Abstract

The early discovery of covalent drugs is frequently inspired by, or derived from, natural sources, with such compounds often showing favorable safety profiles and a comparatively lower risk of clinical failure. However, a straightforward, high-throughput technique for screening covalent-binding molecules directly from complex medicinal plant extracts remains unavailable. In this study, we introduce an integrated strategy that combines protein microarrays with bioorthogonal click chemistry (Ccc-Chip). This platform includes a differential scanning fluorimetry (DSF)-based pre-screening step to enhance efficiency, with the Ccc-Chip serving as the core confirmation tool. It provides simple and intuitive readouts, enabling synchronous, high-throughput screening of covalent ligands targeting multiple proteins through detection of their competitive binding with cysteine-reactive probes. To validate the approach, we constructed a mutant isocitrate dehydrogenase 1 (mIDH1) protein microarray and used the integrated workflow to screen 110 medicinal plants. Our results led to the identification of flavokawain C (Flc), a covalent inhibitor of mIDH1, from Piper methysticum Forst. Subsequent in vivo experiments showed that Flc significantly reduced 2-hydroxyglutarate (2-HG) levels in an mIDH1-driven orthotopic tumor model and enhanced CD8⁺ T cell activity. Notably, when combined with a programmed cell death protein 1 (PD-1) blocking antibody, Flc synergistically augmented antitumor immunity, resulting in suppressed tumor growth. This work not only supports the high-throughput utility of the Ccc-Chip strategy but also provides a practical framework for combining bioorthogonal labeling with protein microarray technology, facilitating the discovery of bioactive covalent molecules from plant sources for challenging therapeutic targets. Graphical In this study, we develop a flexible Ccc