REVIEWS Targeting phospholipase D in cancer, infection and neurodegenerative disorders H. Alex Brown1–4, Paul G. Thomas5 and Craig W. Lindsley1–3,6 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. Phospholipase D Phospholipase D (PLD; KEGG enzyme commission converge. They are known to participate in cellular func- (PLD). A class of number 3.1.4.4) enzymes are phosphodiesterases that tions that require membrane remodelling or biogenesis, phosphodiesterases that serve as key components of multiple signalling and such as vesicular transport, endocytosis, degranulation hydrolyse phosphatidylcholine metabolic pathways. They are encoded by a super- and cell cycle progression. The substrate for PLD1 and and other amine-containing 1 glycerophospholipids to family of genes and can be defined by several highly PLD2 is typically phosphatidylcholine, but the enzymes generate phosphatidic acid conserved motifs. These enzymes catalyse the removal are also able to hydrolyse other amine-containing and a free head group. of head groups from glycerophospholipids to generate glycerophospholipids, including phosphatidyletha- phosphatidic acid (PtdOH), a reaction that results in the nolamine, phosphatidylserine and, to a lesser extent, stoichiometric release of the free head group1–7. One of phosphatidylglycerol. the four subgroups of PLD enzymes is characterized by Many HKD motif-containing PLD enzymes also a conserved H-X-K-X4-D-X6-G-(G/S) catalytic motif catalyse an alternative reaction to hydrolysis (that is, that is commonly known as an HKD motif. Members of transphosphatidylation), in which short-chain primary this subgroup hydrolyse phosphodiester bonds via the alcohols compete with water as a nucleophile, gener- HKD catalytic motif using a generally similar reaction ating a phosphatidyl alcohol product, such as phos- mechanism; however, some family members also exhibit phatidylbutanol (PtdBuOH) or phosphatidylethanol 1Department of lipid hydrolase activity, whereas others do not. In addi- (PtdEtOH). This alcohol-mediated transphosphatidyl- Pharmacology, Vanderbilt (FIG. 1) University School of Medicine, tion, several PLD enzymes that lack HKD motifs have ation reaction uses physiological substrates and 5 Nashville, Tennessee been described that also generate PtdOH . has catalysis rates comparable to those of hydrolysis. In 37232–6600, USA. In mammalian cells, the HKD-containing iso- some cases, the phosphatidyl alcohol products mimic Correspondence to H.A.B. enzymes PLD1 and PLD2, which share highly conserved PtdOH binding to downstream targets, thereby acti- [email protected] phox and pleckstrin homology (PX–PH) domains, are vating some signalling pathways downstream of PLD 5 doi:10.1038/nrd.2016.252 almost ubiquitous . These two isoenzymes frequently enzymes, while blocking others. Erroneously, primary Published online 17 Feb 2017 serve as nodes at points where signalling pathways alcohols have widely been referred to as PLD ‘inhibitors’ NATURE REVIEWS | DRUG DISCOVERY VOLUME 16 | MAY 2017 | 351 ©2017 Mac millan Publishers Li mited, part of Spri nger Nature. All ri ghts reserved. REVIEWS Author addresses kinase (DGK) signalling. From this perspective, one or both of the isoenzymes may be involved both temporally 1 Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, and spatially in a specific cellular function, but PtdOH Tennessee 37232–6600, USA. depletion can be compensated for if an isoenzyme is inac- 2The Vanderbilt Institute of Chemical Biology, 896 Preston Building, Vanderbilt University, tivated. If the causal misregulation step is due to a signal- Nashville, Tennessee 37232–6304, USA. 3The Vanderbilt Ingram Cancer Center, 691 Preston Building, Vanderbilt University, ling pathway that modulates the specific PLD isoenzyme Nashville, Tennessee 37232–6838, USA. in a physiological circuit, then one of the parallel PtdOH- 4Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, generating pathways may not be similarly affected. As Tennessee 37232–6600, USA. an example, purinergic receptors can induce PtdOH 5Department of Immunology, St. Jude Children’s Research Hospital, Memphis, Tennessee production via both PLD and PLC–DGK signalling in 38105–3678, USA. astrocytomas11. These pathways are interconnected and 6Vanderbilt Center for Neuroscience Drug Discovery and Department of Chemistry, seem to constitutively cross-regulate one another in the Vanderbilt University, Nashville, Tennessee 37232, USA. production of primary PtdOH lipid products as well as that of downstream products such as diacylglycerol in publications, and it is likely that some roles previously (DAG). When one pathway is blocked the other com- ascribed to PLD enzymes in studies that used alcohols as pensates, thereby ensuring the continued production of ‘inhibitors’ are really attributable to nonspecific effects essential lipid signalling products. However, the resulting and should be re-examined2. Details of the sequence molecular species differ in acyl composition depending homology among members of the PLD superfamily, and on whether they were generated via the PLD pathway or the enzymology, signalling and functions of respective the PLC–DGK pathway. In some cases, acyl composition PLD proteins, have been reviewed previously 3–6. may be important for the activation of a specific target. Recently, theoretical work was presented that For example, a recent report showed that only unsatu- describes the possible mechanisms underlying the cat- rated fatty acids containing PtdOH dissociate the dishev- alytic activity of HKD motif-containing PLD enzymes elled, EGL‑10 and pleckstrin (DEP) domain-containing using computational methods and models that are mTOR-interacting protein (DEPTOR) from mechanistic based on reaction kinetics, thermodynamics and quan- target of rapamycin complex 1 (mTORC1)12. This find- titative insights from studies of the Streptomyces spp. ing extends previous suggestions that PLD enzymes may 7 strain PMF PLD enzyme (PLDPMF) . The mechanism have a primary role in the regulation of mTORC1 and of catalytic activity includes the following steps: first, that other enzymatic sources of PtdOH have a secondary the formation of a five-coordinate phosphohistidine role. By extension, if an individual isoform of PLD is dys- intermediate and initial phosphoryl transfer during functional and thus contributes to a disease process, then which the head group is cleaved; second, the hydrolysis it is likely that isoform-specific small-molecule inhibi- of the phosphohistidine intermediate and bond disso- tors would have less adverse consequences than would ciation of the hydrolysed substrate; and third, the for- pan-reactive inhibitors. mation of a thermodynamically stable four-coordinate Interest in targeting PLD isoenzymes with small-­ 7 Phosphatidic acid phosphohistidine intermediate . These individual steps molecule inhibitors has steadily grown since PLD (PtdOH). A molecule that are highly conserved among enzymes that contain the family members were implicated in a variety of human functions as both a lipid HKD motif, which supports speculation that the large diseases, ranging from cancer to neurodegeneration and second messenger and a key number of highly diverse PLD enzymes evolved as a con- viral infections. Moreover, viruses such as human cyto­ intermediate in glycerophos- sequence of differences in the mechanism of regulation megalovirus (HCMV)13, influenza virus14 and HIV‑1 pholipid metabolism. The (REF. 15) phospholipase D pathway is by constituents of distinct cell signalling and metabolic have been shown to use PtdOH in various one pathway that is used to pathways to fulfil a variety of cellular functions for which aspects of cellular entry, intracellular trafficking and the generate receptor-mediated PtdOH generation is vital. disruption of innate immune responses. PtdOH. A major pathway of Despite their essential roles in cellular function, Understanding the mechanism of action of PtdOH metabolism is conversion to diacylglycerol by it is curious that one or more PLD isoenzymes can be the HKD motif-containing