Download Document 8919101

Graduate
Category: Physical and Life Sciences
Degree Level: PhD
Abstract ID# 1160
Proteomic Characterization of Recombinant Human α/β Hydrolase Domain 6
The biochemistry behind making a better Marijuana
Christina Miyabe, Nikolai Zvonok, Alexander Zvonok and Alexandros Makriyannis
Center for Drug Discovery (Northeastern University) Boston, MA
Abstract:
Background:
Human α/β Hydrolase Domain 6 (hABHD6) is an endocannabinoidmetabolizing serine hydrolase that has been found to inactivate 2arachidonoylglycerol (2-AG), a potent agonist at both cannabinoid
receptors. ABHD6 is tethered to the membrane post-synaptically,
allowing it to control 2-AG concentrations at a unique subcellular
localization as compared to its relative, monoacylglyerol lipase
(MGL). Selective ABHD6 inhibitors have been shown to have
therapeutic potential towards addiction, pain, inflammation, cancer,
and metabolic, neuroinflammatory, and neurodegenerative
disorders, but more structural data is needed to develop ABHD6selective inhibitors. Recombinant hABHD6 overexpressed in E. coli
and purified by single step immobilized metal affinity
chromatography is catalytically active and has comparable 2-AG
hydrolysis parameters to the native enzyme. A novel highthroughput assay based on the fluorogenic substrate arachidonoyl,
7-hydroxy-6-methoxy-4-methylcoumarin ester (AHMMCE) was
developed and used to identify potent ABHD6 inhibitors. Matrixassisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI TOF MS) was used to analyze hABHD6 by
proteomic peptide fingerprinting and the identification covalent
interactions between the active-site serine and potent inhibitors.
AM6701, which has been shown to carbamylate the active-site
serine of hMGL, acts through the same mechanism to inhibit
ABHD6. The ABHD6-selective inhibitor WWL70 formed covalent
attachment by SN2 addition; this is the first reported evidence for
the molecular mechanism of action of WWL70. The further
structural characterization of the binding pocket of ABHD6 is critical
to identifying and developing more potent, selective inhibitors as
new potential therapeutic agents.
The psychoactive component of the marijuana plant
Cannabis sativa, Δ9-tetrahydrocannabinol, was
discovered and isolated in the 1960’s, while the
endogenous ligands (endocannabinoids) and the
receptors they interact with were not identified until
much later in 1992. Since then, research has
pushed towards understanding the underlying
mechanism behind marijuana’s myriad of effects by
better elucidating the function of the endogenous
system it interacts with in an effort to design potent,
selective therapeutic drugs that act upon the
proteins in the system.
References:
arachidonic acid
glycerol
S148
2-AG
catalytic triad
transmembrane
domain
Results:
Conclusion:
Mass Spectrometry
Protein Purification
Human ABHD6 is a new, relatively unstudied
endocannbinoid-metabolizing enzyme that has
promising potential a therapeutic drug target. We
have successfully genetically modified and purified
an active variant of hABHD6 recombinantly
expressed in the E. coli host system. We have
also identified the mechanism of inhibition for two
covalent inhibitors: AM6701 and WWL70.
hABHD6 IC50 AM6701
+71
IC50 = 0.9 nM
5037-
1. Ahn, K., McKinney, M. K., and Cravatt, B.F. (2008) Chem. Rev. 108, 1687-1707.
2. Blankman, J. L., Simon, G. M., and Cravatt, B.F. (2007) Chem. Biol. 14, 1347-1356.
3. Saario, S. M., and Laitinen, J. T. (2007) Basic Clin. Pharmacol. Toxicol. 101, 287-293.
+224
IC50 = 295 nM
Acknowledgements:
This work was supported by the NIH grant DA003801 (A.M.) and a PhD Candidate Fellowship
Diversity Supplement from the National Institute on Drug Abuse.
homology model
of hABHD6
H306
D278
Digest protein
with trypsin
Analyze/identify specific
peptides