COVID-19 Drugs: Yale Researchers Exploring Using Merofloxacin As Inhibitor of SARS-CoV-2 RNA Translation (-1 PRF Inhibitor)

COVID-19 Drugs: Researchers from Yale University School of Medicine in a new study exploring the inhibitory effects of the drug merofloxacin on frameshifting during SARS-CoV-2 replication have reported positive results in vitro studies. The study findings are an important proof-of-concept that supports the potential for targeting −1 PRF (programmed −1 ribosomal frameshifting) as an effective antiviral therapy in this current COVID-19 pandemic as well as against other beta-coronaviruses.


 
It has been found that translation of open reading frame 1b (ORF1b) in the SARS-CoV-2 virus requires programmed −1 ribosomal frameshifting (−1 PRF) promoted by an RNA pseudoknot.( A pseudoknot is a nucleic acid secondary structure containing at least two stem-loop structures in which half of one stem is intercalated between the two halves of another stem.)
 
The extent to which SARS-CoV-2 replication may be sensitive to changes in −1 PRF efficiency is currently unknown. Through an unbiased, reporter based high-throughput compound screen, the study team identified merafloxacin, a fluoroquinolone antibacterial, as a −1 PRF inhibitor of SARS-CoV-2. Frameshift inhibition by merafloxacin is robust to mutations within the pseudoknot region and is similarly effective on −1 PRF of other beta coronaviruses. Importantly, frameshift inhibition by merafloxacin substantially impedes SARS-CoV-2 replication in Vero E6 cells, thereby providing the proof of principle of targeting −1 PRF as an effective antiviral strategy for SARS-CoV-2.
 
To date the only other drug that has been studied as a -1 PRF Inhibitor is ivermection.
 
The study findings are published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2020.10.21.349225v1
 
The SARS-CoV-2 virus binds to the host cell via its spike protein and begins to replicate in order to establish infection. The translation of the viral proteins begins from the ORF1ab fragment, which is by far the largest, being over 21 kb long, and comprising 2/3 of the whole single-stranded genome.
 
The genome encodes a polyprotein, which is then cleaved into 16 nonstructural proteins (nsp) by the action of two proteases. Many proteins on the second half of the polyprotein play a vital part in viral transcription and replication. This includes nsp12, an RNA-dependent RNA polymerase (RdRp), RNA helicase (Hel/nsp13), and a proofreading exoribonuclease, among others.

The translation of ORF1b, the 3' side of ORF1ab, requires a frameshift to occur in all coronaviruses. When the ribosomes reach the end of ORF1a, some of them need to move in the retrograde direction by one nucleotide. This is the -1 programmed ribosomal frameshift (-1 PRF).
 
It has been found that following this frameshift, they continue to translate the rest of the ORF in the new -1 reading frame until the whole large polyprotein has been translated. If not, they will soon arrive at a stop codon in the 0 frame, which will end the translation prematurely. This will disable viral replication.
 
Importantly the −1 PRF region is often found to contain a 7-nucleot ide slippery sequence (UUUAAAC in SARS-CoV-2) and a downstream stable secondary structure that functions as a frameshift-stimulating element (FSE). This latter brings about brief pauses in the ribosome's onward movement, allowing the tRNAs time to achieve the correct alignment with respect to the slippery sequence. This is partly required for the -1 Programmed Ribosomal Frameshifting (PRF).
 
It has been known that one common FSE is the RNA pseudoknot. In SARS-CoV, SARS-CoV-2, and other betacoronaviruses, it is thought that perhaps a three-stem pseudoknot may act as the FSE. This structure is not very common in host mRNAs. It is, therefore often used to test the effect of explicitly disrupting viral gene expression, as by mutations and drugs that inhibit -1 PRF.
 
Interestingly this has been demonstrated in HIV replication, both by an exogenous -1 PRF inhibitor and by clues indicating that it is part of the host's antiviral response.
 
The researchers in the study identified the fluoroquinolone antibiotic merofloxacin by high-throughput screening for −1 PRF inhibitors of SARS-CoV-2. They study team showed that incubation with this compound suppressed SARS-CoV-2 replication in Vero cells.
 
Although ivermectin is also a -1 PRF inhibitor, it is thought to be slightly cytotoxic and researchers are still not sure of the dosing and the pharma-kinetics and research is still underway on this.
 
However merofloxacin has only modest cytostatic effects even at high levels. It is a specific betacoronavirus -1 PRF inhibitor, unlike other fluoroquinolones that lack this activity. This suggests that the structural groups at certain specific sites may be responsible for the frameshift inhibition.
 
