Optimization of EphA4 antagonists for CNS disorders

描述（由申请人提供）：EphA4受体酪氨酸激酶在发育中和成人神经系统中高表达，神经系统损伤后上调。许多重要的功能都归因于神经细胞中的这种受体。在胚胎神经系统中，EphA4与其膜相关配体（ephrins）的相互作用对于轴突引导和组织边界的形成至关重要。在成人中枢神经系统（CNS）中，EphA4调节神经元连接（突触）的结构和可塑性，在胶质细胞和神经元之间的通讯中起重要作用。例如，我们最近将EphA4及其配体之一ephrin-A3与神经胶质谷氨酸转运蛋白的表达减少联系起来。这表明抑制EphA4-ephrin-A3相互作用可能有利于治疗细胞外谷氨酸水平升高的疾病，如癫痫和肌萎缩侧索硬化症（ALS）。在损伤神经系统中，EphA4通过促进神经胶质瘢痕形成和抑制损伤轴突的发芽来抑制神经再生和功能恢复。与此一致，最近的研究表明，抑制EphA4-ephrin相互作用可能有助于脊髓损伤的治疗。最后，最近的研究表明ephrin-A3和另一种EphA4配体ephrin-A2在维持成人大脑神经前体细胞的静止中起作用。EphA4是脑内主要的ephrin-A2和ephrin-A3受体，这表明EphA4的拮抗剂可能对神经元细胞替代疗法有用。因此，EphA4是治疗中枢神经系统疾病的一个有希望的新靶点。很少有靶向EphA4的分子可用。它们包括靶向EphA4激酶结构域（以及许多其他酪氨酸激酶）的小分子拮抗剂，以及EphA4和ephrin细胞外结构域的可溶性部分，它们抑制ephrin与EphA4（以及其他Eph受体）的结合。我们发现了几种多肽和两种同分异构体小分子，它们可以拮抗EphA4-ephrin的结合，是更具选择性的EphA4拮抗剂。作为药物开发的候选者，小分子具有比多肽更理想的药物特性。然而，我们已经鉴定并广泛表征的两种化合物，虽然对EphA4和相关的EphA2受体具有高选择性，但效力较低。在这个应用中，我们建议优化我们通过高通量筛选确定的EphA4-ephrin结合的小分子拮抗剂。为了开发更有效的小分子拮抗剂，我们提出了药物化学和快速类比的迭代，以产生用于生化/细胞培养试验的化合物，然后验证优化的探针化合物并表征其在神经细胞中的活性。优化后的EphA4拮抗剂可作为研究工具，深入了解EphA4的生理和病理功能，也可作为药物开发的先导化合物，用于治疗中枢神经系统疾病。

DESCRIPTION (provided by applicant): The EphA4 receptor tyrosine kinase is highly expressed in the developing and adult nervous system, and is upregulated following nervous system injury. A number of important functions have been ascribed to this receptor in neural cells. In the embryonic nervous system, EphA4 interaction with its membrane-associated ligands - the ephrins - are critical for axon guidance as well as for the formation of tissue boundaries. In the adult central nervous system (CNS), EphA4 regulates the structure and plasticity of neuronal connections (synapses) and plays an important role in the communication between glial cells and neurons. For example, we have recently linked EphA4 and one of its ligands, ephrin-A3, to decreased expression of glial glutamate transporters. This suggests that inhibition of EphA4-ephrin-A3 interaction may be beneficial for the treatment of pathologies involving elevated extracellular glutamate levels, such as epilepsy and amyotrophic lateral sclerosis (ALS). In the injured nervous system, EphA4 inhibits nerve regeneration and functional recovery by promoting glial scar formation and inhibiting sprouting of injured axons. Consistent with this, recent studies suggest that inhibiting EphA4-ephrin interaction could be useful for the treatment of spinal cord injuries. Finally, recent work has implicated ephrin-A3 and another EphA4 ligand, ephrin-A2, in maintaining the quiescence of neural precursor cells in the adult brain. This suggests that antagonists of EphA4, which is a major ephrin-A2 and ephrin-A3 receptor in the brain, may be useful for neuronal cell replacement therapies. Thus, EphA4 represents a promising new target for the treatment of CNS disorders. Few molecules that target EphA4 are available. They include small molecule antagonists that target the kinase domain of EphA4 (but also many other tyrosine kinases), and soluble portions of EphA4 and ephrin extracellular domains, which inhibit ephrin binding to EphA4 (but also other Eph receptors). Several peptides and two isomeric small molecules that we identified antagonize EphA4-ephrin binding and are much more selective EphA4 antagonists. Small molecules have more desirable pharmaceutical profiles than peptides as candidates for drug development. However, the two compounds that we have identified and extensively characterized, while highly selective for EphA4 and the related EphA2 receptor, have low potency. In this application, we propose to optimize small molecule antagonists of EphA4-ephrin binding that we identified by high throughput screening. To develop more potent small molecule antagonists, we propose iterations of medicinal chemistry and rapid analoging to generate compounds for testing in biochemical/cell culture assays, and then validate the optimized probe compounds and characterize their activities in neural cells. The optimized EphA4 antagonists will be useful as research tools to obtain valuable insight into the physiological and pathological functions of EphA4, and may also serve as lead compounds for drug development to treat CNS disorders. PUBLIC HEALTH RELEVANCE: EphA4 is a cell surface receptor that plays an important role in the developing and adult nervous system. Evidence suggests that EphA4 inhibits recovery following nervous system injury and may contribute to several nervous system disorders. In this project we propose to improve compounds that we have previously identified and characterized as antagonists of EphA4 function. The optimized antagonists will be useful to further study the physiological and pathological functions of EphA4 and may serve as starting points for drug development to treat disorders of the nervous system.