QFRET-based biochemical high throughput primary assay to identify inhibitors of human group III secreted phospholipase A2 enzyme (HGIII-sPLA2)
Name: QFRET-based biochemical high throughput primary assay to identify inhibitors of human group III secreted phospholipase A2 enzyme (HGIII-sPLA2). ..more
BioActive Compounds: 2088
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center
Affiliation: The Scripps Research Institute, TSRI
Assay Provider: Michael H. Gelb, University of Washington
Network: Molecular Library Probe Production Centers Network (MLPCN)
Grant Proposal Number R37HL036235
Grant Proposal PI: Michael H. Gelb, University of Washington
External Assay ID: HGIII-SPLA2_INH_QFRET_1536_1X%INH PRUN
Name: QFRET-based biochemical high throughput primary assay to identify inhibitors of human group III secreted phospholipase A2 enzyme (HGIII-sPLA2).
Phospholipases A2 (PLA2s) are a class of enzymes that hydrolyze the sn-2 ester of glycerophospholipids to release a free fatty acid and a lysophospholipid . Secreted PLA2s (sPLA2s) have been implicated in the pathogenesis of inflammatory disease in humans. Inhibitors of many of the sPLA2s in the human and mouse genome have been made, but to date no inhibitor of human group III sPLA2 (hGIII-sPLA2) has been discovered. The amino acid sequence of hGIII-sPLA2 is similar to that of the sPLA2 in honey bee venom. This enzyme was discovered about 10 years ago, cloned, its enzymatic properties studied, and a knockout mouse constructed . Over the past few years it has been shown that this enzyme is highly expressed in mast cells . The enzyme is secreted from human and mouse mast cells and, together with lipocalin-type prostaglandin D2 synthase, leads to PGD2 production. Presumably the sPLA2 liberates arachidonic acid from the sn-2 position of membrane phospholipids to provide the arachidonic acid for PGD2 production. PGD2 then binds to its receptor (DP1) on the surface of mast cells leading to mast cell maturation. Mice lacking mGIII-sPLA2 produce immature mast cells . These mice show a dramatic reduction in anaphylatic responses, a process that is well known to be mediated in part by mast cells. The results provide a strong rational for the development of GIII-sPLA2 inhibitors to treat mast cell-mediate diseases.
1. Lambeau G, Gelb MH. Biochemistry and physiology of mammalian secreted phospholipases A2. Annu Rev Biochem. 2008;77:495-520.
2. Valentin E, Ghomashchi F, Gelb MH, Lazdunski M, Lambeau G. Novel human secreted phospholipase A(2) with homology to the group III bee venom enzyme. J Biol Chem. 2000 Mar 17;275(11):7492-7496.
3. Triggiani M, Giannattasio G, Calabrese C, Loffredo S, Granata F, Fiorello A, Santini M, Gelb MH, Marone G. Lung mast cells are a source of secreted phospholipases A2. J Allergy Clin Immunol. 2009 Sep;124(3):558-565.
4. Taketomi Y, Ueno N, Kojima T, Sato H, Murase R, Yamamoto K, Tanaka S, Sakanaka M, Nakamura M, Nishito Y, Kawana M, Kambe N, Ikeda K, Taguchi R, Nakamizo S, Kabashima K, Gelb MH, Arita M, Yokomizo T, Nakamura M, Watanabe K, Hirai H, Nakamura M, Okayama Y, Ra C, Aritake K, Urade Y, Morimoto K, Sugimoto Y, Shimizu T, Narumiya S, Hara S, Murakami M. Mast cell maturation is driven via a group III phospholipase A2-prostaglandin D2-DP1 receptor paracrine axis. Nat Immunol. 2013 Jun;14(6):554-563.
