Department of Drug Discovery and Development, Harrison School of Pharmacy, 3306 Walker Building, Auburn University, Auburn, AL 36849, USA.
Department of Chemistry and Biochemistry, 179 Chemistry Building, Auburn University, Auburn, AL 36849, USA.
Biochim Biophys Acta Proteins Proteom. 2018 May-Jun;1866(5-6):731-739. doi: 10.1016/j.bbapap.2018.04.007. Epub 2018 Apr 12.
Ilimaquinone (IQ), a marine sponge metabolite, has been considered as a potential therapeutic agent for various diseases due to its broad range of biological activities. We show that IQ irreversibly inactivates Mycobacterium tuberculosis shikimate kinase (MtSK) through covalent modification of the protein. Inactivation occurred with an apparent second-order rate constant of about 60 M s. Following reaction with IQ, LC-MS analyses of intact MtSK revealed covalent modification of MtSK by IQ, with the concomitant loss of a methoxy group, suggesting a Michael-addition mechanism. Evaluation of tryptic fragments of IQ-derivatized MtSK by MS/MS demonstrated that Ser and Thr residues were most frequently modified with lesser involvement of Lys and Tyr. In or near the MtSK active site, three residues of the P-loop (K15, S16, and T17) as well as S77, T111, and S44 showed evidence of IQ-dependent derivatization. Accordingly, inclusion of ATP in IQ reactions with MtSK partially protected the enzyme from inactivation and limited IQ-based derivatization of K15 and S16. Additionally, molecular docking models for MtSK-IQ were generated for IQ-derivatized S77 and T111. In the latter, ATP was observed to sterically clash with the IQ moiety. Out of three other enzymes evaluated, lactate dehydrogenase was derivatized and inactivated by IQ, but pyruvate kinase and catalase-peroxidase (KatG) were unaffected. Together, these data suggest that IQ is promiscuous (though not entirely indiscriminant) in its reactivity. As such, the potential of IQ as a lead in the development of antitubercular agents directed against MtSK or other targets is questionable.
异绵马醌（IQ）是一种海洋海绵代谢物，由于其广泛的生物活性，已被认为是治疗各种疾病的潜在治疗剂。我们表明，IQ 通过蛋白质的共价修饰不可逆地使结核分枝杆菌莽草酸激酶（MtSK）失活。失活反应的表观二级速率常数约为 60 M s。与 IQ 反应后，通过 LC-MS 分析完整的 MtSK 发现 IQ 共价修饰 MtSK，同时失去一个甲氧基，表明存在迈克尔加成机制。通过 MS/MS 对 IQ 衍生的 MtSK 肽段进行评估，表明 Ser 和 Thr 残基最常被修饰，而 Lys 和 Tyr 残基的参与程度较小。在 MtSK 的活性位点内或附近，P 环的三个残基（K15、S16 和 T17）以及 S77、T111 和 S44 显示出依赖于 IQ 的衍生化的证据。因此，在含有 MtSK 的 IQ 反应中加入 ATP 可部分保护酶免受失活，并限制 IQ 衍生化 K15 和 S16。此外，还生成了用于 MtSK-IQ 的分子对接模型，用于 IQ 衍生化的 S77 和 T111。在后一种情况下，观察到 ATP 与 IQ 部分发生空间冲突。在所评估的其他三种酶中，IQ 衍生化和失活了乳酸脱氢酶，但丙酮酸激酶和过氧化氢酶过氧化物酶（KatG）不受影响。综上所述，这些数据表明 IQ 在反应性方面是混杂的（尽管并非完全无差别）。因此，IQ 作为针对 MtSK 或其他靶标开发抗结核药物的先导化合物的潜力是值得怀疑的。
