Covalent inhibitors of human monoacylglycerol lipase: ligand-assisted characterization of the catalytic site by mass spectrometry and mutational analysis.
Covalent inhibitors of human monoacylglycerol lipase: ligand-assisted characterization of the catalytic site by mass spectrometry and mutational analysis.
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DOI：
10.1016/j.chembiol.2008.06.008
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发表时间：
2008-08-25
影响因子：
--
通讯作者：
Makriyannis A
中科院分区：
文献类型：
--
作者：
Zvonok N;Pandarinathan L;Williams J;Johnston M;Karageorgos I;Janero DR;Krishnan SC;Makriyannis A
丝氨酸酯酶单酰基甘油脂肪酶（MGL）主要负责使信号传导脂质2-花生四烯酸酰甘油（2-AG）失活，2-AG是一种在两种主要大麻素受体上具有完全激动剂活性的内源性大麻素。虽然MGL被认为是一个潜在的治疗目标，这种酶的结构信息的缺乏阻碍了MGL选择性抑制剂的发展。以前，我们过表达和纯化人MGL作为六组氨酸标记的重组蛋白（hMGL），并表明它催化水解的2-AG和新的荧光报告。我们现在使用两种化学上不同的抑制剂作为直接探针通过质谱表征hMGL活性位点：5-（（联苯-4-基）甲基）-N，N-二甲基-2H-四唑-2-甲酰胺（AM 6701）和N-花生四烯酰马来酰亚胺（NAM）。建立了AM 6701抑制hMGL的适宜条件，并对底物处理的酶进行胰蛋白酶消化。通过MALDI-TOF和串联MS分析了AM 6701抑制的hMGL的胰蛋白酶消化物，结果表明AM 6701已将推定的MGL催化三联体的GXSXG基序中的丝氨酸氨甲酰化。这些结果首次直接证实了该丝氨酸残基在催化中的重要作用，并确立了AM 6701作为高亲和力共价hMGL抑制剂的机制。当应用于NAM处理的hMGL时，我们的直接配体辅助方法显示半胱氨酸残基215和/或249的部分烷基化足以实现约80%的hMGL抑制。在半胱氨酸39处的进一步烷基化不增加酶抑制的程度。虽然Cys 215和/或Cys 249突变为丙氨酸不影响hMGL水解报告底物的能力，但与未突变的hMGL相比，C215 A突变体对NAM更敏感，而C249 A突变降低了酶对NAM的敏感性。这些数据最终证明了这种脂肪烷基-马来酰亚胺底物类似物抑制MGL的巯基基机制，其中Cys 249是至关重要的。对hMGL的催化和药理学调节至关重要的氨基酸的鉴定提供了在设计作为潜在药物的选择性MGL抑制剂中有用的信息。
The serine esterase monoacylglycerol lipase (MGL) is primarily responsible for deactivating the signaling lipid 2-arachidonoylglycerol (2-AG), an endocannabinoid with full agonist activity at both principal cannabinoid receptors. Although MGL is recognized as a potential therapeutic target, the paucity of structural information on this enzyme has hindered development of MGL-selective inhibitors. Previously, we overexpressed and purified human MGL as the hexa-histidine-tagged recombinant protein (hMGL) and showed that it catalyzed the hydrolysis of both 2-AG and novel fluorogenic reporters. We now characterize by mass spectroscopy the hMGL active site using two chemically distinct inhibitors as direct probes: 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-arachidonylmaleimide (NAM). Suitable conditions were established for hMGL inhibition by AM6701, and the inhibitor-treated enzyme was subjected to trypsin digestion. The tryptic digest of AM6701-inhibited hMGL was analyzed by MALDI-TOF and tandem MS, which showed that AM6701 had carbamylated the serine in a GXSXG motif of the putative MGL catalytic triad. These results provide the first direct confirmation of the essential role of this serine residue for catalysis and establish the mechanism of AM6701 as a high-affinity, covalent hMGL inhibitor. When applied to NAM-treated hMGL, our direct, ligand-assisted approach revealed that partial alkylation of cysteine residues 215 and/or 249 was sufficient to achieve ~ 80% hMGL inhibition. Further alkylation at cysteine 39 did not increase the extent of enzyme inhibition. Although Cys215 and/or Cys249 mutations to alanine(s) did not affect hMGL’s ability to hydrolyze reporter substrate, as compared to nonmutated hMGL the C215A mutant was more sensitive to NAM, whereas the C249A mutation reduced the enzyme’s sensitivity to NAM. These data conclusively demonstrate a sulfhydryl-based mechanism underlying MGL inhibition by this fatty alkyl-maleimide substrate analog in which Cys249 is of paramount importance. Identification of amino acids critical to catalysis by and pharmacological modulation of hMGL provides information useful in the design of selective MGL inhibitors as potential drugs.
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影响因子：
3.6
作者：
Dinh, TP;Kathuria, S;Piomelli, D
通讯作者：
Piomelli, D
影响因子：
4.4
作者：
Zvonok, Nikolai;Yaddanapudi, Suma;Makriyannis, Alexandros
通讯作者：
Makriyannis, Alexandros
DOI：
10.1006/bbrc.1997.6072
发表时间：
1997-02-03
影响因子：
3.1
作者：
Deutsch, DG;Lin, S;Makriyannis, A
通讯作者：
Makriyannis, A
影响因子：
2.7
作者：
SAKURADA, T;NOMA, A
通讯作者：
NOMA, A
影响因子：
--
作者：
Blankman, Jacqueline L.;Simon, Gabriel M.;Cravatt, Benjamin F.
通讯作者：
Cravatt, Benjamin F.