CN114788830B - 一种能够抑制弓形虫增殖的小分子抑制剂的应用 - Google Patents
一种能够抑制弓形虫增殖的小分子抑制剂的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域，公开了一种能够抑制弓形虫增殖的小分子抑制剂的应用。本发明能够抑制弓形虫增殖的小分子抑制剂包括小分子化合物CGI‑1746，CGI‑1746的分子式为分子量为579.69。本发明所述CGI‑1746是一个低毒且高效的抗弓形虫药物，细胞活力实验表明10μM浓度下CGI‑1746对宿主细胞无毒，空斑实验证实5μM浓度下CGI‑1746明显抑制了弓形虫的增殖，而宿主细胞生长正常，胞内增殖实验进一步表明在5μMCGI‑1746作用下，弓形虫在宿主细胞内增殖缓慢甚至不分裂。不仅如此，出胞实验表明CGI‑1746还抑制弓形虫出胞能力。
Description
技术领域
本发明涉及生物医药技术领域，具体是涉及一种能够抑制弓形虫增殖的小分子抑制剂的应用。
背景技术
刚地弓形虫(Toxoplasma gondii)是一种专性寄生于几乎所有有核细胞内的寄生性原虫，与疟原虫同属顶复门、孢子虫纲。目前，全球约1/3人口感染弓形虫，严重者可致死。宿主免疫功能受损或抑制时，造成全身多器官炎症反应，是导致感染者死亡的主要原因之一。对于妇女而言，当孕期首次感染时可引起流产、死胎或畸型等不良后果。
弓形虫感染其他哺乳类动物后同样可致病畜死亡、流产等(如猪弓形虫病暴发时，整个猪场死亡率可高达70％以上)，给畜牧业带来巨大经济损失。不容忽视的是，感染的动物食/制品还是该病重要的传播途径，约30％～63％人群感染是通过食入含弓形虫包囊的肉类所致。防控弓形虫病在公共卫生和畜牧生产上均有重要意义。
目前并没有有效用于人群的弓形虫疫苗，对弓形虫病的治疗仍以化学药物为主，如乙胺嘧啶、磺胺嘧啶、克林霉素、乙酰螺旋霉素、阿托伐醌等。然而，这些药物都存在副作用强、根治率低、疗程长等缺陷，且耐药株的报道逐年增加。并且这些药物虽能抑制虫体增殖，但却同样具有诱导缓殖子形成的作用，增加了隐性感染的机率。显然，找到一种高效、低毒的药物是弓形虫病防治工作中亟需解决的问题。
发明内容
本发明的目的是为了克服上述背景技术的不足，提供一种高效、低毒抗弓形虫的药物，该药物作为小分子抑制剂能够有效抑制弓形虫的生长，具有应用于治疗刚地弓形虫病的前景。
为达到本发明的目的，本发明能够抑制弓形虫增殖的小分子抑制剂包括小分子化合物CGI-1746，CGI-1746的分子式为
分子量为579.69。
针对国家新药筛选中心的37874个小分子药物，本发明前期通过对数据库信息分析筛选出152个药物，再结合细胞学实验最终筛选出小分子化合物 CGI-1746，确定这种药物对宿主细胞无毒(或低毒)、而对弓形虫具有杀伤作用。
与现有技术相比，本发明的优点如下：
(1)由于弓形虫属于胞内寄生虫，抗弓形虫药物应满足膜通透性好、特异性高、对宿主细胞毒性小等条件。本发明中的CGI-1746属于分子量小于 1000的小分子药物，具有使用广泛、理论成熟等优势，其结构具有良好的空间分散性，其化学性质决定了其良好的成药性能和药物代谢动力学性质。
(2)本发明中的CGI-1746为Bruton酪氨酸激酶(Btk)的强效高选择性小分子抑制剂，可有效地抑制刚地弓形虫的增殖，目前主要用于治疗B细胞恶性肿瘤，属于“老药新用”范畴，相比创新药物，缩短研发周期，后期研发成本更经济、风险更低。且相比新药研发，采用“老药新用”策略具有副作用小等优点。
附图说明
图1为本发明针对国家新药筛选中心的37874个小分子药物进行数据库信息分析后筛选出了152个小分子药物进行细胞学实验，以虫体抑制率60％为下限，筛选出了38个小分子抑制剂；
图2为本发明针对38个小分子抑制剂进一步进行细胞活力试验，以细胞活力95％为下限，筛选出了7个对细胞低毒的小分子抑制剂；
图3为本发明具体实施例空斑实验评价CGI-1746抑制弓形虫增殖能力图；
图4为本发明具体实施例胞内增殖实验评价CGI-1746抑制弓形虫细胞内增殖能力图，附图中1、2、4、8及16表示每一个纳虫泡内虫体数量，由于弓形虫为二分裂增殖，所以含虫体数量多的纳虫泡占比越高，表示其胞内增殖能力越强；
