COVID-19, an infectious respiratory disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has led to a global pandemic with profound public health implications and significant economic impacts worldwide. The Type-II transmembrane serine protease (TMPRSS2) has been identified as the proteolytic driver of SARS-CoV-2 activation and replication, making the dysregulation of TMPRSS2 activity a highly effective host-directed therapeutic strategy.

The previously reported TMPRSS2 inhibitor N-0385 exhibits unfavorable pharmacokinetic properties, including excessively high bioavailability (99%). To address the issue of significant systemic exposure, we designed a small library of peptidomimetic compounds with P3 site modifications by substituting proteinogenic amino acids and further evaluated their potency, in vitro efficacy, and pharmacokinetic profiles.

Exceptionally, compound 9, with Asp at the P3 position, resulted in a 2-fold increase in TMPRSS2 sub-nanomolar inhibitory potency (Ki of 13 ± 0.03 nM), while achieving >700-fold selectivity over Factor Xa and a superior selectivity profile against other proteases (matriptase, TMPRSS6, thrombin, and furin). An in vitro air-liquid interface (ALI) model of pulmonary epithelium revealed that compound 9 demonstrated a 1.5-fold decrease in permeability compared to N-0385, with sustained lung (11 h) and plasma (13 h) stability, suggesting its potential for daily prophylactic or therapeutic intranasal administration.