Chemical biology approach for investigating molecular mechanisms of isothiocyanates-inducing bioactivities

亲电饮食异硫氰酸酯(ITCs)的硫代氨基甲酰化是必需的，以便它们发挥癌症化学预防活性。在这项研究中，我们开发了一种新的方法来识别ITCs的靶向分子，使用两种研究得很好的ITCs，苯基ITC(BITC)和苯乙基ITC(PEITC)。这种方法的原理是基于识别BITC和PEITC之间的差异模式，因为它们表现出相似的化学和生物行为。为了验证方法的有效性，将以游离硫醇为模型分子的二硫苏糖醇还原的牛胰岛素与BITC或PEITC孵育，然后用内切酶Glu-C消化。UPLC-TOF/MS和LC-Q-TOF/MS分析表明，NYCN、FVNQHLCGSHLVE和ALYLVCGE三种多肽在半胱氨酸残基上分别与BITC或PEITC结合。通过反相柱层析，每组与BITC或PEITC结合的多肽的保留时间(RT_&lt；BITC&gt；&lt；RT_&lt；PEITC&gt；)具有分子质量差异(Δ14.01565)。在这些发现的基础上，构建了计算数学方案来提取满足上述标准的MS离子集。将所建立的方法应用于ITCS处理的人结肠癌HCT 116细胞的提取液中，成功地将谷胱甘肽半胱氨酸残基的硫代氨基甲酰化和巨噬细胞移动抑制因子的PMFIVNTNVPR的N末端脯氨酸残基确定为ITCs的胞内靶点。此外，该方法还检测了ITCs与胞内游离半胱氨酸和赖氨酸的硫代氨基甲酰化偶合物。这一新发展的方法将有助于识别与癌症化学预防ITCs结合的新的靶分子。

Thiocarbamoylation by electrophilic dietary isothiocyanates(ITCs) is necessary in order for them to exert cancer chemopreventive activities. In this study, we developed a novel method to identify ITCs-targeted molecules using two well-studied ITCs, benzyl ITC(BITC) and phenethyl ITC(PEITC). The principle of this method is based on identifying a pattern of difference between BITC and PEITC, since they show similar chemical and biological behaviors. For the method validation, dithiothreitol-reduced bovine insulin having free thiols as a model molecule was incubated with either BITC or PEITC, and then digested with endoprotease Glu-C. The generated peptides were analyzed by UPLC-TOF/MS and LC-Q-TOF/MS. Three peptides, NYCN, FVNQHLCGSHLVE and ALYLVCGE, were identified to be bound with BITC or PEITC on their cysteine residues. Each set of peptides bound with either BITC or PEITC showed retention times(RT_<BITC><RT_<PEITC>) by reverse-phase column chromatography with a difference of molecular mass(Δ14.01565). On the basis of these findings, computational mathematical schemes were constructed to extract sets of MS ions satisfying the abovecriteria. Application of the developed method to an extract of ITCs-treated human colon cancer HCT 116 cells, thiocarbamoylation of cysteine residues of glutathione and the N-terminal proline residues of PMFIVNTNVPR from macrophage migration inhibitory factor were successfully identified as the intracellular targets of ITCs. Moreover, the method also detected the thiocarbamoylated conjugates of ITCs with intracellular free cysteine and lysine. This novel developed method should be useful for identifying new target molecules which bind with cancer chemopreventive ITCs.