Study: RK-33, a small molecule inhibitor of host RNA helicase DDX3, suppresses multiple variants of SARS-CoV-2. Image Credit: Pixaprime/Shutterstock
Background
Vaccines are considered the first line of defense to prevent global transmission. To date, all the available COVID-19 vaccines have been developed targeting the Spike protein of the original SARS-CoV-2 variant that was first reported from Wuhan, China, in 2019. In due course, the virus underwent genomic mutations, especially in the spike region, which resulted in many variants with different features compared to the original strain.
Two of the main concerns of the available vaccines include waning of vaccine-induced immune response over time and decline of its effectiveness against some variants, particularly the SARS-CoV-2 Omicron variant, which has been classified as a variant of concern (VOC). This is the reason why more effective COVID-19 therapeutics are needed urgently. Researchers have developed direct-acting antiviral agents (DAAs) and host-targeted antivirals (HTAs) as alternatives to vaccine resistance or used simultaneously with vaccines.
A new study has been published on the bioRxiv* preprint server that has focused on developing a novel anti-SARS-CoV-2 therapy using HTA directed against the human host DEAD (Asp, Glu, Ala, Asp)-box RNA helicase, i.e., DDX3 (DDX3X).
Previous studies have shown that SARS-CoV-2 replication occurs through a 5’cap-dependent translation utilizing the host’s translation complexes. These studies have also revealed that SARS-CoV-2 possesses a 5’capped mRNA genome that requires cap-dependent translation.
Further, it has also been shown that DDX3 is utilized in the translation of the SARS-CoV-2 genome. A recent proteomic analysis also revealed that DDX3 colocalized with SARS-CoV-2 viral particles and is essential for virion production. Additionally, DDX3 interacts with SARS-CoV-2 RNA and is associated with the SARS-CoV-2 interactome.
About a new study
Researchers designed a small molecule inhibitor, namely, RK-33, that has been developed to be a competitive inhibitor of DDX3’s ATP binding site. This compound can abrogate the RNA helicase function of the host, which is essential for the translation of complex structured 5’capped mRNAs.
Earlier studies revealed that several viruses require DDX3 for efficient virion production. Considering previous studies, DDX3 has been considered to be a prime host protein target. Scientists have used RK-33 to target DDX3 and abrogate virion production in Respiratory Syncytial, Dengue, Zika, West Nile, and human Parainfluenza Type-3 viral infections.
Key findings
Scientists revealed that RK-33 treatment has been found to be effective regardless of the SARS-CoV-2 strain, i.e., Lineage A and Lineage B (Alpha, Beta, and Delta variants). They observed that treatment of Calu-3 cells, infected with the above-mentioned isolates, with RK-33, significantly reduced viral load by one to three log orders. These findings were supported by proteomics and RNA-seq analyses, where the infected Calu-3 cells treated with RK-33 exhibited the downregulation of most of the SARS-CoV-2 proteins and genes, such as ORF1 ab, ORF3A, Spike, Membrane, ORF7A, and Nucleoprotein. Additionally, the host TMPRSS2 expression was found to be reduced.
The authors used plaque assay to reveal that RK-33 could effectively reduce the viral titer by over 10,000 times, associated with Lineage A variant. Similarly, the RK-33 treatment reduced viral titers in the Alpha variant by 200,000-fold, Beta by 125,000-fold, and Delta by 2,500-fold. These results were also validated via real-time polymerase chain reaction reverse transcription (RT-qPCR) Lineage of the intra- and extracellular SARS-CoV-2 RNA of the RK-33 treated A infected Calu-3 cells. The authors stated that the use of RK-33 even at CC50 had decreased the SARS-CoV-2 infection rate by 50%.
Principal Components Analysis (PCA) of host RNA demonstrated a significant effect of RK-33 on the infected cells, i.e., RK-33 treated virus-infected samples were segregated from the untreated infected samples. The authors also observed that perturbing DDX3 by RK-33 did not affect most of the host cellular functions while suppressing SARS-CoV-2 replication. Researchers constructed volcano plots to determine how RK-33 altered the gene expression patterns caused by the virus infection.
The authors revealed that by targeting DDX3 with RK-33, SARS-CoV-2 virulence and pathogenicity could be reduced by two different mechanisms. Firstly, the SARS-CoV-2 infection rate could be decreased by downregulating TMPRSS2 protein expression. Secondly, pathogenicity could be reduced by suppressing SARS-CoV-2 replication.
Conclusion
The findings of this study strongly indicate that RK-33 could be used to abrogate host DDX3 functions. Hence, this compound could be a possible option for the treatment of SARS-CoV-2 infection and, most importantly, the treatment could remain viable against the future SARS-CoV-2 variants.
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