摘要
昆虫表皮是由几丁质及蛋白质等组成的复杂且高度有序的结构。由于昆虫必须周期性蜕皮以满足其生长发育的需要,使得其负责其蜕皮过程的关键酶成为有潜力的绿色农药靶标。蛋白质组研究表明,N-乙酰己糖胺酶Hex1是昆虫蜕皮液中几丁质水解酶"211"组合中的一员,RNA干扰该酶会导致昆虫蜕皮异常而死亡。我们解析了亚洲玉米螟N-乙酰己糖胺酶OfHex1的晶体结构,并且发现其活性口袋入口处特殊的Loop结构及关键氨基酸残基Val327、Glu368及Trp490决定了其生理功能特异性,同时也是基于结构选择性抑制剂设计的重要靶点~([1])。基于以上发现,我们通过对天然产物TMG-chitotriomycin及催化反应中间体类似物NAG-thiazoline的结构改造,获得了高活性及高选择性的OfHex1抑制剂TMG-(GlcNAc)_2及NMAGT,其中前者的K_i值为70 nM~([2])。接下来,我们对选择性抑制人类N-乙酰己糖胺酶HsHex的抑制剂M-31850,通过理性设计实现了选择性逆转,获得的选择性抑制OfH ex1的萘酰亚胺衍生物Q2~([3])。本研究为基于靶标结构的抑制剂设计提供了思路,也为针对昆虫蜕皮过程的绿色农药开发提供了先导化合物。
NAG-thiazoline(NGT) and its derivatives are well-known inhibitors against most β-acetylglucosaminidases(β-GlcNAcases) except for insect and bacterial chitinolytic β-GlcNAcases,e.g.,the molting-indispensable OfHex1 from the insect Ostrinia furnacalis.Here,we report the co-crystal structure of OfHex1-NGT,revealing a rather large active pocket in OfHex1 that may account for the poor inhibitory activity of NGT.Therefore,a bulky substituent on the thiazoline ring of NGT was designed and synthesized.The resulting compound(NMAGT) was determined to be a submicromolar inhibitor of OfHex1 with a K_i value of 0.13 μM,which is 600-fold lower than K_i value of NGT.Molecular dynamics simulation analysis supported the good fit of NMAGT to the active pocket.
引文
[1]Liu,T.,Zhang,H.,Liu,F.,et al.,J.Biol.Chem.2011,286,4049-4058.
[2]Liu,T.,Xia,M.,Zhang,H.,et al.,FEBS lett.2015,589,110-116.
[3]Liu,T.,Guo,P.,Zhou,Y.,et al.,Sci.Rep.2014,4,6188.