A Hetero-multivalent Covalent Strategy for Enhanced Pulmonary Fibrosis Therapy

Drug combination therapy offers substantial advantages over single-agent treatments. However, discovering synergistic drug pairs remains challenging, as conventional approaches are often time-consuming, costly, and ill-suited for high-throughput screening. Here, we present an innovative strategy that enables efficient discovery of synergistic covalent drug combinations through hetero-multivalent engagement of KEAP1, a cysteine-rich redox sensor in mammalian cells. First, a conformation-sensitive fluorescent reporter for real-time monitoring of microenvironmental changes in KEAP1 was developed, enabling high-throughput discovery of a synergistic covalent drug combination, omaveloxolone (OXO) and cryptotanshinone (CTS). This combination (CTS and OXO) markedly activated the NRF2 cascade and attenuated fibrotic phenotypes in lung fibroblasts, lung organoids, and animal models. Mechanistically, OXO and CTS hetero-bivalently modify two distinct cysteine residues on KEAP1, which in turn synergistically disrupt the KEAP1-NRF2 interaction through a unique allosteric mechanism. Collectively, this work establishes a scalable framework for efficient discovery of covalent drug combinations and offers a new paradigm for enhanced anti-fibrosis therapy.