Discovery of a series of 2-phenylnaphthalenes as firefly luciferase inhibitors

Abstract

As the most convenient and efficient bioluminescence system, the firefly luciferase/luciferin complex has been widely used in life science research and high-throughput screening (HTS). Nonetheless, the interpretation of firefly luciferase-based assay data is often complicated by the occurrence of "false positives," in part because firefly luciferase (Fluc) is subject to direct inhibition by HTS compounds that might inadvertently act as inhibitors of its catalytic site. Here we report a series of 2-phenylnaphthalenes as Fluc inhibitors with suitable potency both in vitro and in vivo. Besides, our compound 5 showed significant systemic inhibition in transgenic mice. Enzymatic kinetics study reveals that compound 5 is competitive for substrate aminoluciferin and noncompetitive for the second substrate ATP. Furthermore, compound 5 exhibited good performance as a quenching agent in a dual-luciferase reporter assay. We anticipate that these Fluc inhibitors will contribute to the broader utilization of bioluminescence in life science research while circumventing or at least reducing the number of "false positives".Scheme 1 Mechanism of firefly bioluminescence and structure of D-luciferin, D-aminoluciferin, and resveratrol. This journal isScheme 2 Synthesis of 2-phenylnaphthalenes 4-14. Reagents and conditions: (a) bis(pinacolato)diboron, potassium acetate, PdCl 2 (dp.pf), dioxane, 60 C, 6 h. (b) Potassium fluoride PdCl 2 (dppf), dioxane, 90 C, 12h. (c) NaOH, EtOH, reflux then HCl. (d) Boron tribromide, DCM, À78 C, 12 h. 63452 | RSC Adv., 2015, 5, 63450-63457 This journal is

Inhibiting Firefly Bioluminescence by Chalcones

“…Auld et al reported PTC124 (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]­benzoic acid) as a potent Fluc inhibitor with an IC 50 of 7 nM. , Rui Fontes et al indicated that the bioluminescence of luciferin could be inhibited by inorganic pyrophosphate and tripolyphosphate due to their reaction with L-AMP . So far, many compounds containing different structures have been reported that could inhibit firefly luciferase, such as ions, (E)-2-fluoro-4′-methoxystilbene, N-pyridin-2-ylbenzamides, 2-phenylnaphthalenes, aryltriazoles, 2-phenylnaphthalenes, 5-benzyl-3-phenyl-4,5-dihydroisoxazoles, and 5-benzyl-3-phenyl-1,4,2-dioxazoles …”

“…15,16 Rui Fontes et al indicated that the bioluminescence of luciferin could be inhibited by inorganic pyrophosphate and tripolyphosphate due to their reaction with L-AMP. 17 So far, many compounds containing different structures have been reported that could inhibit firefly luciferase, such as ions, 18 (E)-2-fluoro-4′methoxystilbene, 19 N-pyridin-2-ylbenzamides, 20 2-phenylnaphthalenes, 21 aryltriazoles, 22 2-phenylnaphthalenes, 23 5-benzyl-3phenyl-4,5-dihydroisoxazoles, and 5-benzyl-3-phenyl-1,4,2-dioxazoles. 11 In the current study, we found that some chalcone derivatives could potently inhibit the firefly luciferase activity.…”

Examining multiple cellular pathways at once using multiplex hextuple luciferase assaying

“…Also, experiments should exclude the possibility that the tested drugs may inhibit the enzymatic activities of one or more luciferases in the assay, as previously reported for Pifithrin-α 36 . This becomes especially important in high-throughput drug screens, where numerous candidates will be identified of which some may result in possible false positive hits due to interference with luciferase activity 36,54,55 , careful followup studies, as illustrated in this work, should be performed to exclude interference of isolated compounds on luciferase activity. Finally, while the current study involves intracellularly-localized luciferases, endpoint postlysis experimentation only allows for a single multiplex measurement.…”

1‐Aminobenzocyclobutene‐1‐phosphonic Acid and Related Compounds as Inhibitors of Phenylalanine Ammonia‐Lyase

“…[26] PBr 3 (11 ml; 0.113 mol) in dry Et 2 O (10 ml) was added dropwise to a solution of 1,8-bis(hydroxymethyl)naphthalene (6.10 g; 0.0323 mol) in dry Et 2 O (140 ml) at À10 to À2°C over the course of 45 min. [31] [32] A solution of bromomethylbenzene derivatives 13 and 14 (0.02 mol) or 15 (0.040 mol), triethyl phosphonoacetate (16; 5.2 ml, 0.026 mol) and tetrabutylammonium bromide (1.290 g, 0.004 mol) in toluene (20 ml) were stirred and cooled to 14°C. Then the mixture was refluxed for 7 h and then poured onto crushed ice (250 ml), after which CHCl 3 (150 ml) was added.…”

“…[17] Recently in the literature the synthesis of derivatives of diethyl malonate were obtained in other conditions than in this work. [31] [32] A solution of bromomethylbenzene derivatives 13 and 14 (0.02 mol) or 15 (0.040 mol), triethyl phosphonoacetate (16; 5.2 ml, 0.026 mol) and tetrabutylammonium bromide (1.290 g, 0.004 mol) in toluene (20 ml) were stirred and cooled to 14°C. Powdered NaOH (2.4 g, 0.06 mol) was added in about 25 portions over a period of 5 h at a temperature of 14 -18°C.…”