Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.

Enteroviruses (e.g., poliovirus, enterovirus-A71, coxsackievirus, enterovirus-D68, rhinovirus) include many human pathogens causative of various mild and more severe diseases, especially in young children. Unfortunately, antiviral drugs to treat enterovirus infections have not been approved yet. Over the past decades, several direct-acting inhibitors have been developed, including capsid binders, which block virus entry, and inhibitors of viral enzymes required for genome replication. Capsid binders and protease inhibitors have been clinically evaluated, but failed due to limited efficacy or toxicity issues. As an alternative approach, host-targeting inhibitors with potential broad-spectrum activity have been identified. Furthermore, drug repurposing screens have recently uncovered promising new inhibitors with disparate viral and host targets. Together, these findings raise hope for the development of (broad-range) anti-enteroviral drugs.

SummaryEnteroviruses reorganize cellular endomembranes into replication organelles (ROs) for genome replication. Although enterovirus replication depends on phosphatidylinositol 4-kinase type IIIβ (PI4KB), its role, and that of its product, phosphatidylinositol 4-phosphate (PI4P), is only partially understood. Exploiting a mutant coxsackievirus resistant to PI4KB inhibition, we show that PI4KB activity has distinct functions both in proteolytic processing of the viral polyprotein and in RO biogenesis. The escape mutation rectifies a proteolytic processing defect imposed by PI4KB inhibition, pointing to a possible escape mechanism. Remarkably, under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses was observed. Together, our findings challenge the notion that ROs are indispensable for enterovirus genome replication and immune evasion.

Similar to all other positive-strand RNA viruses, enteroviruses reorganize host cellular membranes for efficient genome replication. A host lipid kinase, PI4KB, plays an important role in this membrane rearrangement. The exact mechanism of how enteroviruses recruit PI4KB was unclear. Here, we revealed a role of a Golgi-residing protein, ACBD3, as a mediator of PI4KB recruitment upon enterovirus replication. ACBD3 is responsible for proper localization of enteroviral 3A proteins in host cells, which is important for 3A to recruit PI4KB. By testing ACBD3 and PI4KB mutants that abrogate the ACBD3-PI4KB interaction, we showed that this interaction is crucial for enterovirus replication. The importance of specific domains of ACBD3 was evaluated for the first time, and the domains that are essential for enterovirus replication were identified. Our findings open up a possibility for targeting ACBD3 or its interaction with enteroviruses as a novel strategy for the development of broad-spectrum antienteroviral drugs.

Globally accessible preventive and therapeutic molecules against SARS-CoV-2 are urgently needed. DARPin molecules are an emerging class of novel therapeutics based on naturally occurring repeat proteins (∼15 kDa in size) and can be rapidly produced in bacteria in large quantities. Here, we report the identification of 380 DARPin molecules specifically targeting the SARS-CoV-2 spike protein selected from a naïve library of 1012 DARPin molecules. Using extensive biophysical and biochemical characterization, (pseudo)virus neutralization assays and cryo-EM analysis, 11 mono-DARPin molecules targeting either the receptor binding domain (RBD), the S1 N-terminal-domain (NTD) or the S2 domain of the SARS-CoV-2 spike protein were chosen. Based on these 11 mono-DARPin molecules, 31 anti-SARS-CoV-2 multi-DARPin molecules were constructed which can broadly be grouped into 2 types; multi-paratopic RBD-neutralizing DARPin molecules and multi-mode DARPin molecules targeting simultaneously RBD, NTD and the S2 domain. Each of these multi-DARPin molecules acts by binding with 3 DARPin modules to the SARS-CoV-2 spike protein, leading to potent inhibition of SARS-CoV-2 infection down to 1 ng/ml (12 pM) and potentially providing protection against viral escape mutations. Additionally, 2 DARPin modules binding serum albumin, conferring an expected half-life of about 3 weeks in humans, were included in the multi-DARPin molecules. The protective efficacy of one multi-DARPin molecule was studied in a Golden Syrian hamster SARS-CoV-2 infection model, resulting in a significant reduction in viral load and pathogenesis. In conclusion, the multi-DARPin molecules reported here display very high antiviral potency, high-production yield, and a long systemic half-life, and thereby have the potential for single-dose use for prevention and treatment of COVID-19.