One Step Beyond: Design of Substrates Spanning Primed Positions of Zika Virus NS2B-NS3 Protease

Gruba, Natalia; Martinez, Jose Ignacio Rodriguez; Grzywa, Renata; Wysocka, Magdalena; Skoreński, Marcin; Dąbrowska, Agnieszka; Łęcka, Maria; Suder, Piotr; Sieńczyk, Marcin; Pyrć, Krzysztof; Lesner, Adam

doi:10.1021/acsmedchemlett.8b00316

Cited by 11 publications

(5 citation statements)

References 17 publications

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“…The Zika virus protease NS2B-NS3 (ZiPro) is responsible for the procession of a viral precursor-polyprotein that is essential for viral replication in host cells . Following the nomenclature for protease subsites introduced by Schechter and Berger, the proteases from Zika and related flaviviruses recognize basic side chain residues (arginine or lysine) in the P 1 –P 3 and smaller uncharged residues in the P 4 and P 1 ′ positions . Macrocyclic peptides that act as allosteric inhibitors of ZiPro have recently been reported, but no cyclic substrate-analogs that bind to the active site are known.…”

mentioning

confidence: 99%

Biocompatible Macrocyclization between Cysteine and 2-Cyanopyridine Generates Stable Peptide Inhibitors

et al. 2019

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Peptides featuring an N-terminal cysteine residue and the unnatural amino acid 3-(2-cyano-4-pyridyl)alanine (Cpa) cyclize spontaneously in aqueous solution at neutral pH. Cpa is readily available and easily introduced into peptides using standard solid-phase peptide synthesis. The reaction is orthogonal to all proteinogenic amino acids, including cysteine residues that are not at the N-terminus. A substrate peptide of the Zika virus NS2B-NS3 protease cyclized in this way produced an inhibitor of high affinity and proteolytic stability. 1a CGKRKSCpa-NH2 1b 98 8.3 ± 0.2 2a CCKRKSCpa-NH2 2b 90 2.5 ± 0.1 3a CFKRKSCpa-NH2 3b 97 3.4 ± 0.2 4a CEKRKSCpa-NH2 4b 93 3.3 ± 0.1 5a CGKRKSCpaLI-NH2 5b 96 4.4 ± 0.1 6a CKRKSCpa-NH2 6b 92 8.5 ± 0.1 7a CKRKWCpa-NH2 7b 97 7.3 ± 0.1 8a CGKRKCpa-NH2 8b 97 8.4 ± 0.1 9a CRKSCpa-NH2 9b 91 2.7 ± 0.1 10a CKRKCpa-NH2 10b 94 2.8 ± 0.1 a Peptides synthesized by Fmoc solid-phase peptide synthesis using Rink amide resin. b Cpa, L-3-(2-cyano-4-pyridyl)alanine. c Cyclization was monitored by LC-MS in 10 mM Tris-HCl pH 7.5, 1 mM TCEP buffer at a peptide concentration of 75 µM. d Yields were calculated from LC-MS data. e Half-times were calculated from LC-MS data assuming first-order reaction kinetics.

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mentioning

confidence: 99%

Biocompatible Macrocyclization between Cysteine and 2-Cyanopyridine Generates Stable Peptide Inhibitors

et al. 2019

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“…30 The most obvious temperature sensitive processing by DENVpro was noted in phosphate buffer for substrate 13 , a previously reported substrate for ZIKVpro. 41 An influence of the His 6 -tag could be excluded by using the non-tagged DENV2pro. The influence of the increase in temperature on the pH profile of 13 is minimal, similar to substrate 17 (Supporting Information, Figures S8 and S9).…”

Section: Resultsmentioning

confidence: 99%

“…Substrate 6 is previously reported for DENV2 protease, 40 and substrate 13 for ZIKV and WNV proteases. 41 Substrate 17 is commonly used for DENV2 and ZIKV proteases.…”

Section: Methodsmentioning

confidence: 99%

Alternate recognition by dengue protease: Proteolytic and binding assays provide functional evidence beyond an induced-fit

Behnam,

Klein

2024

Preprint

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Proteases are key enzymes in viral replication, and interfering with these targets is the basis for therapeutic interventions. We previously introduced a hypothesis about conformational selection in the protease of dengue virus and related flaviviruses, based on conformational plasticity noted in X-ray structures. The present work presents the first functional evidence for alternate recognition by the dengue protease, in a mechanism based primarily on conformational selection rather than induced-fit. Recognition of distinct substrates and inhibitors in proteolytic and binding assays varies to a different extent, depending on factors known to influence the dengue protease structure such as pH and salinity. Furthermore, the buffer type and temperature cause a change in binding, proteolysis, or inhibition behavior. Using representative inhibitors with distinct structural scaffolds, we identify two contrasting binding profiles to dengue protease. Noticeable effects are observed in the binding assay upon inclusion of a non-ionic detergent in comparison to the proteolytic assay. The findings highlight the impact of the selection of testing conditions on the observed ligand affinity or inhibitory potency. From a broader scope, the dengue protease presents an example, where the induced-fit paradigm appears insufficient to explain binding events with the biological target. Furthermore, this protein reveals the complexity of comparing or combining biochemical assay data obtained under different conditions. This can be particularly critical for artificial intelligence (AI) approaches in drug discovery that rely on large datasets compiled from different sources.

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“…Orthosteric inhibitors are a class of active compounds against DENV and ZIKV NS2B/NS3 proteases that bind to the active site of the enzyme, differently from the allosteric inhibitors [27,36,[42][43][44]. All inhibitors bear two basic residues (arginine, lysine, or a mimetic) to address the dibasic substrate recognition motif [2,45].…”

Section: Orthosteric Inhibitors Dengue Ns2b/ns3 Protease Inhibitorsmentioning

confidence: 99%

The Inhibition of NS2B/NS3 Protease: A New Therapeutic Opportunity to Treat Dengue and Zika Virus Infection

Starvaggi,

Previti,

Zappalà

et al. 2024

IJMS

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In the global pandemic scenario, dengue and zika viruses (DENV and ZIKV, respectively), both mosquito-borne members of the flaviviridae family, represent a serious health problem, and considering the absence of specific antiviral drugs and available vaccines, there is a dire need to identify new targets to treat these types of viral infections. Within this drug discovery process, the protease NS2B/NS3 is considered the primary target for the development of novel anti-flavivirus drugs. The NS2B/NS3 is a serine protease that has a dual function both in the viral replication process and in the elusion of the innate immunity. To date, two main classes of NS2B/NS3 of DENV and ZIKV protease inhibitors have been discovered: those that bind to the orthosteric site and those that act at the allosteric site. Therefore, this perspective article aims to discuss the main features of the use of the most potent NS2B/NS3 inhibitors and their impact at the social level.

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One Step Beyond: Design of Substrates Spanning Primed Positions of Zika Virus NS2B-NS3 Protease

Cited by 11 publications

References 17 publications

Biocompatible Macrocyclization between Cysteine and 2-Cyanopyridine Generates Stable Peptide Inhibitors

Biocompatible Macrocyclization between Cysteine and 2-Cyanopyridine Generates Stable Peptide Inhibitors

Alternate recognition by dengue protease: Proteolytic and binding assays provide functional evidence beyond an induced-fit

The Inhibition of NS2B/NS3 Protease: A New Therapeutic Opportunity to Treat Dengue and Zika Virus Infection

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