- Email: [email protected]
High-throughput screening reveals enzyme and GPCR targets as putative binding sites for d -deprenyl
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Abstracts / Scandinavian Journal of Pain 8 (2015) 47–54
High-throughput screening reveals enzyme and GPCR targets as putative binding sites for d-deprenyl A. Lesniak a,∗ , A. Jonsson a , M. Aarnio b , T. Norberg c , F. Nyberg a , T. Gordh b a
Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden b Uppsala University Hospital, Department of Surgical Sciences, Anaesthesiology and Intensive Care, Uppsala, Sweden c Uppsala University, Department of Chemistry, Uppsala, Sweden E-mail address: [email protected] (A. Lesniak). 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 selective MAO-A inhibitor pirlindole mesylate. 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-B might be a candidate target for d-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. Reference [1] Linnman C, Appel L, Fredrikson M, Gordh T, Soderlund A, Langstrom B, Engler H. Elevated [11 C]-d-deprenyl uptake in chronic Whiplash Associated Disorder suggests persistent musculoskeletal inflammation. PLoS ONE 2011;6:e19182.
http://dx.doi.org/10.1016/j.sjpain.2015.04.016
New players in the mechanism of spinal cord stimulation for neuropathic pain A. Lind a,∗ , P. Emami b , M. Sjödin c , L. Katila a , M. Wetterhall c , T. Gordh a , K. Kultima b a
Department of Surgical Sciences, Anaesthesiology and Intensive Care, Uppsala University, SE-75185 Uppsala, Sweden b Department of Medical Sciences, Cancer Pharmacology and Computational Medicine, Uppsala University, SE-75185 Uppsala, Sweden c Department of Chemistry-BMC, Analytical Chemistry, Uppsala University, GE Healthcare, SE-75124, Uppsala, Sweden E-mail address: [email protected] (A. Lind). Aims: The aim of this study was to uncover possible proteins involved in the mechanism for neuropathic spinal cord stimulation (SCS) pain relief in humans. Methods: Using two different proteomic protocols we compared the protein concentration in cerebrospinal fluid (CSF) from 14 SCS responsive neuropathic pain patients using a shotgun proteomic approach. The comparison was made between samples from the same individual taken when the stimulator had been off for 48 h, and after the stimulator had been used as normal for three weeks. Mass spectrometry raw files from both experimental setups were analysed using MS Quant and DeCyder softwares and proteins were identified using MASCOT search against Swissprot database. After median normalization moderate paired t-test statistics were used in R limma package to find up-and down regulated proteins. Results: In total, 255 and 216 proteins could be identified by the dimethyl and label free methods respectively and relatively quantified. Of these several were significantly up- and downregulated. Conclusions: These novel proteins add to the SCS mechanism in patients with neuropathic pain. These results strengthen the evidence for SCS as altering extracellular matrix components and affecting central nervous system plasticity. http://dx.doi.org/10.1016/j.sjpain.2015.04.017 Hyperalgesia after experimental and work-related sleep restriction K.B. Nilsen a,b,c , D. Matre a,∗ a
National Institute of Occupational Health, Department of Work Psychology and Physiology, Oslo, Norway b Norwegian University of Science and Technology, Department of Neuroscience, Trondheim, Norway c Oslo University Hospital – Ullevål, Department of Neurology, Section for Clinical Neurophysiology, Norway E-mail address: dagfi[email protected] (D. Matre). Aims: Sleep restriction (SR) increases pain sensitivity. The aim of this study was to compare the effects of night work on pain sensitivity with experimental SR. Methods: In study I 22 healthy volunteers (14 females) and in study II 24 nurses (17 females) received pain stimuli in the laboratory twice; after 2 nights with habitual sleep (study I and II) and after 2 nights of experimental 50% SR (study I) or after 2 nights of work (study II). Order of sleep conditions was randomized. Heat pain at intensity 6/10 (pain-6), the pressure pain threshold (PPT), and subjective sleepiness with Karolinska sleepiness scale (KSS)
Abstracts / Scandinavian Journal of Pain 8 (2015) 47–54
High-throughput screening reveals enzyme and GPCR targets as putative binding sites for d-deprenyl A. Lesniak a,∗ , A. Jonsson a , M. Aarnio b , T. Norberg c , F. Nyberg a , T. Gordh b a
Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden b Uppsala University Hospital, Department of Surgical Sciences, Anaesthesiology and Intensive Care, Uppsala, Sweden c Uppsala University, Department of Chemistry, Uppsala, Sweden E-mail address: [email protected] (A. Lesniak). 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 selective MAO-A inhibitor pirlindole mesylate. 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-B might be a candidate target for d-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. Reference [1] Linnman C, Appel L, Fredrikson M, Gordh T, Soderlund A, Langstrom B, Engler H. Elevated [11 C]-d-deprenyl uptake in chronic Whiplash Associated Disorder suggests persistent musculoskeletal inflammation. PLoS ONE 2011;6:e19182.
