PLD2 Cellular isoenzyme activity (PLD2_Assay_1B_Lib32)
Phospholipase D (PLD) generates the lipid second messenger phosphatidic acid as part of a plethora of cellular functions. Phosphatidic acid (PtdOH) is a critical signaling lipid that is generated by the hydrolysis of phosphatidylcholine and other amine containing phospholipids. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction more ..
BioActive Compounds: 9
Phospholipase D (PLD) generates the lipid second messenger phosphatidic acid as part of a plethora of cellular functions. Phosphatidic acid (PtdOH) is a critical signaling lipid that is generated by the hydrolysis of phosphatidylcholine and other amine containing phospholipids. Phosphatidic acid participates in both G protein-coupled receptor and receptor tyrosine kinase signal transduction networks. Two mammalian isoforms of PLD have been identified, PLD1 and PLD2, which share 53% sequence identity and are subject to different regulatory mechanisms (1). Inhibition of PLD enzymatic activity leads to increased cancer cell apoptosis, decreased cancer cell invasion, and decreased metastasis of cancer cells (2,3); therefore, the development of isoform-specific PLD inhibitors is a novel approach for the treatment of cancer.
In addition to the importance of PLD in cancer cell survival it has been shown that this enzyme is the major source of signaling PtdOH in many cells, including neutrophils, macrophages, and airway epithelial cells that play key roles in defense against infection. PLD1 was recently confirmed as a primary regulator of Fc-gamma receptor-stimulated ROS production in neutrophils (4) using a strategic combination of genetic knockouts and pharmacological inhibitors. A recent report in Nature identified PLD2 as a gene that when downregulated drastically reduced viral replication of a panel of influenza strains including avian H5N1, endemic H1N1 and the pandemic swine flu of 2009 (5), although two related reports (6,7) using similar approaches failed to identify PLD isoenzymes as being relevant in influenza. Also because of PLD's role in vesicular trafficking, more specifically the link between PLD activity and influenza virus haemagglutinin (HA) trafficking to the plasma membrane (8), we hypothesize that this enzyme is a critical target to combat influenza infection. Current therapeutics strategies rely on vaccines or molecules targeting viral neuraminidase, both of which are subject to rapid resistance development.
In preliminary findings, PLD1 and PLD2 have been strongly implicated as playing essential roles in the entry of influenza virus into human airway epithelial cells. Given that PLD1-and PLD2-knockout animals are viable and appear to lack any obvious deficits, we hypothesize that PLD is a novel and therapeutically attractive target for blocking influenza infection. We propose to optimize potency, isoenzyme selectivity, and drug metabolism/pharmacokinetic parameters to develop chemical tool compounds into a preclinical lead compound for therapeutic development.
The development of these tool compounds will be essential in understanding the roles played by PLD in modulating cell responses to oxidative stress that is involved in a variety of human diseases including cancer, infectious disease, cardiovascular diseases and other conditions that are consequences of chronic, pathological inflammation. The development of these tool compounds has empowered the PLD signaling field. Still, major challenges remain to develop more potent and selective compounds that differentiate between PLD1 and PLD2 isoenzymes.
As previously described (2) this is accomplished through a set of parallel assays:
The primary screen will be a parallel single dose screen in two different cell lines. To determine if compounds are active against cellular PLD1, phorbol 12-myristate 13-acetate (PMA) stimulated Calu-1 cells will be used. It has previously been shown that under these conditions the PLD activity in these cells is strictly due to the PLD1 isozyme. A parallel PLD2 centric assay is also carried out using HEK293 cells stably overexpressing gfp-PLD2. Data is expressed as percent activity which is calculated from the vehicle control samples present in each assay with background lipid signals subtracted.
1. Selvy, P.E., et al., Phospholipase D: enzymology, functionality, and chemical modulation. Chem Rev, 2011. 111(10): p. 6064-119.
2. Scott, S.A., et al., Design of isoform-selective phospholipase D inhibitors that modulate cancer cell invasiveness. Nat Chem Biol, 2009. 5(2): p. 108-17.
3. Lavieri, R.R., et al., Design, synthesis, and biological evaluation of halogenated N-(2-(4-oxo-1-phenyl-1,3,8-triazaspiro[4.5]decan-8-yl)ethyl)benzamides: discovery of an isoform-selective small molecule phospholipase D2 inhibitor. J Med Chem, 2010. 53(18): p. 6706-19.
4. Norton, L., et al., PLD1, rather than PLD2, regulates phorbol ester-, adhesion dependent-, and Fcgamma receptor stimulated reactive oxygen species production in neutrophils. Journal of cell science, 2011. 124(12): p. 1973-83.
5. Karlas, A., et al., Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication. Nature, 2010. 463(7282): p. 818-22.
6. Shapira, S.D., et al., A physical and regulatory map of host-influenza interactions reveals pathways in H1N1 infection. Cell, 2009. 139(7): p. 1255-67.
7. Brass, A.L., et al., The IFITM proteins mediate cellular resistance to influenza A H1N1 virus, West Nile virus, and dengue virus. Cell, 2009. 139(7): p. 1243-54.
8. Bi, K., M.G. Roth, and N.T. Ktistakis, Phosphatidic acid formation by phospholipase D is required for transport from the endoplasmic reticulum to the Golgi complex. Curr. Biol., 1997. 7(5): p. 301-7.
9. Brown, H.A., et al., Biochemical analysis of phospholipase D. Methods Enzymol., 2007. 434: p. 49-87.
PLD2 cellular screens
HEK293-gfpPLD2a cells are plated at 250,000 cell/well into poly-lysine coated 12-well tissue culture plates in DMEM 10% FBS 1% AA (antibiotic-antimycotic). After 48 hours media is then removed and cells are pretreated with DMEM containing either compound (at 200 nM) or vehicle control for 5 minutes at room temperature. After pretreatment media is changed out and cells are treated with DMEM containing screening compound (200 nM, and 0.3% n-butylalcohol-d10 for 30 min at 37 degrees C.
After incubation, treatment media is removed, plates are placed on ice and to each well 600 uL 0.1N HCl:methanol (1:1) is added. Cells are then dislodged using a cell scraper and transferred to bullet tubes on ice. The cellular lipids are extracted via a modified Bligh-Dyer extraction by addition of 300 uL ice cold chloroform and internal standard (50 ng 32:0 PtdMeOH/sample in chloroform). Samples are then mixed and centrifuged for 5 min at 4 degrees C. The lower layer (organic phase) is then isolated. The organic phase is then evaporated in a CentriVap and resulting lipid film is redisolved in 80 uL 90% Methanol 10% chloroform plus 1 uL NH4OH.
Resulting solution is then directly infused into a Thermofinnigan TSQ Quantum triple quadrupole MS and detected using negative ionization mode. Data is collected over a 1 minute infusion of solution. The PLD2 lipid products, 34:1 phosphatidylbutanol-d9 (m/z 738) and 32:1 phosphatidylbutanol-d9 (m/z 710) is quantitated using standard curves and internal standards. Data is expressed as percent activity which is calculated from the vehicle control samples (=100% activity) present in each assay with background lipid signals subtracted (using no butanol treated samples). Assay is performed once in triplicate.
After initial hits are identified using the above parallel primary screens compounds with >50% inhibition for both isoforms, or compounds which display isoform preferential inhibition are further characterized using cellular concentration response curves in each of the previously described cell types.
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Data Table (Concise)