protease activity and viral replication, the negatively charged region D⁸⁰DD and the conserved residue G83 of NS2B were mutated (D⁸⁰DD/E⁸⁰EE, D⁸⁰DD/K⁸⁰KK, D⁸⁰DD/A⁸⁰AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D⁸⁰DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D⁸⁰DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.     

Generation and characterization of proteolytically active and highly stable truncated and full-length recombinant West Nile virus NS3

West Nile virus is a medically significant emerging pathogen for which there is no effective antiviral therapy. The viral protease encoded by NS2B and NS3 is an attractive target for development of an inhibitor and has been the focus of numerous studies. Most have employed recombinant proteases based on an expression strategy we developed which links the essential hydrophilic cofactor domain within NS2B to the NS3 protease domain by a flexible glycine linker. However, autoproteolysis has been a significant problem associated with this construct. The recently resolved crystal structure of the cofactor bound WNV NS3 protease for example, was found to be truncated by 18 residues at its N-terminus. In this study, the autocatalytic cleavage site was identified and removed along with nonessential regions of the glycine linker and cofactor domain. In addition, the optimal size of the NS3 protease was defined. Based on this optimized construct, a recombinant protease incorporating the full length of NS3 was also successfully expressed and purified. Somewhat surprisingly, comparative analysis of the proteolytic activity of this recombinant with that of the protease domain alone revealed little influence of the C-terminal two thirds of NS3 on substrate binding. These modifications have yielded highly stable and constrained recombinant proteases, which are more suitable than existing constructs for both activity and structural studies.     

Structure of West Nile Virus NS3 Protease: Ligand Stabilization of the Catalytic Conformation

Over the last decade, West Nile virus has spread rapidly via mosquito transmission from infected migratory birds to humans. One potential therapeutic approach to treating infection is to inhibit the virally encoded serine protease that is essential for viral replication. Here we report the crystal structure of the viral NS3 protease tethered to its essential NS2B cofactor and bound to a potent substrate-based tripeptide inhibitor, 2-naphthoyl-Lys-Lys-Arg-H (K_i = 41 nM), capped at the N-terminus by 2-naphthoyl and capped at the C-terminus by aldehyde. An important and unexpected feature of this structure is the presence of two conformations of the catalytic histidine suggesting a role for ligand stabilization of the catalytically competent His conformation. Analysis of other West Nile virus NS3 protease structures and related serine proteases supports this hypothesis, suggesting that the common catalytic mechanism involves an induced-fit mechanism.     

Serpin mechanism of hepatitis C virus nonstructural 3 (NS3) protease inhibition: induced fit as a mechanism for narrow specificity

Hepatitis C virus (HCV) nonstructural 3 (NS3) serine protease disrupts important cellular antiviral signaling pathways and plays a pivotal role in the proteolytic maturation of the HCV polyprotein precursor. This recent discovery has fostered the search for NS3 protease inhibitors. However, the enzyme's unusual induced fit behavior and peculiar molecular architecture have imposed considerable obstacles to the development of small molecule inhibitors. In this article, we demonstrate that such unique induced fit behavior and the chymotrypsin-like catalytic domain can provide the structural plasticity necessary to generate protein-based inhibitors of the NS3 protease. We took advantage of the macromolecular scaffold of a Drosophila serpin, SP6, which intrinsically supports chymotrypsin-like enzyme inhibition, to design a novel class of potent and selective inhibitors. We show that altering the SP6 reactive site loop (RSL) resulted in the development of the first effective (K(i) of 34 nm) and selective serpin, SP6(EVC/S), directed at the NS3 protease. SP6(EVC/S) operates as a suicide substrate inhibitor, and its partitioning between the complex-forming and proteolytic pathways for the NS3 protease is HCV NS4A cofactor-dependent and -specific. Once bound to the protease active site, SP6(EVC/S) partitions with equal probability to undergo proteolysis by NS3 at the C-terminal site of the engineered RSL, (P(6))Glu-Ile-(P(4))Val-Met-Thr-(P(1))Cys- downward arrow -(P(1)')Ser, or to form a covalent acyl-enzyme complex characteristic of cognate protease-serpin pairs. Our results also reveal a novel cofactor-induced serpin mechanism of enzyme inhibition that could be explored for developing effective and selective inhibitors of other important induced fit viral proteases of the Flaviviridae family such as the West Nile virus NS3 endoprotease.     

