The guanosine analog AT-527 represents a promising candidate against Severe Acute Respiratory Syndrome coronavirus type 2 (SARS-CoV-2). AT-527 recently entered phase III clinical trials for the treatment of COVID-19. Once in cells, AT-527 is converted into its triphosphate form, AT-9010, that presumably targets the viral RNA-dependent RNA polymerase (RdRp, nsp12), for incorporation into viral RNA. Here we report a 2.98 Å cryo-EM structure of the SARS-CoV-2 nsp12-nsp7-nsp82-RNA complex, showing AT-9010 bound at three sites of nsp12. In the RdRp active-site, one AT-9010 is incorporated at the 3′ end of the RNA product strand. Its modified ribose group (2′-fluoro, 2′-methyl) prevents correct alignment of the incoming NTP, in this case a second AT-9010, causing immediate termination of RNA synthesis. The third AT-9010 is bound to the N-terminal domain of nsp12 - known as the NiRAN. In contrast to native NTPs, AT-9010 is in a flipped orientation in the active-site, with its guanine base unexpectedly occupying a previously unnoticed cavity. AT-9010 outcompetes all native nucleotides for NiRAN binding, inhibiting its nucleotidyltransferase activity. The dual mechanism of action of AT-527 at both RdRp and NiRAN active sites represents a promising research avenue against COVID-19.
Enteroviruses
(family Picornaviridae) comprise a large group of
human pathogens against which no licensed antiviral therapy exists.
Drug-repurposing screens uncovered the FDA-approved drug fluoxetine
as a replication inhibitor of enterovirus B and D species. Fluoxetine
likely targets the nonstructural viral protein 2C, but detailed mode-of-action
studies are missing because structural information on 2C of fluoxetine-sensitive
enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral
activity of fluoxetine is stereospecific concomitant with binding
to recombinant 2C. (S)-Fluoxetine inhibits with a
5-fold lower 50% effective concentration (EC50) than racemic
fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive
enterovirus coxsackievirus B3 (CVB3) based upon a recently elucidated
structure of a fluoxetine-insensitive enterovirus, we predicted stable
binding of (S)-fluoxetine. Structure-guided mutations
disrupted binding and rendered coxsackievirus B3 (CVB3) resistant
to fluoxetine. The study provides new insights into the anti-enteroviral
mode-of-action of fluoxetine. Importantly, using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely
reducing side effects.
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