The µ-opioid receptor (µ-OR) is the key target for morphine in the treatment of pain. Endogenous opioids at the µ-OR have been implicated in multiple physiological processes, including pain relief. However, the extent to which µ-OR occupancy of morphine is influenced by endogenous opioids and µ-OR internalization is unclear. The aim of this study was to investigate the impact of endogenous opioids and µ-OR internalization on morphine µ-OR occupancy. To this end we developed a mathematical binding kinetic model incorporating information on binding kinetic parameters, µ-OR expression, µ-OR internalization rates, and levels of multiple endogenous opioids, in rat hypothalamus. µ-OR occupancies were simulated using morphine brain extracellular fluid concentration data, for different static levels of the endogenous opioids, including beta-endorphin, Dynorphin-A 1-17, Endomorphin-2, Leu-enkephalin, and Met-enkephalin. Simulations were performed in absence or presence of µ-OR internalization. We show that beta-endorphin has a strong impact on morphine µ-OR occupancy, followed by limited impact of met-enkephalin. Other endogenous opioids did not demonstrate any significant effect. µ-OR internalization reduced the impact of beta-endorphin on morphine µ-OR occupancy to a limited extent. We conclude that beta-endorphin plays an important role in morphine µ-OR occupancy, which could therefore play a key role in explaining interindividual variability in the analgesic effects of morphine. Finally, this study demonstrates the utility of a mathematical model workflow in deciphering the role of endogenous ligands in morphine's µ-OR occupancy.

2025-03-01·Journal of Veterinary Research

Influence of neurokinin B, dynorphin A and kisspeptin-10 on in vitro gonadotropin secretion by anterior pituitary cells isolated from pubescent ewes

Article

作者: Szysiak, Natalia ; Junkuszew, Andrzej ; Nowakiewicz, Aneta ; Patkowski, Krzysztof ; Longo, Vincenzo ; Bochniarz, Mariola ; Greguła-Kania, Monika ; Kosior-Korzecka, Urszula

Abstract:

Introduction:

The hypothalamic neuropeptides kisspeptin-10 (KiSS-10), neurokinin B (NKB), and dynorphin A (Dyn A) play roles in the endocrine regulation of the hypothalamic–pituitary–ovarian (HPO) axis in puberty. Livestock’s timely attainment of sexual maturity increases reproductive efficiency and raises profitability. The pituitary relationship between these neuropeptides and gonadotropins in puberty in ewes being undercharacterised. The aim of the study was to analyse their direct effect on gonadotropin secretion by pituitary cells isolated from pubescent ewes.

Material and Methods:

Cells were incubated in McCoy’s 5A medium, either without neuropeptides (as the control) or with 10−11, 10−10, 10−9, 10−8 and 10−7 M of KiSS-10, NKB and Dyn A. After 4, 12 and 24 h, the luteinising hormone (LH) and follicle-stimulating hormone (FSH) concentrations were analysed by ELISA using species-specific antibodies.

Results:

Greater LH and FSH secretion was observed after the 4–24 h exposure to respective 10−11–10−8 M and 10−11–10−7 M concentrations of KiSS-10. Moreover, NKB and Dyn A applied in the concentration range elevated the secretion of both LH and FSH throughout the experiment. Dynorphin A had the most significant effect on gonadotropin secretion at all the concentrations used. In contrast, the most pronounced dose-dependent neuropeptide effect throughout the experiment on the FSH secretion was attributed to NKB.

Conclusion:

Kisspeptin-10, NKB and Dyn A had a direct impact on gonadotropin secretion by ovine pituitary cells. However, a detailed explanation of their role in gonadotropin secretion by the anterior pituitary gland in sheep and of their impact on the regulation of the HPO axis during sexual maturation or in the pathomechanism of delayed puberty require further studies.

2025-02-01·Molecular Metabolism

Enhanced dynorphin expression and secretion in pancreatic beta-cells under hyperglycemic conditions

Article

作者: Blandino-Rosano, Manuel ; Louzada, Ruy A ; Movahed, Miranda

OBJECTIVE:

Dynorphin, an endogenous opioid peptide predominantly expressed in the central nervous system and involved in stress response, pain, and addiction, has intrigued researchers due to its expression in pancreatic β-cells. In this study, we aimed to characterize dynorphin expression in mouse and human islets and explore the mechanisms regulating its expression.

METHODS:

We used primary mouse and human islets with unbiased published datasets to examine how glucose and other nutrients regulate dynorphin expression and secretion in islets.

RESULTS:

The prodynorphin gene is significantly upregulated in β-cells under hyperglycemic conditions. In vitro studies revealed that increased glucose concentrations correlate with increased dynorphin expression, indicating a critical interplay involving Ca²⁺, CamKII, and CREB pathways in β-cells. Perifusion studies allowed us to measure the dynamic secretion of dynorphin in response to glucose from mouse and human islets for the first time. Furthermore, we confirmed that increased dynorphin content within the β-cells directly correlates with enhanced dynorphin secretion. Finally, our findings demonstrate a synergistic effect of palmitate in conjunction with high glucose, further amplifying dynorphin levels and secretion in pancreatic islets.

CONCLUSIONS:

This study demonstrates that the opioid peptide prodynorphin is expressed in mouse and human β-cells. Prodynorphin levels are regulated in parallel with insulin in response to glucose, palmitate, and amino acids. Our findings elucidate the signaling pathways involved, with CamKII playing a key role in regulating prodynorphin levels in β-cells. Finally, our findings are the first to demonstrate active dynorphin secretion from mouse and human islets in response to glucose.

