Allosteric activation of Trx1 by antagonizing nitrative modification at tyrosine 49 confers neuroprotection against ischemic stroke

Ischemic stroke remains a leading cause of mortality and chronic disability worldwide, with limited therapeutic options. Tetramethylpyrazine (TMP) is a natural product with well-established clinical efficacy against ischemic stroke, yet its molecular target and mechanism of action remain elusive. By integrating a bifunctional photoaffinity TMP probe with stable isotope labeling by amino acids in cell culture and activity-based protein profiling (SILAC-ABPP), we identified thioredoxin 1 (Trx1) as a direct target of TMP. We demonstrate that TMP binds specifically to tyrosine 49 (Y49) on Trx1, antagonizes its nitrative modification, and functions as an allosteric activator. This binding enhances Trx1's reductase activity, strengthens its interaction with apoptosis signal-regulating kinase 1 (ASK1), and consequently suppresses the ASK1-p38/JNK signaling cascade. Genetic ablation of Trx1 or ASK1, or pharmacological induction of Trx1 nitration, completely abolishes TMP-mediated neuroprotection. Our findings not only decipher the mechanistic basis for TMP's clinical efficacy but also identify Y49 of Trx1 as a druggable allosteric site, unveiling a novel anti-nitrative therapeutic strategy for ischemic stroke and related disorders involving nitrative stress.