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.
Cui et al. (2026) studied this question.