Molecular and Cellular PharmacologySelectivity of the diacylglycerol kinase inhibitor 3-{2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl}-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) among diacylglycerol kinase subtypes
Section snippets
Materials
[γ-32P]ATP was from DuPont New England Nuclear. Octyl-β-d-glucopyranoside (octylglucoside), sodium deoxycholate, PMSF, Triton X-100, Triton X-114, leupeptin, aprotinin, DTPA, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (C16 sulfobetaine, C16SB), EDTA, EGTA, dihexadecyl phosphate, and sorbitan trioleate were from the Sigma Chemical Co. BHT was from the Aldrich Chemical Co. sn-1-Palmitoyl-2-oleoyl phosphatidylserine (PS) was from Avanti Polar Lipids. sn-1-Palmitoyl-2-oleoylglycerol
Selectivity of R59949 for Ca2+-activated DGKs
R59022 and R59949 (Fig. 1) were initially described as inhibitors of DAG phosphorylation in intact cells 12, 13. However, in vitro inhibition of DGK activities has not been observed uniformly. One potential difficulty with such measurements is complexation of cationic moieties on these compounds with anionic lipid cofactors employed in DGK assays. We have also noted these agents to be poorly soluble in octylglucoside and deoxycholate, which are commonly used to disperse the DAG substrate.
Discussion
This work demonstrates that R59949 and R59022 interact directly with DGKs. A few studies have examined highly purified DGK preparations for in vitro inhibition by these agents. R59022 inhibited DGKα assayed in the presence of PS or sphingosine [21]. In this same study, another DGK prepared from thymus was not inhibited. In a Triton micelle assay, neither R59022 nor R59949 inhibited an arachidonoyl DAG-specific DGK from testis [22]. R59022 inhibited three platelet DGK activities from 20 to 80%
Acknowledgements
This work was performed while Dr. James P. Walsh was a Pfizer Scholar. Additional support was provided by a grant from the US Veterans Health Administration Medical Research Service to Dr. James P. Walsh. We thank Drs. Tom Kovala and Anna Depaoli-Roach for assistance with transient expression of DGKs in COS-1 cells. We also thank Dr. Kaoru Goto for providing DGKζ cDNA.
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