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BioAssay: AID 588854

Summary AID for PLD inhibitor development project

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 ..
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AID: 588854
Data Source: Vanderbilt Specialized Chemistry Center (PLD1_PLD2_Project_Summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-12-06
Modify Date: 2012-02-29

Data Table ( Complete ):           Active    All
BioActive Compounds: Chemical Probe: 3    Active: 3
Depositor Specified Assays
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602144Cellular PLD1 isoenzyme activity assays at set concentrationscreeningCellular PLD1 isoenzyme activity assays at set concentration
602143Cellular PLD2 isoenzyme activity assays at a set concentrationscreeningCellular PLD2 isoenzyme activity assays at a set concentration
602151Cellular PLD1 concentration responseconfirmatoryCellular PLD1 concentration response
602147Cellular PLD2 concentration responseconfirmatoryCellular PLD2 concentration response
602150PLD1 purified enzyme concentration responseconfirmatoryPLD1 purified enzyme concentration response
602149PLD2 purified enzyme concentration responseconfirmatoryPLD2 purified enzyme concentration response
602153ML270, ML271, ML272 Competition in Radioligand Binding assays (Ricerca)otherML270, ML271, ML272 Competition in Radioligand Binding assays (Ricerca)
602175Developing isoenzyme selective inhibitors of phospholipase D as an antiviral therapeutic - Secondary Assay PanelotherPLD_Panel_01
623933ML298, ML299 Competition in Radioligand Binding assays (Ricerca)other
624000PLD2 purified enzyme concentration response (PLD2_Assay_3B_Lib32)confirmatory
624001PLD1 purified enzyme concentration response (PLD1_Assay_3A_Lib32)confirmatory
624003PLD1 Cellular concentration response (PLD1_Assay_2A_Lib32)confirmatory
624004PLD2 Cellular concentration response (PLD2_Assay_2B_Lib32)confirmatory
624008PLD1 Cellular isoenzyme activity (PLD1_Assay_1A_Lib32)screening
624009PLD2 Cellular isoenzyme activity (PLD2_Assay_1B_Lib32)screening
743388On Hold
743389On Hold
743390On Hold
743391On Hold
743400On Hold
743409On Hold
743437On Hold
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.

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. A concentration response curve is generated using GraphPad Prism 4 software and IC50 values are obtained from the non-linear curve fitting (sigmoidal dose reponse) of the data.

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.
Result Definitions
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50_PLD1_uMPotency for inhibition of PLD1 in Exogenous protein-based assayFloatμM
2IC50_PLD2_uMPotency for inhibition of PLD2 in Exogenous protein-based assayFloatμM
3IC50_PLD1_Cell_uMPotency for inhibition of PLD1 in cell-based assayFloatμM
4IC50_PLD2_Cell_uMPotency for inhibition of PLD2 in cell-based assayFloatμM
Additional Information
Grant Number: P01 ES013125

Data Table (Concise)