项与 Dynorphin A 相关的新闻（医药）

2024-11-29

·GoDesign

【蛋白设计】David Baker无序蛋白binder设计（上）

——简介—— 自然界进化出了众多机理来结合无序蛋白或无序肽段，例如抗体antibody、主要组织相容性复合体MHCs、TPR蛋白结构域等，尽管可以看到自然界的众多手段，但是人工设计无序肽段（intrinsically disordered regions，IDRs）的binder仍然是极大的挑战。寻找到针对IDRs强结合能力、高特异性binder的一般方法在生物学研究，疾病治愈与诊断中具有重大意义。诺奖得主David Baker在BioRxiv上发表两篇文章"Sequence-specific targeting of intrinsically disordered protein regions"和"Diffusing protein binders to intrinsically disordered proteins"，开发了IDRs的binder设计的一般方法。这两篇文章在预印本上发表数月有余，但仍然未有对其内容的详细介绍。本文将一探究竟在"Sequence-specific targeting of intrinsically disordered protein regions"中，Wu等人会如何实现对任意IDR序列设计强亲和力和高特异性binder。 ——方法思路—— 图1. 关于IDRs的binder设计的主要流程 Wu等人假设IDRs靶标均能以舒展的构象与形状匹配的binder形成稳定复合物。为此，他们设计了一个复合物模板库，库中IDRs（靶标）以舒展的拉伸构象参与结合，binder以形状匹配的沟槽型口袋参与结合（图1）。模板中IDRs参与结合的残基数在4-30个，因此在实际应用时，他们将会沿IDRs靶标的序列搜索最适于靶向的靶标模板，再对该模板的复合物进行结构和序列优化，优化后的复合物中的binder即为所设计的binder。要该方法发挥预期效果，需要满足两个条件：1）binder与IDRs靶标结合的特异性可以由足够的氢键提供，2）模板库中有足够的模板为任意IDRs靶标提供至少一种其偏好的结合态构象。具体来说，该方法分为四步。第一步为“口袋生成”。作者为多种二肽重复序列构建特异性、高亲和力的口袋，再将这些口袋和对应的二肽重复序列切断，进而得到结合单氨基酸、二肽或更长序列的口袋。这些被设计的口袋的侧链不仅可以与结合的氨基酸残基的侧链有相互作用，并且可以与其主链骨架存在氢键，以提供结构的特异性及补偿去溶剂化效应。第二步为“口袋组装”。作者将上步得到的口袋组装，得到了能够识别舒展构象的沟槽型口袋。使用RFdiffusion重组生成口袋间的相互作用界面，并且要求产生整体刚性的结构。至此产生了众多口袋所组成的有着不同顺序和几何结构的模块。第三步为“穿连装入”。将IDR靶标序列映射到每个模版的靶标骨架上，去寻找靶标序列中最合适的结合模式。最后第四步“精修改善”。将产生的最佳匹配使用ProteinMPNN优化序列并用AF2评估结构。第一、二步的模板文库构建完成后可被重复使用。针对新IDRs靶标的binder设计则仅需进行第三、四步，大大减少了计算的花费。 ——细节与结果—— 口袋生成基于之前超螺旋匹配的方式，聚焦于更加扩展的氨基酸残基构象，多肽与蛋白的对接使用双齿（bidentate）氢键相互作用，重复蛋白剩余序列使用ProteinMPNN优化，设计的复合物使用Rosetta和AlphaFold2结构预测匹配模型。刚开始选择了LK、RT、YD、PV和GA等二肽，使用实验验证后又用RFdiffusion对方法进行了改进，最后将二肽口袋拆分为单氨基酸口袋以覆盖20种氨基酸残基。为实现对非重复序列的识别，将定制的结合口袋组装到新骨架上，用参数定位和 RFdiffusion连接生成设计，最后用口袋组装构建含识别极性残基口袋的嵌合骨架，经序列设计多样化等操作，得到含340个模板的库并扩展至1000个成员。针对内在无序蛋白或区域，将其4-30氨基酸序列段匹配到目标骨架上，使用ProteinMPNN优化binder序列，对 AF2指标不佳的用RFdiffusion改进。用此方法获 21种相关靶标的binder（含GPCR、胰岛素、胰蛋白酶肽等配体），经表达、纯化及 BLI测结合，多数有纳摩尔至皮摩尔亲和力，还对部分binder进一步优化。这其中解析了dynorphin A结合复合物的共晶结构（结合力为7 nM）。图 2. 选择的无序结构及设计的binder的结合测定除此之外还通过Pull-Down等实验验证了设计配体具有很好的特异性。 ——总结—— 总的来说，Baker在"Sequence-specific targeting of intrinsically disordered protein regions"这篇文章中描述了以高亲和力和特异性设计结合IDRs蛋白的方法，在43个靶标中设计成功了39个，其中36个binder通过实验得到，其中大部分亲和力优于100 nM。在下一篇推送中将会介绍Baker针对IDP的binder设计的另一篇文章"Diffusing protein binders to intrinsically disordered proteins"。参考文献： [1] Wu, K., Jiang, H., Hicks, D. R., Liu, C., Muratspahić, E., Ramelot, T. A., ... & Baker, D. (2024). Sequence-specific targeting of intrinsically disordered protein regions. bioRxiv, 2024-07. 作者：程北溟审稿：王瀚平编辑：王宇哲 GoDesign ID：Molecular_Design_Lab （扫描下方二维码可以订阅哦！）

基因疗法

100 项与 Dynorphin A 相关的药物交易

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