A Novel Capsid Binding Inhibitor Displays Potent Antiviral Activity against Enterovirus D68

Abstract

Enterovirus D68 (EV-D68) is a respiratory viral pathogen that primarily infects children under the age of 8. Although EV-D68 infection typically leads to moderate to severe respiratory illnesses, recent years have seen increasing cases of EV-D68 triggered neurological complications such as the acute flaccid myelitis (AFM). There is currently no vaccine or antiviral available for EV-D68, we therefore aimed to develop potent and specific small molecule antivirals against EV-D68. In this study, we report our discovery of a viral capsid inhibitor R856932 that inhibits multiple contemporary EV-D68 strains with single-digit to submicromolar efficacy. Mechanistic studies have shown that the tetrazole compound R856932 binds to the hydrophobic pocket of viral capsid protein VP1, thereby preventing viral uncoating and releasing of viral genome in the infected cells. The mechanism of action of R856932 was confirmed by time-of-addition, western blot, RT-qPCR, viral heat inactivation, serial viral passage and reverse genetics experiments. A single mutation located at VP1, A129V, confers resistance against R856932. However, recombination virus encoding VP1-A129V appeared to have compromised fitness of replication compared to the wild type EV-D68 virus as shown by the competition growth assay. Overall, the hit compound identified in this study, R856932, represents a promising starting point with a confirmed mechanism of action that can be further developed into EV-D68 antivirals.

Ebselen, disulfiram, carmofur, PX-12, tideglusib, and shikonin are non-specific promiscuous SARS-CoV-2 main protease inhibitors

“…Specific binding of a small-molecule to the native state of a protein usually stabilizes the protein, leading to a shift of the melting temperature (ΔT m ). 32 – 34 Here we measured T m change upon addition of these six compounds in the presence or absence of 4 mM DTT against six viral cysteine proteases including M pro All compounds were tested at 40 μM except shikonin, which was tested at 10 μM as it quenches the SYPRO orange dye fluorescence signal at 40 μM. Compounds were pre-incubated with 3 μM protease in its corresponding enzymatic reaction buffer at 30 °C for 30 min, then a 20 to 90 °C temperature gradient was applied and T m was calculated.…”

“…The thermal shift binding assay (TSA) was carried out using a Thermal Fisher QuantStudio TM 5 Real-Time PCR System as described previously. [32][33] Native mass spectrometry (MS) was performed as previously described using Q-Exactive HF quadrupole-Orbitrap mass spectrometer with the Ultra-High Mass Range (UHMR) research modifications (Thermo Fisher Scientific). All of the samples were ionized in positive ion mode using 0.9 kV capillary voltage with the temperature set to 200 °C.…”

“…The EC 50 and CC 50 values for the protease inhibitors investigated in this study were measured using RD cells as described previously. 32 Briefly, RD cells were seeded and grown overnight to ~90% confluence in 96-well plate at 37 °C 5% CO 2 incubator. For EV-D68 virus infection, cells were washed with PBS saline and infected with virus diluted in DMEM medium with 2% FBS and 30 mM MgCl 2 .…”

“…The thermal shift binding assay (TSA) was carried out using a Thermal Fisher QuantStudio™ 5 Real-Time PCR System as described previously. 32 – 33 Briefly, 3 μM protease in its enzymatic reaction buffer (see the above Enzymatic assays section for the reaction buffer components) in the presence of 4 mM DTT or in the absence of DTT, was incubated with testing compounds at 30 °C for 30 min in 96-well PCR plate. 1X SYPRO orange dye was added and fluorescence of the well was monitored under a temperature gradient range from 20 °C to 90 °C with 0.05 °C/s incremental step.…”

“…Specific binding of a small-molecule to the native state of a protein usually stabilizes the protein, leading to a shift of the melting temperature (ΔT m ). [32][33][34] Here we measured T m change upon addition of these six compounds in the presence or absence of 4 mM DTT against six viral cysteine proteases including M pro .…”