Department of Biochemistry and Neurobiology, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, Mickiewicza 30, 30-059 Krakow, Poland.
Polish Academy of Sciences, Maj Institute of Pharmacology, Laboratory of Proteomics and Mass Spectrometry, Smetna 12, 31-343 Krakow, Poland.
Molecules. 2020 Feb 13;25(4):813. doi: 10.3390/molecules25040813.
This work presents the synthesis of the novel covalent inhibitor of cysteine proteases where epoxide has been replaced by the iodoacetyl functional group. The molecule, similar in action to E-64 and DCG-04, the commonly applied inhibitors, is additionally biotinylated and contains tyrosyl iodination sites. The Fmoc solid phase synthesis has been applied. Conjugation of iodoacetic acid with the peptide was optimized by testing different conjugation agents. The purity of the final product was verified by mass spectrometry and its bioactivity was tested by incubation with a model cysteine protease-staphopain C. Finally, it was shown that the synthesized inhibitor binds to the protein at the ratio of 1:1. More detailed analysis by means of tandem mass spectrometry proved that the inhibitor binds to the cysteine present in the active site of the enzyme.
这项工作提出了新型半胱氨酸蛋白酶共价抑制剂的合成，其中环氧化物被碘乙酰胺官能团取代。该分子的作用类似于常用抑制剂 E-64 和 DCG-04，同时还进行了生物素化，并含有酪氨酸碘化位点。采用了 Fmoc 固相合成。通过测试不同的偶联剂来优化碘乙酸与肽的偶联。通过质谱法验证了最终产物的纯度，并通过与模型半胱氨酸蛋白酶-葡萄球菌蛋白酶 C 孵育来测试其生物活性。最后，结果表明合成的抑制剂以 1:1 的比例与蛋白质结合。通过串联质谱分析更详细的分析证明，抑制剂与酶活性位点中的半胱氨酸结合。
