Progress for dengue virus diseases

Melino, Sonia; Paci, Maurizio

doi:10.1111/j.1742-4658.2007.05831.x

Cited by 41 publications

(16 citation statements)

References 137 publications

(171 reference statements)

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“…Two hepatitis C virus protease inhibitors recently approved for therapeutic use have likewise become the standard of care (28). Thus one of the most prominent attempted strategies for dengue virus therapy has been development of small compounds that target the active site NS2B-NS3pro (29–33). …”

Section: Introductionmentioning

confidence: 99%

Allosteric Inhibition of the NS2B-NS3 Protease from Dengue Virus

et al. 2013

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Dengue virus is the flavivirus that causes dengue fever, dengue hemorrhagic disease, and dengue shock syndrome, which are currently increasing in incidence worldwide. Dengue virus protease (NS2B-NS3pro) is essential for dengue virus infection and is thus a target of therapeutic interest. To date, attention has focused on developing active-site inhibitors of NS2B-NS3pro. The flat and charged nature of the NS2B-NS3pro active site may contribute to difficulties in developing inhibitors and suggests that a strategy of identifying allosteric sites may be useful. We report an approach that allowed us to scan the NS2B-NS3pro surface by cysteine mutagenesis and use cysteine reactive probes to identify regions of the protein that are susceptible to allosteric inhibition. This method identified a new allosteric site utilizing a circumscribed panel of just eight cysteine variants and only five cysteine reactive probes. The allosterically sensitive site is centered at Ala125, between the 120s loop and the 150s loop. The crystal structures of WT and modified NS2B-NS3pro demonstrate that the 120s loop is flexible. Our work suggests that binding at this site prevents a conformational rearrangement of the NS2B region of the protein, which is required for activation. Preventing this movement locks the protein into the open, inactive conformation, suggesting that this site may be useful in the future development of therapeutic allosteric inhibitors.

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Section: Introductionmentioning

confidence: 99%

Allosteric Inhibition of the NS2B-NS3 Protease from Dengue Virus

et al. 2013

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“…An increase of geographical spread, incidence and severity of diseases over the past decade has now stimulated intensive efforts to develop effective antiviral therapeutics which are eventually useful for the prevention and cure of dengue virus infections. The development of small molecule drugs directed at inhibition of replication and maturation of the virus is now considered as promising route for the treatment of acute dengue diseases [ for review see [3-5] and references herein ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2010

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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. Methods Alanine substitutions were introduced by site-directed mutagenesis at residues L115, D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli . Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters K m , k cat and k cat / K m were determined. Results Kinetic data for mutant derivatives in the active site of the dengue virus NS3 protease were essentially in agreement with a functional role of the selected residues for substrate binding and/or catalysis. Only the L115A mutant displayed activity comparable to the wild-type enzyme, whereas mutation of residues Y150 and G151 to alanine completely abrogated enzyme activity. A G133A mutant had an approximately 10-fold reduced catalytic efficiency thus suggesting a critical role for this residue seemingly as part of the oxyanion binding hole. Conclusions Kinetic data obtained for mutants in the NS3 protease have confirmed predictions for the conformation of the active site S1 and S2 pockets based on earlier observations. The data presented herein will be useful to further explore structure-activity relationships of the flaviviral proteases important for the structure-guided design of novel antiviral therapeutics.

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“…Indeed, this complex, also known as "flavivirin" [7], plays a key role in posttranslational processing of the viral polyprotein by mediating several cleavages [8,9]. Therefore, it is considered as a promising target for antiviral therapy [10,11]. In this complex, the ∼180 amino acid long N-terminal domain of NS3, called NS3pro, contains the conserved catalytic triad of trypsin-like serine proteases [12,13], whereas NS2B is a membrane-anchored protein required as a cofactor [14,15].…”

Section: Introductionmentioning

confidence: 99%