Skip to main content

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


With CA16, enterovirus-71 is the causative agent of hand foot and mouth disease (HFMD) which occurs mostly in children under 5 years-old and responsible of several outbreaks since a decade. Most of the time, HFMD is a mild disease but can progress to severe complications such as meningitis, brain stem encephalitis, acute flaccid paralysis (AFP) and even death; EV71 has been identified in all severe cases. Therefore, it is actually one of the most public health issues that threatens children’s life. \(3{\rm C}^{{\rm pro}}\) is a protease which plays important functions in EV71 infection. To date, a lot of \(3{\rm C}^{{\rm pro}}\) inhibitors have been tested but none of them has been approved yet. Therefore, a drug screening is still an utmost importance in order to treat and/or prevent EV71 infections. This work highlights the EV71 life cycle, \(3{\rm C}^{{\rm pro}}\) functions and \(3{\rm C}^{{\rm pro}}\) inhibitors recently screened. It permits to well understand all mechanisms about \(3{\rm C}^{{\rm pro}}\) and consequently allow further development of drugs targeting \(3{\rm C}^{{\rm pro}}\). Thus, this review is helpful for screening of more new \(3{\rm C}^{{\rm pro}}\) inhibitors or for designing analogues of well known \(3{\rm C}^{{\rm pro}}\) inhibitors in order to improve its antiviral activity.


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 [6,7,8,9]. 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.

Review on EV-71 life cycle

Fig. 1
figure 1

Illustration of EV71 life cycle and virus-host interactions. EV71 replication steps: from attachment to release (a). 3C-host proteins interactions are blocked by \(3{\rm C}^{{\rm pro}}\) inhibitors (b)

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).

\(3{\rm C}^{{\rm pro}}\) functions

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.

\(3{\rm C}^{{\rm pro}}\) inhibitors

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

Peptidomimetic compounds

  1. (a)

    Rupintrivir and analogues: Rupintrivir (AG7088) is probably the well-known \(3{\rm C}^{{\rm pro}}\) inhibitors to date. More than being a safe compound for the cells, it is able to bind to the active site of \(3{\rm C}^{{\rm pro}}\) [21]. It was firstly identified as \(3{\rm C}\) Human Rhinovirus (HRV) inhibitors, later Zhang et al. [34] shown that it also had a strong antiviral activity against EV71 \(3{\rm C}^{{\rm pro}}\) in both cell lines and animal models. In fact, AG7088 inhibits the antiviral activity at \({\rm EC}_{50}=0.01\,\upmu {\rm M}\) and protease activity at \({\rm IC}_{50}=2.5\pm 0.5\,\upmu {\rm M}\) with \({\rm CC}_{50}=1000\,\upmu {\rm M}\); in-vivo a low dose of 0.1 mg/kg prevent severe symptoms in suckling mice. Since the discovery of this compound, several analogues have been designed in order to increase its efficiency against EV71 infection [21]. To improve the anti-EV71 activity of rupintrivir, Kuo et al. has designed several inhibitor analogues (compound 1 to \(10{\rm b}\)) by replacing the P3 group of AG7088 with a series of cinnamoyl derivates. The compound \(10{\rm b}\) seemed to be potentially effective against EV71 among all the analogues, with an \({\rm EC}_{50}\) and \({\rm CC}_{50}\) of \(0.018\,\upmu {\rm M}\) and \(>25\,\upmu {\rm M}\) respectively [35]. Then later Shang et al. [36] replaced the cinnamoyl of compound 1 to 2-chloride-phenylacetyl and noticed that the efficiency of it antiviral activity has been increased twice\({\rm IC}_{50}=1.89\pm 0.25\,\upmu {\rm M}\). Another method to further improve rupintrivir action is to combine it with IFN\((\alpha /\beta )\) . In fact, it was proved that rupintrivir and Interferon had an synergistic inhibition against EV71 infection [37].

