The Discovery of Antivirals and Targets for SARS‐CoV‐2 and EV‐A71

Positive Strand RNA (PSR) viruses, such as coronaviruses and enteroviruses, cause serious health and economic threats worldwide, as currently seen with the COVID‐19 pandemic. This has drawn attention to the importance of identifying new antivirals and molecular targets in RNA viruses. The multifunctionality of PSR genomes make them desirable targets for therapeutic intervention. Here, we present a class of antivirals that can inhibit SARS‐CoV‐2 replication invitro by targeting conserved viral RNA structures at the 5’‐end. Specifically, stem loops 1, 4, 5a, and 6 of the viral 5’‐region have shown a degree of binding with these small molecules as determined by NMR structural analysis. These results open the door to potentially develop specific small molecules against SARS‐CoV‐2 and related coronaviruses. Additionally, Enterovirus A71 (EV‐A71), which is the etiological agent of the hand, foot, and mouth disease, has caused severe morbidity and high mortality rates in children for decades. Thus, understanding the mechanisms by which EV‐A71 replicates within the cellular environment can bring to light efficient drug targets for viral inhibition. The multifunctional viral protein, 3C protease (3Cpro), is essential for viral protein and RNA synthesis. Here, we investigate how RNA binding allosterically modulates the enzymatic activity of 3Cpro. We identify an overlooked dimerization surface on 3Cpro that is proximal to its active site and distal to its RNA binding domain. Our data show that RNA binding is allosterically coupled to 3Cpro dimerization, and we posit that this is a novel mechanism to regulate its enzymatic function. To that point, single, double, and triple point mutations in the 3Cpro dimerization domain attenuates viral growth and kinetics. Taken together, we present compelling data that demonstrates novel targeting surfaces on 3Cpro that can be pursued as antiviral targets.

Amilorides inhibit SARS-CoV-2 replication in vitro by targeting RNA structures.

The SARS-CoV-2 pandemic, and the likelihood of future coronavirus pandemics, emphasized the urgent need for development of novel antivirals. Small-molecule chemical probes offer both to reveal aspects of virus replication and to serve as leads for antiviral therapeutic development. Here, we report on the identification of amiloride-based small molecules that potently inhibit OC43 and SARS-CoV-2 replication through targeting of conserved structured elements within the viral 5'-end. Nuclear magnetic resonance­based structural studies revealed specific amiloride interactions with stem loops containing bulge like structures and were predicted to be strongly bound by the lead amilorides in retrospective docking studies. Amilorides represent the first antiviral small molecules that target RNA structures within the 5' untranslated regions and proximal region of the CoV genomes. These molecules will serve as chemical probes to further understand CoV RNA biology and can pave the way for the development of specific CoV RNA­targeted antivirals.

Secondary structure determination of conserved SARS-CoV-2 RNA elements by NMR spectroscopy.

The current pandemic situation caused by the Betacoronavirus SARS-CoV-2 (SCoV2) highlights the need for coordinated research to combat COVID-19. A particularly important aspect is the development of medication. In addition to viral proteins, structured RNA elements represent a potent alternative as drug targets. The search for drugs that target RNA requires their high-resolution structural characterization. Using nuclear magnetic resonance (NMR) spectroscopy, a worldwide consortium of NMR researchers aims to characterize potential RNA drug targets of SCoV2. Here, we report the characterization of 15 conserved RNA elements located at the 5' end, the ribosomal frameshift segment and the 3'-untranslated region (3'-UTR) of the SCoV2 genome, their large-scale production and NMR-based secondary structure determination. The NMR data are corroborated with secondary structure probing by DMS footprinting experiments. The close agreement of NMR secondary structure determination of isolated RNA elements with DMS footprinting and NMR performed on larger RNA regions shows that the secondary structure elements fold independently. The NMR data reported here provide the basis for NMR investigations of RNA function, RNA interactions with viral and host proteins and screening campaigns to identify potential RNA binders for pharmaceutical intervention.

Integrated approaches to reveal mechanisms by which RNA viruses reprogram the cellular environment.

RNA viruses are major threats to global society and mass outbreaks can cause long-lasting damage to international economies. RNA and related retro viruses represent a large and diverse family that contribute to the onset of human diseases such as AIDS; certain cancers like T cell lymphoma; severe acute respiratory illnesses as seen with COVID-19; and others. The hallmark of this viral family is the storage of genetic material in the form of RNA, and upon infecting host cells, their RNA genomes reprogram the cellular environment to favor productive viral replication. RNA is a multifunctional biomolecule that not only stores and transmits heritable information, but it also has the capacity to catalyze complex biochemical reactions. It is therefore no surprise that RNA viruses use this functional diversity to their advantage to sustain chronic or lifelong infections. Efforts to subvert RNA viruses therefore requires a deep understanding of the mechanisms by which these pathogens usurp cellular machinery. Here, we briefly summarize several experimental techniques that individually inform on key physicochemical features of viral RNA genomes and their interactions with proteins. Each of these techniques provide important vantage points to understand the complexities of virus-host interactions, but we attempt to make the case that by integrating these and similar methods, more vivid descriptions of how viruses reprogram the cellular environment emerges. These vivid descriptions should expedite the identification of novel therapeutic targets.