Dato Florian M, Maaßen Andreas, Goldfuß Bernd, Pietsch Markus
Institute II of Pharmacology, Center of Pharmacology, Medical Faculty, University of Cologne, Gleueler Str. 24, D-50931 Cologne, Germany; Institute of Organic Chemistry, Department of Chemistry, University of Cologne, Greinstr. 4, D-50939 Cologne, Germany.
Institute of Organic Chemistry, Department of Chemistry, University of Cologne, Greinstr. 4, D-50939 Cologne, Germany.
Anal Biochem. 2018 Apr 1;546:50-57. doi: 10.1016/j.ab.2018.01.026. Epub 2018 Jan 31.
Fatty acid amide hydrolase (FAAH) is involved in many human diseases, particularly cancer, pain and inflammation as well as neurological, metabolic and cardiovascular disorders. Therefore, FAAH is an attractive target for the development of low-molecular-weight inhibitors as therapeutics, which requires robust assays that can be used for high-throughput screening (HTS) of compound libraries. Here, we report the development of a fluorometric assay based on FAAH's ability to effectively hydrolyze medium-chain fatty acid amides, introducing N-decanoyl-substituted 5-amino-2-methoxypyridine (D-MAP) as new amide substrate. D-MAP is cleaved by FAAH with an 8-fold larger specificity constant than the previously reported octanoyl-analog Oc-MAP (V/K of 1.09 and 0.134 mL min mg, respectively), with both MAP derivatives possessing superior substrate properties and much increased aqueous solubility compared to the respective p-nitroaniline compounds D-pNA and Oc-pNA. The new assay with D-MAP as substrate is highly sensitive using a lower enzyme concentration (1 μg mL) than literature-reported fluorimetric FAAH assays. In addition, D-MAP was validated in comparison to the substrate Oc-MAP for the characterization of FAAH inhibitors by means of the reference compounds URB597 and TC-F2 and was shown to be highly suitable for HTS in both kinetic and endpoint assays (Z' factors of 0.81 and 0.78, respectively).
脂肪酸酰胺水解酶（FAAH）与多种人类疾病相关，尤其是癌症、疼痛和炎症以及神经、代谢和心血管疾病。因此，FAAH是开发低分子量抑制剂作为治疗药物的一个有吸引力的靶点，这需要能够用于化合物库高通量筛选（HTS）的可靠检测方法。在此，我们报告了一种基于FAAH有效水解中链脂肪酸酰胺能力的荧光检测方法的开发，引入了N-癸酰基取代的5-氨基-2-甲氧基吡啶（D-MAP）作为新的酰胺底物。FAAH对D-MAP的切割特异性常数比先前报道的辛酰基类似物Oc-MAP大8倍（V/K分别为1.09和0.134 mL min mg），与相应的对硝基苯胺化合物D-pNA和Oc-pNA相比，两种MAP衍生物都具有优异的底物特性且水溶性大大增加。以D-MAP为底物时，新检测方法使用的酶浓度（1 μg mL）比文献报道的荧光FAAH检测方法低，因此具有高灵敏度。此外，通过参考化合物URB597和TC-F2，将D-MAP与底物Oc-MAP进行比较，以验证其用于FAAH抑制剂的表征，结果表明它在动力学和终点检测中都非常适合高通量筛选（Z'因子分别为0.81和0.78）。
