Karlsson Jessica, Morgillo Carmine M, Deplano Alessandro, Smaldone Giovanni, Pedone Emilia, Luque F Javier, Svensson Mona, Novellino Ettore, Congiu Cenzo, Onnis Valentina, Catalanotti Bruno, Fowler Christopher J
Department of Pharmacology and Clinical Neuroscience, Pharmacology Unit, Umeå University, Umeå, Sweden.
Department of Pharmacy, Università degli Studi di Napoli Federico II, Napoli, Italy.
PLoS One. 2015 Nov 13;10(11):e0142711. doi: 10.1371/journal.pone.0142711. eCollection 2015.
Combined fatty acid amide hydrolase (FAAH) and cyclooxygenase (COX) inhibition is a promising approach for pain-relief. The Flu-AM1 and Ibu-AM5 derivatives of flurbiprofen and ibuprofen retain similar COX-inhibitory properties and are more potent inhibitors of FAAH than the parent compounds. However, little is known as to the nature of their interaction with FAAH, or to the importance of their chirality. This has been explored here.
METHODOLOGY/PRINCIPAL FINDINGS: FAAH inhibitory activity was measured in rat brain homogenates and in lysates expressing either wild-type or FAAH(T488A)-mutated enzyme. Molecular modelling was undertaken using both docking and molecular dynamics. The (R)- and (S)-enantiomers of Flu-AM1 inhibited rat FAAH with similar potencies (IC50 values of 0.74 and 0.99 μM, respectively), whereas the (S)-enantiomer of Ibu-AM5 (IC50 0.59 μM) was more potent than the (R)-enantiomer (IC50 5.7 μM). Multiple inhibition experiments indicated that both (R)-Flu-AM1 and (S)-Ibu-AM5 inhibited FAAH in a manner mutually exclusive to carprofen. Computational studies indicated that the binding site for the Flu-AM1 and Ibu-AM5 enantiomers was located between the acyl chain binding channel and the membrane access channel, in a site overlapping the carprofen binding site, and showed a binding mode in line with that proposed for carprofen and other non-covalent ligands. The potency of (R)-Flu-AM1 was lower towards lysates expressing FAAH mutated at the proposed carprofen binding area than in lysates expressing wild-type FAAH.
CONCLUSIONS/SIGNIFICANCE: The study provides kinetic and structural evidence that the enantiomers of Flu-AM1 and Ibu-AM5 bind in the substrate channel of FAAH. This information will be useful in aiding the design of novel dual-action FAAH: COX inhibitors.
联合抑制脂肪酸酰胺水解酶（FAAH）和环氧化酶（COX）是一种很有前景的止痛方法。氟比洛芬和布洛芬的Flu-AM1和Ibu-AM5衍生物保留了相似的COX抑制特性，并且比母体化合物更有效地抑制FAAH。然而，关于它们与FAAH相互作用的性质以及它们手性的重要性，人们了解甚少。本文对此进行了探讨。
方法/主要发现：在大鼠脑匀浆和表达野生型或FAAH（T488A）突变酶的裂解物中测量FAAH抑制活性。使用对接和分子动力学进行分子建模。Flu-AM1的（R）-和（S）-对映体对大鼠FAAH的抑制效力相似（IC50值分别为0.74和0.99 μM），而Ibu-AM5的（S）-对映体（IC50 0.59 μM）比（R）-对映体（IC50 5.7 μM）更有效。多重抑制实验表明，（R）-Flu-AM1和（S）-Ibu-AM5均以与卡洛芬相互排斥的方式抑制FAAH。计算研究表明，Flu-AM1和Ibu-AM5对映体的结合位点位于酰基链结合通道和膜通道之间，与卡洛芬结合位点重叠的区域，并显示出与卡洛芬和其他非共价配体所提出的结合模式一致的结合模式。（R）-Flu-AM1对在所提出的卡洛芬结合区域发生突变的FAAH表达裂解物的效力低于对表达野生型FAAH的裂解物的效力。
结论/意义：该研究提供了动力学和结构证据，表明Flu-AM1和Ibu-AM5的对映体在FAAH的底物通道中结合。这些信息将有助于新型双作用FAAH：COX抑制剂的设计。
