Towards the design of antiviral inhibitors against flaviviruses: The case for the multifunctional NS3 protein from Dengue virus as a target

Lescar, Julien; Luo, Dahai; Xu, Ting; Sampath, Aruna; Lim, Siew Pheng; Canard, Bruno; Vasudevan, Subhash G.

doi:10.1016/j.antiviral.2008.07.001

Cited by 196 publications

(201 citation statements)

References 66 publications

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“…This is not true for the helicase and ATPase activities that appear to be influenced by the presence of the protease domain (16). Inconsistencies in the literature could stem from variations between constructs used to measure the various enzymatic activities, but overall there seems to be a general agreement that the protease domain influences the activities of helicase domain (11,26). The two domains of NS3 are linked by a polypeptide segment spanning residues 169 -179.…”

Section: Discussionmentioning

confidence: 99%

“…The NS3 protein (618 amino acids in DENV4) contains a serine-protease domain at its N terminus (whose activity requires the formation of a noncovalent complex with the central 40-residue hydrophilic segment of the membrane-bound NS2B protein cofactor) and an ATP-driven helicase and RNA triphosphatase at its C-terminal end. Atomic structures for the active NS3 protease domain (NS2B 47 NS3pro) (2)(3)(4), the ATPase/helicase domain (NS3hel) (5)(6)(7)(8)(9), and the full-length NS3 molecule fused to 18 residues of the NS2B cofactor (NS2B 18 NS3) (10) have been reported and reviewed (11). Together, these structures provide an explanation for the lack of protease activity and solubility observed for NS3pro domains expressed in Escherichia coli (6,12,13).…”

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confidence: 99%

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Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

Luo

Wei

Doan

et al. 2010

Journal of Biological Chemistry

126

161

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The dengue virus (DENV) NS3 protein is essential for viral polyprotein processing and RNA replication. It contains an N-terminal serine protease region (residues 1-168) joined to an RNA helicase (residues 180 -618) by an 11-amino acid linker (169 -179). The structure at 3.15 Å of the soluble NS3 protein from DENV4 covalently attached to 18 residues of the NS2B cofactor region (NS2B 18 NS3) revealed an elongated molecule with the protease domain abutting subdomains I and II of the helicase (Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., and Lescar, J. (2008) J. Virol. 82, 173-183). Unexpectedly, using similar crystal growth conditions, we observed an alternative conformation where the protease domain has rotated by ϳ161°with respect to the helicase domain. We report this new crystal structure bound to ADP-Mn 2؉ refined to a resolution of 2.2 Å . The biological significance for interdomain flexibility conferred by the linker region was probed by either inserting a Gly residue between Glu 173 and Pro 174 or replacing Pro 174 with a Gly residue. Both mutations resulted in significantly lower ATPase and helicase activities. We next increased flexibility in the linker by introducing a Pro 176 to Gly mutation in a DENV2 replicon system. A 70% reduction in luciferase reporter signal and a similar reduction in the level of viral RNA synthesis were observed. Our results indicate that the linker region has evolved to an optimum length to confer flexibility to the NS3 protein that is required both for polyprotein processing and RNA replication. Dengue virus (DENV)4 is an important vector-borne virus that causes a spectrum of illnesses in humans ranging from asymptomatic to severe disease, including dengue hemorrhagic fever and dengue shock syndrome. More than half of the ϳ70 members of the flavivirus genus that includes the four serotypes of DENV (DENV1-4), yellow fever virus, Japanese encephalitis virus, tick-borne encephalitis virus, and West Nile virus, are important human pathogens (1). The positive-sense flavivirus RNA genome of ϳ11 kb contains a single open reading frame that is translated into a polyprotein precursor, consisting of the structural proteins C, prM, and E and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. During viral replication, the polyprotein is cleaved by host proteases in the endoplasmic reticulum lumen and viral proteases at the cytoplasmic face (1). The NS3 protein (618 amino acids in DENV4) contains a serine-protease domain at its N terminus (whose activity requires the formation of a noncovalent complex with the central 40-residue hydrophilic segment of the membrane-bound NS2B protein cofactor) and an ATP-driven helicase and RNA triphosphatase at its C-terminal end. Atomic structures for the active NS3 protease domain (NS2B 47 NS3pro) (2-4), the ATPase/helicase domain (NS3hel) (5-9), and the full-length NS3 molecule fused to 18 residues of the NS2B cofactor (NS2B 18 NS3) (10) have been reported and reviewed (11). Together, these structures provide an explana...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

Luo

Wei

Doan

et al. 2010

Journal of Biological Chemistry

126

161

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“…41 To exhibit the artificial heterocomplex, the NS2B-NS3, is depicted in the enlarged section (figure was adopted from Lescar et al). 50 To generate a stable complex of NS2B and NS3 for enzymatic studies and crystallography, an artificial sequence consisting of a (Gly) 4 -Ser-(Gly) 4 sequence was introduced between both domains. 51,52 A crystal structure of the recombinant NS2B40-G4SG4-NS3 protein domain has been resolved illustrating that NS2B40 wraps around NS3 protein in a belt-like manner.…”

