Recent advances of enterovirus 71 \(3{\rm C}^{{\rm pro}}\) targeting Inhibitors

Enterovirus 71, belongs to human enterovirus A species, Picornaviridae family, was discovered in a patient with central nervous system (CNS), in California, 1969 [1]. In term of structure, EV71 is a non-enveloped virus with a capsid made up of 60 protomers of envelop proteins and contains a single-stranded RNA positive [2, 3]. Each protomer contains four envelop proteins: VP1–VP2–VP3, located in the external part and are exposed to the host antibodies and cell receptors; and VP4 which is completely hidden in the internal part. The RNA genome is small \((7.5\,{\rm kb})\) and constituted by 3 parts: Pl, P2 and P3, flanked by 2 UTRs (non-translated regions) located in \(5^{\prime }\) and \(3^{\prime }\) [4]. Several outbreaks and fatal cases, caused by this virus, make it a major public health issue mainly in the Asia-Pacific region. Indeed, China has experienced the latest and largest outbreaks with more than 1.7 million cases, 27.000 patients with severe neurological complications and 905 deaths, in 2010 [5]; while a cyclical and seasonal pattern occurs in Sarawak, Japan, Taiwan and Vietnam [6‐9]. To manage such infections and epidemics is primordial, and the best way to eradicate this infection is the combination of a valuable vaccine and drugs [10]. Nevertheless, vaccine research has progressed more than drugs discovery because to date there is no approved drug against EV71 while 3 vaccines have completed their clinical trials III and are in following-up stage [11]. For this reason, the treatment is only symptomatic along with public surveillance systems [12]. Many plant extracts and chemical compounds have been discovered as having a potential effects against the virus and might be used as drugs against enterovirus 71 infections but none of them has been approved yet [13]. Thus, the finding of an approved and valuable drug is still an utmost importance. \(3{\rm C}^{{\rm pro}}\) represent a valuable target because it has primordial functions in both virulence and virus-host interactions. This review highlights the important functions and recent progress of \(3{\rm C}^{{\rm pro}}\) inhibitors and permit to acknowledge that \(3{\rm C}^{{\rm pro}}\) is a valuable target for EV71 drug development, which should be deeply investigated.

The EV71 life cycle goes through an attachment and entry, via a recognition and binding of surface protein to the cell receptors (SCARB2, PSGL-I, Anx2, Heparan Sulfate, Sialylated glycan) [14], to the release of the new virions by cell lysis (Fig. 1a). The mechanism of entry is known as through clathrin-mediated endocytosis (real events remain unclear) but recent investigation has showed that multiple pathways may be used by EV71 to enter the host cells [15, 16]. Then, a series of conformational changes occurs at low pH and let the virus to leave his icosahedral capsid structrure to an A-particle: loss of VP4 and formation of a channel followed by a release of RNA in cell cytoplasm [17]. Once the RNA is located in the cytoplasm, the viral genome, as a positive sense, act as an mRNA, so directly translated into a polyprotein (Pl, P2, P3) of 2193 AA. The polyprotein processing is assured by two main proteins \(2{\rm A}^{{\rm pro}}\) and \(3{\rm C}^{{\rm pro}}\). Thus, \(2{\rm A}^{{\rm pro}}\) and \(3{\rm C}^{{\rm pro}}\) cleaved the polyprotein into VP1–VP4 (structural protein) and 2A–2C, 3A–3D (non-structural protein) [18]. When a considerable number of the 11 mature proteins are synthesized, the RNA replication take place after the interactions of IRES-specific-trans-acting factors (ITAFs), which are translocated from the nucleus to the cytoplasm, with the internal ribosome site (IRES) at its stem-loop [19, 20]. A negative-RNA is first synthesis within using the viral genome as template, and then followed by synthesis of numerous positive-strands using in turn the negative-strand as template. RNA-dependent RNA- polymerase (RdRp) or \(3{\rm D}^{{\rm po}1}\) is the viral enzyme responsible of the RNA synthesis [18]. Finally, the structural proteins and the genome is encapsidated to form a new virion which is released during lysis of the cell (apoptosis).

