Zoonotic coronaviruses that spread from animals to humans have caused three pandemics during the last century. Beginning with the severe acute respiratory syndrome coronavirus (SARS-CoV) outbreak in 2002 and the Middle East respiratory syndrome coronavirus epidemic in 2012, these viruses have shown a propensity to affect the respiratory system.
Study: A Novel Frameshifting Inhibitor Having Antiviral Activity against Zoonotic Coronaviruses. Image Credit: Gorodenkoff/ Shutterstock
The current coronavirus disease 2019 (COVID-19) pandemic, which has resulted in 4.43 million global fatalities over the last 18 months, is caused by the rapid and extensively transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Few specific and effective antivirals have been identified so far.
A recent study in the journal Viruses describes a novel compound, KCB261770, with frameshifting capability against MERS-CoV, inhibiting viral propagation within infected cells. The same compound also had similar, though more limited, activity against SARS-CoV-2, indicating promising treatment against coronavirus infections.
Background
All coronaviruses are single-stranded ribonucleic acid (RNA) viruses, with approximately 30,000 bases in their genomes. The RNA strand has two untranslated regions (UTR), at 5’ and 3’, and open‐reading frames (ORFs). From the 5’ end, two-thirds of the genome contains the large polyprotein (pp)-encoding regions called ORFs 1a and 1b.
While ORF1a encodes pp1a, pp1ab is encoded by both, made possible by a backward shift of the RNA reading frame on the ribosome. The ribosome begins the new reading frame from one nucleotide behind the site of ribosome stalling on the mRNA secondary structure during translation of pp1a.
ORF1b does not have its own translation initiation site and makes use of this programmed −1 ribosomal frameshift (−1 PRF), occurring at the junction where the two ORFs overlap. The two pps are cleaved by viral proteases encoded within them, yielding non-structural proteins (nsps) 1-16.
The nsps 12-16 make up the replication‐transcription complex (RTC) that is responsible for the replication, transcription, and translation of the viral RNA—comprising of RNA‐dependent RNA polymerase (RdRp or nsp12), helicase (HEL or nsp13), exonuclease (ExoN or nsp14), endonuclease (NendoU or nsp15), and methyltransferase (2´‐O‐MT or nsp16).
The latter three are essential for viral RNA to replicate efficiently. The inhibition of -1PRF will therefore interrupt the viral life cycle, and an altered ORF1a to ORF1b protein ratio will also impact the propagation and infectivity of the virus.
Inhibition of frameshifting
The coronavirus frameshifting signals contain a heptameric slippery sequence and a highly conserved pseudoknot RNA structure. Any disruption of the latter by mutation affects frameshifting negatively, making it a target for anti-coronavirus agents.
While antisense peptide nucleic acids (PNAs) have been found to inhibit efficient SARS-CoV-2 replication via disruption of the -1PRF signal, the current study was focused on small molecule -1PRF inhibitors against MERS-CoV.
The ORF1b of this virus includes a replicase gene cluster containing replication-required viral elements. Of the seven CoVs known to infect humans, the -1PRFs are approximately 91-97 nucleotides in length and have slippery sequences at the frameshifting site.
When these slippery sequences from the different human-infecting coronaviruses were compared, relatively less conservation of SARS-CoV and MERS-CoV was found. Despite that, the -1PRF sequences of SARS-CoV and SARS-CoV-2 being homologous.
Using a firefly and Renilla luciferase gene assay to demonstrate the occurrence of frameshifting through the pseudoknot, the researchers evaluated the efficiency of frameshifting to be approximately 8%, 14%, and 16% for MERS‐CoV, SARS‐CoV, and SARS‐CoV‐2, respectively. The SARS-CoV value shows agreement with that of earlier studies.
What were the findings?
Chemical inhibitors were screened for MERS-CoV frameshifting. Of the final six compounds selected, compound KCB261770 had the highest inhibitory effects. The aforementioned compound is a newly synthesized furo-quinoline derivative belonging to a group of compounds with anti‐inflammatory, anti‐malarial, and antimicrobial activities.
This compound does not directly inhibit luciferase activity. It is the strongest inhibitor among 88 similar furo-quinoline derivatives, which in turn showed about a fifth greater inhibition than the library of approximately 10,000 compounds initially used—possibly signifying the baseline inhibition offered by the furol(2,3-b) quinoline backbone.
Broad range of inhibition
This compound was compared with two others for frameshifting inhibition and indirect effects, such as blocking transcription or translation. It was found that 6‐thioguanine (a purine analog and DNA synthesis inhibitor) had a dose-dependent inhibitory effect. Still, there was no change in the frameshifting ratio.
KCB261770 inhibited firefly luciferase in a dose-dependent manner but not Renilla luciferase, reducing the frameshifting ratio steeply, indicating that inhibition of frameshifting of MERS-CoV -1PRF is not due to the indirect inhibition of translation elongation or other cytotoxic effects.
Compound KCB261770 showed selectivity for MERS-CoV and suppressed the propagation of MERS-CoV, as shown by a drastic reduction in viral RNA and a significant reduction in virus-induced cell death.
The compound acts by disrupting replicase genes rather than inhibiting viral proteins. Being a more delayed action, it may not be effective in suppressing virus-induced cell death after infection. Despite the steep drop in viral load, infected cells may show limited recovery even in early infection.
What are the implications?
The researchers found that targeting efficient frameshifting can modulate the propagation and infectivity of the virus by disrupting genomic and subgenomic RNA synthesis. A novel small molecule inhibitor was identified, with robust suppression of frameshifting in MERS-CoV and reduced but still efficient reduction for SARS-CoV and SARS-CoV-2.
Many different mechanisms are possible, such as interference with the RNA-translation complex interaction via binding of the compound to the non-active sites of the ribosome translation complexes.
The advantage of the frameshifting signal targeting approach is the expression of several viral proteins is reduced simultaneously. Secondly, the conserved nature of the RNA pseudoknot structure of the -1PRF of MERS-CoV and SARS-CoV, as well as SARS-CoV-2, indicates that this compound may have pan-coronavirus activity. Thirdly, it is difficult for escape mutations to arise since the frameshifting involves essential viral functions. Finally, it can be combined with other therapies employing other mechanisms, such as RdRp inhibitors, thus enhancing the efficacy and broadening the reach of coronavirus treatment.
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