Section: The Biological Roles Of the Nonstructural Viral Proteinsmentioning

confidence: 99%

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

et al. 2013

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A series of new substrate analogue inhibitors of the WNV NS2B–NS3 protease containing decarboxylated arginine mimetics at the P1 position was developed. Among the various analogues, trans‐(4‐guanidino)cyclohexylmethylamide (GCMA) was identified as the most suitable P1 residue. In combination with dichloro‐substituted phenylacetyl groups at the P4 position, three inhibitors with inhibition constants of <0.2 μM were obtained. These GCMA inhibitors have a better selectivity profile than the previously described agmatine analogues, and possess negligible affinity for the trypsin‐like serine proteases thrombin, factor Xa, and matriptase. A crystal structure in complex with the WNV protease was determined for one of the most potent inhibitors, 3,4‐dichlorophenylacetyl‐Lys‐Lys‐GCMA (Ki=0.13 μM). The inhibitor adopts a horseshoe‐like conformation, most likely due to a hydrophobic contact between the P4 phenyl ring and the P1 cyclohexyl group, which is further stabilized by an intramolecular hydrogen bond between the P1 guanidino group and the P4 carbonyl oxygen atom. These inhibitors are stable, readily accessible, and have a noncovalent binding mode. Therefore, they may serve as suitable lead structures for further development.

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“…6 DENV protease is regarded as a similarly promising target for drug discovery efforts against dengue virus infections. 7 The enzyme is critical for the viral replication cycle. It posttranslationally cleaves the viral polyprotein, encoded by a single stranded RNA, into three structural and seven nonstructural (NS) proteins.…”

mentioning

confidence: 99%

C-Terminal Residue Optimization and Fragment Merging: Discovery of a Potent Peptide-Hybrid Inhibitor of Dengue Protease

Behnam

Nitsche

Vechi

et al. 2014

ACS Med. Chem. Lett.

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Dengue virus protease is a promising target for the development of antiviral drugs. We describe here a two-step rational optimization that led to the discovery of the potent inhibitor 35 with nanomolar binding affinity at dengue protease serotype 2 (IC 50 = 0.6 μM, K i = 0.4 μM). First, a large number of natural and non-natural amino acids were screened at the C-terminal position of the previously reported, canonical peptide sequence (Cap-Arg-Lys-Nle-NH 2 ). Compared to the reference compound 1 (Bz-Arg-Lys-Nle-NH 2 , IC 50 = 13.3 μM), a 4-fold higher inhibitory potential was observed with the incorporation of a C-terminal phenylglycine (compound 9, IC 50 = 3.3 μM). Second, we applied fragment merging of 9 with the previously reported thiazolidinedione peptide hybrid 33 (IC 50 = 2.5 μM). This approach led to the fusion of two inhibitor-fragments with micromolar affinity into a 20-fold more potent, competitive inhibitor of dengue protease. KEYWORDS: Dengue virus, protease inhibitor, peptide, fragment merging D engue virus (DENV), with its four common serotypes (DENV 1−4), is considered the most important diseasecausing arbovirus in tropical and subtropical regions and a major public health concern. A recent study estimates a global infection burden of 390 million cases in 2010, a more than 3-fold higher number than reported previously by the World Health Organization. 1,2 Increasing prevalence of dengue infections worldwide has been associated with the geographical spread of Aedes mosquitoes, the primary transmitting vector, probably as a result of global warming and climate changes. 3 Currently, vaccines or antiviral agents against DENV remain unavailable. Viral proteases are extremely relevant for the development of antiviral drugs. 4 Highly successful examples are the hepatitis C virus (HCV) NS3 protease inhibitors, such as boceprevir and telaprevir, 5 and the HIV protease inhibitors. 6 DENV protease is regarded as a similarly promising target for drug discovery efforts against dengue virus infections. 7 The enzyme is critical for the viral replication cycle. It posttranslationally cleaves the viral polyprotein, encoded by a single stranded RNA, into three structural and seven nonstructural (NS) proteins. The proteolytically competent NS3-NS2B complex consists of the NS3 serine protease domain and the hydrophilic core sequence of NS2B as cofactor. 8,9 Published inhibitors against DENV protease range from small molecule nonpeptidic inhibitors, 10−13 so far not capable of achieving sufficient target inhibition, to substrate-mimicking peptidic inhibitors. 14−18 The latter class benefits from improved molecular recognition because of its similarity to the natural substrate, which offers higher selectivity and activity, reaching the low micromolar range and even the nanomolar range when combined with an electrophilic warhead. 18 However, the promising in vitro binding affinities come at the expense of pharmacokinetic properties, such as membrane permeability and metabolic stability, which is challenged by ...

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Towards the design of antiviral inhibitors against flaviviruses: The case for the multifunctional NS3 protein from Dengue virus as a target

Cited by 196 publications

References 66 publications

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

Flexibility between the Protease and Helicase Domains of the Dengue Virus NS3 Protein Conferred by the Linker Region and Its Functional Implications

Development and Characterization of New Peptidomimetic Inhibitors of the West Nile Virus NS2B–NS3 Protease

C-Terminal Residue Optimization and Fragment Merging: Discovery of a Potent Peptide-Hybrid Inhibitor of Dengue Protease

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