Università degli Studi di Firenze, Laboratorio di Chimica Bioinorganica, Rm. 188, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Florence, Italy.
J Am Chem Soc. 2009 Mar 4;131(8):3057-62. doi: 10.1021/ja809683v.
The X-ray crystal structure of the adduct between the zinc metalloenzyme carbonic anhydrase II (CA, EC 4.2.1.1) with the recently discovered natural product coumarin derivative 6-(1S-hydroxy-3-methylbutyl)-7-methoxy-2H-chromen-2-one showed the coumarin hydrolysis product, a cis-2-hydroxy-cinnamic acid derivative, and not the parent coumarin, bound within the enzyme active site. The bound inhibitor exhibits an extended, two-arm conformation that effectively plugs the entrance to the enzyme active site with no interactions with the catalytically crucial zinc ion. The inhibitor is sandwiched between Phe131, with which it makes an edge-to-face stacking, and Asn67/Glu238sym, with which it makes several polar and hydrogen bonding interactions. This unusual binding mode, with no interactions between the inhibitor molecule and the active site metal ion is previously unobserved for this enzyme class and presents a new opportunity for future drug design campaigns to target a mode of inhibition that differs substantially from classical inhibitors such as the clinically used sulfonamides and sulfamates. Several structurally simple coumarin scaffolds were also shown to inhibit all 13 catalytically active mammalian CA isoforms, with inhibition constants ranging from nanomolar to millimolar. The inhibition is time dependent, with maximum inhibition being observed after 6 h.
锌金属酶碳酸酐酶 II（CA，EC 4.2.1.1）与最近发现的天然产物香豆素衍生物 6-（1S-羟基-3-甲基丁基）-7-甲氧基-2H-色烯-2-酮的加合物的 X 射线晶体结构显示，香豆素水解产物顺式-2-羟基肉桂酸衍生物而不是母体香豆素结合在酶活性部位内。结合的抑制剂呈现出伸展的双臂构象，有效地阻塞了酶活性部位的入口，与催化关键的锌离子没有相互作用。抑制剂夹在 Phe131 之间，与 Phe131 形成边缘到面的堆积，与 Asn67/Glu238sym 形成几个极性和氢键相互作用。这种不寻常的结合模式，抑制剂分子与活性部位金属离子之间没有相互作用，以前在这种酶类中没有观察到，为未来的药物设计活动提供了一个新的机会，可以针对与经典抑制剂（如临床使用的磺胺类和磺胺酸盐）有很大不同的抑制模式。还显示几种结构简单的香豆素支架抑制所有 13 种催化活性的哺乳动物 CA 同工酶，抑制常数范围从纳摩尔到毫摩尔。抑制是时间依赖性的，在 6 小时后观察到最大抑制。
