CN111671741A - 一种化合物hss-8的医药用途 - Google Patents
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Abstract
本发明公开了一种化合物HSS‑8的医药用途。本发明首次发现化合物HSS‑8具有CREB激动活性，可以用于制备CREB激动剂药物。本领域技术人员知道：CREB与神经元保护息息相关；CREB在神经元的发育、再生、突触形成及修复等中起着关键性的作用，是调节中枢神经系统功能的关键性因子之一；CREB的激活与促进学习记忆有关；CREB的激活与抗焦虑、抗抑郁有关。因此，化合物HSS‑8具有开发成神经元保护，促进神经元发育、再生、突触形成及修复，促进学习记忆，以及抗焦虑或抗抑郁药物的前景。
Description
技术领域
本发明属于医药领域，涉及已知化合物新用途，具体涉及一种化合物HSS-8的医药用途。
背景技术
化合物HSS-8是一种酰胺类化合物，其化学结构式如下：
CREB是cAMP应答元件结合蛋白(cAMP response element binding protein)的简称，它于80年代后期被发现。CREB由341个氨基酸残基组成，分子量43000。分子结构分两个区域，N端区域与调节转录的功能有关，C端区域是与启动子结合的部位。CREB是一种细胞核内调控因子，它通过自身磷酸化实现调节转录的功能。研究发现，CREB在一些神经生理活动中如神经元的发育、再生、突触形成及修复等中起着关键性的作用，是调节中枢神经系统功能的关键性因子之一。具体作用包括：
※CREB与学习记忆的关系
CREB在学习记忆中作用的最初研究来自海兔。多次对海兔尾部的电刺激可使参与其缩尾反射的感觉和运动神经元之间形成突触连接的长时程易化，并可持续数天；与之相反的是单次刺激引起的的短时程易化持续时间却很短。长时程易化过程可通过引起cAMP增加来介导CREB磷酸化，进而诱导相关基因表达，最终导致感觉神经元中产生依赖于蛋白质合成的结构改变。随后在学习记忆和可塑性分子机制的研究中发现，海兔显示出的长时程易化类似于记忆的敏化行为，而且这种长时程易化需要cAMP-CREB信号通路的参与，首次提出关于学习记忆活动依赖性基因表达的机制。在动物学习记忆过程中或学习训练后抑制mRNA和蛋白的表达将阻断记忆的储存和长时程记忆的获得。另外，对果蝇、小鼠、大鼠等学习记忆的研究表明，长时记忆的形成从非脊椎动物到哺乳动物CREB起到重要作用。果蝇长时程记忆研究中，研究者利用热冲击条件诱导一个CREB的抑制蛋白表达后，便阻断了长时程记忆的形成，相反，诱导一个CREB激活蛋白的表达，则促进长时程记忆的形成。(文献：北京大学生命科学学院余瑞元等，CREB研究进展，中国生物工程杂志，2003；上海大学生命科学学院葛军等，CREB转录因子及其磷酸化信号通路的研究进展，安徽农业科学，2010年；宋海龙等，CREB信号通路在神经系统中的调控及功能，中国生物化学与分子生物学报，2013)。
※CREB与焦虑、抑郁等情绪的关系
国内外相关研究发现，CREB在维持正常情绪中起着重要的调控作用；动物实验结果也显示，海马组织中CREB合成不足或磷酸化程度下降均可使动物出现焦虑、抑郁等一系列情绪障碍。有研究发现抑郁症患者大脑神经细胞内cANP信号通路功能紊乱，抑郁患者额叶皮层CREB的mRNA表达降低，抗抑郁剂的通过提高细胞内CREB的含量发挥抗抑郁作用。Nakagawa通过对抑郁症发病机制的研究发现，应激和抑郁症下调了cAMP CREB系统，减少了CREB及p-CREB的表达，并从而减少了脑源性神经生长因子及其受体酪氨酸激酶B的表达(参考文献：CREB与学习记忆及情绪关系，医药论坛杂志，2008)。