Ligand-bound structures of the dengue virus protease reveal the active conformation

Dengue is a mosquito-borne viral hemorrhagic disease that is a major threat to human health in tropical and subtropical regions. Here we report crystal structures of a peptide covalently bound to dengue virus serotype 3 (DENV-3) protease as well as the serine-protease inhibitor aprotinin bound to the same enzyme. These structures reveal, for the first time, a catalytically active, closed conformation of the DENV protease. In the presence of the peptide, the DENV-3 protease forms the closed conformation in which the hydrophilic β-hairpin region of NS2B wraps around the NS3 protease core, in a manner analogous to the structure of West Nile virus (WNV) protease. Our results confirm that flavivirus proteases form the closed conformation during proteolysis, as previously proposed for WNV. The current DENV-3 protease structures reveal the detailed interactions at the P4' to P3 sites of the substrate. The new structural information explains the sequence preference, particularly for long basic residues in the nonprime side, as well as the difference in substrate specificity between the WNV and DENV proteases at the prime side. Structural analysis of the DENV-3 protease-peptide complex revealed a pocket that is formed by residues from NS2B and NS3; this pocket also exists in the WNV NS2B/NS3 protease structure and could be targeted for potential antivirus development. The structural information presented in the current study is invaluable for the design of specific inhibitors of DENV protease.     

Yellow fever virus NS2B/NS3 protease: Hydrolytic Properties and Substrate Specificity

Here we report the hydrolytic behavior of recombinant YFV NS2B/NS3 protease against FRET substrates mimicking the prime and non-prime region of the natural polyprotein cleavage sites. While the P2-P′1 motif is the main factor associated with the catalytic efficiency of Dengue (DV) and West Nile Virus (WNV) protease, we show that the k_cat/K_m of YFV NS2B/NS3 varied by more than two orders of magnitude, despite the presence of the same motif in all natural substrates. The catalytic significance of this homogeneity – a unique feature among worldwide prominent flavivirus – was kinetically analyzed using FRET peptides containing all possible combinations of two and three basic amino acids in tandem, and Arg and Lys residues produced distinct effects on k_cat/K_m. The parallel of our data with those obtained in vivo by Chambers et al. (1991) restrains the idea that these sites co-evolved with the NS2B/NS3 protease to promote highly efficient hydrolysis and supports the notion that secondary substrate interaction distant from cleavage sites are the main factor associated with the different hydrolytic rates on YFV NS2B-NS3pro natural substrates.     

Structure-guided mutagenesis of active site residues in the dengue virus two-component protease NS2B-NS3

Background  

The dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is now intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme-substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of designing inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed.     

Dengue virus NS3 serine protease. Crystal structure and insights into interaction of the active site with substrates by molecular modeling and structural analysis of mutational effects

The mosquito-borne dengue viruses are widespread human pathogens causing dengue fever, dengue hemorrhagic fever, and dengue shock syndrome, placing 40% of the world's population at risk with no effective treatment. The viral genome is a positive strand RNA that encodes a single polyprotein precursor. Processing of the polyprotein precursor into mature proteins is carried out by the host signal peptidase and by NS3 serine protease, which requires NS2B as a cofactor. We report here the crystal structure of the NS3 serine protease domain at 2.1 A resolution. This structure of the protease combined with modeling of peptide substrates into the active site suggests identities of residues involved in substrate recognition as well as providing a structural basis for several mutational effects on enzyme activity. This structure will be useful for development of specific inhibitors as therapeutics against dengue and other flaviviral proteases.     

Conformational changes in human hepatitis C virus NS3 protease upon binding of product-based inhibitors.

One of the most promising approaches to anti-hepatitis C virus drug discovery is the development of inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is essential for the maturation of the viral polyprotein, and processing requires complex formation between NS3 and its cofactor NS4A. Recently, we reported on the discovery of potent cleavage product-derived inhibitors [Ingallinella et al. (1998) Biochemistry 37, 8906-8914]. Here we study the interaction of these inhibitors with NS3 and the NS3/cofactor complex. Inhibitors bind NS3 according to an induced-fit mechanism. In the absence of cofactor different binding modes are apparent, while in the presence of cofactor all inhibitors show the same binding mode with a small rearrangement in the NS3 structure, as suggested by circular dichroism spectroscopy. These data are consistent with the hypothesis that NS4A complexation induces an NS3 structure that is already (but not entirely) preorganized for substrate binding not only for what concerns the S' site, as already suggested, but also for the S site. Inhibitor binding to the NS3/cofactor complex induces the stabilization of the enzyme structure as highlighted by limited proteolysis experiments. We envisage that this may occur through stabilization of the individual N-terminal and C-terminal domains where the cofactor and inhibitor, respectively, bind and subsequent tightening of the interdomain interaction in the ternary complex.     

Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.