  2. (b)

    NK-1.8k: is a peptidyl aldehyde discovered to have strong anti-viral activity against not only EV71 but also the Enterovirus 68. The mechanism of action is known as the same as rupintrivir which targeted the \(3{\rm C}^{{\rm pro}}\) EV71 in dependent-concentration manner. However, structurally, they are different because NK-1.8k is a dipeptide with six-member-ring lactam and rupintrivir, a tripeptide with five-member-ring lactam. Thus, its structure confers to NK-1.8k a better stability and drug features than rupintrivir which is always taken as reference. Indeed, NK-1.8k decrease the viral RNA production at \({\rm EC}_{50}=34.5\, {\rm nM}\). Moreover, it is potent in all the 3 genotypes of EV71 in different cell lines (RD and T293 \({\rm EC}_{50}=0.108\,\upmu {\rm M}\) ; Vero \({\rm EC}_{50}=2.41\,\upmu {\rm M}\)) [24]. NK-1.8k represents a new peptidomimetic-compound which might take the place of rupintrivir as an achetype in EV71 drug screening.

  3. (c)

    SG85: the \(3{\rm C}^{{\rm pro}}\) inhibitors SG85 is a peptidic Michael acceptor compound. It has been tested against Enterovirus 68, EV71, echovirus 11 and various rhinovirus serotypes. However, it was found to be more potent against HRV11 and EV71 with \({\rm EC}_{50}=60\,{\rm nM}\), \({\rm EC}_{50}=180\,{\rm nM}\) respectively [38]. Furthermore, it has screened to have strong antiviral activity against all the 11 EV71 strains with \({\rm EC}_{50}\) between 0.039 and \(0.200\,\upmu {\rm M}\) [39]. Deep study of SG85 is needed in order to progress the drug discovery of EV71.

  4. (d)

    (R)-1: is proved to be one of the most efficient \(3{\rm C}^{{\rm pro}}\) inhibitors screened to date with an \({\rm EC}_{50}=0.088\pm 0.006\,\upmu {\rm M}\). However, the presence of cyanohydrins, which is labile, gives it unstable and toxic properties [40].

  5. (e)

    4e and 4g: are compounds resulted from improvement of (R)-1. In fact, acyl cyanohydrins which make unstable (R)-1 have been replaced by 4-iminooxazolidin-2-one. After a series of test, 4e and 4g were the compound having the most potent antiviral activity with \({\rm EC}_{50}=0.21\pm 0.005\) and \(0.033\pm 0.008\,\upmu {\rm M}\) respectively. Moreover, those compounds are safe towards the cell (\({\rm CC}_{50}>100\,\upmu {\rm M}\)). Thus, they can be used as base for EV71 drug therapy [41].

  6. (f)

    8v, 8w and 8x: are alpha-keto-amid inhibitors against EV71 \(3{\rm C}^{{\rm pro}}\). Zeng et al. noticed that the pivotal function of \(3{\rm C}^{{\rm pro}}\) makes it the ideal target to fight against EV71 infection. Then, they synthesized several alpha-keto-amids as 3C inhibitors via Passerini reaction. Hence, the compounds 8v, 8w and 8x were exhibiting the most potent antiviral activity against enterovirus 71 with \({\rm EC}_{50}=1.32\pm 0.26, 1.88\pm 0.35\,\,{\hbox {and}}\,\, 1.52\pm 0.31\,\upmu {\rm M}\) respectively. Nevertheless, those compounds should be more improved and studied in order to contribute for EV71 drug discovery which is currently in need [42].

Non-peptidyl compound: DC07090

Recently identified as novel small potent molecule \(3{\rm C}\) inhibitor, it is a non-peptidyl compound designed by docking-based virtual screening and able to bind with \(3{\rm C}\) through its binding site and reversible inhibits its protease activity at \({\rm EC}_{50}=22.09\pm 1.07\,\upmu {\rm M}\). Besides, DC07090 has a very low cytotoxicity rate \(({\rm CC}_{50}>200\,\upmu {\rm M})\) which makes it an attractive compound for further drug development [43].


Flavonoids, originally synthesized by the plants as abiotic stresses: in order to protect themselves against ultraviolet radiation, pathogens and herbivores are a group of natural compounds largely distributed in fruits, vegetables, tea, soy foods and herbs. Most importantly, they have huge therapeutic bioactivities: anti-oxidative, anti-inflammatory and antiviral properties. Researchers used them as a base of drug and dietary supplement in several diseases [44]. They present an attractive therapy for Enterovirus 71 due to their low toxicity towards host cells and their strong antiviral activity.