目前未见化合物HSS-8对CREB激活作用的报道。
发明内容
本发明旨在提供化合物HSS-8用于激活CREB进而用于制备CREB激动剂药物、用于制备神经元保护药物、用于制备促进神经元的发育、再生、突触形成及修复药物、用于制备促进学习记忆药物和用于制备抗焦虑或抗抑郁药物的用途。
本发明上述目的通过如下技术方案实现：
如下化学结构的化合物HSS-8用于制备CREB激动剂药物的医药用途：
上述化合物HSS-8用于制备神经元保护的药物的医药用途。
上述化合物HSS-8用于制备促进神经元的发育、再生、突触形成及修复的药物的医药用途。
上述化合物HSS-8用于制备促进学习记忆的药物的医药用途。
上述化合物HSS-8用于制备抗焦虑或抗抑郁的药物的医药用途。
有益效果：
本发明首次发现化合物HSS-8具有CREB激动活性，可以用于制备CREB激动剂药物。本领域技术人员知道：CREB与神经元保护息息相关；CREB在神经元的发育、再生、突触形成及修复等中起着关键性的作用，是调节中枢神经系统功能的关键性因子之一；CREB的激活与促进学习记忆有关；CREB的激活与抗焦虑、抗抑郁有关。因此，化合物HSS-8具有开发成神经元保护，促进神经元发育、再生、突触形成及修复，促进学习记忆，以及抗焦虑或抗抑郁药物的前景。
附图说明
图1为CREB相对荧光强度。
具体实施方式
下面结合附图和实施例具体介绍本发明实质性内容，但并不以此限定本发明的保护范围。
一、实验简介：
荧光素酶报告基因是指以荧光素(luciferin)为底物来检测萤火虫荧光素酶(fireflyluciferase)活性的一种报告系统。荧光素酶可以催化luciferin氧化成oxyluciferin，在luciferin氧化的过程中，会发出生物荧光(bioluminescence)。Luciferase报告基因系统是以荧光素(luciferin)为底物来检测萤火虫荧光素酶(fireflyluciferase)活性的一种报告系统。然后可以通过荧光测定仪也称化学发光仪(luminometer)或液闪测定仪测定luciferin氧化过程中释放的生物荧光。荧光素和荧光素酶这一生物发光体系，可以极其灵敏、高效地检测基因的表达。是检测转录因子与目的基因启动子区DNA相互作用的一种检测方法。
转录因子是一种具有特殊结构、行使调控基因表达功能的蛋白质分子，也称为反式作用因子。某些转录因子仅与其靶启动子中的特异序列结合，这些特异性的序列被称为顺式作用元件，转录因子的DNA结合域和顺式作用元件实现共价结合，从而对基因的表达起抑制或增强的作用。荧光素酶报告基因实验(luciferase assay)是检测这类转录因子和其靶启动子中的特异顺序结合的重要手段。
二、原理简介：
(1)构建一个将靶启动子的特定片段插入到荧光素酶表达序列前方的报告基因质粒，如pGL3-basic等。
(2)将要检测的转录因子表达质粒与报告基因质粒共转染293细胞或其它相关的细胞系。如果此转录因子能够激活靶启动子，则荧光素酶基因就会表达，荧光素酶的表达量与转录因子的作用强度成正比。
(3)加入特定的荧光素酶底物，荧光素酶与底物反应，产生荧光，通过检测荧光的强度可以测定荧光素酶的活性，从而判断转录因子是否能与此靶启动子片段有作用。
三、实验材料
待测酰胺类化合物HSS-8(化学结构如下)，HPLC纯度不低于98％。
Lucifercase Assay System Freezer Pack(报告基因试剂盒，美国Progmega,REF:E8130,LOT：0000408247)PolyJetTM In Vitro DNATransfection Reagent(DNA体外转染试剂，思科生物科技有限公司，Cat:SL100688)工具细胞：HEK293T(人肾上皮细胞)。
