Elsevier

Biochimie

Volume 166, November 2019, Pages 132-141
Biochimie

Review
Exploiting the unique features of Zika and Dengue proteases for inhibitor design

https://doi.org/10.1016/j.biochi.2019.05.004Get rights and content

Highlights

  • Zika and Dengue viruses have caused recent outbreaks; no specific treatments exist.

  • Zika and Dengue proteases are embedded in a multienzyme NS3 protein.

  • NS3 needs the hydrophilic part of membrane-anchored NS2B as an activating cofactor.

  • Several types of constructs have been used for in vitro studies and crystallization.

  • Active-site, allosteric and precursor-binding inhibitors have been identified.

Abstract

Zika and Dengue viruses have attracted substantial attention from researchers in light of recent outbreaks of Dengue fever and increases in cases of congenital microcephaly in areas with Zika incidence. This review summarizes the current state of knowledge about Zika and Dengue proteases. These enzymes have several interesting features: 1) NS3 serine protease requires the activating co-factor NS2B, which is anchored in the membrane of the endoplasmic reticulum; 2) NS2B displays extensive conformational dynamics; 3) NS3 is a multidomain protein with proteolytic, NTPase, RNA 5′ triphosphatase and helicase activity and has many protein-protein interaction partners; 4) NS3 is autoproteolytically released from its precursor. Attempts to design tight-binding and specific active-site inhibitors are complicated by the facts that the substrate pocket of the NS2B-NS3 protease is flat and the active-site ligands are charged. The ionic character of potential active-site inhibitors negatively influences their cell permeability. Possibilities to block cis-autoprocessing of the protease precursor have recently been considered. Additionally, potential allosteric sites on NS2B-NS3 proteases have been identified and allosteric compounds have been designed to impair substrate binding and/or block the NS2B-NS3 interaction. Such compounds could be specific to viral proteases, without off-target effects on host serine proteases, and could have favorable pharmacokinetic profiles. This review discusses various groups of inhibitors of these proteases according to their mechanisms of action and chemical structures.

Introduction

Despite advances in infectious disease surveillance, the global public health community has been surprised in recent years by outbreaks of previously unknown viruses, such as SARS in 2002 [1] and MERS in 2012 [2], and variants of known viruses with altered genotypes and/or patterns of spread. Examples of the latter include the Ebola outbreaks in Africa in 2014–2016 [3] and 2018 [4] and the global spread of H1N1 influenza (swine flu) in 2009 [5]. The mosquito-borne Zika and Dengue flaviviruses belong to the group of viruses that have emerged or re-emerged in recent years [6]. More than 70 flaviviruses have been identified. These viruses have varied geographical distribution and cause human illnesses with various levels of severity. Many of them also infect wild animals, including rodents, bats, birds and monkeys, which can become hidden viral reservoirs in a sylvatic cycle [7]. Knowledge about these animal reservoirs can aid responses to current and potential future flaviviral outbreaks.

Flaviviruses are positive-strand RNA viruses. RNA synthesis is error-prone, enabling production of many mutated variants in a large population of viral progeny. This enables rapid, continual viral evolution and adaptation [8]. Travel and concentrations of people in specific geographical places also introduces new opportunities for viral transmission and spread [9].

Zika virus offers an example of how human movement can influence the spread of infection. Zika virus was originally found in sentinel monkeys in Uganda in 1947 [10] and was considered to cause a mild tropical fever. Sixty years later, in 2007, Zika virus emerged on Yap Island in Micronesia. A second Zika outbreak occurred in French Polynesia in 2013–14. The Zika virus was transferred to Brazil by a traveler and spread during the World Sprint Championship Va'a canoe race in August 2014 [11,12]. Now, Zika occurs in all South American areas where Aedes mosquitoes, the Zika virus vectors, live [12]. Higher incidence of Guillain-Barré syndrome and microcephaly among newborns have been documented in areas with Zika virus outbreaks [13,14]. The virus also can be transmitted sexually, and a chronic infection can develop [15]. WHO has added Zika infection to its Research and Development (R&D) Blueprint, the list of diseases recommended for intensive research [16].

Dengue virus spread to the Americas from Africa during the 16th century. During the 21st century, the number of global cases has increased each year: from 33 million cases in 2005 to 80 million in 2015. In recent years, there have been an estimated up to 390 million cases annually, [17,18]. Dengue virus causes a febrile illness with rash and intense myalgia. Most people recover spontaneously within 4–7 days, acquiring immunity against the particular Dengue serotype. However, four Dengue serotypes exist (denoted 1, 2, 3 and 4). Subsequent infection with a different serotype increases the probability of life-threatening symptoms, including hemorrhagic fever and cytokine storm leading to Dengue shock syndrome due to antibody-dependent enhancement (ADE)—the production of non-neutralizing antibodies by memory B lymphocytes. These non-neutralizing antibodies are cross-reactive, binding to the newly infecting virus of different serotype and facilitating infection of myeloid cells. This leads to suppression of effective host antiviral immune responses [19,20].

