Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme, plays a crucial role in the energy metabolism of cancer cells and has been proposed as a valuable target for the development of anticancer agents. Among a series of 5-substituted 3-bromo-4,5-dihydroisoxazole (BDHI) derivatives, we identified the spirocyclic compound 11, which is able to covalently inactivate recombinant human GAPDH (hGAPDH) with a faster reactivity than koningic acid, one of the most potent hGAPDH inhibitors known to date. Computational studies confirmed that conformational rigidification is crucial to stabilize the interaction of the inhibitor with the binding site, thus favoring the subsequent covalent bond formation. Investigation of intrinsic warhead reactivity at different pH disclosed the negligible reactivity of 11 with free thiols, highlighting its ability to selectively react with the activated cysteine of hGAPDH with respect to other sulfhydryl groups. Compound 11 strongly reduced cancer cell growth in four different pancreatic cancer cell lines and its antiproliferative activity correlated well with the intracellular inhibition of hGAPDH. Overall, our results qualify 11 as a potent hGAPDH covalent inhibitor with a moderate drug-like reactivity that could be further exploited to develop anticancer agents.

甘油醛-3-磷酸脱氢酶 (GAPDH) 是一种关键的糖酵解酶，在癌细胞的能量代谢中起着至关重要的作用，已被提议作为开发抗癌剂的重要靶点。在一系列 5-取代的 3-溴-4,5-二氢异恶唑 (BDHI) 衍生物中，我们鉴定了螺环化合物11 ，它能够共价灭活重组人 GAPDH (hGAPDH)，其反应性比康宁酸更快，康宁酸是其中一种迄今为止已知的最有效的 hGAPDH 抑制剂。计算研究证实，构象硬化对于稳定抑制剂与结合位点的相互作用至关重要，从而有利于随后的共价键的形成。对不同 pH 值下固有弹头反应性的研究揭示了11与游离硫醇的反应性可以忽略不计，突出了它相对于其他巯基与 hGAPDH 的活化半胱氨酸选择性反应的能力。化合物11强烈减少四种不同胰腺癌细胞系中的癌细胞生长，其抗增殖活性与 hGAPDH 的细胞内抑制密切相关。总体而言，我们的结果使11成为一种有效的 hGAPDH 共价抑制剂，具有适度的药物样反应性，可进一步用于开发抗癌剂。