Validating Enterovirus D68-2A ^pro as an Antiviral Drug Target and the Discovery of Telaprevir as a Potent D68-2A ^pro Inhibitor

Abstract

Enterovirus D68 (EV-D68) is a viral pathogen that leads to severe respiratory illness and has been linked with the development of acute flaccid myelitis (AFM) in children. No vaccines or antivirals are currently available for EV-D68 infection, and treatment options for hospitalized patients are limited to supportive care. Here, we report the expression of the EV-D68 2A protease (2Apro) and characterization of its enzymatic activity. Furthermore, we discovered that telaprevir, an FDA-approved drug used for the treatment of hepatitis C virus (HCV) infections, is a potent antiviral against EV-D68 by targeting the 2Apro enzyme. Using a fluorescence resonance energy transfer-based substrate cleavage assay, we showed that the purified EV-D68 2Apro has proteolytic activity selective against a peptide sequence corresponding to the viral VP1-2A polyprotein junction. Telaprevir inhibits EV-D68 2Apro through a nearly irreversible, biphasic binding mechanism. In cell culture, telaprevir showed submicromolar-to-low-micromolar potency against several recently circulating neurotropic strains of EV-D68 in different human cell lines. To further confirm the antiviral drug target, serial viral passage experiments were performed to select for resistance against telaprevir. An N84T mutation near the active site of 2Apro was identified in resistant viruses, and this mutation reduced the potency of telaprevir in both the enzymatic and cellular antiviral assays. Collectively, we report for the first time the in vitro enzymatic activity of EV-D68 2Apro and the identification of telaprevir as a potent EV-D68 2Apro inhibitor. These findings implicate EV-D68 2Apro as an antiviral drug target and highlight the repurposing potential of telaprevir to treat EV-D68 infection. IMPORTANCE A 2014 EV-D68 outbreak in the United States has been linked to the development of acute flaccid myelitis in children. Unfortunately, no treatment options against EV-D68 are currently available, and the development of effective therapeutics is urgently needed. Here, we characterize and validate a new EV-D68 drug target, the 2Apro, and identify telaprevir—an FDA-approved drug used to treat hepatitis C virus (HCV) infections—as a potent antiviral with a novel mechanism of action toward 2Apro. 2Apro functions as a viral protease that cleaves a peptide sequence corresponding to the VP1-2A polyprotein junction. The binding of telaprevir potently inhibits its enzymatic activity, and using drug resistance selection, we show that the potent antiviral activity of telaprevir was due to 2Apro inhibition. This is the first inhibitor to selectively target the 2Apro from EV-D68 and can be used as a starting point for the development of therapeutics with selective activity against EV-D68.

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

“…They can be used either alone or in combination with polymerase inhibitors such as remdesivir as a means to achieve potential synergic antiviral effect as well as to suppress drug resistance. 2Apro and 3Cpro were expressed in the pET28b(+) vector as previously described 15,32,33 .…”

“…14 Both compounds 64 and 65 were not active against the EV-A71 2A protease, but showed potent inhibition against the EV-A71 3C protease, which is consistent with previously reported results. 12,15,16 When plotting the IC50 values (log scale) of the inhibitors against M pro from the FRET enzymatic assay with the melting temperature shifts (ΔTm) from thermal shift binding assay (Fig. 3A), a linear correlation was observed, and the r 2 of the linear regression fitting is 0.94.…”

“…The protease inhibitors are grouped based on their targets and mechanism of action and include proteasome inhibitors (1-8); HIV protease inhibitors (9)(10)(11)(12)(13)(14); γ-secretase inhibitors (15)(16)(17)(18)(19)(20)(21)(22); HCV NS3-4A protease inhibitors (23)(24)(25)(26)(27)(28)(29); DPP-4 inhibitors (30)(31)(32)(33)(34)(35); miscellaneous serine protease inhibitors (36)(37)(38)(39); cathepsin and calpain protease inhibitors (40-43); miscellaneous cysteine protease inhibitors (44-48); matrix metalloprotease inhibitors (49-51); and miscellaneous protease inhibitors (52-55). The inhibitors were pre-incubated with 100 nM of M pro at 30 °C for 30 minutes in the presence of 4 mM 1,4-dithiothreitol (DTT) before the addition of 10 µM FRET substrate.…”

Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

“…17 Both compounds (64 and 65) were inactive against the EV-A71 2A protease, but showed potent inhibition against the EV-A71 3C protease, which is consistent with previously reported results. 15,18,19 When plotting the IC 50 values (log scale) of the inhibitors against M pro from the FRET enzymatic assay with the melting temperature shifts (ΔT m ) from TSA ( Fig. 3a), a linear correlation was observed, and the r 2 of the linear regression fitting is 0.94.…”

“…With the established FRET assay condition, we screened a collection of protease inhibitors from the Selleckchem bioactive compound library to identify potential SARS-CoV-2 M pro inhibitors ( Table 1). The protease inhibitors are grouped based on their targets and mechanism of action, including proteasome inhibitors (1)(2)(3)(4)(5)(6)(7)(8), HIV protease inhibitors (9)(10)(11)(12)(13)(14), γ-secretase inhibitors (15)(16)(17)(18)(19)(20)(21)(22), HCV NS3-4A protease inhibitors (23)(24)(25)(26)(27)(28)(29), DPP-4 inhibitors (30)(31)(32)(33)(34)(35), miscellaneous serine protease inhibitors (36)(37)(38)(39), cathepsin and calpain protease inhibitors (40)(41)(42)(43), miscellaneous cysteine protease inhibitors (44)(45)…”