Functional Profiling of Recombinant NS3 Proteases from All Four Serotypes of Dengue Virus Using Tetrapeptide and Octapeptide Substrate Libraries

Li, Jun; Lim, Siew Pheng; Beer, David; Patel, Viral; Wen, Daying; Tumanut, Christine; Tully, David C.; Williams, Jennifer; Jiricek, Jan; Priestle, John P.; Harris, Jennifer L.; Vasudevan, Subhash G.

doi:10.1074/jbc.m500588200

Cited by 229 publications

(335 citation statements)

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“…NS2B-NS3pro can be activated readily by adding a widely used synthetic substrate AC-LKKR-AMC [10]. Using Michaelis-Menten kinetics we determined that the K m and k cat are 85.3 ± 2.1 μM and 1.149 ± 0.069 s -1 , respectively (Supplementary information, Figure S1D), which are comparable to the dengue virus (DENV) and the West Nile virus (WNV) NS2B-NS3pros [11,12], but different from ZIKV NS2B-NS3 (R95A/ R29G), which has an increased activity [8]. To discover potential inhibitors of ZIKV, we screened small molecule compounds against ZIKV NS2B-NS3pro.…”

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confidence: 82%

“…Since residues 152-167 are the key components for S1 subsite, their conformation is critical for enzymatic activity. The configuration of this loop in our structure may represent an auto-inhibitory mode ( Figure 1E), which is distinct from DENV and WNV apo NS2B-NS3pro structures [12,14]. In our crystal structure, this loop (in molecule A) is pushed away by residues 29-32 from the neighboring molecule (molecule B) ( Figure 1E, middle panel), resulting in a conformation that is unfavorable for catalysis.…”

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confidence: 99%

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Mechanisms of activation and inhibition of Zika virus NS2B-NS3 protease

Chen

Yang

et al. 2016

Cell Res

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confidence: 82%

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confidence: 99%

Mechanisms of activation and inhibition of Zika virus NS2B-NS3 protease

Chen

Yang

et al. 2016

Cell Res

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“…These were two basic residues, particularly two arginines, as P 1 and P 2 , 20 lysine as P 3 , and norleucine as P 4 . 21 Against this background, we investigated the activity of different retro, retro-inverse, semiretro-inverse, and nonretro dipeptides and tripeptides. 22 The retro tetrapeptide Arg-Arg-Lys-Nle-NH 2 was found to be less active than the tripeptide Arg-Lys-Nle-NH 2 with only one arginine residue.…”

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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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“…Cloning of DENV2 CF40-gly-NS3pro185 W mutant constructs All Trp mutants were generated with overlapping PCR using the plasmid DENV2 TSV01 pET15b-CF40-gly-NS3pro185 as a template [22]. To obtain cNS2B with Trp mutations, PCR was carried out using the forward primer NS2BcfXhoI-F [22] and reverse primers W5A-REV, W50A-REV, W69A-REV, W83A-REV, and W89A-REV, respectively (see Table S1 in Supplementary material).…”

Section: Chemistrymentioning

confidence: 99%

“…To obtain cNS2B with Trp mutations, PCR was carried out using the forward primer NS2BcfXhoI-F [22] and reverse primers W5A-REV, W50A-REV, W69A-REV, W83A-REV, and W89A-REV, respectively (see Table S1 in Supplementary material). To obtain NS3pro185 with W mutations, PCR was carried out using the forward primers W5A-FOR, W50A-FOR, W69A-FOR, W83A-FOR, and W89A-FOR, respectively (see Table S1) and reverse primer NS3pro185BamHI-R [22]. The two products were joined in the second round of PCR using the primer pair NS2BcfXhoI-F and NS3pro185BamHI-R to generate the individual CF40-glyNS3pro185 W mutants.…”

Section: Chemistrymentioning

confidence: 99%

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

Bodenreider

Beer

Keller

et al. 2009

Analytical Biochemistry

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In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC (50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions. b s t r a c tIn drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC 50 values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC 50 values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other proteinligand interactions. Ó 2009 Elsevier Inc. All rights reserved.High-throughput screening (HTS) 2 is a major method in drug discovery for new small lead molecules. In high-throughput enzyme inhibition assays, more than a million compounds are usua...

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Functional Profiling of Recombinant NS3 Proteases from All Four Serotypes of Dengue Virus Using Tetrapeptide and Octapeptide Substrate Libraries

Cited by 229 publications

References 36 publications

Mechanisms of activation and inhibition of Zika virus NS2B-NS3 protease

Mechanisms of activation and inhibition of Zika virus NS2B-NS3 protease

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

A fluorescence quenching assay to discriminate between specific and nonspecific inhibitors of dengue virus protease

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