Elsevier

Antiviral Research

Volume 141, May 2017, Pages 91-100
Antiviral Research

Inhibition of enterovirus 71 replication by an α-hydroxy-nitrile derivative NK-1.9k

https://doi.org/10.1016/j.antiviral.2017.01.002Get rights and content

Highlights

  • Peptidomimetics with hydroxy-nitrile warheads were found to inhibit the EV71 3C protease.

  • An α-hydroxy-nitrile derivative NK-1.9k potently inhibits EV71 with low cytotoxicity.

  • The hydroxy-nitrile group provides better potency, selectivity, and pharmacological properties for further development.

Abstract

Enterovirus 71 (EV71) is one of the major etiological agents of human hand-foot-and-mouth disease (HFMD) worldwide. EV71 infection in young children and people with immunodeficiency causes severe symptoms with a high fatality rates. However, there is still no approved drugs to treat such infections. Based on our previous report of a peptide-aldehyde anti-EV71 protease, we present here a highly specific α-hydroxy-nitrile derivative NK-1.9k, which inhibited the proliferation of multiple EV71 strains and coxsackievirus A16 (CVA16) in various cells with EC50 of 37.0 nM with low cytotoxicity (CC50 > 200 μM). The hydroxy-nitrile covalent warhead conferred NK-1.9k high potency and selectivity to interact with the cysteine residue of the active site of the viral protease. We also documented the resistance to NK-1.9k with a N69S mutation in EV71 3Cpro. The combination of NK-1.9k and EV71 polymerase or entry inhibitors produced strong synergistic antiviral effects. Collectively, our findings suggest our compounds can potentially be developed as drugs for the treatment of HFMD.

Introduction

Enterovirus 71 (EV71) is one of the major etiologic agents of hand-foot-and-mouth disease (HFMD) worldwide (McMinn, 2002). Infection of EV71 may cause severe encephalitis, aseptic meningitis, pulmonary edema, acute flaccid paralysis, and myocarditis, and thus leads to a high fatality rate, especially in children and people with immunodeficiency (Huang and Shih, 2014). In recent years, EV71 was reported to cause several large-scale outbreaks of severe complications in children involving the central nervous system, resulting in fatalities (Mao et al., 2016) (Sun et al., 2016). In 2010, China experienced her largest outbreak with an estimated 1.7 million infections of which 27,000 suffered severe neurological complications, resulting in 905 fatalities (Zeng et al., 2012).

EV71 belongs to the Enterovirus genus in the Picornaviridae family. The genome of EV71 contains a single-stranded positive-sense polyadenylated RNA with a length of approximately 7500 nucleotides and guides the translation of a viral polyprotein with a molecular weight of approximately 250 kDa (Chen et al., 2013, Sun et al., 2012). This polyprotein is further processed into P1, P2 and P3 precursors. P1 contains four structural proteins VP1, VP2, VP3 and VP4 to form the viral capsid. P2 and P3 encode seven nonstructural proteins, including 2Apro, 2B, 2C, 3A, 3B, 3Cpro and 3Dpol, which are involved in genome replication and processing. The correct proteolysis processing of polyprotein into individual functional proteins by 2Apro and 3Cpro proteases is one of the essential steps for virus proliferation. Because viral proteases play crucial roles in viral survival and propagation, they are potential drug targets using protease inhibitors (De Clercq and Li, 2016).

Currently, there are no approved small-molecule drugs for treating HFMD (Tan et al., 2016). Until now, several strategies have been launched to discover anti-EV71 inhibitors (Guo et al., 2016, Zhang et al., 2013). For example, NITD008 is an adenosine nucleoside analog that potently inhibits flaviviruses by functioning as a polymerase chain-terminator, displaying high anti-EV71 activity (Deng et al., 2014, Qing et al., 2016, Shang et al., 2014, Yin et al., 2009). Moreover, three capsid binding compounds, i.e., pleconaril, ALD and cyclosporine, block the entry of EV71 (De Colibus et al., 2014). In our previous work, we reported that a peptidyl aldehyde-derivative of PI, NK-1.8k, inhibited EV71 3Cpro activity and showed potent EV71 inhibition (Wang et al., 2015, Zhai et al., 2015). However, the selectivity of NK-1.8k remained imperfect since the strong electrophilic aldehyde group in NK-1.8k reacted with both the thiol group of cysteine proteases and the hydroxyl group of a wide range of serine proteases. Moreover, the EV71-inhibition of NK-1.8k (with an EC50 value of 0.28 μM) could be further optimized.

To increase the selectivity and inhibitory potency, we introduced the α-hydroxy-nitrile electrophile into anti-EV71 PIs. The peptidyl α-hydroxy-nitrile derivative NK-1.9k effectively inhibited various EV71 strains in human (RD and 293T) and monkey (Vero) cell lines with a best EC50 value of 37.0 ± 0.1 nM; NK-1.9k also inhibited CVA16 with an EC50 value of 52.0 ± 0.4 nM. We also characterized the profile of EV71 resistance to NK-1.9k with a mutation in EV71 3Cpro. In addition, NK-1.9k exhibited a strong synergistic anti-EV71 effect when combined with reported polymerase and entry inhibitors. These findings may be relevant to the development of clinical therapeutics to treat EV71 infections.

Section snippets

Cells and viruses

Human rhabdomyosarcoma (RD) cells, African green monkey kidney (Vero) cells and human embryonic kidney 293T (HEK-293T) cells were cultured in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a 5% CO2 humidified incubator. EV71 strain SK-EV006 with a green fluorescent protein reporter (EV71-GFP) was donated by Prof. Satoshi Koike (Tokyo Metropolitan Institute of Medical Science,

Analysis of active group of peptidyl mimic PI

Aldehydes are widely employed as covalent electrophiles in peptidomimetic protease inhibitors, covalently binding to the active cysteine or serine of the target protease. However, aldehydes are rapidly metabolized into alcohols or carboxylic acids in vivo (Leak and Dalton, 1983). As a result, they typically exhibiting poor bioavailability in vivo. In addition, the highly reactive aldehyde warhead results in low selectivity, initiating non-specific covalent interactions with different types of

Discussion

The current lack of antiviral strategies to treat HFMD patients has stimulated work to develop novel inhibitors to combat this growing healthcare problem. Viral proteases are deemed plausible targets due to their substrate specificity (Andany and Loutfy, 2013) (Llibre et al., 2013) (Pearlman, 2012). A number of viruses encode cysteine proteases for virus replication inside host cells to process the polyprotein translated from the viral RNA genome into individual functional proteins. We

Acknowledgements

This work is supported by the National Major Project (2014CB542800 and 2013CB911100), National Natural Science Foundation of China (31370733 and 81322023) and Tsinghua University Initiative Scientific Research Program (20131089228).

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    These authors contribute equally to this work.

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