Fusion Strategy of DNA-Encoded Libraries Drives Discovery of Allosteric Inhibitors of SARS-CoV‑2 RdRp.

Allosteric regulation is a central mechanism for modulating biological functions and offers an attractive strategy in drug discovery, particularly for targets considered challenging or "undruggable." However, the discovery of allosteric inhibitors is hindered by poorly defined binding sites and the lack of effective screening approaches. Here, we present a dual DNA-encoded library (DEL) screening strategy that integrates reversible DEL and covalent DEL (CoDEL) technologies to identify novel allosteric inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp). Using this approach, we discovered the first covalent allosteric inhibitors of RdRp, which engage a previously uncharacterized pocket on the nsp8 subunit and form a covalent bond with Cys114. Subsequent SAR studies and biochemical assays confirmed the allosteric mechanism and elucidated structural determinants of activity. This work highlights the power of integrating reversible DEL screening with CoDEL screening for ligand discovery and establishes a generalizable strategy to identify covalent allosteric modulators for therapeutically important targets for therapy or active probe design.