Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors

Yin, Zheng; Patel, Sonika; Wang, Weiling; Chan, Wai-Ling; Rao, K. R. Ranga; Wang, Gang; Ngew, Xinyi; Patel, Viral; Beer, David; Knox, John E.; Ling, Ngai; Ehrhardt, Claus; Lim, Siew Pheng; Vasudevan, Subhash G.; Keller, Thomas H.

doi:10.1016/j.bmcl.2005.09.049

Cited by 139 publications

(133 citation statements)

References 19 publications

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“…The protease domain is contained in nonstructural protein 3 (NS3) [9], and its activity is greatly enhanced by interactions with the NS2B protein, which acts as its cofactor [10,11]. Several efforts to find inhibitors for different flavivirus proteases (West Nile virus [WNV], dengue, and yellow fever virus [YFV]) have been reported, with several studies focusing on peptidic substrate-based inhibitors [12][13][14][15][16][17]. Nonpeptidic inhibitors have been identified by in vitro screening [18][19][20] or in silico throughput docking [21].…”

Section: B S T R a C Tmentioning

confidence: 99%

“…Nonpeptidic inhibitors have been identified by in vitro screening [18][19][20] or in silico throughput docking [21]. Most of these compounds do not possess the appropriate properties for drug development, either because the scaffold is too labile [15][16][17]19] or because the inhibitors bind too weakly [18,20].…”

Section: B S T R a C Tmentioning

confidence: 99%

“…The assay was used to test peptidic inhibitors against DENV and WNV proteases [13,15,16]. We adapted it to a 1536-well format and used it to screen our in-house library comprising approximately 10 6 compounds.…”

Section: Hts/choice Of Lead Compound/structure Of Inhibitormentioning

confidence: 99%

“…1). Binding of 1 to the catalytic pocket was verified by nuclear magnetic resonance (NMR) spectroscopy, where the presence of 1 led to a dramatically improved 15 N-HSQC (heteronuclear single quantum correlation) spectrum of DENV2 protease (see Fig. S1 in Supplementary material).…”

Section: Hts/choice Of Lead Compound/structure Of Inhibitormentioning

confidence: 99%

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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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Section: B S T R a C Tmentioning

confidence: 99%

Section: B S T R a C Tmentioning

confidence: 99%

Section: Hts/choice Of Lead Compound/structure Of Inhibitormentioning

confidence: 99%

Section: Hts/choice Of Lead Compound/structure Of Inhibitormentioning

confidence: 99%

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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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“…Combining the cyclic amino acid to an aromatic ring (10) restored a part of the activity. The peptide sequence incorporating serine 2 had an 17 reported equipotent inhibitors in the case of substitutions at P 4 with either alanine or phenylalanine, which implied minimal contribution of P 4 in the enzyme binding. Inversion of configuration into D-norleucine resulted in a nearly 2-fold lower binding affinity.…”

mentioning

confidence: 96%

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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RNA‐virus proteases counteracting host innate immunity

Lei

Hilgenfeld

2017

FEBS Letters

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Virus invasion triggers host immune responses, in particular, innate immune responses. Pathogen‐associated molecular patterns of viruses (such as dsRNA, ssRNA, or viral proteins) released during virus replication are detected by the corresponding pattern‐recognition receptors of the host, and innate immune responses are induced. Through production of type‐I and type‐III interferons as well as various other cytokines, the host innate immune system forms the frontline to protect host cells and inhibit virus infection. Not surprisingly, viruses have evolved diverse strategies to counter this antiviral system. In this review, we discuss the multiple strategies used by proteases of positive‐sense single‐stranded RNA viruses of the families Picornaviridae, Coronaviridae, and Flaviviridae, when counteracting host innate immune responses.

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Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors

Cited by 139 publications

References 19 publications

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

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

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

RNA‐virus proteases counteracting host innate immunity

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