Pardon Marie, Claes Pieter, Druwé Sarah, Martini Murielle, Siekierska Aleksandra, Menet Christel, de Witte Peter A M, Copmans Daniëlle
Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium.
Confo Therapeutics, Ghent, Belgium.
Front Pharmacol. 2022 Oct 10;13:1012622. doi: 10.3389/fphar.2022.1012622. eCollection 2022.
New pharmacological approaches that target orexin receptors (OXRs) are being developed to treat sleep disorders such as insomnia and narcolepsy, with fewer side effects than existing treatments. Orexins are neuropeptides that exert excitatory effects on postsynaptic neurons the OXRs, and are important in regulating sleep/wake states. To date, there are three FDA-approved dual orexin receptor antagonists for the treatment of insomnia, and several small molecule oral OX2R (OXR type 2) agonists are in the pipeline for addressing the orexin deficiency in narcolepsy. To find new hypnotics and psychostimulants, rodents have been the model of choice, but they are costly and have substantially different sleep patterns to humans. As an alternative model, zebrafish larvae that like humans are diurnal and show peak daytime activity and rest at night offer several potential advantages including the ability for high throughput screening. To pharmacologically validate the use of a zebrafish model in the discovery of new compounds, we aimed in this study to evaluate the functionality of a set of known small molecule OX2R agonists and antagonists on human and zebrafish OXRs and to probe their effects on the behavior of zebrafish larvae. To this end, we developed an IP-One Homogeneous Time Resolved Fluorescence (HTRF) immunoassay, and locomotor assays that record the locomotor activity of zebrafish larvae under physiological light conditions as well as under dark-light triggers. We demonstrate that the functional IP-One test is a good predictor of biological activity . Moreover, the behavioral data show that a high-throughput assay that records the locomotor activity of zebrafish throughout the evening, night and morning is able to distinguish between OXR agonists and antagonists active on the zebrafish OXR. Conversely, a locomotor assay with alternating 30 min dark-light transitions throughout the day is not able to distinguish between the two sets of compounds, indicating the importance of circadian rhythm to their pharmacological activity. Overall, the results show that a functional IP-one test in combination with a behavioral assay using zebrafish is well-suited as a discovery platform to find novel compounds that target OXRs for the treatment of sleep disorders.
目前正在研发针对食欲素受体（OXRs）的新型药理学方法，用于治疗失眠和发作性睡病等睡眠障碍，且副作用比现有治疗方法更少。食欲素是对突触后神经元（即OXRs）产生兴奋作用的神经肽，在调节睡眠/觉醒状态中起重要作用。迄今为止，有三种已获美国食品药品监督管理局（FDA）批准的双重食欲素受体拮抗剂用于治疗失眠，还有几种小分子口服OX2R（2型食欲素受体）激动剂正在研发中，用于解决发作性睡病中的食欲素缺乏问题。为了寻找新型催眠药和精神兴奋药，啮齿动物一直是首选模型，但它们成本高昂，且睡眠模式与人类有很大不同。作为替代模型，与人类一样昼行性、白天活动高峰且夜间休息的斑马鱼幼虫具有几个潜在优势，包括高通量筛选的能力。为了从药理学角度验证斑马鱼模型在发现新化合物中的应用，我们在本研究中旨在评估一组已知小分子OX2R激动剂和拮抗剂对人和斑马鱼OXRs的功能，并探究它们对斑马鱼幼虫行为的影响。为此，我们开发了一种IP-One均相时间分辨荧光（HTRF）免疫测定法，以及在生理光照条件下以及明暗触发条件下记录斑马鱼幼虫运动活性的运动测定法。我们证明功能性IP-One测试是生物活性的良好预测指标。此外，行为数据表明，一种记录斑马鱼整个晚上、夜间和早晨运动活性的高通量测定法能够区分对斑马鱼OXR有活性的OXR激动剂和拮抗剂。相反，一种在一天中交替进行30分钟明暗转换的运动测定法无法区分这两组化合物，这表明昼夜节律对其药理活性的重要性。总体而言，结果表明功能性IP-One测试与使用斑马鱼的行为测定法相结合，非常适合作为一个发现平台，以寻找靶向OXRs用于治疗睡眠障碍的新型化合物。
