A multicomponent reaction-enabled discovery platform reveals a TXNRD1-targeted ferroptosis inducer

<p>Covalent inhibitors offer durable target engagement, but their discovery is often serendipitous and constrained by limited structural diversity. We introduce a multicomponent reaction (MCR)–based platform that combines modular synthesis, phenotypic screening, and proteome-level target deconvolution to identify HF-4aa, a covalent inhibitor of thioredoxin reductase 1 (TXNRD1). Stereodivergent synthesis enabled access to all four HF-4aa stereoisomers using Rh(II)/chiral phosphoric acid cocatalysis. HF-4aa displays nanomolar potency in cancer cells and modifies TXNRD1 <i>via</i> selenocysteine/cysteine targeting, supported by chemoproteomics and intact-protein mass spectrometry. TXNRD1 inhibition is functionally linked to redox collapse characterized by ROS accumulation, Fe²⁺ elevation, and lipid peroxidation, thereby inducing ferroptotic cell death. <i>In vivo</i>, HF-4aa suppresses tumor growth, and its efficacy is reversed by Ferrostatin-1, confirming ferroptosis dependence. Liposomal encapsulation overcomes the short metabolic half-life of free HF-4aa, and the resulting formulation demonstrates potent antitumor activity including in paclitaxel-resistant tumor models. HF-4aa serves as a proof-of-concept covalent chemical probe for TXNRD1-directed ferroptosis induction, and this study establishes a scalable MCR-based framework for systematic covalent inhibitor discovery.</p>