Firefly Luciferase Inhibitor-Conjugated Peptide Quenches Bioluminescence: A Versatile Tool for Real Time Monitoring Cellular Uptake of Biomolecules Pekka K. Poutiainen,* ,† Teemu Rö nkkö , † Ari E. Hinkkanen, § Jorma J. Palvimo, ‡ Ale Na ̈ rva ̈ nen, † Petri Turhanen, † Reino Laatikainen, † Janne Weisell, † and Juha T. Pulkkinen † † 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 * S Supporting Information 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 (IC 50 = 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. ■ INTRODUCTION Problems with formulations and bioavailability are one of the main reasons promising drugs fail in clinical trials. Conjugation of small molecules to, e.g., cell penetrating peptides can be used to make lipophilic drugs more water-soluble and more importantly transported to target tissue. 1 Most of the transporter molecules, such as peptides and nucleotides, are using mainly endocytotic pathways to penetrate the cell membrane. However, during the endocytosis, therapeutic macromolecules may be trapped in the endosomal/lysosomal compartment which reduces their bioavailability. 2-4 Several methods have been reported for elucidation of cellular uptake mechanisms and for tracking of the location of macromolecules inside the cell. One of the most widely used methods is the conjugation of macromolecules with a suitable fluorophore. 2,5 The main drawback of this method is, however, the difficulty discriminating between truly cytosol-internalized peptides from those bound to the membrane or trapped in the endosomes. Thus, the real time experiments in vitro and in vivo are not possible. 2 Fluorescence quenching methods have been used to investigate the delivery of therapeutic molecules to the cytosol by cell penetrating peptides (CPPs), but these assays represent end point studies. 6 One widely used method is to inhibit the potential route of the uptake pathway by using small molecule uptake inhibitors. 7 The main challenge with the uptake of inhibitors is the existence of parallel uptake mechanisms, which can substitute the inhibited delivery route. 8 Overall, novel techniques are needed to ensure the cytosol entry of the biomolecules to verify that the cargo is transported and released inside the cell. One of the most studied imaging and transporter technique is the so-called controlled release technique, which takes advantage of the bioluminescence. 9 In this method the conjugate is actively cleaved from the transporter inside the cell to produce easily detectable light but it has also some down sides. 10,11 Briefly, the natural substrate of luciferase, D-luciferin, has been conjugated to peptides 9 or other macromolecules 12 using cleavable linkers that release the substrate after penetration into the cytosol, where the light producing enzyme, luciferase, is situated. However, the linker may already be cleaved inside the endosome, thus releasing only the easy membrane penetrating substrate D-luciferin, while the delivery vector remains entrapped inside the vesicle which could lead to Received: August 5, 2013 Revised: December 16, 2013 Published: December 16, 2013 Communication pubs.acs.org/bc © 2013 American Chemical Society 4 dx.doi.org/10.1021/bc4003713 | Bioconjugate Chem. 2014, 25, 4-10