In addition to its polyprotein processing activity, the non-structural protein \(3{\rm C}\) plays a role in numerous biological mechanisms. Recent discovery of the \(3{\rm C}\) crystal structure has permit to identify the sites of its substrat binding affinity (between 2 similar \(\beta\)-ribbon) and confirmed its cleavage activity of the viral polyprotein but also several host proteins in order to optimize viral replication and spreading [21]. EV71 infection symptoms range from mild to severe diseases which depend on both the viral genetic sequence and the host immune system. In fact, the relationship between \(3{\rm C}\) genome sequence and the corresponding clinical symptoms (mild or severe) revealed that the 79th residue is the responsible sequence that leads to severe diseases [22]. Besides, Li et al. [23] have found another residue associated with the virulence of EV71, their finding suggests that the 69th residue is the virulent determinant because a single mutation of the hydrogen bond between Asn69 and Glu71 causes a significant decrease in the EV71 infection. The same result was found during the study of NK-1.8k compound where the substitution of asparagine at 69th residue by serine has decreased the fitness of the virus but on the other hand causes total resistance towards the tested compound. Indeed, the 69th residue plays an important role in \(3{\rm C}^{{\rm pro}}\) functions even if it is not directly part of the active site according to the crystal structure [24]. EV71 interacts with the innate immune system through PRRs (Pattern-recognition receptors) such as TLRs which is involved in \({\rm IFN}-{\rm I}\) production, RLRs responsible for detection of RNA virus infection and NLRs which function is to form cytosolic inflammasome [25]. In fact, concomitantly with the virus invasion, different host-immune responses occur such as production of type I interferon (IFN\((\alpha /\beta )\)) ; then to escape and to impair the immunity, the virus uses the proteolytic activity of \(3{\rm C}^{{\rm pro}}\) by cleaving numerous needed host proteins: KPNA-I in order to suppress the signaling pathway STAT/KPNA-I [26], \({\rm TAK}1/{\rm TAB}1/{\rm TAB}2/{\rm TAB}3\) complex [27], TRIF, shut-off \({\rm IR}3/7\) [28] and consequently block the production of IFN\((\alpha /\beta )\) Likewise, to permit the release and spread of virus progeny, \(3{\rm C}\) induced apoptosis of host cells through the capsase-3 pathway [29], cleavage of hnRNPA1 [30] and PinXl [31]. Finally, \(3{\rm C}\) is able to enter the nuclei through its precursor \(3{\rm CD}\) [32] and cleaves the polyadenylation factor CstF-64. As a result, the host mRNA 3' polyadenylation ,which is essential for its translocation, stability and translation, is shut off [33] (Fig. 1b). Due to such functions, 3C is definitely an excellent target for drug screening.

\(3{\rm C}^{{\rm pro}}\) is an important target to block EV71 replication. Indeed, several \(3{\rm C}^{{\rm pro}}\) inhibitors have been deeply investigated (Table 1, Fig. 1b)

The unavailable of approved clinical drug makes the finding of a potent compound against EV71 really important. \(3{\rm C}^{{\rm pro}}\) is an essential protein for EV71 life cycle and infection, moreover, it has strict subtract and does not have a lot of homologues in mammalian cells [35]. Thus, it is an excellent and attractive target for development of potent drugs. In this review, we summarized several classes of compound recently screened and also rupintrivir which is the drug of reference against \(3{\rm C}^{{\rm pro}}\). Actually, rupintrivir and analogues are considered as the most potent \(3{\rm C}^{{\rm pro}}\) inhibitors. However, NK-1.8k has almost the same potency and efficiency as rupintrivir (Table 1), and as more stable, it can take the place of rupintrivir as archetype of \(3{\rm C}^{{\rm pro}}\) inhibitors. In fact, peptidomimetic compounds represent the most potent class with the minimal effective concentration (180 nM to 2.89 μM, Table 1). It might be due to the fact that they are synthetically designed to fit in the \(3{\rm C}^{{\rm pro}}\) active pocket. Nevertheless, flavonoids class, which is composed of active compounds from plants, has satisfactory antiviral activity as well. Indeed, nowadays, the trend of using bioactive compounds as drug candidates is done more and more, because of their broad biological and pharmacological activities, their availability and safety towards the host cells. Besides, the screening of non-peptidyl compound has been tempted but only DC07090 among 50 other compounds has given a satisfactory result [43]. Peptidomimetic compounds might be more potent and interesting than non-peptidyl-compounds. Hence, deep investigation, mainly in an appropriate animal model, should be done for luteoloside, quercentin and CPI which could be approved as EV71 therapy; while more and more peptidomimetic compounds should be designed and/or improved by using the revelation of \(3{\rm C}^{{\rm pro}}\) structure as reference. Following the drug screening work, the 69th residue of \(3{\rm C}^{{\rm pro}}\), which plays important role in conferring EV71 resistance, could be investigated in order to make sure that the virus will not develop a resistance mutation toward the potent drug as investigated by Wang et al. [24]. Finally, the last recent strategy is the use of RNAi. In fact, there are few investigation about siRNA as therapy against EV71 infection; however, it has been successful against a wide range of viruses: Human immunodeficiency virus, hepatitis B/C virus, Influenza virus [50‐53]. Therefore, even if it is a challenging technique, investigating this strategy is worth it.

Coupling an effective vaccine and drugs against Enterovirus 71 is the most prominent manner to eradicate EV71 infection. The prevention will be secure by the vaccine and the treatment by an effective drug. However, the drug progress has not been as developed as for vaccines. In fact, currently only a surveillance is set up to control the disease. EV71 is a threat for children’s life; therefore, the screening of an effective drug is quite indispensable as soon as possible. For that, \(3{\rm C}^{{\rm pro}}\) represent an excellent target due to the several key functions that it plays in both virulence and interaction of the virus to the host. More \(3{\rm C}^{{\rm pro}}\) inhibitors should be exploited. Besides, as \(3{\rm C}^{{\rm pro}}\) and \(2{\rm A}^{{\rm pro}}\) play role in early stage of the viral replication through cleaving the EV71 polyprotein, a combination of \(2{\rm A}^{{\rm pro}}\) and \(3{\rm C}^{{\rm pro}}\) inhibitors in order to act in a synergetic manner may represent a valuable strategy. Indeed, the 3C X-ray structure is already defined so it would promotes further studies of its protease activity inhibitions by a compound. Meanwhile, all drugs screening must be tested in an appropriate animal model which will be compare to the in-vitro screening in order to achieve the goals of using it as treatment against EV71 infections.