Neolymphostin A Is a Covalent Phosphoinositide 3‑Kinase (PI3K)/Mammalian Target of Rapamycin (mTOR) Dual Inhibitor That Employs an Unusual Electrophilic Vinylogous Ester

Using a novel chemistry-based
assay for identifying electrophilic
natural products in unprocessed extracts, we identified the PI3-kinase/mTOR
dual inhibitor neolymphostin A from Salinispora arenicola CNY-486. The method further showed that the vinylogous ester substituent
on the neolymphostin core was the exact site for enzyme conjugation.
Tandem MS/MS experiments on PI3Kα treated with the inhibitor
revealed that neolymphostin covalently modified Lys802 with a shift
in mass of +306 amu, corresponding to addition of the inhibitor and
elimination of methanol. The binding pose of the inhibitor bound to
PI3Kα was modeled, and hydrogen–deuterium exchange mass
spectrometry experiments supported this model. Against a panel of
kinases, neolymphostin showed good selectivity for PI3-kinase and
mTOR. In addition, the natural product blocked AKT phosphorylation
in live cells with an IC50 of ∼3 nM. Taken together,
neolymphostin is the first reported example of a covalent kinase inhibitor
from the bacterial domain of life.

本研究采用一种基于化学的新型检测方法，用于从未经处理的提取物中识别亲电性天然产物，最终从盐生盐单胞菌（Salinispora arenicola）CNY-486中筛选得到了磷脂酰肌醇3-激酶/哺乳动物雷帕霉素靶蛋白（PI3-kinase/mTOR）双重抑制剂新淋巴菌素A（neolymphostin A）。该方法进一步证实，新淋巴菌素母核上的插烯酯取代基是其与酶共价结合的精确位点。对经该抑制剂处理的磷脂酰肌醇3-激酶α亚型（PI3Kα）开展串联质谱（MS/MS）实验，结果显示新淋巴菌素以共价方式修饰了赖氨酸802（Lys802），质量偏移量为+306原子质量单位（amu），对应抑制剂的加成与甲醇的消除过程。我们对该抑制剂与PI3Kα的结合构象进行了分子建模，氢-氘交换质谱（hydrogen–deuterium exchange mass spectrometry）实验结果验证了该模型的合理性。在一组激酶筛选面板中，新淋巴菌素对PI3-激酶与mTOR展现出良好的选择性。此外，该天然产物可在活细胞中抑制蛋白激酶B（AKT）的磷酸化，半抑制浓度（IC50）约为3纳摩尔（nM）。综上，新淋巴菌素是首个被报道的源自细菌域的共价激酶抑制剂。