The nucleocapsid protein Np of SARS-CoV-2 is involved in the replication, transcription, and packaging of the viral genome, but it also plays a role in the modulation of the host cell innate immunity and inflammation response. Ectopic expression of Np alone was able to induce significant changes in the proteome of human cells. The cellular RNA helicase DDX1 was among the proteins whose levels were increased by Np expression. DDX1 and its related helicase DDX3X were found to physically interact with Np and to increase 2- to 4-fold its affinity for double-stranded RNA in a helicase-independent manner. Conversely, Np inhibited the RNA helicase activity of both proteins. These functional interactions among Np and DDX1 and DDX3X highlight novel possible roles played by these host RNA helicases in the viral life cycle.

Current therapy against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are based on the use of Remdesivir 1, Molnupiravir 2, and the recently identified Nirmatrelvir 3. Unfortunately, these three drugs showed some limitations regarding potency and possible drug–drug interactions. A series of derivatives coming from a decoration approach of the privileged scaffold s-triazines were synthesized and evaluated against SAR-CoV-2. One derivative emerged as the hit of the series for its micromolar antiviral activity and low cytotoxicity. Mode of action and pharmacokinetic in vitro preliminary studies further confirm the role as candidates for a future optimization campaign of the most active derivative identified with this work.

The SARS-CoV-2 nucleocapsid protein (Np) plays multifunctional roles in the viral life cycle. By interacting with host cellular proteins, Np regulates viral RNA transcription, replication, and immune evasion. It controls genome packaging and counteracts host RNA interference mediated antiviral responses through its RNA binding activity. Previous studies revealed a physical interaction between Np and DDX3X, a human DEAD-box RNA helicase that facilitates the replication of several viruses. This interaction enhances Np affinity for double-stranded RNA and inhibits DDX3X helicase activity. Since Np-RNA binding activity promotes ribonucleoprotein complex formation, targeting this interaction is a promising antiviral strategy. We generated truncated protein variants to define interaction regions between Np and DDX3X. Using AlphaFold modelling, we identified RecA2 as the key DDX3X domain involved in Np binding. Finally, to disrupt Np-RNA complex formation, we screened a small molecule library of putative binders of Np N-terminal region and identified two candidate inhibitors for further development.

The SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase essential for viral replication and immune evasion. Its conservation across coronaviruses makes it an attractive target for the development of broad-spectrum antiviral agents. Hence, we rationally designed and synthesised 42 novel 2-iminothiazolidin-4-one derivatives as potential inhibitors. Several compounds displayed low-micromolar IC₅₀ values against both nsp13 NTPase and unwinding activities. Among these, 3i emerged as the lead compound, combining enzymatic inhibition (IC_50unwinding 2.65 ± 0.51 μM and IC_{50 NTPase} 8.52 ± 0.68 μM) with a favourable antiviral activity/cytotoxicity profile in two complementary cell models of SARS-CoV-2 infection (EC₅₀ 23.80 ± 1.80 μM in VeroE6-GFP, SI 18.15; EC₅₀ 6.7 ± 0.86 μM in Calu-3 cells, a human lung epithelial model). 3i also showed antiviral activity against hCoV-229E and MERS-CoV, with EC₅₀ values of 5.7 ± 0.1 and 4.6 ± 1.1 μM, respectively, supporting activity across human coronaviruses. The compound further demonstrated no marked hepatocellular cytotoxicity under the tested conditions in HepG2 cells, negligible inhibition of human helicases DDX3X and DDX5. Experimental biomimetic HPLC analyses provided an initial assessment of its physicochemical and distribution properties. Binding affinity evaluation by SPR revealed a K_D value of 6.20 μM, while enzyme kinetic studies indicated mixed-type NTPase inhibition. Exploratory docking and molecular dynamics analyses predicted possible binding regions that may account for the simultaneous modulation of the two nsp13 enzymatic functions. Finally, 3i showed a favourable antiviral combination profile with Remdesivir or Nirmatrelvir, mirroring established combination strategies in antiviral therapy.