Synthetic Flavonoid Analogues as CatB/CatL Inhibitors with Potential Anti-SARS-CoV‑2 Activity

The COVID-19 pandemic profoundly affected global public health. Intensive efforts have led to the development of vaccines and antiviral drugs. However, the emergence of SARS-CoV-2 variants has raised concerns about resistance to therapies, making the exploration of alternative molecular pathways a promising strategy. Cathepsins stand out in this context due to their crucial role in mediating SARS-CoV-2 entry via endosomes. Structurally, these enzymes share high similarity with proteases from other organisms such as Leishmania CPB. Previously, we reported the synthesis and activity of 17 flavonoid derivatives as CPB inhibitors with robust antimicrobial activity. Motivated by the high resemblance between the active sites of CPB and CatB, we evaluated the anti-SARS-CoV-2 activity of these compounds and validated cathepsins as their main molecular target. Among them, nine flavonoid derivatives exhibited potent simple-linear noncompetitive dual CatB/CatL inhibition, with K _i values ranging from 3.20 to 29.05 μM for CatB and from 2.41 to 12.44 μM for CatL. Chalcones 1c, 2a, and 2b translated this enzymatic activity into significant reductions in SARS-CoV-2 titers (IC₅₀ = 12.06, 1.05, and 2.12 μM, respectively), while demonstrating low toxicity in both Vero-CCL81 and L929 cells. Their potential direct effects on viral replication were also evaluated using SARS-CoV-2 main protease assays and virucidal tests. No compound showed activity on viral targets, suggesting that cathepsin inhibition is their potential antiviral mechanism. Although additional mechanistic assays are still required, these data provide initial support for the involvement of dual CatB/CatL as antiviral targets and offer insights for the development of chalcone-based leads against COVID-19.