Synthesis, Biological Evaluation And Application Of Fluorescent Opiate Probes

LS Schembri1, PJ Scammells1, B Graham1, LA Stoddart2, M Canals1, B Kellam2, SJ Briddon2. 1Monash Institute of Pharmaceutical Sciences, Melbourne, Victoria, Australia, 2University of Nottingham, Nottingham, Nottinghamshire, UK

Recent literature has shown an increasing interest in fluorescent G protein- coupled receptor (GPCR) ligands, from use as imaging agents to study receptor localisation and trafficking (1), to use in binding assays (2). A fluorescent GPCR ligand/probe consists of a known agonist or antagonist connected to a fluorophore via an appropriate linker. They hold several advantages over traditionally used radioligands. They are safer for both the user and the environment, which makes them more amenable to high throughput screening. The assays they are employed in follow a continuous format and are extremely sensitive, with the ability to be detected in single cells or even subsection of cells (3, 4). This work is centered on the design, synthesis and pharmacological evaluation of fluorescent opioid probes.

The ligand oripavine (5) was structurally elaborated to include an appropriate linker moiety. A range of fluorophores were then connected via amide coupling to give rise to five new fluorescent probes.

The affinity and efficacy of the fluorescent ligands at the Mu-opioid receptor (MOR) were measured using the Alphascreen ERK phosphorylation assay. The binding affinities (pA2 values) were determined by Schild plot analysis of their ability to shift the DAMGO agonist concentration response curve. Imaging studies were carried out on HEK293 cells expressing the MOR and images were captured using a Zeiss LSM 5 Exciter confocal microscope with a Plan-Apochromat 63x oil objective lens and a 633 nm excitation laser. A displacement curve for naloxone was constructed using oripavine-Cy5 as per Stoddart et al (6). All data was analysed using GraphPad Prism 6 and all data shown is mean ± SEM.

It was found that all five compounds acted as competitive antagonists. As shown in table 1, all compounds maintained high affinity at the MOR, with pA2 values ranging from 7.38 to 8.31.

Table 1 Fluorescent probes developed and resulting pA2 values.

Oripavine-Bodipy and oripavine-Cy5 were taken forward for imaging studies. Both displayed clear, displaceable membrane binding on cells expressing the MOR. In high content imaging studies, the constructed naloxone displacement curves gave a pKi of 8.95 ± 0.12 (n=3), similar to that reported in the literature (9.03) (7). These studies indicate that oripavine based fluorescent ligands retain affinity at the MOR and oripavine-Cy5 shows favorable imaging properties for HCS based studies. These fluorescent ligands have the potential to be used to study novel aspects of MOR receptor pharmacology.

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