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Synthesis of polyfluoro ketones for selective inhibition of human phospholipase A2 enzymes.

ABSTRACT: 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 alpha' position of a trifluoromethyl ketone constitutes an important strategy for the development of new potent GVIA iPLA(2) inhibitors.

SUBMITTER: Baskakis C

PROVIDER: S-EPMC2649009 | biostudies-literature | 2008 Dec

REPOSITORIES: biostudies-literature

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Synthesis of polyfluoro ketones for selective inhibition of human phospholipase A2 enzymes.

Baskakis Constantinos C Magrioti Victoria V Cotton Naomi N Stephens Daren D Constantinou-Kokotou Violetta V Dennis Edward A EA Kokotos George G

Journal of medicinal chemistry 20081201 24

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 c ...[more]

PMID: 19053783

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Project description:BACKGROUND:Phospholipase A2s mediate the rate-limiting step in the formation of eicosanoids such as cysteinyl leukotrienes (CysLTs). Group IVA cytosolic PLA2? (cPLA2?) is thought to be the dominant PLA2 in eosinophils; however, eosinophils also have secreted PLA2 (sPLA2) activity that has not been fully defined. OBJECTIVES:To examine the expression of sPLA2 group X (sPLA2-X) in eosinophils, the participation of sPLA2-X in the formation of CysLTs, and the mechanism by which sPLA2-X initiates the synthesis of CysLTs in eosinophils. METHODS:Peripheral blood eosinophils were obtained from volunteers with asthma and/or allergy. A rabbit polyclonal anti-sPLA2-X antibody identified sPLA2-X by Western blot. We used confocal microscopy to colocalize the sPLA2-X to intracellular structures. An inhibitor of sPLA2-X (ROC-0929) that does not inhibit other mammalian sPLA2s, as well as inhibitors of the mitogen-activated kinase cascade (MAPK) and cPLA2?, was used to examine the mechanism of N-formyl-methionyl-leucyl-phenylalanine (fMLP)-mediated formation of CysLT. RESULTS:Eosinophils express the mammalian sPLA2-X gene (PLA2G10). The sPLA2-X protein is located in the endoplasmic reticulum, golgi, and granules of eosinophils and moves to the granules and lipid bodies during fMLP-mediated activation. Selective sPLA2-X inhibition attenuated the fMLP-mediated release of arachidonic acid and CysLT formation by eosinophils. Inhibitors of p38, extracellular-signal-regulated kinases 1/2 (p44/42 MAPK), c-Jun N-terminal kinase, and cPLA2? also attenuated the fMLP-mediated formation of CysLT. The sPLA2-X inhibitor reduced the phosphorylation of p38 and extracellular-signal-regulated kinases 1/2 (p44/42 MAPK) as well as cPLA2? during cellular activation, indicating that sPLA2-X is involved in activating the MAPK cascade leading to the formation of CysLT via cPLA2?. We further demonstrate that sPLA2-X is activated before secretion from the cell during activation. Short-term priming with IL-13 and TNF/IL-1? increased the expression of PLA2G10 by eosinophils. CONCLUSIONS:These results demonstrate that sPLA2-X plays a significant role in the formation of CysLTs by human eosinophils. The predominant role of the enzyme is the regulation of MAPK activation that leads to the phosphorylation of cPLA2?. The sPLA2-X protein is regulated by proteolytic cleavage, suggesting that an inflammatory environment may promote the formation of CysLTs through this mechanism. These results have important implications for the treatment of eosinophilic disorders such as asthma.

Project description:The long-standing proposal that phospholipase A2 (PLA2) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA2 activity from the pathogens Rickettsia prowazekii and R. typhi. However, pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 Rickettsia genomes revealed 1) pat1 and pat2 loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) pat2 has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous pat1 forms two divergent groups (pat1A and pat1B) with strong evidence for recombination between pat1B and plasmid-encoded homologs. In light of these findings, we extended the characterization of R. typhi Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during R. typhi infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA2 activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in R. typhi infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in R. typhi infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of R. typhi blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that R. typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.

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Synthesis of polyfluoro ketones for selective inhibition of human phospholipase A2 enzymes.

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Synthesis of polyfluoro ketones for selective inhibition of human phospholipase A2 enzymes.

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