Fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of alpha/beta hydrolase domain containing 4 (ABHD4).
Name: Fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of alpha/beta hydrolase domain containing 4 (ABHD4). ..more
BioActive Compounds: 2005
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute
Assay Provider: Ben Cravatt, The Scripps Research Institute
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: R01 DA033760
Grant Proposal PI: Ben Cravatt, The Scripps Research Institute
External Assay ID: ABHD4_INH_FP_1536_1X%INH PRUN
Name: Fluorescence polarization-based biochemical high throughput primary assay to identify inhibitors of alpha/beta hydrolase domain containing 4 (ABHD4).
Serine hydrolases play key roles in mammalian signaling networks and are particularly enriched in the nervous system, where they have been shown to regulate the metabolism of neurotransmitters such as acetylcholine and the endocannabinoids. The development of selective inhibitors for such hydrolytic enzymes has facilitated our understanding of the role that neurotransmitters play in vivo . Recently, alpha/beta hydrolase domain containing 4 (ABHD4) was identified as the principle biosynthetic enzyme of the endocannabinoid anandamide . Anandamide is an endogenous ligand of cannabinoid receptors and a well-characterized mediator of many physiological processes including inflammation, pain, and appetite . Despite considerable advances in our understanding of anandamide degradation system, the enzymes responsible for the biosynthesis of anandamide have remained enigmatic. ABHD4 produces Glycerophospho-N-acyl Ethanolamine (GP-NAE), a precursor of anandamide . Since disruption of anandamide signaling has proven therapeutically beneficial , the development of the first potent and selective ABHD4 inhibitors would not only provide crucial tools to advance our understanding of the complex metabolic network that supports the endocannabinoid system in vivo, but also offer a new drug target for a range of human disorders such as obesity.
2. Simon, G.M. and B.F. Cravatt, Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alphalpha/betaeta-hydrolase 4 in this pathway. J Biol Chem, 2006. 281(36): p. 26465-72.
3. Di Marzo, V., Targeting the endocannabinoid system: to enhance or reduce? Nat Rev Drug Discov, 2008. 7(5): p. 438-55.
4. Natarajan, V., et al., N-Acylation of ethanolamine phospholipids in canine myocardium. Biochim Biophys Acta, 1982. 712(2): p. 342-55.
5. Natarajan, V., et al., Catabolism of N-acylethanolamine phospholipids by dog brain preparations. J Neurochem, 1984. 42(6): p. 1613-9.
6. Okamoto, Y., et al., Molecular characterization of a phospholipase D generating anandamide and its congeners. J Biol Chem, 2004. 279(7): p. 5298-305.
7. Leung, D., et al., Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids. Biochemistry, 2006. 45(15): p. 4720-6.
PRUN, primary, ABHD4, mABHD4, wil type ABHD4, mutant ABHD4, alpha/beta hydrolase domain containing 4, FLJ12816, primary, endocannabinoid, serine hydrolase, anandamide, neurotransmitters, FP-Rh, inhibitor FP, fluorescence polarization, absorbance, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC inhibitor, fluorescence polarization, HTS, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this biochemical assay is to identify compounds that act as inhibitors of alpha/beta hydrolase domain containing 4 (ABHD4). In this assay, ABHD4 protein is incubated with test compounds and fluorophosphonate-rhodamine (FP-Rh) activity-based probe. The reaction is excited with linear polarized light and the intensity of the emitted light is measured as the polarization value (mP). As designed, test compounds that act as ABHD4 inhibitors will prevent ABHD4-probe interactions, thereby increasing the proportion of free (unbound) fluorescent probe in the well, leading to low fluorescence polarization. Compounds are tested in singlicate at a nominal test concentration of 8.92 micromolar.
Prior to the start of the assay, 4 microliters of assay buffer (50 mM HEPES pH 7.0, 150 mM NaCl and 0.01% Pluronic F-127) were dispensed into column 2 only. Next, 4 microliters of assay buffer containing 46.9 ug/mL of ABHD4 mutant protein (S159A) were dispensed into columns 1 and 3 of 1536 microtiter plates and 4 microliters of assay buffer containing 46.9 ug/mL of ABHD4 wild type protein were dispensed into columns 4 thru 48. Then, 45 nL of test compound in DMSO or DMSO alone (0.89% final concentration) were added to the appropriate wells and incubated for 45 minutes at 25 degrees Celsius.
The assay was started by dispensing 1.0 microliter of 188 nM FP-Rh in assay buffer to all wells. Plates were centrifuged and after 60 minutes of incubation at 25 degrees Celsius, fluorescence polarization was read on a Viewlux microplate reader (PerkinElmer, Turku, Finland) using a BODIPY TMR FP filter set and a BODIPY dichroic mirror (excitation = 525nm, emission = 598nm) for 30 seconds for each polarization plane (parallel and perpendicular).
Prior to further calculations, the following formula was used to calculate fluorescence polarization (FP):
FP = ( Raw1 - Raw2 ) / ( Raw1 + Raw2 )
Raw1 is defined as the S channel.
Raw2 is defined as the P channel.
The percent inhibition for each compound was calculated as follows:
100 *( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Test_Compound is defined as wells containing ABHD4 wild type protein in the presence of test compound and FP-Rh.
High_Control is defined as wells containing ABHD4 mutant protein, DMSO and FP-Rh.
Low_Control is defined as the median of the wells containing test compounds.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Two values were calculated: (1) the average percent inhibition of all compounds tested, and (2) three times their standard deviation. The sum of these two values was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition than the cutoff parameter was declared active.
The reported PubChem Activity Score has been normalized to 100% observed primary inhibition. Negative % inhibition values are reported as activity score zero.
The activity score range for active compounds is 100-6, for inactive 6-0.
List of Reagents:
mABHD4 wild type protein (supplied by Assay Provider)
mABHD4 (S159A) mutant protein (supplied by Assay Provider).
FP-Rh probe (supplied by Assay Provider)
HEPES (Sigma, part 83264)
NaCl (Sigma, part S6546)
Pluronic F-127 (Invitrogen, part P6866)
1536-well plates (Corning, part 7261)
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR.
Categorized Comment - additional comments and annotations
** Test Concentration.
Data Table (Concise)