Targeting phospholipase D in cancer, infection and neurodegenerative disorders

Abstract

Lipid second messengers have essential roles in cellular function and contribute to the molecular mechanisms that underlie inflammation, malignant transformation, invasiveness, neurodegenerative disorders, and infectious and other pathophysiological processes. The phospholipase D (PLD) isoenzymes PLD1 and PLD2 are one of the major sources of signal-activated phosphatidic acid (PtdOH) generation downstream of a variety of cell-surface receptors, including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and integrins. Recent advances in the development of isoenzyme-selective PLD inhibitors and in molecular genetics have suggested that PLD isoenzymes in mammalian cells and pathogenic organisms may be valuable targets for the treatment of several human diseases. Isoenzyme-selective inhibitors have revealed complex inter-relationships between PtdOH biosynthetic pathways and the role of PtdOH in pathophysiology. PLD enzymes were once thought to be undruggable owing to the ubiquitous nature of PtdOH in cell signalling and concerns that inhibitors would be too toxic for use in humans. However, recent promising discoveries suggest that small-molecule isoenzyme-selective inhibitors may provide novel compounds for a unique approach to the treatment of cancers, neurodegenerative disorders and other afflictions of the central nervous system, and potentially serve as broad-spectrum antiviral and antimicrobial therapeutics.

Figure 1. Phospholipase D enzymes as therapeutic targets and their mechanism of action.

a | Recent findings have implicated phospholipase D (PLD) enzymes as therapeutic targets in a variety of human diseases. b | Most PLD enzymes mediate both a hydrolysis reaction that generates phosphatidic acid (PtdOH) directly and a transphosphatidylation reaction in which primary alcohols serve as alternative substrates for the generation of a phosphatidyl alcohol lipid product. Allosteric small-molecule inhibitors block both reactions. PtdOH is metabolized to diacylglycerol (DAG) by lipid phosphate phosphatase (LPP) enzymes. PtdOH species are also generated downstream of PLC enzymes, which directly produce DAG; subsequent phosphorylation of DAG by DAG kinases (DGKs) generates PtdOH. The mechanism of transphosphatidylation has been reviewed in detail elsewhere. BuOH, butanol; PtdBuOH, phosphatidylbutanol. *denotes long-chain fatty acid residues. PowerPoint slide

Figure 2. Metabolic pathways that lead to the generation of phosphatidic acid.

Metabolic pathways that lead to the generation of phosphatidic acid (PtdOH) include de novo biosynthesis as well as the phospholipase D (PLD) and PLC–diacylglycerol kinase (DGK)-mediated signalling pathways. The generation of PtdOH seems to be broadly exploited by pathogens as part of the infection process and thus represents a novel therapeutic opportunity; the design of novel small-molecule inhibitors may lead to new treatments for infection. The figure illustrates three routes of cellular PtdOH generation via the indicated enzyme-mediated pathways. ATX, autotaxin; FA, fatty acid; G3P, glycerol-3-phosphate; GK, glycerol kinase; GPAT, glycerol-3-phosphate acyltransferase; LPP, lipid phosphate phosphatase; LPAAT, lyso-PtdOH acyltransferase; LPCAT, lysophosphatidylcholine acyltransferase; MGAT, monoacylglycerol acyltransferase; MGK, monoacylglycerol kinase; MGL, monoacylglycerol lipase; PI, phosphatidylinositol; PIP, PI phosphate; PI(4,5)P₂, PI 4,5-bisphosphate; R1, R2, fatty acyl moieties. PowerPoint slide

Figure 3. Inhibitors of phospholipase D enzymes.

a | Representative early indirect and direct inhibitors of phospholipase D (PLD). b | Second-generation PLD inhibitors that are based on halopemide (providing the first isoenzyme-selective PLD1 inhibitors). IC₅₀, concentration that inhibits PLD activity by 50%; FIPI, 5-fluoro-2 indolyl des-chlorohalopemide. PowerPoint slide

Figure 4. Representative examples of phospholipase D1-preferring inhibitors with a benzimidazolone scaffold.

