Elsevier

Biochemical Pharmacology

Volume 59, Issue 7, 1 April 2000, Pages 763-772
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Selectivity 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

https://doi.org/10.1016/S0006-2952(99)00395-0Get rights and content

Abstract

Diacylglycerol kinases (DGKs) attenuate diacylglycerol-induced protein kinase C activation during stimulated phosphatidylinositol turnover. This reaction also initiates phosphatidylinositol resynthesis. Two agents, 3-{2-(4-[bis-(4-fluorophenyl)methylene]-1-piperidinyl)ethyl}-2,3-dihydro-2-thioxo-4(1H)quinazolinone (R59949) and 6-{2-{4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl}ethyl}-7-methyl-5H-thiazolo(3,2-a)pyrimidin-5-one (R59022), inhibit diacylglycerol phosphorylation in several systems. To examine the mechanism of this effect, we developed a mixed micelle method suitable for in vitro study of DGK inhibition. Animal cells express multiple DGK isoforms. In a survey of DGK isotypes, these agents selectively inhibited Ca2+-activated DGKs. R59949 was the more selective of the two. To map the site of interaction with the enzyme, a series of DGKα deletion mutants were prepared and examined. Deletion of the Ca2+-binding EF hand motif, which is shared by Ca2+-activated DGKs, had no effect on inhibition. Consistent with this observation, inhibition kinetics were noncompetitive with Ca2+. A construct expressing only the catalytic domain was also inhibited by R59949. Studies of substrate kinetics demonstrated that MgATP potentiated R59949 inhibition, indicating synergy of inhibitor and MgATP binding. These results indicate that R59949 inhibits DGKα by binding to its catalytic domain.

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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