A Novel Aromatic Carboxylic Acid Inactivates Luciferase by Acylation of an Enzymatically Active Regulatory Lysine Residue

Abstract

Firefly luciferase (Luc) is widely used as a reporter enzyme in cell-based assays for gene expression. A novel aromatic carboxylic acid, F-53, reported here for the first time, substantially inhibited the enzymatic activity of Luc in a Luc reporter screening. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and tandem mass spectrometry (MS/MS) analyses showed that F-53 modifies Luc at lysine-529 via amidation of the F-53 carboxyl group. The lysine-529 residue of Luc, which plays a regulatory catalytic role, can be acetylated. Luc also has a long-chain fatty acyl-CoA synthase activity. An in vitro assay that involved both recombinant Luc and mouse liver microsomes identified F-53-CoA as the reactive form produced from F-53. However, whereas the inhibitory effect of F-53 is observed in Hela cells that transiently expressed Luc, it is not observed in an in vitro assay that involves recombinant Luc alone. Therefore, insights into the activities of certain mammalian transferases can be translated to better understand the acylation by F-53. The insights from this study about the novel inhibitory modification mechanism might help not only to avoid misinterpretation of the results of Luc-based reporter screening assays but also to explain the pharmacological and toxicological effects of carboxylic acid-containing drugs.

Synthesis of Probe Molecules, 6-(Dimethylamino)-2-phenylisoindolin-1-ones, for Mechanistic Studies of Firefly Luciferase Inhibition

“…A reported synthesis 7 of the isoindolinone ring was modified for the construction of the core fragment of 1 and 2. However, installation of the two side chains on the aryl ring fragment of 2 exposed a challenge.…”

“…Methyl 2-(bromomethyl)-5-nitrobenzoate (4). 7 A solution of 2.0 g (10.6 mmol) of methyl 2-methyl-5-nitrobenzoate, 2.07 g (11.7 mmol) of N-bromosuccinimide, and 51 mg of benzoyl peroxide in 80 mL of CH2Cl2, under argon was heated under reflux for 15 h. The reaction mixture was neutralized with saturated aqueous sodium hydrogencarbonate, and the organic and aqueous layers were separated.…”

“…6 Previously, it was found that molecule F-53 (Figure 1) inactivated luciferase by acylating the regulatory lysine residue 529 in live mammalian cells and it didn't inactivate luciferase by acylating the regulatory lysine residue 529 in vitro reconstruction system. 7 Presumably, the carboxylic acid group of F-53 activates by luciferase to give the corresponding thioester F53-CoA, which in turn reacts with the amino function of lysine-529 residue of luciferase to give an amide, F53-Lys529(luciferase), catalyzed by some transferase(s) of acyl-CoA in cells. 7 Since Lys529 is needed in the enzymatic reactions, 8 this acylation resulted in an inhibition of luciferase.…”

“…7 Presumably, the carboxylic acid group of F-53 activates by luciferase to give the corresponding thioester F53-CoA, which in turn reacts with the amino function of lysine-529 residue of luciferase to give an amide, F53-Lys529(luciferase), catalyzed by some transferase(s) of acyl-CoA in cells. 7 Since Lys529 is needed in the enzymatic reactions, 8 this acylation resulted in an inhibition of luciferase. Carboxylic acid containing drugs including valproic acid, a drug for the treatment of bipolar disease, can be catalyzed by acyl-CoA synthetase using ATP, Mg 2+ and CoASH to give the corresponding acyl-CoA thioester, which has been suggested to produce serious adverse effects.…”