图5为本发明具体实施例出胞实验评价CGI-1746抑制弓形虫出胞能力图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。应当理解，以下描述仅仅用以解释本发明，并不用于限定本发明。
本文中所用的术语“包含”、“包括”、“具有”、“含有”或其任何其它变形，意在覆盖非排它性的包括。例如，包含所列要素的组合物、步骤、方法、制品或装置不必仅限于那些要素，而是可以包括未明确列出的其它要素或此种组合物、步骤、方法、制品或装置所固有的要素。
当量、浓度、或者其它值或参数以范围、优选范围、或一系列上限优选值和下限优选值限定的范围表示时，这应当被理解为具体公开了由任何范围上限或优选值与任何范围下限或优选值的任一配对所形成的所有范围，而不论该范围是否单独公开了。例如，当公开了范围“1至5”时，所描述的范围应被解释为包括范围“1至4”、“1至3”、“1至2”、“1至2和4至5”、“1至3和5”等。当数值范围在本文中被描述时，除非另外说明，否则该范围意图包括其端值和在该范围内的所有整数和分数。
本发明要素或组分前的不定冠词“一种”和“一个”对要素或组分的数量要求(即出现次数)无限制性。因此“一个”或“一种”应被解读为包括一个或至少一个，并且单数形式的要素或组分也包括复数形式，除非所述数量明显只指单数形式。
此外，下面所描述的术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不是必须针对相同的实施例或示例。而且，本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
本发明的具体实施例是用于制备抑制弓形虫增殖的小分子抑制剂的应用，具体步骤如下：
(一)初筛实验之虫体抑制率实验
1、HFF细胞铺96孔板，每孔加100μl 10％FBS无酚红培养液(完全培养液)，含1000个细胞，同时β-Gal株虫体培养液换成10％FBS无酚红培养液。
2、补加完全培养液42μl/孔。
3、加药物：18μl药物(药物终浓度10μM)，DMSO也加18μl/孔(最终稀释1000倍)。
4、感染β-Gal虫株：用1ml注射器针头抽吸培养液2-3遍，过滤去掉细胞碎片，2000rpm离心10min，计数，每孔加200个虫体，20μl完全培养液，避光封口培养72h。
5、加入CPRG：CPRG先稀释100倍(储存浓度100mM，稀释后1mM)，再每孔加入20μl(终浓度100μM)，继续避光封口培养24h。
6、570nm分光光度计检测每孔吸光度。
7、实验结果显示CGI-1746实验组OD值为0.167，对照组OD值为0.452，虫体抑制率为63.14％(实验组OD值/对照组OD值)，表明该药物对弓形虫生长具有抑制作用。
(二)初筛实验之细胞活力实验
1、HFF细胞铺96孔板，每孔加100μl完全培养液，含1000个细胞。
2、每孔补加80μl完全培养液，再每孔加20μl药物(终浓度10μM)，培养4天。
3、预稀释CellTiter96R(1份原液：4份完全培养液)，吸去孔中液体，每孔加100μl稀释好的CellTiter96R，继续培养3-4h，490nm检测OD值
4、实验结果显示CGI-1746实验组OD值为0.96，对照组OD值为0.913，细胞活力为105.15％(实验组OD值/对照组OD值)，表明该药物对宿主细胞无毒。
(三)空斑实验评价CGI-1746抑制弓形虫增殖能力
1、准备一块铺满HFF细胞的96孔板，加入约300个RH△HX虫株，每个样品做三个重复。
2、2小时后，吸取舍弃96孔板中的培养液及未入胞的虫体，加入新鲜的1％的弓形虫培养液，并加入5μM的CGI-1746，96孔板静置5天。
3、将96孔板取出，镜下观察是否形成可视化的空斑。
4、吸取舍弃孔板中的培养液，用PBS洗一次，用预冷的100％甲醇于-20 度固定10min，吸取舍弃甲醇后，孔板在室温条件下干燥过夜。