As the FSE of the SARS-CoV sequence is almost identical to that of SARS-CoV-2, merofloxacin inhibits 1- frameshift in both almost equally. The half-maximal inhibitory concentration (IC50) is ~20 μM, which is superior to the 25% inhibition produced by another inhibitor called SHFL.
 
It should be noted that Merafloxacin is a specific inhibitor of betacoronavirus FSEs, but not of other coronaviruses or other common viruses. Other seasonal coronaviruses have a more elaborate pseudoknot structure, unlike the 3-stem pseudoknot of the betacoronaviruses.
 
The study team also found that merofloxacin-induced frameshift inhibition persists despite mutations in the viral genome. RNA viruses are known to have a rapid mutation rate, some of which may render the virus resistant to the antiviral agent. The study included mutations already known to exist in the virus.
 
The study team found very few mutations in the FSE sequence, which is to be expected in the light of the great importance of -1 PRF to viral replication.
 
Importantly among 20 mutations in this region, no less than 16 have occurred only once. Of the remaining four, two are in stem 1, and one each in stem 2 and 3.
 
However being on the stem, none of these could result in an alteration in the three-stem structure of the pseudoknot. However, the researchers found that -1 PRF inhibition by merofloxacin occurred with all these variants without loss of efficacy.
 
The team proceeded from these already existing mutations that did not change the pseudoknot structure and  then explored the effect of other mutations that disturbed the structure. They found that these caused a reduction in frameshifting by 10% to 77%, depending on their disruption.
 
Significantly one of the most significant reductions was caused by a mutation in single unpaired uridine, indicating that it has an unknown function. Both this mutation, U13485, and the following G13486 are completely conserved among human coronaviruses.


 A high-throughput screen identifies SARS-CoV-2 −1 PRF inhibitors

Although these mutations generate a wide range of structural perturbations, the inhibition of frameshift produced by merofloxacin acting on all these variants remained almost the same. This indicates that this action persists, and escape mutations are unlikely to emerge.
 
The study team then examined the effect of the inhibitor on viral replication. They found that merofloxacin inhibited replication from the lowest concentration of 1.25 μM, at which the half-maximal effective concentration (EC50) was 2.6 μM. When the drug concentration was raised to 40 μM or above, the virus became undetectable. The drop in viral yield was almost exponentially related to the -1 PRF inhibition. This indicates that the rate of replication of the virus depends on frameshift efficiency.
 
It is already known that the FSEs of coronaviruses are highly efficient, and this study explores the measure to which this attribute relates to viral viability by using a newly identified frameshift inhibitor.
 
Corresponding author Professor Dr Brett D. Lindenbach from the department of Microbial Pathogenesis, Yale University School of Medicine told Thailand Medical News, "The near-exponential relationship between the viral titer and −1 PRF efficiency points to a simple model in which ORF1b translation sets the limit of SARS-CoV-2 growth rate."
 
Significantly this justifies the development of −1 PRF inhibitors to effectively antagonize SARS-CoV-2 growth and other RNA viruses of the same type of PRF. More research remains to be carried out to elucidate the mechanism of inhibition of -1 PRF by merofloxacin.
 
The study findings could also be explained by the absolute need for highly efficient PRF to achieve the right stoichiometric correlation between the replication complex enzymes. The loss of this balance by reducing or increasing the efficiency of -1 PRF by even a little bit impacts viral growth severely.
 
The study team said that another possibility is that the small reductions in each of the translated rate-limiting enzymes of the replicase-transcriptase complex accumulate to cause a marked reduction in viral replication.
 
The team said that it could be due to direct binding of the FSE, disturbing the stability of the pseudoknot and thus decreasing the necessary pausing of the ribosome which is required for frameshifting. Another view is that this binding confers extra stability to the pseudoknot so that the arriving ribosomes are stuck too long, collide or have to queue, all disturbing the smooth translation elongation process.
 
Among other possibilities are the stabilization of an incorrect FSE conformation, which precludes frameshifting. Such structures have been recently demonstrated in cells infected by the virus. If merofloxacin is found to interact with one or more of such unproductive structures, it could reduce the FSE efficiency by the resulting smaller fraction of productive RNAs.
 
Merofloxacin also may act on modulatory host factors that act on the reverse frameshift, such as host RNA helicases, which might participate in pseudoknot unfolding following the -1 PRF but prior to the continuation of translation. Fluoroquinolones are known to target bacterial DNA topoisomerases. Further study will reveal the analogous enzymes in higher organisms, including RNA analogs.
 
The study team is planning to start vivo studies involving animal models soon before proceeding to actual human trials
 
It should be noted that Merofloxacin is currently only approved by the FDA for the treatment of individual pets and domestic animals in the United States and goes under the name of enrofloxacin  sold by the Bayer Corporation under the trade name Baytril. The drug is not to be consumed by humans as yet.
 
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