Substrate profiling of Zika virus <scp>NS</scp>2B‐<scp>NS</scp>3 protease

Gruba, Natalia; Martinez, Jose Ignacio Rodriguez; Grzywa, Renata; Wysocka, Magdalena; Skoreński, Marcin; Burmistrz, Michał; Łęcka, Maria; Lesner, Adam; Sieńczyk, Marcin; Pyrć, Krzysztof

doi:10.1002/1873-3468.12443

Cited by 48 publications

(51 citation statements)

References 42 publications

(50 reference statements)

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“…Proteases containing a glycine linker between NS2B CF and NS3 pro are denoted as 'linked', even when they undergo autocatalytic cleavage during purification. Kinetic data are derived from a protease assay (25°C) employing a fluorogenic tripeptide (Boc-GRR-AMC) [20], melting temperatures are based on tryptophan fluorescence measurements ( Figs 2B and S2C). For detailed information see Table S1.…”

Section: Resultsmentioning

confidence: 99%

“…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 . This finding is intriguing for multiple reasons: The ZIKV NS2B CF ‐NS3 pro is believed to be specific toward dibasic residues . However, in the frequently used ZIKV NS2B CF ‐NS3 pro construct, cleavage occurred at a single basic residue R95 NS2B which is preceded by a methionine.…”

Section: Discussionmentioning

confidence: 99%

“…specific toward dibasic residues [20]. However, in the frequently used ZIKV NS2B CF -NS3 pro construct, cleavage occurred at a single basic residue R95 NS2B which is preceded by a methionine.…”

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: Resultsmentioning

confidence: 99%

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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“…This might strongly influence how side chains, specially at the P4 position, bind into the different active site pockets. Indeed, using different capping group in PS-CSL of substrates has been shown to result in different amino acid preferences in the S4 pocket of the Zika virus NS2B-NS3 protease [53,54]. Also, if cooperativity exist between the P4 and other positions, the influence of the capping group on the S4 pocket specificity will also influence that of other pockets.…”

Section: Correlation Between Ps-scl Of Substrates and Inhibitors For mentioning

confidence: 99%

Characterization ofP. falciparumdipeptidyl aminopeptidase 3 specificity reveals structural factors responsible for differences in amino acid preferences between peptide-based substrates and covalent inhibitors

Vries

Groborz

et al. 2018

Preprint

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Dipeptidyl aminopeptidases (DPAPs) are druggable cysteine proteases that cleave dipeptides from the N-terminus of proteins and oligopeptides. Plasmodium DPAPs have been shown to be important for the asexual replication of the malaria parasite and are therefore potential antimalarial targets. DPAP1 seems to be important for parasite growth within infected red blood cells, and DPAP3 plays a critical role in the invasion of erythrocytes by the malaria parasite. An inhibitor able to block both of these proteases will target the parasite at different developmental stages thus making the emergence of drug resistance more difficult.Understanding the substrate specificity of these proteases is important to understand their molecular functions and to help develop potent and selective inhibitors. In this study, we used peptide-based libraries of substrates and inhibitors to determine the specificity of DPAP3 and compared it to that of DPAP1 and human cathepsin C (mammalian DPAP homologue). We then used the structure activity relationships information from these screens to develop optimal DPAP3 fluorogenic substrates as well as highly specific DPAP1 and DPAP3 inhibitors.Interestingly, while the substrate specificity of a protease is often used to develop potent inhibitors, in this study we show that equally potent and highly specific inhibitors can be developed based on the structure of poor substrates. Overall, this study illustrates that focusing the development of peptidic inhibitors solely on the substrate specificity of a protease might overlook important structural features that can be exploited to developed highly potent and selective compounds.not peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was . http://dx.doi.org/10.1101/246124 doi: bioRxiv preprint first posted online Jan. 11, 2018; 3 Malaria is a devastating infectious parasitic disease causing close to half a million deaths every year 1 . Over the last 15 years the world has seen a very significant drop in malaria incidence, mainly due to the global distribution of insecticide-impregnated bed nets and the use of artemisinin-based combination therapies as the standard of care for uncomplicated malaria 2 .However, malaria remains a major global health burden with half of the world population at risk and around 200 million clinical cases per year. Unfortunately, mosquitoes are becoming increasingly resistance to insecticides 3 and artemisinin resistance is on the rise 4 , thus making the identification of antimalarial targets and the development of drugs with novel mechanisms of action extremely urgent 5 .Malaria is caused by parasites of the Plasmodium genus and is transmitted by Anopheles mosquitoes during a blood meal. Upon infection, malaria parasites first establish an asymptomatic infection in the liver before reaching the blood stream where they replicate asexually through multiple rounds of red blood cell (RBC) invasion, intracellular growth and divis...

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“…Regarding NS2A, to our knowledge, there are no specific studies addressing the function of this protein in ZIKV. On the other hand, NS3 is a trypsin-like serine protease involved in polyprotein processing (Gruba et al, 2016;Lei et al, 2016) that also exhibits helicase activity, which plays a pivotal role in viral RNA replication enabling RNA unwinding . Apart from its involvement in viral polyprotein processing, NS3 can cleave cellular factors such as FAM134B, hence modulating the autophagic response within ZIKV-infected cells (Lennemann and Coyne, 2017).…”

Section: Proteinsmentioning

confidence: 99%

Zika Virus: What Have We Learnt Since the Start of the Recent Epidemic?

2018

Frontiers Research Topics

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Zika is a viral disease transmitted mainly by mosquitoes of the genus Aedes. In recent years, it has expanded geographically, changing from an endemic mosquito-borne disease across equatorial Asia and Africa, to an epidemic disease causing large outbreaks in several areas of the world. With the recent Zika virus (ZIKV) outbreaks in the Americas, the disease has become a focus of attention of public health agencies and of the international research community, especially due to an association with neurological disorders in adults and to the severe neurological and ophthalmological abnormalities found in fetuses and newborns of mothers exposed to ZIKV during pregnancy. A large number of studies have been published in the last 3 years, revealing the structure of the virus, how it is transmitted and how it affects human cells. Many different animal models have been developed, which recapitulate several features of ZIKV disease and its neurological consequences. Moreover, several vaccine candidates are now in active preclinical development, and three of them have already entered phase I clinical trials. Likewise, many different compounds targeting viral and cellular components are being tested in in vitro and in experimental animal models. This review aims to discuss the current state of this rapidly growing literature from a multidisciplinary perspective, as well as to present an overview of the public health response to Zika and of the perspectives for the prevention and treatment of this disease.

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Substrate profiling of Zika virus NS2B‐NS3 protease

Cited by 48 publications

References 42 publications

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

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

Characterization ofP. falciparumdipeptidyl aminopeptidase 3 specificity reveals structural factors responsible for differences in amino acid preferences between peptide-based substrates and covalent inhibitors

Zika Virus: What Have We Learnt Since the Start of the Recent Epidemic?

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