Full Mass Spectrometric Characterization of Human Monoacylglycerol Lipase Generated by Large-Scale Expression and Single-Step Purification

Zvonok, Nikolai; Williams, Josh; Johnston, Meghan; Pandarinathan, Lakshmipathi; Janero, David R.; Li, Jing; Krishnan, Srinivasan; Makriyannis, Alexandros

doi:10.1021/pr700839z

Cited by 39 publications

(73 citation statements)

References 29 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Recombinant rat FAAH and recombinant hexa-histidine-tagged human monoacylglycerol lipase (MAGL) were expressed in E. coli as previously described (Patricelli et al 1998; Ramarao et al 2005; Zvonok et al 2008). FAAH and MAGL activities were performed in 96-well plates using the fluorogenic substrates arachidonoyl 7-amino-4-methylcoumarin amide for FAAH, and arachidonoyl, 7-hydroxy-6-methoxy-4-methylcoumarin ester for MAGL.…”

Section: Methodsmentioning

confidence: 99%

A New Generation Fatty Acid Amide Hydrolase Inhibitor Protects Against Kainate-Induced Excitotoxicity

et al. 2010

Self Cite

View full text Add to dashboard Cite

Endocannabinoids, including anandamide (AEA), have been implicated in neuroprotective on-demand responses. Related to such a response to injury, an excitotoxic kainic acid (KA) injection (i.p.) was found to increase AEA levels in the brain. To modulate the endocannabinoid response during events of excitotoxicity in vitro and in vivo, we utilized a new generation compound (AM5206) that selectively inhibits the AEA deactivating enzyme fatty acid amide hydrolase (FAAH). KA caused calpain-mediated spectrin breakdown, declines in synaptic markers, and disruption of neuronal integrity in cultured hippocampal slices. FAAH inhibition with AM5206 protected against the neurodegenerative cascade assessed in the slice model 24 h postinsult. In vivo, KA administration induced seizures and the same neurodegenerative events exhibited in vitro. When AM5206 was injected immediately after KA in rats, the seizure scores were markedly reduced as were levels of cytoskeletal damage and synaptic protein decline. The pre- and postsynaptic proteins were protected by the FAAH inhibitor to levels comparable to those found in healthy control brains. These data support the idea that endocannabinoids are released and converge on pro-survival pathways that prevent excitotoxic progression.

show abstract

Section: Methodsmentioning

confidence: 99%

A New Generation Fatty Acid Amide Hydrolase Inhibitor Protects Against Kainate-Induced Excitotoxicity

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…In aggregate, these high-resolution structural and molecular enzymology data suggest that the activated Ser 122 nucleophile of the (h)MGL catalytic triad is critical to both the enzyme's catalytic mechanism and the structural and topological dynamics associated with the (h)MGL catalytic cycle. This suggestion has gained direct experimental support from studies in our laboratory involving mass spectroscopy (MS) characterization of hMGL and its inhibition by various covalent inhibitors, 17,18 functional hMGL mutagenesis, 18 and identification by nuclear magnetic resonance spectroscopy (NMR) of a dynamic, active-site hydrogenbond network. 19 Although crystal structures of hMGL in apo and enzymeligand forms are available, questions remain concerning the structural and dynamic details associated with the opening and closing of the enzyme's lid domain, the process of enzymemembrane (dis)association, and the interaction profiles of small-molecule inhibitors.…”

mentioning

confidence: 99%

“…We and others have engineered assorted recombinant hMGL deletion mutants as well as single, double, and triple point mutants in order to increase expression yield, enhance enzyme aqueous solubility, and probe the structural basis of hMGL catalysis/inhibition. 15,17,18 In the present work, we have employed site-directed mutagenesis and targeted ligand synthesis as a molecular-pharmacology approach for gaining further insight into the mechanism of hMGL catalysis and the molecular features involved in reversible and irreversible hMGL inactivation by inhibitors. In order to enhance hMGL solubility, we constructed a Leu- ( [4,5-b]pyridin-1-ylmethanone (AM6580), 20 and the reversible inhibitor 2-cyclohexyl-6-{[3-(4-pyrimidin-2-yl-piperazin-1-yl)azetidin-1-yl]carbonyl}-1,3-benzoxazole (AM10212) 21 ( Figure 1).…”

mentioning

confidence: 99%

Endocannabinoid Enzyme Engineering: Soluble Human Thio-Monoacylglycerol Lipase (sol-S-hMGL)

Karageorgos

Zvonok

Janero

et al. 2012

ACS Chem. Neurosci.

