From N‑0385 to N‑0920: Unveiling a Host-Directed Protease Inhibitor with Picomolar Antiviral Efficacy against Prevalent SARS-CoV‑2 Variants

The worldwide spread of new SARS-CoV-2 variants emphasizes
the
need to diversify existing therapeutic strategies. TMPRSS2, a host
protease crucial for SARS-CoV-2 entry, has garnered significant research
attention as a potential target for therapeutic intervention. Here,
we optimized N-0385, a previously reported TMPRSS2 ketobenzothiazole-based
peptidomimetic inhibitor, by screening 135 derivatives for target
affinity and antiviral potency. Among the top candidates, N-0695 exhibited
low nanomolar Ki values
against three TTSPs associated with respiratory virus entry: TMPRSS2,
matriptase, and TMPRSS13. Notably, N-0920 demonstrated exceptional
potency in reducing SARS-CoV-2 variants EG.5.1 and JN.1 entry in Calu-3
cells, representing the first in cellulo picomolar inhibitor with
EC50 values of 300 and 90 pM, respectively. Additionally,
molecular modeling provided insights into the binding interactions
between the compounds and their targets. This study underscores the
effectiveness of our screening approach in refining an existing peptidomimetic
scaffold to enhance selectivity and antiviral activity.