The phospholipase D 1 (PLD1) IC₅₀ values (concentration that inhibits PLD activity by 50%) were determined in cellular PLD1 assay with Calu-1 cells. The PLD2 IC₅₀ values were determined in a cellular assay with HEK293-GFP PLD2 cells. Each IC₅₀ was determined in triplicate measurements. X denotes H or halogen, and R¹ denotes the aryl- or cyclopropylaryl moieties (as listed in the table). PowerPoint slide

Figure 5. Second-generation phospholipase D2-selective and dual phospholipase D1–phospholipase D2 inhibitors within the triazaspirone series.

VU0364739 (also known as JWJ; compound 12) emerged as an important phospholipase D2 (PLD2)-selective compound. IC₅₀, concentration that inhibits PLD activity by 50%. PowerPoint slide

Figure 6. Third-generation phospholipase D2-selective and dual phospholipase D1–phospholipase D2 inhibitors within the triazaspirone series.

These compounds have a superior ancillary pharmacology profile compared with PLD inhibitors 8-13 (Figs 3,5). IC₅₀, concentration that inhibits PLD activity by 50%; PLD, phospholipase D. PowerPoint slide

Figure 7. Selective oestrogen receptor modulator chemotypes.

17–20 selective oestrogen receptor modulator (SERM) chemotypes have inhibitory activity against mammalian phospholipase D1 (PLD1), PLD2 and Pseudomonas aeruginosa PLD (PldA). IC₅₀, concentration that inhibits PLD activity by 50%. PowerPoint slide

Figure 8. Synthesis of the first universal phospholipase D inhibitor (compound 21).

Chemical optimization of desketoraloxifene (compound 20) delivered compound 21, which is the first universal inhibitor of phylogenetically and structurally diverse PLD enzymes that is devoid of selective oestrogen receptor modulator (SERM) activity. IC₅₀, concentration that inhibits PLD activity by 50%; NAPE-PLD, N-acyl phosphatidylethanolamine-specific phospholipase D. PowerPoint slide

Figure 9. Illustration of lipid involvement in host cell infection with influenza virus.

The phospholipase D2 (PLD2) signalling axis is involved in both the entry and egress of influenza virus, as well as the process of autophagy, which can have antiviral function. PLD activity facilitates viral entry, whereas PLD inhibition delays the kinetics of viral endocytosis, thus widening the window of time in which the host cell can effectively prevent virus replication by producing antiviral effector molecules, such as interferon-induced transmembrane protein 3 (IFITM3) and MxA. Similarly, viral egress appears to be impaired by PLD inhibition. The regulation of AKT activity by PLD2 has also been shown to activate the autophagy pathway, which can limit virus production. PLD-generated phosphatidic acid (PtdOH) recruits AKT to cellular membranes, and AKT subsequently phosphorylates beclin 1 at serine 295. This leads to dissociation of the beclin 1–rubicon complex, a process that promotes autophagy. PLD inhibitors reduce PtdOH concentrations, reduce AKT-mediated phosphorylation of beclin 1 and stabilize the beclin 1–rubicon complex to stall autophagy. The changes depicted in membrane lipid composition are based on previous work that investigated the contributions of PtdOH to membrane curvature^,. PowerPoint slide

Figure 10. PLD isoenzyme pathways with antiviral function.

Left panel: phospholipase D2 (PLD2)-generated phosphatidic acid (PtdOH) recruits and binds to AKT and subsequently promotes autophagy via beclin 1, as detailed in Fig. 9. During the process of autophagy, autophagosomes fuse with endosomes and lysosomes, which leads to their maturation into autolysosomes. These display the autophagy markers LC3 (light chain 3, also known as MAP1LC3) and p62 (also known as sequestosome 1). This process results in acidification and allows the autolysosome to digest captured cellular material such as viruses. The respective details of PLD1 and PLD2 activation are discussed elsewhere. Right panel: this shows a PLD1 pathway that affects pyrimidine biosynthesis and intracellular deoxyribonucleotide triphosphate (dNTP) levels via the enzyme CAD (which contains carbamoyl aspartate synthase, aspartate transcarbamylase and dihydro-orotase domains). PLD1 allosterically modulates the activity of mechanistic target of rapamycin (mTOR), which leads to the phosphorylation of CAD and enhances its catalytic activity. The subsequent production of pyrimidine nucleotides via the activation of CAD is independent of AKT. PLD1 inhibition, in turn, can lead to reductions in dNTP production. PowerPoint slide