Rational design of highly potent broad-spectrum enterovirus inhibitors targeting the nonstructural protein 2C

Abstract

There is a great need for antiviral drugs to treat enterovirus (EV) and rhinovirus (RV) infections, which can be severe and occasionally life-threatening. The conserved nonstructural protein 2C, which is an AAA+ ATPase, is a promising target for drug development. Here, we present a structure-activity relationship study of a previously identified compound that targets the 2C protein of EV-A71 and several EV-B species members, but not poliovirus (PV) (EV-C species). This compound is structurally related to the Food and Drug Administration (FDA)-approved drug fluoxetine—which also targets 2C—but has favorable chemical properties. We identified several compounds with increased antiviral potency and broadened activity. Four compounds showed broad-spectrum EV and RV activity and inhibited contemporary strains of emerging EVs of public health concern, including EV-A71, coxsackievirus (CV)-A24v, and EV-D68. Importantly, unlike (S)-fluoxetine, these compounds are no longer neuroactive. By raising resistant EV-A71, CV-B3, and EV-D68 variants against one of these inhibitors, we identified novel 2C resistance mutations. Reverse engineering of these mutations revealed a conserved mechanism of resistance development. Resistant viruses first acquired a mutation in, or adjacent to, the α2 helix of 2C. This mutation disrupted compound binding and provided drug resistance, but this was at the cost of viral fitness. Additional mutations at distantly localized 2C residues were then acquired to increase resistance and/or to compensate for the loss of fitness. Using computational methods to identify solvent accessible tunnels near the α2 helix in the EV-A71 and PV 2C crystal structures, a conserved binding pocket of the inhibitors is proposed.

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

“…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 . Viruses resistant to this inhibitor contained mutations in, or adjacent to, the α2 helix, which is distal to the catalytic site.…”

“…Mutations in this loop have been previously identified in CV-B3 resistant to 2C-targeting compounds 18,33 . In addition, we previously demonstrated that substitutions C179Y, C179F and F190L also provide resistance to SFX 29 . Notably, these latter mutations, as well as 224-AGSINA-229 loop mutations, provide cross-resistance against dibucaine (Fig S7 ).…”

“…To gain more insight into the binding pocket of SFX, we raised resistant CV-B3 via a clonal selection procedure as described previously 29 . Several SFX-resistant viruses were obtained, containing single (I227V), double (I227V/A229V) or triple (A224V/I227V/A229V) mutations in the 224-AGSINA-229 loop (Figure S6).…”

“…Mode-of-action studies revealed that only the S-enantiomer of fluoxetine potently binds 2C and inhibits EV-B and D replication 26,27 . Despite the growing number of identified 2C-targeting compounds, the molecular basis for their antiviral activity remains unknown 28,29 . Such information would provide an invaluable resource for structurebased design of potent, pan-enterovirus antivirals with low toxicity.…”

Rhinovirus Inhibitors: Including a New Target, the Viral RNA

“…Contemplating the various drug targets and the corresponding drug candidates in Figure 1 and Table 1 , Table 2 and Table 3 , it becomes clear that one, namely the viral RNA genome that has attracted attention as a point of attack in the case of influenza A virus, Zika virus, and herpes simplex virus 1 [ 126 , 127 , 128 ], has so far not been considered in the case of picornaviruses [ 94 , 129 , 130 ]. In the following, we shall thus discuss the possibility of impacting on the stability of specific secondary structural elements of the viral RNA.…”