  1. (a)

    Luteoloside: is a flavonoid distributed mainly in Lonicera japonica, plant used in traditionnal Chinese medicine, and has got broad activities such as anti-microbial, anti-cancer and antiviral activity against influenza virus, human rhinovirus, coxsackievirus B4 and enterovirus 71. The real mechanisms against EV71 remain unknown and need further deep to elucidate but it is sure that it blocked the pathway at \(3{\rm C}\) protease activity stage, \({\rm IC}_{50}=0.36{\rm mM}\) with a selectivity index of 5.3 according to the investigation of Cao et al. Therefore, it is an excellent candidate for drug development [45].

  2. (b)

    Ouercetin: is a member of the flavonol subgroup of flavonoid found in many plants, fruits, grains and vegetables with anti-inflammatory, anti-cancer and anti-viral properties. It is probably one of the latest \(3{\rm C}\) inhibitor tested. Without toxicity towards the cells, our group’s recent finding reveals that quercentin exhibits a prominent effectivity against the protein \(3{\rm C}\) of enterovirus 71 by binding its substrate-binding pocket. Moreover, quercentin seems to have a preventive action. Indeed, cells pre-treated by quercetin present a high survival rate when infected by EV71 virus. Consequently, quercetin may be used both in preventive and in therapeutic application [46]. Therewith, a drug library composing of 1430 FDA approved drugs were previously screened from our laboratory. Interestingly, we found that the compound 3 had significantly anti-EV71 effect among them. Further mechanism study revealed that it targeted viral 3\({\rm C}\) protease and block viral replication (unpublished data).

  3. (c)

    Diisopropyl Chrysin-7-i1 Phosphate (CPI): is a phosphate ester of chrysin, a natural flavonoid found in many plants. CPI is able to bind in the pocket site of hydrophobic and polar residue of \(3{\rm C}\) protease like LEU4- 8, SER-I I I, MET-112. PHE-113 and PRO-115 and inhibits the protease activity at \({\rm EC}_{50}=4.03\,{\rm mM}\). Indeed, \(3{\rm C}^{{\rm pro}}\) is unable to cleave human interferon regulator factor 9(IRF9) in the presence of CPI [47].


siRNA is a powerful tool which can be used to target a specific gene in order to suppress it. Small interfering RNA therapeutics has been explored against several human viral infections including Enterovirus due to its specificity and promising effect both in-vitro and in-vivo [48]. Indeed, siRNA recognize, bind and degrade the target mRNA. It is a challenging strategy by the potential risk of mutation, inflammation or immune responses. However, Yang et al. showed that there is any toxicity of the siRNA targeted \(3{\rm C}^{{\rm pro}}\) and \(3{\rm D}^{{\rm pol}}\) during their investigation. They have designed a novel minicircle vector through \(3{\rm C}^{{\rm pro}}\) and \(3{\rm D}^{{\rm pol}}\) sequence available in Genbank. In fact, the siRNA did not affect the growth and viability of the cell. Moreover, it has reduced the protein levels to \(10.8\pm 6.7\%\), the viral mRNAs to \(12.4 \pm 1.75\%\) and the progeny virion production to 15% in infected cells. More importantly, it has protected the infected-suckling mice of a significant weight loss and hind limbs paralysis. Hence, further investigation must be conducted about silencing gene strategy within using \(3{\rm C}^{{\rm pro}}\) as target [49].


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 [50,51,52,53]. Therefore, even if it is a challenging technique, investigating this strategy is worth it.

Table 1 Detailed list and classification of 3Cpro inhibitors: chemical structure, classes, effectivity, test in cell lines and animal models


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.

Availability of data and materials

Not applicable.



