title: Firefly luciferase inhibitor-conjugated peptide quenches bioluminescence: a versatile tool for real time monitoring cellular uptake of biomolecules.
abstract: In this paper, novel firefly luciferase-specific inhibitor compounds (FLICs) are evaluated as potential tools for cellular trafficking of transporter conjugates. As a proof-of-concept, we designed FLICs that were suitable for solid phase peptide synthesis and could be covalently conjugated to peptides via an amide bond. The spacer between inhibitor and peptide was optimized to gain efficient inhibition of recombinant firefly luciferase (FLuc) without compromising the activity of the model peptides. The hypothesis of using FLICs as tools for cellular trafficking studies was ensured with U87Fluc glioblastoma cells expressing firefly luciferase. Results show that cell penetrating peptide (penetratin) FLIC conjugate 9 inhibited FLuc penetrated cells efficiently (IC50 = 1.6 μM) and inhibited bioluminescence, without affecting the viability of the cells. Based on these results, peptide-FLIC conjugates can be used for the analysis of cellular uptake of biomolecules in a new way that can at the same time overcome some downsides seen with other methods. Thus, FLICs can be considered as versatile tools that broaden the plethora of methods that take advantage of the bioluminescence phenomena.
authors.affiliation: School of Pharmacy, ‡Institute of Biomedicine, and §A. I. Virtanen Institute for Molecular Sciences, University of Eastern Finland , P.O. Box 1627, FI-70211 Kuopio, Finland.
authors.forename: Pekka K
authors.initials: PK
authors.lastname: Poutiainen
authors.forename: Teemu
authors.initials: T
authors.lastname: Rönkkö
authors.forename: Ari E
authors.initials: AE
authors.lastname: Hinkkanen
authors.forename: Jorma J
authors.initials: JJ
authors.lastname: Palvimo
authors.forename: Ale
authors.initials: A
authors.lastname: Närvänen
authors.forename: Petri
authors.initials: P
authors.lastname: Turhanen
authors.forename: Reino
authors.initials: R
authors.lastname: Laatikainen
authors.forename: Janne
authors.initials: J
authors.lastname: Weisell
authors.forename: Juha T
authors.initials: JT
authors.lastname: Pulkkinen
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D057846:Cell-Penetrating Peptides; D004305:Dose-Response Relationship, Drug; D048888:Fireflies / Q000201:enzymology*; D006801:Humans; D007555:Isoxazoles / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology*; D007700:Kinetics; D049409:Luciferases, Firefly / Q000037:antagonists & inhibitors / Q000378:metabolism; D049449:Luminescence; D008163:Luminescent Measurements; D015394:Molecular Structure; D013329:Structure-Activity Relationship; D013997:Time Factors; D014018:Tissue Distribution
chemical_list: D002352:Carrier Proteins; D057846:Cell-Penetrating Peptides; D007555:Isoxazoles; C414409:penetratin; D049409:Luciferases, Firefly