Phospholipase synthesis

LY311727 is an indole acetic acid based selective inhibitor of human non-pancreatic secretory phospholipase A2 (hnpsPLA2) under development by Lilly as a potential treatment for sepsis. The synthesis of LY311727 involved a Nenitzescu indolization reaction as a key step. The Nenitzescu condensation of quinone 4 with the p-aminoacrylate 39 was carried out in CH3NO2 to provide the desired 5-hydroxylindole 40 in 83% yield. Protection of the 5-hydroxyl moiety in indole 40 was accomplished in H2O under phase transfer conditions in 80% yield. Lithium aluminum hydride mediated reduction of the ester functional group in 41 provided the alcohol 42 in 78% yield. 

There are three groups of eicosanoids that are synthesized from C20 eicosanoic acids derived from the essential fatty acids linoleate and a-linolenate, or directly from dietary arachidonate and eicosapentaenoate (Figure 23-5). Arachidonate, usually derived from the 2 position of phospholipids in the plasma membrane by the action of phospholipase Aj (Figure 24-6)—but also from the diet—is the substrate for the synthesis of the PG2, 1X2 series (prostanoids) by the cyclooxygenase pathway, or the LT4 and LX4 series by the lipoxygenase pathway, with the two pathways competing for the arachidonate substrate (Figure 23-5). 

Therefore, it is currently believed that anandamide is formed from membrane phospholipids (Fig. 4) through a pathway that involves (1) a trans-acylation of the amino group of phosphatidylethanolamine with arachidonate from the sn-1 position of phosphatidylcholine and (2) a D-type phosphodiesterase activity on the resulting A-arachidonylphosphati-dylethanolamide (NAPE). Synthesis of anandamide is presumably regulated at the levels of both enzymes, the A-acyltranferase and the phospholipase D, by stimuli that raise intracellular calcium or by receptors linked with cAMP and PKA. It has been shown that anandamide is formed when neurons are depolarized and, therefore, the intracellular calcium ion levels are elevated (Cadas, 1996). 

Zaikova, T. (2001). Synthesis of fluorogenic substrates for continuous assay of phosphatidylinositol-specific phospholipase C. Bioconjug. Chem. 12, 307-313. 

P2Y receptors that are found on endothelial cells elicit a Ca2+-dependent release of endothelium-dependent relaxing factor (EDRF) and vasodilation. A secondary activation of a Ca2+-sensitive phospholipase A2 increases the synthesis of endothelial prostacyclin, which limits the extent of intravascular platelet aggregation following vascular damage and platelet stimulation. The P2Y-mediated vasodilation opposes a vasoconstriction evoked by P2X receptors located on vascular smooth muscle cells. The latter elicit an endothelial-independent excitation (i.e. constriction). P2Y receptors are also found on adrenal chromaffin cells and platelets, where they modulate catecholamine release and aggregation respectively. 

Topical corticosteroids (Table 16-1) may halt synthesis and mitosis of DNA in epidermal cells and appear to inhibit phospholipase A, lowering the amounts of arachidonic acid, prostaglandins, and leukotrienes in the skin. These effects, coupled with local vasoconstriction, reduce erythema, pruritus, and scaling. As antipsoriatic agents, they are best used adjunc-tively with a product that specifically functions to normalize epidermal hyperproliferation. 

Figure 1 The MAPK pathway and its connections to other signals A negative feedback loop connects the phosphorylated endpoint of the pathway ERK (Extracellular-signal Regulated Kinase) to the transcriptionally-driven synthesis of the phosphatase, MKP MAP kinase phosphatase. MKP then de-phosphorylates ERK to shut down the signaling cascade. The positive feedback loop again starts with the terminal kinase ERK which activates cPLA2 (cytosolic phospholipase A2). This leads to the synthesis of arachidonic acid, which, in turn activates protein kinase C (PKC). PKC is a positive regulator of RAS (Please see Color Plate Section in the back of this book).

Anandamide is believed to be synthesized from a phospholipid precursor, /V-arachidonoyl-phosphatidylethanolamine, catalysed by phospholipase D (Di Marzo et al. 1998). The other proposed route of synthesis is from condensation of arachidonic acid and ethanolamine, although this has yet to be demonstrated in living cells. 2-AG is formed in a calcium-dependent manner, and mediated by the enzymes phospholipase C and diacylglycerol lipase (Kondo et al. 1998 Stella et al. 1997). 

In summary, the release of nenrotransmitter from a presynaptic neurone into a synaptic cleft occurs via the process of exocytosis, which is regulated by the increase in Ca " ion concentration in the presynaptic terminal. The increase in Ca " ion concentration is achieved by release of Ca " ions by opening of the Ca ion channel in the endoplasmic reticulum, which is controlled by the concentration of IP3. Failure to release inositol from the inositol phosphates reduces the free inositol concentration, which interferes in the synthesis of PIP2. The phospholipase no longer catalyses a zero order reaction. Consequently, sufficient IP3 to activate the ion channel is released in the presynaptic neurone, so that less nenrotransmitter is released into the synaptic cleft (Figure 12.19). 

The cells in the hypothalamus that control body temperature respond to the cytokines by stimulating the activity of the membrane bound phospholipase, which results in the formation of arachidonic acid, the substrate for the enzyme cyclooxygenase-2 (COX-2) which is the rate-limiting step in the pathway for synthesis of prostaglandins. Prostaglandins influence cells in the hypothalamus that are responsible for temperature regulation. 

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