High-throughput screening reveals enzyme and GPCR targets as putative binding sites for d-deprenyl

Aims: In PET studies of patients suffering from chronic pain following whip lash trauma, d-deprenyl was shown to bind to painful sites in the neck [1]. High uptake points towards an existence of an inflammation-specific binding site. Thus, the aim of this study was to identify the binding site for d-deprenyl employing radioligand receptor binding and high-throughput analysis of its activity towards 165 G-protein coupled receptors and 84 enzyme targets commonly used in drug discovery and development. Methods: d-Deprenyl activity towards GPCR targets was assessed by DiscoverX in CHO-K1 EDG1 -arrestin EFC cell line utilizing the PathHunterTM technique. Enzyme inhibition by Ddeprenyl was identified in the EnzymeProfilingTM screening panel provided by Eurofins Cerep Panlabs. [H3]d-deprenyl binding studies with specific GPCR agonists and enzyme inhibitors at newly identified targets were also performed. Results: Our investigation revealed that a 10 M concentration of d-deprenyl inhibited MAO-B and MAO-A activity by 99% and 55%, respectively. In addition, a 70% inhibition of angiotensin converting enzyme (ACE) activity in rabbit lung preparations was found. Furthermore, binding studies in rat mitochondrial membrane homogenates confirmed a submicromolar [H3]d-deprenyl competition with a selective MAO-B inhibitor seligiline, but not with the selectiveMAO-A inhibitor pirlindolemesylate. No evident hits among GPCR targets were identified. However, attention was drawn towards the histamine HRH1 and HRH3 receptors to which d-deprenyl showed a 20% and 42% antagonistic activity. Conclusions:MAO-Bmightbeacandidate target ford-deprenyl, as many other studies documented higher d-deprenyl uptake in activated astrocytes, non-secreting pituitary adenomas and brown adipose tissue, where MAO-B is overexpressed. Moreover, ACE inhibition was shown to hamper down-regulation of transcription factors preventing ROS-mediated cartilage damage.