5、向干燥好的96孔板中加入1.5％结晶紫染色5min，待宿主细胞上的空斑变得清晰明显，进行拍照并利用Fiji软件(图片处理数据计算常用软件)计数空斑面积。
6、实验结果表明，静置5天后，与对照组相比，实验组的空斑面积明显减少，说明CGI-1746抑制弓形虫增殖的能力显著。
(四)胞内增殖实验评价CGI-1746抑制弓形虫胞内增殖能力
1、在虫株即将出胞的前一天消化一瓶长满HFF的25cm2细胞，对其进行计数及记录，将细胞悬液接种于带有爬片的24孔板中，每孔3.5*10^4个细胞， 37℃培养24h。
2、24h后，向24孔板中分别加入比例为细胞：虫株＝1:4的GFP-ATG8 虫株，2h后，吸取舍弃未入胞虫体，培养液更换为含相应药物浓度的1％ FBS-DMEM培养液，孔板放进37℃培养箱。
3、24h后，将24孔板从培养箱中取出，弃去培养液，用预冷的甲醇固定 10min，瑞吉氏染色20min，取8μL 50％甘油滴于载玻片上，将24孔板中的爬片倒扣于载玻片上，油镜下观察100个纳虫泡，计数每个纳虫泡内虫体数量。
4、结果显示，虫体在5μM的药物作用下，感染宿主细胞后纳虫泡内均只有1或2个虫体，几乎处于不分裂的状态，说明CGI-1746具有显著抑制弓形虫胞内增殖的作用。
(五)出胞实验评价CGI-1746抑制弓形虫出胞能力
1、观察含有大量纳虫泡的时候，更换虫体培养液，洗去已出胞的虫体，继续培养；HFF铺96孔板，每孔5000个细胞，每组三个重复，培养过夜。
2、计数虫体数量，每孔直接加入5*104个虫子，4h后，洗去未入胞的虫体，分别向孔板中加入含相应药物浓度的1％FBS-DMEM培养液，37℃培养 24h。
3、37℃预热的Ringer buffer轻轻洗孔3遍，再用Ringer buffer稀释(扎普司特)Zaprinast至57μM，每孔加入45μl，37℃5％CO2孵育20min。20min 后将96孔板立即置于冰上，将上清液分别吸入离心管，4℃500*g离心5min，进行乳酸脱氢酶(LDH)检测。
4、数据显示，实验组LDH水平明显低于对照组，P值小于0.05，结果有统计学意义。这说明在Zaprinast诱导虫体出胞条件下，实验组虫体经5μM CGI-1746处理后，相比对照组，出胞能力减弱，虫体出胞行为导致的宿主细胞膜破裂减少，所以分泌入培养液中的宿主细胞乳酸脱氢酶(LDH)减少。
综上，本发明利用空斑试验、胞内增殖实验及出胞实验进一步证实 CGI-1746能够显著抑制弓形虫生长。此外，细胞活力实验已验证CGI-1746 对宿主细胞没有毒性。
本领域的技术人员容易理解，以上所述仅为本发明的实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
Claims (1)
1.一种能够抑制弓形虫增殖的药物在制备抑制弓形虫生长的药物中的用途，其特征在于，所述药物中包含小分子化合物CGI-1746；所述药物中CGI-1746的浓度不大于10μM；所述药物中CGI-1746的浓度不小于5μM；
CGI-1746的分子式为
分子量为579.69；CGI-1746能够抑制弓形虫生长。
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| CN105102000A (zh) * | 2012-11-01 | 2015-11-25 | 无限药品公司 | 使用pi3激酶亚型调节剂的癌症疗法 |
| CN113813268A (zh) * | 2020-06-18 | 2021-12-21 | 苏州亚盛药业有限公司 | 包含Bcl-2抑制剂或Bcl-2/Bcl-xL抑制剂的组合产品及其用途 |
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| CN113813268A (zh) * | 2020-06-18 | 2021-12-21 | 苏州亚盛药业有限公司 | 包含Bcl-2抑制剂或Bcl-2/Bcl-xL抑制剂的组合产品及其用途 |
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