Comparative analysis of the molecular mechanism of resistance to vapendavir across a panel of picornavirus species

Q: What are the key findings of this study?

Vapendavir resistance selection across picornaviruses yielded VP1 mutations (EC50 >10 μM) with >10-fold cross-resistance to pleconaril/pirodavir and a drug-dependent hRV2 G149C mutant.

Q: What is the clinical evidence from this study?

Population: Picornavirus infection (rhinovirus 2, rhinovirus 14, enterovirus-D68, and poliovirus 1 Sabin). Intervention: Vapendavir vs. Untreated wild-type virus controls and other WIN-like capsid binders (pleconaril, pirodavir). Primary outcome: In vitro antiviral resistance (EC50 fold-change in multicycle CPE-reduction assay) and identification of VP1 resistance mutations (p=<0.0005).

Q: What does this research mean for the field?

Resistance to the picornavirus inhibitor vapendavir can arise from mutations outside the VP1 drug-binding pocket, including an hRV2 G149C substitution that confers cross-resistance to other capsid binders and makes viral replication drug-dependent. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.CHALLENGES_CONSENSUS.

Q: What question did this study set out to answer?

To identify and compare the molecular mechanisms and phenotypes of resistance to the capsid inhibitor vapendavir across multiple picornavirus species.

Kristina Lanko; Liang Sun; Matheus Froeyen; Pieter Leyssen; Leen Delang; Carmen Mirabelli; Johan Neyts

doi:10.1016/j.antiviral.2021.105177

September 10, 2021Antiviral ResearchOpen Access

Comparative analysis of the molecular mechanism of resistance to vapendavir across a panel of picornavirus species

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Key result

Vapendavir resistance selection induces VP1 mutations conferring >10-fold cross-resistance to other capsid binders.

P<0.0005

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Why the study?

The molecular mechanisms of resistance to the capsid binder vapendavir across clinically relevant Picornavirus species remained to be characterized and compared.

Key Points

To identify and compare the molecular mechanisms and phenotypes of resistance to the capsid inhibitor vapendavir across multiple picornavirus species.
Applied an in vitro 5-step resistance selection protocol in human rhinovirus 2 (hRV2) and 14 (hRV14), enterovirus-D68 (EV-D68 CU70), and poliovirus 1 Sabin (PV1).
Assessed cross-resistance to pleconaril and pirodavir using multicycle cytopathic effect (CPE) reduction assays, followed by capsid VP1 sequencing and molecular dynamics modeling.
Vapendavir selection generated resistant isolates with EC50 >10 μM carrying VP1 drug-binding pocket mutations (C199R/Y in hRV14, I194F in PV1, and M252L/A156T in EV-D68), conferring cross-resistance (>10-fold reduction in activity) to pleconaril and pirodavir.
Identified mutations outside the binding pocket, including K167E in EV-D68 and G149C in hRV2, which shifted vapendavir EC50 from 0.04 ± 0.003 μM to >10 μM (>250-fold change).
The hRV2 G149C variant demonstrated drug dependency, requiring 1 μM vapendavir to produce infectious viral particles and achieving a 1 log10 increase in viral genome copies from 24 h to 48 h post-transfection (p < 0.0005).

Structured PICO

P

Population

In vitro viral cell cultures (HeLa, BGM, and RD cells) infected with clinically relevant picornavirus species (hRV2, hRV14, EV-D68 CU70, and PV1 Sabin) to select for vapendavir-resistant isolates.

I

Intervention

In vitro 5-step resistance selection and antiviral testing with vapendavir (BTA798, concentrations including 1 μM, 5 μM, and serial dilutions), site-directed mutagenesis of VP1 residues, and cross-resistance evaluation against pleconaril and pirodavir.

C

Comparator

Wild-type (WT) viral strains and DMSO solvent/untreated controls.

O

Outcome

Development of antiviral resistance (measured by EC50 in μM and fold-change in EC50 via multicycle CPE-reduction assay), identification of VP1 capsid amino acid substitutions by sequencing, and viral infectivity/replication kinetics (quantified by end-point titration and qRT-PCR of viral RNA copies at 24 h and 48 h).surrogate

Main Result

p-value: p=<0.0005

Picornaviruses rapidly develop resistance to vapendavir via both pocket and non-pocket VP1 mutations—including a novel drug-dependent hRV2 G149C phenotype and broad cross-resistance to other capsid binders—highlighting the need to consider combination antiviral therapies.

Limitations

Carry-over effect of remaining vapendavir in hRV2_C3 stocks cannot be excluded in the thermostability assay at 52 °C
Molecular dynamics simulation time (60 ns) may be too short to observe the structural effects of the G149C mutation
In vitro cell culture and molecular modeling study without in vivo validation
Potential carry-over effect of residual vapendavir in hRV2_C3 virus stocks during the 52 °C thermostability assay
Molecular dynamics simulation duration (60 ns) may have been insufficient to capture conformational effects of the G149C mutation
Preclinical in vitro design requiring further structural studies to fully explain the mechanism of G149C drug dependency

Implication

Preclinical resistance via VP1 mutations signals caution for capsid binders; leaves open whether combinations can overcome this in trials.

Abstract

Vapendavir is a rhino/enterovirus inhibitor that targets a hydrophobic pocket in the viral capsid preventing the virus from entering the cell. We set out to study and compare the molecular mechanisms of resistance to vapendavir among clinically relevant Picornavirus species. To this end in vitro resistance selection of drug-resistant isolates was applied in rhinovirus 2 and 14, enterovirus-D68 and Poliovirus 1 Sabin. Mutations in the drug-binding pocket in VP1 (C199R/Y in hRV14; I194F in PV1; M252L and A156T in EV-D68), typical for this class of compounds, were identified. Interestingly, we also observed mutations located outside the pocket (K167E in EV-D68 and G149C in hRV2) that contribute to the resistant phenotype. Remarkably, the G149C substitution rendered the replication of human rhinovirus 2 dependent on the presence of vapendavir. Our data suggest that the binding of vapendavir to the capsid of the G149C isolate may be required to stabilize the viral particle and to allow efficient dissemination of the virus. We observed the dependency of the G149C isolate on other compounds of this class, suggesting that this phenotype is common for capsid binders. In addition the VP1 region containing the G149C substitution has not been associated with antiviral resistance before. Our results demonstrate that the phenotype and genotype of clinically relevant vapendavir-resistant picornavirus species is more complex than generally believed.

Discussion

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Cite This Study

Lanko et al. (2021) studied Picornavirus infection (rhinovirus 2, rhinovirus 14, enterovirus-D68, and poliovirus 1 Sabin). Vapendavir vs. Untreated wild-type virus controls and other WIN-like capsid binders (pleconaril, pirodavir) was evaluated on In vitro antiviral resistance (EC50 fold-change in multicycle CPE-reduction assay) and identification of VP1 resistance mutations (p=<0.0005). Vapendavir resistance selection across picornaviruses yielded VP1 mutations (EC50 >10 μM) with >10-fold cross-resistance to pleconaril/pirodavir and a drug-dependent hRV2 G149C mutant.

synapsesocial.com/papers/6a78b98cc5ab23c76ced79ff https://doi.org/10.1016/j.antiviral.2021.105177

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Comparative analysis of the molecular mechanism of resistance to vapendavir across a panel of picornavirus species

Figures

Key Points

Structured PICO

Main Result

Limitations

Implication

Abstract

Discussion

Cite This Study

Comparative analysis of the molecular mechanism of resistance to vapendavir across a panel of picornavirus species

Figures

Key Points

Structured PICO

Main Result

Limitations

Implication

Abstract

Discussion

Cite This Study

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