Enterovirus 71


Coxsackievirus A16


Hand, foot and mouth disease


Acute flaccid paralysis

3Cpro :

3C protease


Central Nervous System


Ribonucleic acid


non-translated regions


Scavenger receptor class B member 2


P-selectin glycoprotein ligand-1



2Apro :

2A protease

3Dpol :

3D polymerase


IRES-specific-trans-acting factors


Internal ribosome site


RNA-dependent RNA- polymerase


Pattern-recognition receptors


Toll-like receptors


Interferon type I


RIG-I-like receptors


NOD-like receptors

IFNα/β :

Interferon alpha/beta


Karyopherin subunit Alpha-1


Signal transducer and activator of transcription


Transforming growth factor beta activated kinase


TGF-beta activated kinase 1/2/3


Interferon regulator 3/7


heterogeneous nuclear ribonucleoprotein


PIN2 interacting telomerase inhibitor-1


messenger Ribonucleic acid


Human Rhinovirus

EC50 :

50% Effective Concentration

IC50 :

50% Inhibition concentration

CC50 :

50% Cytotoxic Concentration


Food and Drug Administration


Chrysin-7-il Phosphate












Small Intefering RNA


RNA interference




Verda Reno


Buffalo green monkey kidney cells


Henrietta Lack


  1. Schmidt NJ, Lennette EH, Ho HH. An apparently new enterovirus isolated from patients with disease ofthe central nervous system. J Infect Dis. 1974;129(3):304–9.

    Article  CAS  PubMed  Google Scholar 

  2. Putnak JR, Phillips BA. Picornaviral structure and assembly. Microbiol Rev. 1981;45(2):287–315.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Yuan J, et al. Enterovirus A71 proteins: structure and function. Front Microbiol. 2018;9:286.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis. 2010;10(11):778–90.

    Article  PubMed  Google Scholar 

  5. Zeng M, et al. Seroepidemiology of Enterovirus 71 infection prior to the 2011 season in children in Shanghai. J Clin Virol. 2012;53(4):285–9.

    Article  PubMed  Google Scholar 

  6. Podin Y, et al. Sentinel surveillance for human enterovirus 71 in Sarawak, Malaysia: lessons from the first 7 years. BMC Public Health. 2006;6:180.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Mizuta K, et al. Molecular epidemiology of enterovirus 71 strains isolated from children in Yamagata, Japan, between 1990 and 2013. J Med Microbiol. 2014;63:1356–62.

    Article  PubMed  Google Scholar 

  8. Te Lee J, et al. Enterovirus 71 seroepidemiology in Taiwan in 2017 and comparison of those rates in 1997, 1999 and 2007. PLoS ONE. 2019;14(10):e0224110.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Van Le T, Nguyen VTT, Nguyen QH, Pham DT. Molecular epidemiology analysis of enterovirus 71 strains isolated in Dak Lak, Vietnam, 2011–2016. J Med Virol. 2019;91(1):56–64.

    Article  PubMed  Google Scholar 

  10. Liang Z, Wang J. EV71 vaccine, an invaluable gift for children. Clin Transl Immunol. 2014;3(10):e28.

    Article  CAS  Google Scholar 

  11. Lin JY, Kung YA, Shih SR. Antivirals and vaccines for Enterovirus A71. J Biomed Sci. 2019;26(1):65.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Lin C, Chen KH, Tong Chen K. Update on enterovirus 71 infections: epidemiology, molecular epidemiology, and vaccine development. J Infect Dis Ther. 2018.

    Article  Google Scholar 

  13. Wang H, Li Y. Recent progress on functional genomics research of enterovirus 71. Virol Sin. 2019;34(1):9–21.

    Article  CAS  PubMed  Google Scholar 

  14. Yamayoshi S, Fujii K, Koike S. Receptors for enterovirus 71. Emerg Microbes Infect. 2014;3:e53.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Xu Y, Liu Q, Zhang Z. Human EV71 invades human neuroblastoma SK-N-SH cells by clathrin-mediated endocytosis. Xi Bao Yu Fen Zi Mian Yi XueZaZhi. 2017;33(6):761–6.

    Google Scholar 

  16. Yuan M, et al. Enhanced human enterovirus 71 infection by endocytosis inhibitors reveals multiple entry pathways by enterovirus causing hand-foot-and-mouth diseases. Virol J. 2018;15(1):1.

    Article  PubMed  PubMed Central  Google Scholar 

  17. Danthi P, Tosteson M, Li Q, Chow M. Genome delivery and ion channel properties are altered in VP4 mutants of poliovirus. J Virol. 2003;77(9):5266–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Baggen J, Thibaut HJ, Strating JR, van Kuppeveld FJ. The life cycle of non-polio enteroviruses and how to target it. Nat Rev Microbiol. 2018;16(6):368–81.

