No abstract

The Group IVA cytosolic phospholipase A2 (GIVA cPLA2) is a key provider of substrates for the production of eicosanoids and platelet-activating factor. We explored the structure-activity relationship of 2-oxoamide-based compounds and GIVA cPLA2 inhibition. The most potent inhibitors are derived from delta- and gamma-amino acid-based 2-oxoamides. The optimal side-chain moiety is a short nonpolar aliphatic chain. All of the newly developed 2-oxoamides as well as those previously described have now been tested with the human Group V secreted PLA2 (GV sPLA2) and the human Group VIA calcium-independent PLA2 (GVIA iPLA2). Only one 2-oxoamide compound had appreciable inhibition of GV sPLA2, and none of the potent GIVA cPLA2 inhibitors inhibited either GV sPLA2 or GVIA iPLA2. Two of these specific GIVA cPLA2 inhibitors were also found to have potent therapeutic effects in animal models of pain and inflammation at dosages well below the control nonsteroidal anti-inflammatory drugs.

Group VIA calcium-independent phospholipase A2 (GVIA iPLA2) has recently emerged as a novel pharmaceutical target. We have now explored the structure-activity relationship between fluoroketones and GVIA iPLA2 inhibition. The presence of a naphthyl group proved to be of paramount importance. 1,1,1-Trifluoro-6-(naphthalen-2-yl)hexan-2-one (FKGK18) is the most potent inhibitor of GVIA iPLA2 (XI(50) 0.0002) ever reported. Being 195 and >455 times more potent for GVIA iPLA2 than for GIVA cPLA2 and GV sPLA2, respectively, makes it a valuable tool to explore the role of GVIA iPLA2 in cells and in vivo models. 1,1,1,2,2,3,3-Heptafluoro-8-(naphthalene-2-yl) octan-4-one inhibited GVIA iPLA2 with a XI(50) value of 0.001, while inhibiting the other intracellular GIVA cPLA2 and GV sPLA2 at least 90-times less potently. Hexa- and octa-fluoro ketones were also found to be potent inhibitors of GVIA iPLA2; however they are not selective.

Phospholipase A2s generate lipid mediators that constitute an important component of the integrated response of macrophages to stimuli of the innate immune response. Because these cells contain multiple phospholipase A2 forms, the challenge is to elucidate the roles that each of these forms plays in regulating normal cellular processes and in disease pathogenesis. A major issue is to precisely determine the phospholipid substrates that these enzymes use for generating lipid mediators. There is compelling evidence that group IVA cytosolic phospholipase A2 (cPLA2α) targets arachidonic acid–containing phospholipids but the role of the other cytosolic enzyme present in macrophages, the Ca2+-independent group VIA phospholipase A2 (iPLA2β) has not been clearly defined. We applied mass spectrometry–based lipid profiling to study the substrate specificities of these two enzymes during inflammatory activation of macrophages with zymosan. Using selective inhibitors, we find that, contrary to cPLA2α, iPLA2β spares arachidonate-containing phospholipids and hydrolyzes only those that do not contain arachidonate. Analyses of the lysophospholipids generated during activation reveal that one of the major species produced, palmitoyl-glycerophosphocholine, is generated by iPLA2β, with minimal or no involvement of cPLA2α. The other major species produced, stearoyl-glycerophosphocholine, is generated primarily by cPLA2α. Collectively, these findings suggest that cPLA2α and iPLA2β act on different phospholipids during zymosan stimulation of macrophages and that iPLA2β shows a hitherto unrecognized preference for choline phospholipids containing palmitic acid at the sn-1 position that could be exploited for the design of selective inhibitors of this enzyme with therapeutic potential.

The phospholipase A(2) (PLA(2)) superfamily hydrolyzes phospholipids to release free fatty acids and lysophospholipids, some of which can mediate inflammation and demyelination, hallmarks of the CNS autoimmune disease multiple sclerosis. The expression of two of the intracellular PLA(2)s (cPLA(2) GIVA and iPLA(2) GVIA) and two of the secreted PLA(2)s (sPLA(2) GIIA and sPLA(2) GV) are increased in different stages of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We show using small molecule inhibitors, that cPLA(2) GIVA plays a role in the onset, and iPLA(2) GVIA in the onset and progression of EAE. We also show a potential role for sPLA(2) in the later remission phase. These studies demonstrate that selective inhibition of iPLA(2) can ameliorate disease progression when treatment is started before or after the onset of symptoms. The effects of these inhibitors on lesion burden, chemokine and cytokine expression as well as on the lipid profile provide insights into their potential modes of action. iPLA(2) is also expressed by macrophages and other immune cells in multiple sclerosis lesions. Our results therefore suggest that iPLA(2) might be an excellent target to block for the treatment of CNS autoimmune diseases, such as multiple sclerosis.

Although the role of each PLA(2) is not yet distinct in different diseases, the development and future use of different PLA(2) inhibitors to treat human disease seems very promising.

Autoimmune β-cell death leads to type 1 diabetes, and with findings that Ca2+-independent phospholipase A2β (iPLA2β) activation contributes to β-cell death, we assessed the effects of iPLA2β inhibition on diabetes development. Administration of FKGK18, a reversible iPLA2β inhibitor, to NOD female mice significantly reduced diabetes incidence in association with 1) reduced insulitis, reflected by reductions in CD4+ T cells and B cells; 2) improved glucose homeostasis; 3) higher circulating insulin; and 4) β-cell preservation. Furthermore, FKGK18 inhibited production of tumor necrosis factor-α (TNF-α) from CD4+ T cells and antibodies from B cells, suggesting modulation of immune cell responses by iPLA2β-derived products. Consistent with this, 1) adoptive transfer of diabetes by CD4+ T cells to immunodeficient and diabetes-resistant NOD.scid mice was mitigated by FKGK18 pretreatment and 2) TNF-α production from CD4+ T cells was reduced by inhibitors of cyclooxygenase and 12-lipoxygenase, which metabolize arachidonic acid to generate bioactive inflammatory eicosanoids. However, adoptive transfer of diabetes was not prevented when mice were administered FKGK18-pretreated T cells or when FKGK18 administration was initiated with T-cell transfer. The present observations suggest that iPLA2β-derived lipid signals modulate immune cell responses, raising the possibility that early inhibition of iPLA2β may be beneficial in ameliorating autoimmune destruction of β-cells and mitigating type 1 diabetes development.

The development of selective inhibitors for individual PLA 2 enzymes is necessary in order to target PLA 2 -specific signaling pathways; but it is challenging due to the observed promiscuity of known PLA 2 inhibitors. In the current work, we present the development and application of a variety of synthetic routes to produce pentafluoro, tetrafluoro and trifluoro derivatives of activated carbonyl groups in order to screen for selective inhibitors and characterize the chemical properties that can lead to selective inhibition. Our results demonstrate that the pentafluoroethyl ketone functionality favors selective inhibition of the GVIA iPLA 2 , a very important enzyme for which specific, potent reversible inhibitors are needed. We find that 1,1,1,2,2-pentafluoro-7-phenyl-heptan-3-one (FKGK11) is a selective inhibitor of GVIA iPLA 2 (X I (50) = 0.0073). Furthermore, we conclude that the introduction of an additional fluorine atom at the α′ position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA 2 inhibitors.