Self Cite

View full text Add to dashboard Cite

In the mammalian central nervous system, monoacylglycerol lipase (MGL) is principally responsible for inactivating the endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) and modulates cannabinoid-1 receptor (CB1R) desensitization and signal intensity. MGL is also a drug target for diseases in which CB1R stimulation may be therapeutic. To inform the design of human MGL (hMGL) inhibitors, we have engineered a Leu(Leu 169 ;Leu 176 )-to-Ser(Ser 169 ;Ser 176 ) double hMGL mutant (sol-hMGL) which exhibited enhanced solubility properties, and we further mutated this variant by substituting its catalytic-triad Ser 122 with Cys (sol-S-hMGL). The hMGL variants hydrolyzed both 2-AG and a fluorogenic reporter substrate with comparable affinities. Our results suggest that the hMGL cysteine mutant maintains the same overall architecture as wildtype hMGL. The results also underscore the superior nucleophilic nature of the reactive catalytic Ser 122 residue as compared to that of Cys 122 in the sol-S-hMGL mutant and suggest that the nucleophilic character of the Cys 122 residue is not commensurately enhanced within the three dimensional architecture of hMGL. The interaction of the sol-hMGL variants with the irreversible inhibitors AM6580 and N-arachidonylmaleimide (NAM) and the reversible inhibitor AM10212 was profiled. LC/MS analysis of tryptic digests from sol-S-hMGL directly demonstrate covalent modification of this variant by NAM and AM6580, consistent with enzyme thiol alkylation and carbamoylation, respectively. These data provide insight into hMGL catalysis, the key role of the nucleophilic character of Ser 122 , and the mechanisms underlying hMGL inhibition by different classes of small molecules.

show abstract

“…Medium-throughput fluorimetric screening assays were employed to determine inhibitory potencies of AM4301-3 using recombinant human hMGL and rat rMGL according to established protocols (Zvonok et al 2008a, b). Briefly described, recombinant human MAGL (hMAGL) or rat MAGL (rMAGL) were expressed in Escherichia coli cells and purified (Zvonok et al 2008a, b).…”

Section: Methodsmentioning

confidence: 99%

A comparison of novel, selective fatty acid amide hydrolase (FAAH), monoacyglycerol lipase (MAGL) or dual FAAH/MAGL inhibitors to suppress acute and anticipatory nausea in rat models

et al. 2016

Self Cite

View full text Add to dashboard Cite

Rationale Drugs that block fatty acid amide hydrolase (FAAH, which elevates anandamide [AEA]) and drugs which block monoacylglycerol (MAGL, which elevates 2-arachidonyl glycerol [2-AG]) have promise in treating both acute and anticipatory nausea in human patients. Objective This study aims to evaluate the relative effectiveness of dual MAGL/FAAH inhibition with either alone to reduce acute and anticipatory nausea in rat models. Materials and methods AM4302, a new dual MAGL/FAAH inhibitor, was compared with a new selective MAGL inhibitor, AM4301, and new selective FAAH inhibitor, AM4303, for their potential to reduce acute nausea (gaping in taste reactivity) and anticipatory nausea (contextually elicited conditioned gaping) in two rat models. Results Our in vitro studies indicate that AM4302 blocks human and rat FAAH: IC50 60 and 31 nM, respectively, with comparable potencies against human MAGL (IC50 41 nM) and rat MAGL (IC50 200 nM). AM4301 selectively blocks human and rat MAGL (IC50 8.9 and 36 nM, respectively), while AM4303 selectively inhibits human and rat FAAH (IC50 2 and 1.9 nM), respectively. Our in vivo studies show that the MAGL inhibitor, AM4301, suppressed acute nausea in a CB1-mediated manner, when delivered systemically or into the interoceptive insular cortex. Although the dual FAAH/MAGL inhibitor, AM4302, was equally effective as the FAAH inhibitor or MAGL inhibitor in reducing acute nausea, it was more effective than both in suppressing anticipatory nausea. Conclusions Dual FAAH and MAGL inhibition with AM4302 may be an especially effective treatment for the very difficult to treat symptom of anticipatory nausea.

show abstract

Full Mass Spectrometric Characterization of Human Monoacylglycerol Lipase Generated by Large-Scale Expression and Single-Step Purification

Cited by 39 publications

References 29 publications

A New Generation Fatty Acid Amide Hydrolase Inhibitor Protects Against Kainate-Induced Excitotoxicity

A New Generation Fatty Acid Amide Hydrolase Inhibitor Protects Against Kainate-Induced Excitotoxicity

Endocannabinoid Enzyme Engineering: Soluble Human Thio-Monoacylglycerol Lipase (sol-S-hMGL)

A comparison of novel, selective fatty acid amide hydrolase (FAAH), monoacyglycerol lipase (MAGL) or dual FAAH/MAGL inhibitors to suppress acute and anticipatory nausea in rat models

Contact Info

Product

Resources

About