Vaccines against Zika and Dengue viruses are being vigorously pursued through intensive research [21] and development efforts [22]. One vaccine against Dengue virus, Dengvaxia, has been approved; however, it has some limitations. Due to ADE issues, it is not recommended for dengue-naive persons [23]. It is also less active against the Dengue 2 serotype [24]. No specific drugs against Zika and Dengue viruses have been approved, although multiple candidates are in the pipeline (see chapter 5).

Section snippets

Biological roles of Zika and Dengue proteases

This review focuses on Zika and Dengue proteases, which are key players in viral maturation. Zika and Dengue viruses employ a replication strategy in which viral proteins are produced as a polyprotein. Viral particles become fully infectious upon cleavage of this polyprotein into functional proteins. Flaviviral polyprotein is intertwined through the intracellular membrane of the endoplasmic reticulum (ER). It is cleaved by host proteases on the luminal site of ER and by a viral protease on the

Properties of Zika and Dengue proteases

Zika and Dengue proteases are serine proteases with the classical Asp, His, Ser catalytic triad. This catalytic triad is harbored on the NS3 molecule.

Serine proteases in general need to be stabilized (for example, by disulfide bonds, as in elastase). Dengue and Zika proteases do not require metal ions; Dengue 2 protease is stabilized by a glutamate-lysine salt bridge [49].

Isolated NS3 protease is intrinsically disordered and tends to precipitate. However, the Dengue 2 NS3 protease domain is

Constructs for in vitro study of Zika and Dengue proteases

Due to the necessity of the hydrophilic 40-residue NS2B peptide for the proper function of the protease domain, different fusion protein variants containing both parts of the active protease have been developed for in vitro studies. In vivo, the hydrophilic part of NS2B is anchored in the membrane of the endoplasmic reticulum at both termini, which could result in some steric restrictions. From this point of view, all the soluble constructs are more-or-less artificial.

To avoid processing (and

Active-site inhibitors

Older inhibitors of Dengue protease were summarized in a comprehensive review in 2014 [62]. A vast majority of these active-site-targeted compounds have inhibition constants in the micromolar range. More active compounds are under development [83]. Zika and Dengue proteases have quite similar substrate specificities (Fig. 2B). The general preferred pattern of natural cleavage sites is two basic amino acids in the P1 and P2 positions, followed by a small hydrophobic or uncharged residue in P1'.

Reducing off-target binding

Among the classical broad-spectrum inhibitors of serine proteases (which also inhibit human proteases), only aprotinin is a potent inhibitor of Dengue and Zika proteases, inhibiting Dengue protease in the low nanomolar range [65,113] and Zika protease in the higher nanomolar range [57,122]. The differences between flaviviral and human serine proteases could be key to the design of selective antiflaviviral inhibitors without off-target inhibition of human proteases. Maximal selectivity can be

Inhibition of the precursor

Recent studies of autoprocessing of Dengue protease [136] have demonstrated that at least three sites in the precursor are processed in cis (intramolecularly) but not in trans (intermolecularly). As expected, the NS2B-NS3 junction is cleaved in cis. Surprisingly, the NS3 internal intrahelicase site and NS3-NS4A junction also are exclusively processed in cis [136]. ARDP0006 (1,8-dihydroxy-4,5-dinitroanthraquinone) has been reported as an active-site Dengue protease inhibitor that blocks

Allosteric inhibitors

Allosteric inhibitors may offer greater selectivity for flaviviral proteases over off-target human serine proteases.

A combination of pharmacophore construction and molecular docking using a representative set of diverse nonpeptidic Dengue protease inhibitors [110,111,115,[153], [154], [155], [156]] revealed a deep allosteric binding pocket in the vicinity of the catalytic triad. This pocket is formed by NS3 residues Val 155, Asn 119, Thr120, Asn 167, Ile 165, Glu 88 and Gly 87 on one side and

Drug repurposing

Drugs approved for other indications can offer a rapid, inexpensive way to treat newly emerging diseases, or they can become lead compounds for further optimization. The MicroSource Spectrum Collection was screened against Dengue protease, revealing tyrothricin as a micromolar inhibitor [168]. FDA-approved drugs have been screened against Zika protease, resulting in several positive hits. Among these, novobiocin and lopinavir-ritonavir [169], hydroxychloroquine [170], bromocriptine [171,172],

Concluding remarks

Zika and Dengue proteases are interesting enzymes, embedded in the NS3 protein together with NTPase, RNA 5′ triphosphatase and helicase domains. Very little is known about the structural and functional interplay between these domains. The extramembrane part of the membrane-intertwined NS2B protein is essential for activation and catalytic activity of NS3 serine protease. Both covalent and noncovalent active-site inhibitors and nonpeptide allosteric inhibitors are under intensive development.

Authors are in agreement

Material submitted is original, all authors are in agreement to have the article published.

Authors contributions

T.M., P.N., E.K., and J.K. wrote the manuscript. T.M, E.K., and P.N. conducted literature searches. P.N. and T.M. prepared the figures.

Acknowledgements

We thank Pavel Šácha, Evžen Bouřa, Kamil Hercík, Edward A. Curtis, Martin Hradilek and Pavel Majer for helpful discussions and Hillary Hoffman for language corrections. We acknowledge the financial support of the project InterBioMed LO 1302 from the Ministry of Education, Youth and Sports of the Czech Republic.

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