    Article  CAS  PubMed  Google Scholar 

  19. Huang PN, et al. Far upstream element binding protein 1 binds the internal ribosomal entry site of enterovirus 71 and enhances viral translation and viral growth. Nucleic Acids Res. 2011;39(22):9633–48.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Lin JY, Li ML, Shih SR. Far upstream element binding protein 2 interacts with enterovirus 71 internal ribosomal entry site and negatively regulates viral translation. Nucleic Acids Res. 2009;37(1):47–59.

    Article  CAS  PubMed  Google Scholar 

  21. Wang J, et al. Crystal structures of enterovirus 71 3C protease complexed with rupintrivir reveal the roles of catalytically important residues. J Virol. 2011;85(19):10021–30.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ma HY, et al. Association of EV713C polymorphisms with clinical severity. J Microbiol Immunol Infect. 2018;51(5):608–13.

    Article  CAS  PubMed  Google Scholar 

  23. Bingqing L, et al. A novel enterovirus 71 (EV71) virulence determinant: The 69th residue of 3C protease modulates pathogenicity. Front Cell Infect Microbiol. 2017;7:26.

    Article  CAS  Google Scholar 

  24. Wang Y, Yang B, Zhai Y, Yin Z, Sun Y, Rao Z. Peptidyl aldehyde NK-1.8k suppresses enterovirus 71 and enterovirus 68 infection by targeting protease 3C. Antimicrob Agents Chemother. 2015;59(5):2636–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Chen KR, Ling P. Interplays between enterovirus A71 and the innate immune system. J Biomed Sci. 2019;26(1):95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wang C, et al. Enterovirus 71 suppresses interferon responses by blocking Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling through inducing karyopherin-α1 degradation. J Biol Chem. 2017;292(24):10262–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Lei X, Han N, Xiao X, Jin Q, He B, Wang J. Enterovirus 713C inhibits cytokine expression through cleavage of the TAK1/TAB 1/TAB 2/TAB3 complex. J Virol. 2014;88(17):9830–41.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Lei X, Xiao X, Xue Q, Jin Q, He B, Wang J. Cleavage of interferon regulatory factor 7 by enterovirus 713C suppresses cellular responses. J Virol. 2013;87(3):1690–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Li ML, et al. The 3C protease activity of enterovirus 71 induces human neural cell apoptosis. Virology. 2002;293(2):386–95.

    Article  CAS  PubMed  Google Scholar 

  30. Li ML, et al. EV713C protease induces apoptosis by cleavage of hnRNP Al to promote apaf-l translation. PLoS ONE. 2019;14(9):e0221048.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Li J, et al. Enterovirus 713C promotes apoptosis through cleavage of PinXl, a telomere binding protein. J Virol. 2017.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Sharma R, Raychaudhuri S, Dasgupta A. Nuclear entry of poliovirus protease- polymerase precursor 3CD: implications for host cell transcription shut-off. Virology. 2004;320(2):195–205.

    Article  CAS  PubMed  Google Scholar 

  33. Weng KF, Li ML, Hung CT, Shih SR. Enterovirus 713C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation. PLoS Pathog. 2009;5(9):e1000593.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Zhang XN, Song ZG, Jiang T, Shi BS, Hu YW, Yuan ZH. Rupintrivir is a promising candidate for treating severe cases of Enterovirus-71 infection. World J Gastroenterol. 2010;16(2):201–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kuo CJ, et al. Design, synthesis, and evaluation of 3C protease inhibitors as anti-enterovirus 71 agents. Bioorgan Med Chem. 2008;16(15):7388–98.

    Article  CAS  Google Scholar 

  36. Shang L, et al. Biochemical characterization of recombinant enterovirus 71 3C protease with fluorogenic model peptide substrates and development of a biochemical assay. Antimicrob Agents Chemother. 2015;59(4):1827–36.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Hung HC, Wang HC, Shih SR, Teng IF, Tseng CP, Hsu JTA. Synergistic inhibition of enterovirus 71 replication by interferon and rupintrivir. J Infect Dis. 2011;203(12):1784–90.

