Structural Basis of the Major TMPRSS2 Promoter G-Quadruplex and Its Complex with Berberine

TMPRSS2 plays a crucial role in facilitating the entry of both influenza virus and the SARS-CoV-2 coronavirus into host cells. Recent studies have identified a guanine-rich sequence in the proximal promoter region of the TMPRSS2 gene, which can form G-quadruplex structures (TMPRSS2-G4s) that are potential targets for small molecules to inhibit TMPRSS2 expression. However, the structure details of major TMPRSS2-G4 and its complex with small molecules remain unknown, hindering the development of antivirus drugs targeting TMPRSS2-G4s. This study reports the first high-resolution NMR solution structure of the major TMPRSS2-G4, which consists of a three-tetrad core parallel-stranded G4. Both 3′ and 5′ flanking regions form well-defined capping structures that are stabilized by multiple hydrogen bonds. Importantly, we found berberine, an antiviral alkaloid, strongly binds to the major TMPRSS2-G4 and further determined its binding complex structures with TMPRSS2-G4 in a 2:1 binding stoichiometry. Each berberine recruits an adjacent flanking residue, forming a coplanar structure superimposed on two outer G-tetrads. Moreover, we demonstrated that the major TMPRSS2-G4 can stably form within a longer DNA context and be targeted by small molecules for DNA polymerase inhibition. Overall, this study provides structural insights into the recognition mechanism of small molecules to the major TMPRSS2-G4 and may be beneficial for novel antiviral drug development that targets TMPRSS2-G4.