http://dx.doi.org/10.1016/j.sjpain.2015.04.016
New players in the mechanism of spinal cord stimulation for neuropathic pain A. Lind a,∗ , P. Emami b , M. Sjödin c , L. Katila a , M. Wetterhall c , T. Gordh a , K. Kultima b a
Department of Surgical Sciences, Anaesthesiology and Intensive Care, Uppsala University, SE-75185 Uppsala, Sweden b Department of Medical Sciences, Cancer Pharmacology and Computational Medicine, Uppsala University, SE-75185 Uppsala, Sweden c Department of Chemistry-BMC, Analytical Chemistry, Uppsala University, GE Healthcare, SE-75124, Uppsala, Sweden E-mail address: [email protected] (A. Lind). Aims: The aim of this study was to uncover possible proteins involved in the mechanism for neuropathic spinal cord stimulation (SCS) pain relief in humans. Methods: Using two different proteomic protocols we compared the protein concentration in cerebrospinal fluid (CSF) from 14 SCS responsive neuropathic pain patients using a shotgun proteomic approach. The comparison was made between samples from the same individual taken when the stimulator had been off for 48 h, and after the stimulator had been used as normal for three weeks. Mass spectrometry raw files from both experimental setups were analysed using MS Quant and DeCyder softwares and proteins were identified using MASCOT search against Swissprot database. After median normalization moderate paired t-test statistics were used in R limma package to find up-and down regulated proteins. Results: In total, 255 and 216 proteins could be identified by the dimethyl and label free methods respectively and relatively quantified. Of these several were significantly up- and downregulated. Conclusions: These novel proteins add to the SCS mechanism in patients with neuropathic pain. These results strengthen the evidence for SCS as altering extracellular matrix components and affecting central nervous system plasticity. http://dx.doi.org/10.1016/j.sjpain.2015.04.017 Hyperalgesia after experimental and work-related sleep restriction K.B. Nilsen a,b,c , D. Matre a,∗ a
National Institute of Occupational Health, Department of Work Psychology and Physiology, Oslo, Norway b Norwegian University of Science and Technology, Department of Neuroscience, Trondheim, Norway c Oslo University Hospital – Ullevål, Department of Neurology, Section for Clinical Neurophysiology, Norway E-mail address: dagfi[email protected] (D. Matre). Aims: Sleep restriction (SR) increases pain sensitivity. The aim of this study was to compare the effects of night work on pain sensitivity with experimental SR. Methods: In study I 22 healthy volunteers (14 females) and in study II 24 nurses (17 females) received pain stimuli in the laboratory twice; after 2 nights with habitual sleep (study I and II) and after 2 nights of experimental 50% SR (study I) or after 2 nights of work (study II). Order of sleep conditions was randomized. Heat pain at intensity 6/10 (pain-6), the pressure pain threshold (PPT), and subjective sleepiness with Karolinska sleepiness scale (KSS)