    Article  CAS  PubMed  Google Scholar 

  38. Tan J, et al. 3C protease of enterovirus 68: structure-based design of Michael acceptor inhibitors and their broad-spectrum antiviral effects against picornaviruses. J Virol. 2013;87(8):4339–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Tijsma A, et al. The capsid binder vapendavir and the novel protease inhibitor SG85 inhibit enterovirus 71 replication. Antimicrob Agents Chemother. 2014;58(11):6990–2.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Y Z, et al. Cyanohydrin as an anchoring group for potent and selective inhibitors ofenterovirus 713C protease. J Med Chem. 2015;58(23):9414–20. LK -.

    Article  CAS  Google Scholar 

  41. Ma Y, et al. 4-Iminooxazolidin-2-one as a Bioisostere of the cyanohydrin moiety: inhibitors of enterovirus 71 3C protease. J Med Chem. 2018;61(22):10333–9.

    Article  CAS  PubMed  Google Scholar 

  42. Zeng D, et al. Synthesis and structure-activity relationship of α-keto amides as enterovirus 713C protease inhibitors. Bioorgan Med Chem Lett. 2016;26(7):1762–6.

    Article  CAS  Google Scholar 

  43. Ma GH, et al. Identification and biochemical characterization of DC07090 as a novel potent small molecule inhibitor against human enterovirus 71 3C protease by structure-based virtual screening. Eur J Med Chem. 2016;124:981–91.

    Article  CAS  PubMed  Google Scholar 

  44. Zakaryan H, Arabyan E, Oo A, Zandi K. Flavonoids: promising natural compounds against viral infections. Arch Virol. 2017;162(9):2539–51.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Cao Z, et al. Luteoloside acts as 3C protease inhibitor of enterovirus 71 in vitro. PLoS ONE. 2016;11(2):e0148693.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Yao C, et al. Inhibition of enterovirus 71 replication and viral 3C protease by quercetin. Virol J. 2018;15(1):116.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Wang J, Zhang T, Du J, Cui S, Yang F, Jin Q. Anti-enterovirus 71 effects of chrysin and its phosphate ester. PLoS ONE. 2014;9(3):e89668.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Tan EL, Tan TMC, Chow VTK, Poh CL. Inhibition of enterovirus 71 in virusinfected mice by RNA interference. Mol Ther. 2007;15(11):1931–8.

    Article  CAS  PubMed  Google Scholar 

  49. Yang Z, Li G, Zhang Y, Liu X, Tien P. A novel minicircle vector based system for inhibting the replication and gene expression of enterovirus 71 and Coxsackievirus A16. Antivir Res. 2012;96(2):234–44.

    Article  CAS  PubMed  Google Scholar 

  50. Subramanya S, Kim SS, Manjunath N, Shankar P. RNA interference-based therapeutics for human immunodeficiency virus HIV-I treatment: synthetic siRNA or vectorbased shRNA? Expert Opin Biol Ther. 2010;10(2):201–13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ashfaq UA, Yousaf MZ, Aslam M, Ejaz R, Jahan S, Ullah O. SiRNAs: potential therapeutic agents against Hepatitis C Virus. Virol J. 2011;8:1–6.

    Article  CAS  Google Scholar 

  52. Fujimoto Y, et al. Antiviral effects against influenza a virus infection by a short hairpin RNA targeting the non-coding terminal region of the viral nucleoprotein gene. J Vet Med Sci. 2019;81(3):383–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Wang W, Peng H, Li J, et al. Controllable inhibition of hepatitis B virus replication by a DR1-targeting short hairpin RNA (shRNA) expressed from a DOX-inducible lentiviral vector. Virus Genes. 2013;46:393–403.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references


Not applicable.


Not applicable.

Author information

Authors and Affiliations



RD wrote the review under the lead, supervision and correction of HK. All the authors read and approved the final manuscript.

Corresponding author

Correspondence to Kanghong Hu.

Ethics declarations

Ethics approval and consent for participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

Not applicable.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Diarimalala, R.O., Hu, M., Wei, Y. et al. Recent advances of enterovirus 71 \(3{\rm C}^{{\rm pro}}\) targeting Inhibitors. Virol J 17, 173 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI:


  • Enterovirus 71
  • Enterovirus 71 life cycle
  • \(3{\rm C}^{{\rm pro}}\) functions
  • \(3{\rm C}^{{\rm pro}}\) inhibitors
  • EV71 drugs screening