Table_2_In silico Screening and Behavioral Validation of a Novel Peptide, LCGA-17, With Anxiolytic-Like Properties.DOCX

The aim of the study was to develop better anxiolytics and antidepressants. We focused on GABAA receptors and the α2δ auxiliary subunit of V-gated Ca2+ channels as putative targets because they are established as mediators of efficacious anxiolytics, antidepressants, and anticonvulsants. We further focused on short peptides as candidate ligands because of their high safety and tolerability profiles. We employed a structural bioinformatics approach to develop novel tetrapeptides with predicted affinity to GABAA receptors and α2δ. In silico docking studies of one of these peptides, LCGA-17, showed a high binding score for both GABAA receptors and α2δ, combined with anxiolytic-like properties in a Danio rerio behavioral screen. LCGA-17 showed anxiolytic-like effects in the novel tank test, the light–dark box, and the social preference test, with efficacy comparable to fluvoxamine and diazepam. In binding assays using rat brain membranes, [3H]-LCGA-17 was competed more effectively by gabapentinoid ligands of α2δ than ligands of GABAA receptors, suggesting that α2δ represents a likely target for LCGA-17. [3H]-LCGA-17 binding to brain lysates was unaffected by competition with ligands for GABAB, glutamate, dopamine, serotonin, and other receptors, suggesting specific interaction with α2δ. Dose-finding studies in mice using acute administration of LCGA-17 (i.p.) demonstrated anxiolytic-like effects in the open field test, elevated plus maze, and marble burying tests, as well as antidepressant-like properties in the forced swim test. The anxiolytic effects were effectively blocked by bicuculline. Therefore, LCGA-17 is a novel candidate anxiolytic and antidepressant that may act through α2δ, with possible synergism by GABAA receptors.

本研究旨在开发更优质的抗焦虑药与抗抑郁药。我们聚焦于γ-氨基丁酸A型受体（GABAA receptors）以及电压门控钙离子通道（V-gated Ca2+ channels）的α2δ辅助亚基作为潜在靶点，因为上述靶点已被证实为有效抗焦虑药、抗抑郁药及抗惊厥药的作用介质。我们进一步将候选配体聚焦于短肽，因其具备优异的安全性与耐受性特征。我们采用结构生物信息学方法，开发出对GABAA受体与α2δ亚基具有预测结合亲和力的新型四肽。针对其中一种肽LCGA-17的计算机虚拟对接（in silico docking）研究显示，其对GABAA受体与α2δ亚基均具有较高结合得分，且在斑马鱼（Danio rerio）行为学筛选中展现出类抗焦虑活性。LCGA-17在新水箱实验、明暗箱实验与社交偏好实验中均表现出类抗焦虑效应，其疗效可与氟伏沙明（fluvoxamine）及地西泮（diazepam）相媲美。采用大鼠脑细胞膜进行的结合实验显示，α2δ亚基的加巴喷丁类配体相比GABAA受体配体，能更有效地竞争结合[3H]-LCGA-17，这表明α2δ亚基可能是LCGA-17的作用靶点。[3H]-LCGA-17与脑裂解物的结合不受GABAB受体、谷氨酸、多巴胺、5-羟色胺及其他受体配体的竞争影响，提示其与α2δ亚基存在特异性相互作用。对小鼠进行腹腔注射（i.p.）LCGA-17的剂量探索研究显示，其在旷场实验、高架十字迷宫实验与埋珠实验中均表现出类抗焦虑效应，同时在强迫游泳实验中展现出类抗抑郁活性。荷包牡丹碱（bicuculline）可有效阻断其抗焦虑效应。综上，LCGA-17是一种新型候选抗焦虑与抗抑郁药物，可能通过α2δ亚基发挥作用，且可能与GABAA受体存在协同效应。