Primary, PRUN, human group III secreted phospholipase A2, HGIII-sPLA2, PLA2, Phospholipase A2, HGIII-SPLA2, BODIPY substrate, QFRET, BODIPY PC-A2, FRET, BODIPY FL pentanoic acid, biochemical, fluorescence, inhibition, modulators, HTS, high throughput screen, 1536, Scripps Florida, The Scripps Research Institute Molecular Screening Center, SRIMSC inhibitor, fluorescence, HTS, Molecular Libraries Probe Production Centers Network, MLPCN
The purpose of this biochemical assay is to identify compounds that act as inhibitors of human group III secreted phospholipase A2 enzyme (HGIII-sPLA2). In this assay, HGIII-sPLA2 enzyme is pre-incubated with test compounds, followed by incubation with the BODIPY PC-A2 substrate in a suspension of DOPC:DOPG liposomes. Fluorescence is determined at at specific time point. The cleavage of the BODIPY FL pentanoic acid substituent at the sn-2 position is a PLA2-dependent increase in BODIPY FL fluorescence emission detected in the range 515?545nm. As designed, test compounds that act as HGIII-sPLA2 inhibitors will prevent the cleavage of the BODIPY FL pentanoic acid at the sn-2 position, resulting in a decrease in well fluorescence. Compounds are tested in singlicate at a nominal test concentration of 12.2 micromolar.
Prior to the start of the assay, 2 microliters of assay buffer (50mM Tris-HCl pH 8.9, 100mM NaCl and 1mM CaCl2) were dispensed into columns 1 thru column 3 of 1536 microtiter plates. Next, 2 microliters of assay buffer containing 2.63ug/mL of HGIII-sPLA2 enzyme were dispensed into columns 4 thru 48. Then, 37 nL of test compound in DMSO or DMSO alone (1.2% final concentration) were added to the appropriate wells and incubated for 15 minutes at 25 degrees Celsius. The assay start by the addition of 1 microliter of 9 uM BODIPY PC-A2 substrate incorporated in liposomes containing 45 uM DOPC and 45 uM DOPG in assay buffer to all wells. Plates were centrifuged and after 35 minutes of incubation at 25 degrees Celsius, fluorescence was measured at 485nm excitation and 535nm emission.
The percent inhibition for each compound was calculated as follows:
%Inhibition= 100*( ( Test_Compound - Median_Low_Control ) / ( Median_High_Control - Median_Low_Control ) )
Test_Compound is defined as wells containing HGIII-SPLA2 enzyme in the presence test compound and BODIPY PC-A2 substrate.
Low_Control is defined as the median of the wells containing test compounds.
High_Control is defined as wells containing DMSO, BODIPY PC-A2 but, no HGIII-SPLA2 enzyme.
PubChem Activity Outcome and Score:
A mathematical algorithm was used to determine nominally inhibiting compounds in the primary screen. Four values were calculated: (1) the average percent inhibition of all high controls tested plus three times the standard deviation of the high controls, (2) the average percent inhibition of all low controls tested minus three times the standard deviation of the low controls, (3) the average percent inhibition of all compounds tested between (1) and (2), and (4) three times their standard deviation. The sum of two of these values, (3) and (4), was used as a cutoff parameter, i.e. any compound that exhibited greater % inhibition/activity 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 PubChem Activity Score range for active compounds is 100-20, and for inactive compounds 20-0.
List of Reagents:
HGIII-sPLA2 enzyme (supplied by Assay Provider)
Red/Green BODIPY#PC-A2 (Life Technologies, part A10072)
Dioleoylphosphatidylcholine (DOPC) (Avanti Polar Lipids, part 850375)
Dioleoylphosphatidylglycerol (DOPG) (Avanti Polar Lipids, part 840475)
Tris (Amresco, part 0497)
NaCl (Fisher, part S640)
CaCl2#2H20 (calcium chloride dihydrate) (Fisher, part C79)
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 non-specifically modulate luciferase activity, and compounds that quench or emit luminescence within the well. All test compound concentrations reported are nominal; the specific concentration for a particular test compound may vary based upon the actual sample provided by the MLSMR.
** Test Concentration.
Data Table (Concise)