四、实验方法
1、荧光素酶报告基因检测化合物对CREB的激活作用
采用荧光素酶报告基因法检测，取对数生长期的293T细胞，种植于96孔板中，待细胞铺板融合度大于70％时，采用lip2000将CREB荧光素酶报告基因质粒转入293T细胞中，6h后更换完全培养，给药10μM处理，阳性药为咯利普兰，给药浓度5μM。培养18h后，弃去培养基，加入报告基因裂解液振摇裂解30min，随后加入报告基因裂解底物，使用酶标仪检测各孔的化学发光。计算CREB荧光素酶相对活力：相对活力＝(给药组响应值)/(对照组响应值)。
具体步骤如下：
(1)将293T细胞种于96孔板中，细胞计数，每孔四万，设置三复孔
(2)细胞过夜培养，细胞密度达到70-80％进行转染
(3)转染:计算所需质粒，转染试剂及DMEM稀释液所需体积，96孔板每孔20微升，质粒稀释液和转染试剂PolyJet稀释液体积比1:1，转染试剂稀释液与质粒稀释液分别轻轻混匀后，将转染试剂稀释液加入质粒稀释液中，轻轻混匀，室温静置10-15分钟，每孔加入20微升，转染6小时后换液(细胞完全培养基)
V质粒＝所需转染孔数×每孔所需转染质粒的量(ng)/质粒浓度
V转染试剂＝所需转染孔数×每孔所需转染体积(96孔板每孔0.5微升)
V质粒DMEM稀释液＝(转染孔数×20/2)-V质粒
V PolyJet转染试剂DMEM稀释液＝转染孔数×20/2-VPolyJet转染试剂体积
(4)转染过夜后加药
(5)报告基因测试:①将96孔板细胞上清液吸出；②每孔加入1×细胞裂解液100微升(5×细胞裂解液原液用纯水稀释)；③放置摇床摇30min，摇床转速80-120(摇床速度过快96孔板液体会飞溅，转速过低可能会影响细胞裂解程度)；④每孔吸取30微升用排枪混合均匀后的细胞裂解液，转移到白色不透明白板中；④避光加入1×荧光素酶底物，每孔50微升(3×荧光素酶用纯水避光稀释)；⑤使用OPTIMA软件辅助酶标仪进行化学发光测试；⑥进行数据处理。
2、数据处理和分析
将测试后的结果导入EXCEL表中，取空白组平均值，并与每组数值相比，各组比值求取平均值，即为CREB的荧光素酶相对活力，公式如下：
相对活力＝(给药组响应值)/(对照组响应值)
数据使用Graphpad7.0进行统计分析，结果以
表示，组间比较采用单因素方差分析。
五、实验结果
结果如图1所示，与阳性药一样，化合物HSS-8对CREB具有激活作用，化合物HSS-8具有成为CREB激动剂的潜力。
综上可见，化合物HSS-8具有CREB激动活性，可以用于制备CREB激动剂药物。本领域技术人员知道：CREB与神经元保护息息相关；CREB在神经元的发育、再生、突触形成及修复等中起着关键性的作用，是调节中枢神经系统功能的关键性因子之一；CREB的激活与促进学习记忆有关；CREB的激活与抗焦虑、抗抑郁有关。因此，化合物HSS-8具有开发成神经元保护，促进神经元发育、再生、突触形成及修复，促进学习记忆，以及抗焦虑或抗抑郁药物的前景。
上述实施例的作用在于具体介绍本发明的实质性内容，但本领域技术人员应当知道，不应将本发明的保护范围局限于该具体实施例。
Claims (5)
1.如下化学结构的化合物HSS-8用于制备CREB激动剂药物的医药用途：
2.权利要求1所述化合物HSS-8用于制备神经元保护的药物的医药用途。
3.权利要求1所述化合物HSS-8用于制备促进神经元的发育、再生、突触形成及修复的药物的医药用途。
4.权利要求1所述化合物HSS-8用于制备促进学习记忆的药物的医药用途。
5.权利要求1所述化合物HSS-8用于制备抗焦虑或抗抑郁的药物的医药用途。
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