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High-throughput screening and radioligand binding studies reveal monoamine oxidase-B as the primary binding target for D-deprenyl
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Anaesthesiology and Intensive Care.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
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2016 (English)In: Life Sciences, ISSN 0024-3205, E-ISSN 1879-0631, Vol. 152, p. 231-237Article in journal (Refereed) Published
Abstract [en]

Aims: D-deprenyl is a useful positron emission tomography tracer for visualization of inflammatory processes. Studies with [C-11]-D-deprenyl showed robust uptake in peripheral painful sites of patients with rheumatoid arthritis or chronic whiplash injury. The mechanism of preferential D-deprenyl uptake is not yet known, but the existence of a specific binding site was proposed. Thus, in the present study, we sought to identify the binding site for D-deprenyl and verify the hypothesis about the possibility of monoamine oxidase enzymes as major targets for this molecule. Main methods: A high-throughput analysis of D-deprenyl activity towards 165 G-protein coupled receptors and 84 enzyme targets was performed. Additionally, binding studies were used to verify the competition of [H-3]D-deprenyl with ligands specific for targets identified in the high-throughput screen. Key findings: Our high-throughput investigation identified monoamine oxidase-B, monoamine oxidase-A and angiotensin converting enzyme as potential targets for D-deprenyl. Further competitive [3H] D-deprenyl binding studies with specific inhibitors identified monoamine oxidase-B as the major binding site. No evident high-affinity hits were identified among G-protein coupled receptors. Significance: Our study was the first to utilize a high-throughput screening approach to identify putative D-deprenyl targets. It verified 249 candidate proteins and confirmed the role of monoamine oxidase - B in D-deprenyl binding. Our results add knowledge about the possible mechanism of D-deprenyl binding, which might aid in explaining the increased uptake of this compound in peripheral inflammation. Monoamine oxidase-B will be further investigated in future studies utilizing human inflamed synovium.

Place, publisher, year, edition, pages
2016. Vol. 152, p. 231-237
Keywords [en]
D-deprenyl High-throughput screening Binding site
National Category
Pharmaceutical Sciences Clinical Medicine
Identifiers
URN: urn:nbn:se:uu:diva-291492DOI: 10.1016/j.lfs.2016.03.058ISI: 000375728500028PubMedID: 27058977OAI: oai:DiVA.org:uu-291492DiVA, id: diva2:925668
Funder
Berzelii Centre EXSELENT, 2013-01495Available from: 2016-05-03 Created: 2016-05-03 Last updated: 2018-04-09Bibliographically approved
In thesis
1. Visualization of Peripheral Pain Generating Processes and Inflammation in Musculoskeletal Tissue using [11C]-D-deprenyl PET
Open this publication in new window or tab >>Visualization of Peripheral Pain Generating Processes and Inflammation in Musculoskeletal Tissue using [11C]-D-deprenyl PET
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

An objective visualization and quantification of pain-generating processes in the periphery would alter pain diagnosis and represent an important paradigm shift in pain research. Positron emission tomography (PET) radioligand [11C]-D-deprenyl has shown an elevated uptake in painful inflammatory arthritis and whiplash-associated disorder. However, D-Deprenyl’s molecular binding target and uptake mechanism in inflammation and musculoskeletal injuries are still unknown. The present thesis aimed to gain insight into the mechanisms of D-deprenyl binding and uptake and to verify whether pain-associated sites and inflammation in acute musculoskeletal injury could be visualized, objectively quantified and followed over time with [11C]-D-deprenyl PET-computed tomography (PET/CT).

To identify the D-deprenyl binding target, a high-throughput analysis and competitive radioligand binding studies were performed. D-deprenyl inhibited monoamine oxidase A (MAO-A) activity by 55%, MAO-B activity by 99% and angiotensin-converting enzyme (ACE) by 70%, which identified these enzymes as higher-affinity targets. Furthermore, radioligand receptor binding assays pointed favorably towards the concept of MAO-B as the primary target. To investigate the biochemical characteristics of the binding site, we used radioligand binding assays to assess differences in the binding profile in inflamed human synovial membranes exhibiting varying levels of inflammation. D-deprenyl bound to a single, saturable population of membrane-bound protein in synovial membrane homogenates and the level of inflammation correlated with an increase in D-deprenyl binding affinity.

To verify whether D-deprenyl can visualize pain-generating processes, patients with musculoskeletal injuries were investigated and followed-up with [11C]-D-deprenyl PET/CT. In the study of eight patients with ankle sprain, the molecular aspects of inflammation and tissue injury could be visualized, objectively quantified and followed over time with [11C]-D-deprenyl PET/CT. The pain coexisted with increased [11C]-D-deprenyl uptake. In the study of 16 whiplash patients, an altered [11C]-D-deprenyl uptake in the cervical bone structures and facet joints was associated with subjective pain levels and self-rated disability.

To further evaluate D-Deprenyl’s usefulness as a marker of inflammation, three PET tracers were compared in an animal PET/CT study. Preliminary findings showed that [11C]-D-deprenyl had an almost identical uptake pattern when compared with [11C]-L-deprenyl. The two deprenyl enantiomers showed no signs of specific binding or trapping and therefore may not be useful to study further in models of inflammatory pain, surgical pain, or both.

This thesis demonstrates that D-deprenyl visualizes painful inflammation in musculoskeletal injuries and that the probable underlying mechanism of [11C]-D-deprenyl uptake is binding to MAO.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1456
Keywords
ankle injuries, arthritis, binding site, binding target, carbon-11, deprenyl, high-throughput screening, inflammation, monoamine oxi-dase, pain, PET, whiplash
National Category
Anesthesiology and Intensive Care Radiology, Nuclear Medicine and Medical Imaging
Research subject
Anaesthesiology and Intensive Care
Identifiers
urn:nbn:se:uu:diva-347685 (URN)978-91-513-0313-0 (ISBN)
Public defence
2018-05-25, Universitetshuset, Biskopsgatan 3, Uppsala, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2018-05-02 Created: 2018-04-06 Last updated: 2018-05-02

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Lesniak, AnnaAarnio, MikkoNorberg, ThomasNyberg, FredGordh, Torsten

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