Rational Design of Fatty Acid Amide Hydrolase Inhibitors That Act by Covalently Bonding to Two Active Site Residues
Rational Design of Fatty Acid Amide Hydrolase Inhibitors That Act by Covalently Bonding to Two Active Site Residues
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DOI：
10.1021/ja4014997
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发表时间：
2013-04-24
影响因子：
15
通讯作者：
Boger, Dale L.
中科院分区：
文献类型：
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作者：
Otrubova, Katerina;Brown, Monica;Boger, Dale L.
公开了 α-酮杂环脂肪酸酰胺水解酶 (FAAH) 抑制剂的设计和表征，其另外不可逆地靶向酶胞质端口中发现的半胱氨酸 (Cys269)，同时维持负责其快速和最初可逆酶抑制的可逆共价 Ser241 连接。制备了两种 α-酮恶唑（3 和 4），它们在 2（OL-135）的吡啶基取代基的 C5 位上策略性地放置了亲电子试剂，并作为 FAAH 抑制剂进行了检查。与观察到的时间依赖性非竞争性抑制一致，3与大鼠FAAH人源化变体结合的共晶X射线结构表明，3不仅作为去质子化半缩酮与活性位点催化亲核试剂Ser241共价结合，而且还通过吡啶基C5取代基与Cys269结合，从而提供了在酶活性位点具有双共价连接的抑制剂。小鼠原型抑制剂的体内表征表明，与可逆抑制剂 2 相比，它们能更大程度地提高 FAAH 底物的内源性大脑水平，持续时间更长（>6 小时），这表明抑制剂在大脑中积累并持续存在，可在较长时间内完全抑制 FAAH。与这种行为和靶向不可逆酶抑制一致，3在小鼠神经性疼痛的慢性压迫性损伤模型中逆转冷异常性疼痛，持续时间（>6小时）超过了用可逆抑制剂2观察到的结果，提供了在监测的1-6小时时间过程中没有变化的效果。
The design and characterization of alpha-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two alpha-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent noncompetitive inhibition, the cocrystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrates that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1-6 h time course monitored.