Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study

Tang, Qi; Xu, Zhichao; Jin, Mengyu; Shu, Ting; Chen, Yinuo; Feng, Leilei; Zhang, Qiuhan; Lan, Ke; Wu, Shuwen; Zhou, Hai‐Bing

doi:10.1016/j.ejmech.2020.112310

Cited by 38 publications

(23 citation statements)

References 36 publications

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“…It has often been hypothesized that 2C inhibitors bind directly in the catalytic site. One study suggested that dibucaine analogs target the ATP-binding site ( 38 ), but no direct evidence supporting this hypothesis was presented. Recently, we obtained evidence for an allosteric binding site based on CV-B3, EV-A71, and EV-D68 mutants that were raised against a highly potent 2C inhibitor ( 26 ).…”

Section: Discussionmentioning

confidence: 99%

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex

et al. 2022

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Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing screens identified 2C-targeting compounds such as fluoxetine and dibucaine, but how they inhibit 2C is unknown. Here, we present a crystal structure of the soluble and monomeric fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), revealing an allosteric binding site. To study the functional consequences of SFX binding, we engineered an adenosine triphosphatase (ATPase)-competent, hexameric 2C protein. Using this system, we show that SFX, dibucaine, HBB [2-(-hydroxybenzyl)-benzimidazole], and guanidine hydrochloride inhibit 2C ATPase activity. Moreover, cryo-electron microscopy analysis demonstrated that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition. Collectively, these results provide important insights into 2C inhibition and a robust engineering strategy for structural, functional, and drug-screening analysis of 2C proteins.

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Section: Discussionmentioning

confidence: 99%

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex

et al. 2022

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“…It has often been hypothesized that 2C inhibitors bind directly in the catalytic site. One study suggested that dibucaine analogues target the ATP binding site 42 , but no direct evidence supporting this hypothesis was presented. Recently, we obtained evidence for an allosteric binding site based on CV-B3, EV-A71 and EV-D68 mutants that were raised against a novel and highly potent 2C inhibitor 29 .…”

Section: Discussionmentioning

confidence: 99%

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes the hexameric complex

Hurdiss

Kazzi

Bauer

et al. 2021

Preprint

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The enterovirus genus encompasses many clinically important human pathogens such as poliovirus, coxsackieviruses, echoviruses, numbered enteroviruses and rhinoviruses. These viruses are the etiological agents of several human diseases, including hand-foot-and-mouth disease, neonatal sepsis, encephalitis, meningitis, paralysis and respiratory infections. There is an unmet need for antivirals to treat these diseases. The non-structural protein 2C is a AAA+ helicase and plays a key role in viral replication. As such, it is an attractive target for antiviral drug development. Several repurposing screens with FDA-approved drugs have identified 2C-targeting compounds such as fluoxetine and dibucaine, but the molecular basis of 2C inhibition has remained enigmatic. Here we present the 1.5 Å resolution crystal structure of the soluble fragment of coxsackievirus B3 2C protein in complex with (S)-fluoxetine (SFX), which reveals a conserved, hydrophobic drug-binding pocket which is distal to the ATP binding site. To decipher the molecular mechanism of inhibition by fluoxetine and other 2C-targeting compounds, we engineered a soluble, hexameric and ATPase competent 2C protein. Using this system, we show that SFX, dibucaine, HBB and guanidine hydrochloride inhibit 2C ATPase activity in a dose-dependent manner. Moreover, using cryo-EM analysis, we demonstrate that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition and allowing us to generate the first reconstruction of the oligomeric complex. Taken together, these results provide important structural and mechanistic insights into 2C inhibition and provide a robust engineering strategy which can be used for structural, functional and drug-screening analysis of 2C proteins from current or future enteroviruses.

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“…To date, several anti-viral inhibitors which target the 2C protein have been identified, including guanidine hydrochloride (GuHCl; Pfister and Wimmer, 1999;Sadeghipour et al, 2012), HBB (Hadaschik et al, 1999), MRL-1237 (Shimizu et al, 2000), and TBZE-029 (De Palma et al, 2008a). In addition, several other compounds, such as metrifudil (Arita et al, 2008), N 6benzyladenosine (Arita et al, 2008), quinoline analogues (Musharrafieh et al, 2019), dibucaine derivatives (Tang et al, 2020), fluoxetine analogues (Manganaro et al, 2020), R523062 (Ma et al, 2020), and viperin (Wei et al, 2018), which also target the enterovirus 2C protein, are summarized in Table 3.…”

Section: Effect Of Anti-viral Drugs On the 2c Proteinmentioning

confidence: 99%

The Structure, Function, and Mechanisms of Action of Enterovirus Non-structural Protein 2C

Wang

Zhao

et al. 2020

Front. Microbiol.

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Enteroviruses are a group of RNA viruses belonging to the family Picornaviridae. They include human enterovirus groups A, B, C, and D as well as non-human enteroviruses. Enterovirus infections can lead to hand, foot, and mouth disease and herpangina, whose clinical manifestations are often mild, although some strains can result in severe neurological complications such as encephalitis, myocarditis, meningitis, and poliomyelitis. To date, research on enterovirus non-structural proteins has mainly focused on the 2A and 3C proteases and 3D polymerase. However, another non-structural protein, 2C, is the most highly conserved protein, and plays a vital role in the enterovirus life cycle. There are relatively few studies on this protein. Previous studies have demonstrated that enterovirus 2C is involved in virus uncoating, host cell membrane rearrangements, RNA replication, encapsidation, morphogenesis, ATPase, helicase, and chaperoning activities. Despite ongoing research, little is known about the pathogenesis of enterovirus 2C proteins in viral replication or in the host innate immune system. In this review, we discuss and summarize the current understanding of the structure, function, and mechanism of the enterovirus 2C proteins, focusing on the key mutations and motifs involved in viral infection, replication, and immune regulation. We also focus on recent progress in research into the role of 2C proteins in regulating the pattern recognition receptors and type I interferon signaling pathway to facilitate viral replication. Given these functions and mechanisms, the potential application of the 2C proteins as a target for anti-viral drug development is also discussed. Future studies will focus on the determination of more crystal structures of enterovirus 2C proteins, which might provide more potential targets for anti-viral drug development against enterovirus infections.

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Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study

Cited by 38 publications

References 36 publications

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex

Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes the hexameric complex

The Structure, Function, and Mechanisms of Action of Enterovirus Non-structural Protein 2C

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