Small molecule inhibitor shown to be effective against SARS-CoV-2 variants

In a recently published study in frontiers in microbiologyresearchers developed RK-33, a small molecule inhibitor of DDX3, and evaluated its efficacy against variants of concern (VOCs) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The continuous emergence of SARS-CoV-2 VOCs, despite the effectiveness of COVID-19 vaccines, has led to an increase in cases of coronavirus disease 2019 (COVID-19) worldwide. The emerging VOCs have higher transmissibility and immune evasiveness than previous VOCs, justifying the need to develop anti-SARS-CoV-2 agents to broaden the therapeutic landscape of COVID-19 and reduce the burden of COVID-19.

Host proteins essential for SARS-CoV-2 replication could be targeted to develop novel drugs as alternative or complementary strategies to vaccines. Studies have reported that DDX3 is required by SARS-CoV-2 for virion production and is part of the SARS-CoV-2 interactome. Therefore, molecules that inhibit DDX3 could potentially be effective against SARS-CoV-2.

About the study

In the present study, researchers evaluated the anti-SARS-CoV-2 efficacy of RK-33.

Calu-3 (lung cancer cell line) cells were cultured with non-toxic doses of RK-33 for cell culture experiments and SARS-CoV-2 isolates such as the A-line (reference), B-line, Beta-VOC, Alpha-VOC and , Delta VOC were used for the assays. A cell viability analysis was performed and the 50% cytotoxic concentration (CC₅₀) values ​​determined.

Plaque assays were performed to determine virus titers and mean effective concentration (EC₅₀) values ​​determined. SARS-CoV-2 RNA extracted from cell lysates containing SARS-CoV-2 was subjected to ribonucleic acid (RNA) sequencing (RNA-seq) analysis. In addition, SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and envelope (E) genes were amplified from RNA by quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis to assess copy numbers, and a proteomic analysis was performed.

Protein sequences for SARS-CoV-2 proteins (n=25) were extracted from the genome sequences of SARS-CoV-2 isolates and multiple sequence alignment (MSA) analysis was performed for each protein. Lineage A differences were noted and the evolutionary distances of the proteins assessed. The team obtained three-dimensional (3D) models of A-lineage spike (S) protein trimers in closed and open conformations with protein sequences similar to those of the A-lineage used in the present study and visualized alpha VOC, beta VOC and delta VOC Variations of 3D structures.

Results

Targeting DDX3 with RK-33 reduced the viral load of lineage A, lineage B alpha, beta, and delta SARS-CoV-2 isolates by one log to three logs in Calu-3 cells. Furthermore, RK-33 treatment reduced intracellular SARS-CoV-2 RNA production by 2.5 to 3.0 log10 as shown by RT-qPCR analysis. The results indicated that RK-33 exhibited potent anti-SARS-CoV-2 effects against all virus isolates tested.

In addition, proteomic and RNA-seq analyzes showed that RK-33 downregulated most SARS-CoV-2 genes [S, E, membrane (M), nucleocapsid (N), open reading frames (ORFs)1ab, 3a,7a, 7b, 8, and 10]. In addition, RK-33 reduced the expression of transmembrane serine protease 2 (TMPRSS2) by 50% within two days, possibly due to the ability of DDX3 to resolve G-quadruplex structures present in TMPRSS2.

All samples included 45 million reads; However, MSA analysis showed that virally infected Calu-3 cells had 33% unique human genome matches compared to the other samples (88%). This was a critical finding because nonstructural protein 1 (Nsp1) degenerates host cell RNA to allow for hijacking of host cells by SARS-CoV-2. The reversion of SARS-CoV-2 RNA into host cell RNA in RK-33 treated and SARS-CoV-2 infected cells underscored the importance of RK-33 in DDX3 targeting.

The majority of SARS-CoV-2 proteins (P0DTC3 ORF3a, P0DTC2 S, P0DTC9 N, P0DTC5 M, P0DTD1 replicase polyprotein 1ab, P0DTC8 ORF8, aP59595 N and P0DTD2 ORF9b) showed significant upregulation (>7.5 FC ) in Calu-3 cells post-SARS-CoV-2 infection and significant down-regulation after RK-33 therapy.

The five main significantly enriched mechanisms after SARS-CoV-2 infections are (i) interferon alpha/beta (IFN-α/β signaling), (ii) IFN-stimulated gene 15 (ISG15) antiviral mechanisms, (iii) SARS-CoV-2 mRNA translation, (iv) signal recognition particle (SRP)-dependent targeting of co-translational proteins to membranes, and (v) elongation of peptide chains.

After RK-33 therapy, a shift in the affected pathways was observed, with the five main significantly enriched mechanisms being (i) mitochondrial (mt) translation initiation, (ii) mt translation elongation, (iii) mt translation termination, (iv). antigen presentation [including folding, assembling, and major histocompatibility complex (MHC) class I loading]and (v) SRP-dependent targeting of co-translational proteins to the membrane.

By querying the STRING database, the team found that certain mechanisms were positively enriched in RK-33 treated cells (endosomal/vacuolar pathways and regulation of the complement cascade). In contrast, certain signaling pathways were negatively enriched (mt translation and initiation of mt translation). The results indicated that the small molecule inhibitor suppressed SARS-CoV-2 protein production and the changes were consistent with changes in the host proteome, particularly in relation to innate immunological signaling pathways.

In addition, 1 μM RK-33 reduced human coronavirus (HCoV)-OC43 titers by >2 log or >100-fold. The established CC₅₀ and EC₅₀ Values ​​among the Calu-3 cells were 13.5 μM and <1 μM, resulting in a selectivity index (CC₅₀/ EC₅₀) from 14.5.

Overall, study results indicated that RK-33 could be used as a potent anti-SARS-CoV-2 therapeutic to eliminate host DDX3 functions and curb transmission of novel SARS-CoV-2 VOCs.