Extended substrate specificity and first potent irreversible inhibitor/activity-based probe design for Zika virus NS2B-NS3 protease

Rut, Wioletta; Zhang, Linlin; Kasperkiewicz, Paulina; Poręba, Marcin; Hilgenfeld, Rolf; Drąg, Marcin

doi:10.1016/j.antiviral.2016.12.018

Cited by 57 publications

(62 citation statements)

References 12 publications

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“…In agreement with previous studies [7,15,16,21], we also observed cleavage of linked WT ZIKV NS2B CF -NS3 pro . We therefore set out to identify the cleavage site, whether cleavage occurs in cis, that is, an individual protease cleaves its own linker or in trans, that is, a protease linker is cleaved by another ZIKV NS2B CF -NS3 pro protease in the sample and to compare the melting behaviors of cleaved and uncleaved protease constructs.…”

Section: Resultssupporting

confidence: 93%

“…The sizes of the autocatalytic cleavage products led us and others to conclude that there are two possible cleavage sites in ZIKV NS2B CF ‐NS3 pro , one at the NS2B CF C‐terminal R95 NS2B and one at R29 NS3 in the NS3 pro domain. The double mutation simultaneously removing both arginine residues successfully abrogated autocatalytic cleavage . Here, we mutated these sites individually and thus identified the relevant autocatalytic cleavage site to be at position R95 NS2B in the C‐terminus of the NS2B CF .…”

Section: Discussionmentioning

confidence: 99%

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Cis autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

et al. 2019

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The flaviviral heterodimeric serine protease NS2B‐NS3, consisting of the NS3 protease domain and the NS2B co‐factor, is essential for ZIKA virus maturation and replication in cells. For in vitro studies a ‘linked’ construct, where a polyglycine linker connects NS2BCF and NS3pro, is often used. This construct undergoes autocatalytic cleavage. Here, we show that linked ZIKV NS2BCF‐NS3pro is cleaved in cis in the NS2BCF exclusively at position R95 and not at the previously proposed alternate cleavage site at residue R29 in the NS3pro. Cleavage neither affects protease stability nor activity, despite some observed differences in spectroscopic behavior. This minimally modified construct may thus be useful for future structural and functional studies of the flaviviral protease, for example when testing new inhibitors.

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Section: Resultssupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Cis autocatalytic cleavage of glycine‐linked Zika virus NS2B‐NS3 protease constructs

et al. 2019

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“…This is the case with the N2B-NS3 trypsin-like serine protease, which plays a key role in virus replication by contributing to viral polyprotein processing (30), or with NS3 helicase activity (31). Due to the relevance of NS2B-NS3 function in the ZIKV life cycle, the search for inhibitors of the enzymatic activity of this complex is at the front line of antiviral discovery against ZIKV (30,(32)(33)(34). For instance, taking advantage of a previous work that identified inhibitors of the HCV protease by high-throughput screening of over 40,000 compounds (35), the same group have recently analyzed 71 of these nonpeptidic small molecules against ZIKV and found that 10 of them showed 50% inhibitory concentrations (IC 50 s) lower than 50 M, with IC 50 s of 5.2 M and 4.1 M for compounds 2 and 3, respectively (30).…”

Section: Reference(s) or Sourcementioning

confidence: 99%

The Race To Find Antivirals for Zika Virus

Saiz

Martín-Acebes

2017

Antimicrob Agents Chemother

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Zika virus (ZIKV), a flavivirus transmitted by mosquitoes, was an almost neglected pathogen until its introduction in the Americas in 2015 and its subsequent explosive spread throughout the continent, where it has infected millions of people. The virus has caused social and sanitary alarm, mainly due to its association with severe neurological disorders (Guillain-Barré syndrome and microcephaly in fetuses and newborns). Nowadays, no specific antiviral therapy against ZIKV is available. However, during the past months, a great effort has been made to search for antiviral candidates using different approaches and methodologies, ranging from testing specific compounds with known antiviral activity to the screening of libraries with hundreds of bioactive molecules. The identified antiviral candidates include drugs targeting viral components as well as cellular ones. Here, an updated review of what has been done in this line is presented.

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“…Recently, the HyCoSuL methodology was applied to design a probe, WRPK3, which rapidly labels Zika's NS3 protease at low nanomolar concentrations ( Fig. 3f) (Rut et al 2017). This probe has the potential to be applied to interrogate NS3 activity during Zika infection.…”

Section: Selective Labelling Of Individual Viral Enzymesmentioning

confidence: 99%