title: The phosphoinositide-specific phospholipase C inhibitor U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) spontaneously forms conjugates with common components of cell culture medium.
abstract: Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca(2+) release from inositol 1,4,5-triphosphate-sensitive stores. U73122 (1-(6-((17beta-3-methoxyestra-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), Hanks' buffered saline solution (with 2 mM glutamine), optimized basal nutrient medium (MCDB131, without BSA), complete medium, Dulbecco's modified Eagle's medium (with 2 mM l-glutamine) was approximately 150, 60, 32, 30, and 18 min, 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 Ca(2+) 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.
authors.affiliation: Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Belmont, Surrey, UK. nicola.wilsher@icr.ac.uk
authors.forename: Nicola E
authors.initials: NE
authors.lastname: Wilsher
authors.forename: Will J
authors.initials: WJ
authors.lastname: Court
authors.forename: Ruth
authors.initials: R
authors.lastname: Ruddle
authors.forename: Yvette M
authors.initials: YM
authors.lastname: Newbatt
authors.forename: Wynne
authors.initials: W
authors.lastname: Aherne
authors.forename: Peter W
authors.initials: PW
authors.lastname: Sheldrake
authors.forename: Neil P
authors.initials: NP
authors.lastname: Jones
authors.forename: Matilda
authors.initials: M
authors.lastname: Katan
authors.forename: Suzanne A
authors.initials: SA
authors.lastname: Eccles
authors.forename: Florence I
authors.initials: FI
authors.lastname: Raynaud
mesh_terms: D016477:Artifacts; D001681:Biological Assay; D020013:Calcium Signaling / Q000187:drug effects*; D045744:Cell Line, Tumor; D003470:Culture Media / Q000737:chemistry* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D004815:Epidermal Growth Factor / Q000494:pharmacology; D004963:Estrenes / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D005973:Glutamine / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D006207:Half-Life; D006801:Humans; D011485:Protein Binding; D011760:Pyrrolidinones / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D012710:Serum Albumin, Bovine / Q000737:chemistry; D013997:Time Factors; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors*
chemical_list: D003470:Culture Media; D004791:Enzyme Inhibitors; D004963:Estrenes; D011760:Pyrrolidinones; D005973:Glutamine; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D012710:Serum Albumin, Bovine; D004815:Epidermal Growth Factor; D010738:Type C Phospholipases; D005978:Glutathione