PLD

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Introduction

Phospholipase D (PLD; KEGG enzyme committee number 3.1.4.4) is a phosphodiesterase, encoded by a superfamily of genes, and plays a key role in a variety of signal transduction and metabolic pathways. These enzymes are responsible for catalyzing the removal of head groups in glycerophospholipids to generate phosphatidic acid (PtdOH) for the reaction, and the result of the reaction is the stoichiometric release of the free head groups. One of the four subgroups of PLD enzymes is characterized by the conserved H-X-K-X₄-D-X₆-G-(G/S) catalytic motif, called the HKD motif. Members of this subgroup can use a similar reaction mechanism to hydrolyze the phosphodiester bond through the HKD-catalyzed motif. In addition, some PLD enzymes that lack HKD motif can also produce PtdOH.

In mammalian cells, the HKD-containing isoenzymes PLD1 and PLD2 are almost everywhere, and they often act as nodes at the confluence of signal pathways. They are involved in many cellular processes that require membrane remodeling or biogenesis, including vesicle transport, endocytosis, degranulation, and cell cycle progression. The substrate of PLD1 and PLD2 is usually phosphatidylcholine, but it can also hydrolyze other amine-containing glycerophospholipids, including phosphatidylethanolamine, phosphatidylserine. Many HKD motif-containing PLD enzymes are also responsible for catalyzing the alternative reaction of hydrolysis (transphosphatidylation), in which short-chain primary alcohols compete with water as nucleophiles to produce a phosphatidyl alcohol product.

Figure 1. Recent findings have implicated phospholipase D (PLD) enzymes as therapeutic targets in a variety of human diseases (Brown, H.A.; et al.2017)

PLD isoenzyme inhibitors

Until 2009, there were few chemical tools that could help us directly study and clarify the functions of PLD, and no small molecules could be used in the research of the two main mammalian PLD isoenzymes (PLD1 and PLD2) or other treatment-related PLDs. Research in this field almost relies on overexpression of the catalytically active or inactive forms of the enzymes, or the use of RNA-mediated interference (RNAi) to block their expression. However, these biochemical and genetic methods have hardly attracted the attention of the pharmaceutical industry. In order to develop PLD inhibition as a viable treatment, selective small molecules are needed for target verification. Although early efforts did identify certain small molecules that can modulate PLD function, these ligands ultimately failed to accelerate the development of this field, partly because of their apparent lack of specificity. Recently, the most commonly used class of molecules to study the function of PLD enzymes are primary alcohols. As mentioned above, alcohols such as n-butanol block the production of PtdOH by competing with water as a nucleophile. However, under experimental conditions, the blockade of PtdOH production is usually incomplete. Therefore, caution must be exercised when interpreting research data using primary alcohols (such as n-butanol).

Looking ahead and moving forward

Lipid signaling metabolite intermediates provide potential targets for the therapeutic intervention of many diseases such as cancer, infectious diseases, and central nervous system disorders. Due to the rapid flux of these lipids, the accumulation of PtdOH may be instantaneous because PtdOH is converted by enzymes into DAG, lysophosphatidic acid (lysoPtdOH), and constituents of the Kennedy pathway (see Figure 2). In addition, although the roles of PLD enzymes in cancer, infectious and neurodegenerative diseases are complex and multifaceted, it is easy to realize that this reflects the common role of intermediate metabolism in each of these diseases.

A key issue at present is that PtdOH mainly plays a biophysical role, or affects nuclear transcriptional events or other innate immune processes that are also exploited by bacteria and parasites. The structural role played of lipids in the viral envelope still needs further examination. The tdOH produced via de novo synthesis in the Kennedy pathway, PLD pathway and PLC-DGK pathway requires further experimental research in other pathogenic microorganisms. These results will support whether inhibitors of these pathways can be used as broad-spectrum anti-infectives, which is crucial now that drug-resistant strains continue to emerge.

Figure 2. 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 (Brown, H.A.; et al.2017)