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First published on April 2, 2007; DOI: 10.1124/dmd.106.014498

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Received for publication December 26, 2006.
Revised March 27, 2007.
Accepted for publication March 28, 2007.

The phosphoinositide-specific phospholipase C inhibitor U73122 spontaneously forms conjugates with common components of cell culture medium

Nicola E Wilsher 1*, Will J Court 1, Ruth Ruddle 1, Yvette M Newbatt 1, Wynne Aherne 1, Peter W Sheldrake 1, Neil P Jones 1, Matilda Katan 1, Suzanne A Eccles 1, Florence I Raynaud 1

1 Institute of Cancer Research

* Address correspondence to: E-mail: nicola.wilsher{at}icr.ac.uk

Abstract

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca2+ release from inositol 1,4,5-triphosphate sensitive stores. U73122 (1-(6-((17b-3-methoxyestr-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) has been extensively used as a pharmacological inhibitor of PLC to elucidate the importance of this enzyme family in signal transduction pathways. U73122 has an electrophilic maleimide group, which readily reacts with nucleophiles such as thiols and amines. In the current study the conjugation of U73122 to common components of cell culture medium, namely L-glutamine, glutathione and bovine serum albumin (BSA) was demonstrated. The half-life of U73122 on incubation with phosphate buffered saline (PBS), Hank's buffered saline solution (with 2mM glutamine), optimised basal nutrient medium (MCDB131, without BSA), complete medium and Dulbecco's Modified Eagle's Medium (with 2mM L-glutamine) was ~150, 60, 32, 30 and 18 minutes respectively. However, U73122 was not recoverable from medium supplemented with 0.5% BSA. U73122 underwent hydrolysis of the maleimide group when incubated with PBS. Glutamine conjugates of U73122 were identified in cell culture medium. Furthermore, the inhibition of epidermal growth factor stimulated Ca2+ release in a human epidermoid carcinoma cell-line (A431) by U73122 was substantially reduced by the presence of BSA in a time-dependent manner. In complex cellular assays, the availability of U73122 to inhibit PLC may be limited by its chemical reactivity and lead to the misinterpretation of results in pharmacological assays.


Key words: drug interactions, glutathione conjugates, half-life, mass spectrometry, reactive metabolites


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