CN105687187A - 化合物Pyrrocidines在制备抗结核病的药物中的应用 - Google Patents
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Abstract
本发明提供了化合物Pyrrocidines在制备抗结核病的药物中的应用，即化合物Pyrrocidines或其衍生物或其药学上可接受的盐在作为或制备酪氨酸磷酸酶抑制剂的应用，化合物19-O-methyl-pyrrrocidine B和Pyrrocidine B对结核分枝杆菌酪氨酸磷酸酶B具有抑制活性，其IC50分别为39.1μM，75.5μM。所述化合物能够有效地抑制结核分枝杆菌酪氨酸磷酸酶B的去磷酸化活性，具有结核病治疗的临床应用潜力，另外，化合物Pyrrocidines是从微生物分离的天然代谢活性小分子产物，可以通过化学合成生产，也可通过大规模发酵分离生产，来源丰富，生产成本低，成药可能性高。
Description
技术领域
本发明涉及生物学技术领域，更具体地，涉及化合物Pyrrocidines在制备抗结核病的药物中的应用。
背景技术
结核病是由结核分枝杆菌引起的慢性传染病，是全世界最主要的致命传染病之一。根据世界卫生组织2014全球结核病控制报告，在2013年，共有900万人患结核病，150万人死亡。虽然在过去的二十年，中国结核病例及死亡率持续大幅下降，但中国仍是世界十大结核病高发国家之一，且耐药性结核病呈不断上升的趋势。目前，结核病防治形势仍十分严峻。
结核分枝杆菌是一种胞内病原菌，细菌进入肺部后会被巨噬细胞吞噬，在细胞内生长增殖。结核分枝杆菌能够通过向细胞内分泌效应蛋白，调整宿主的信号途径，让自已能够躲避宿主的免疫反应，成功在宿主内生长繁殖。可逆的蛋白磷酸化调控多种生理生化反应，蛋白磷酸酶是结核分枝菌的关键分子，不仅调控微生物自身的代谢，还干扰宿主细胞的信号转导。这在结核菌逃避宿主的免疫效应、阻止吞噬体与溶酶体融合中起着重要的作用。结核分枝杆菌酪氨酸磷酸酶B（Mycobacteriumtuberculosisproteintyrosinephosphatase,MptpB），是结核分枝杆菌侵染巨噬细胞所必须的分泌型毒力因子，具有酪氨酸磷酸酶活性，介导结核分枝杆菌在宿主中的存活。结核分枝杆菌酪氨酸磷酸酶B的核苷酸序列含有831bp（Rv0153c），编码276个氨基酸，分子量约为31kDa。MptpB拥有磷酸酶保守的CX5R序列，由Cys160、Lys164、Asp165、Arg166构成活性位点P环，其磷酸化功能在干扰宿主信号途径起着巨大作用。MptpB进入巨噬细胞后，干扰P38、ERK1/2，抑制IL-6的产生，降低宿主的免疫应答。另外，对PI3P去磷酸化，抑制吞噬体成熟。MptpB还能调节Caspase-3及Akt的活性，抑制细胞凋亡进程。因此，MptpB为结核分枝杆菌成功在宿主中持留、增殖发挥重要作用。
MptpB目前被认为是抗结核药物筛选的新靶点，抑制结核分枝杆菌分泌MptpB，可以组织结核分枝杆菌对宿主免疫产生抑制作用，有助于宿主对结核杆菌产生免疫，从而达到治疗结核病的目的。现有技术比较缺乏新的抗结核药物的筛选。
发明内容
本发明所要解决的技术问题是克服现有技术中存在的上述缺陷，提供一种新的结核分枝杆菌酪氨酸磷酸酶B的抑制剂，即化合物Pyrrocidines。
本发明提供化合物Pyrrocidines的应用。
本发明的目的是通过以下技术方案予以实现的：
化合物Pyrrocidines或其衍生物或其药学上可接受的盐作为酪氨酸磷酸酶抑制剂的应用，其特征在于，所述化合物Pyrrocidines的结构式为式（I）或式（II），
本发明通过实验研究发现，式（I）或式（II）所述化合物能够抑制酪氨酸磷酸酶的去磷酸化活性，因此，式（I）或式（II）所述化合物及其衍生物或其药学上可接受的盐能够作为酪氨酸磷酸酶抑制剂。本发明所述化合物Pyrrocidines可以是来源于微生物天然代谢活性产物。
化合物Pyrrocidines或其衍生物或其药学上可接受的盐在制备酪氨酸磷酸酶抑制剂的应用，其特征在于，所述化合物Pyrrocidines的结构式为式（I）或式（II），
优选地，所述酪氨酸磷酸酶为结核分枝杆菌酪氨酸磷酸酶B。
本发明还提供化合物Pyrrocidines或其衍生物或其药学上可接受的盐在制备抗结核病的药物中的应用，其特征在于，所述化合物Pyrrocidines的结构式为式（I）或式（II），
当酪氨酸磷酸酶B抑制率为50~70%时，式（I）的浓度为39.1uM~81.8uM，式（II）的浓度为75.5~181.2uM。
与现有技术相比，本发明具有以下有益效果：
本发明提供了化合物Pyrrocidines或其衍生物或其药学上可接受的盐在作为或制备酪氨酸磷酸酶抑制剂的应用，化合物19-O-methyl-pyrrrocidineB和PyrrocidineB对结核分枝杆菌酪氨酸磷酸酶B具有抑制活性，其IC50分别为39.1μM，75.5μM。所述化合物能够有效地抑制结核分枝杆菌酪氨酸磷酸酶B的去磷酸化活性，具有结核病治疗的临床应用潜力，另外，化合物Pyrrocidines是从微生物分离的天然代谢活性小分子产物，其结构已被解析，可以通过化学合成生产，也可通过大规模发酵分离生产，来源丰富，生产成本低，成药可能性高。
附图说明
图1为从结核分枝杆菌H37Ra基因组中PCR扩增MptpB基因片段的琼脂糖凝胶电泳检测图；其中M为核酸标准分子量（fermentas1KbDNAladder），b为831bp的结核分枝杆菌酪氨酸磷酸酶B基因片段。
图2为表达带组氨酸标签的结核分枝杆菌酪氨酸磷酸酶B融合蛋白纯化后的SDS-PAGE分析结果；其中M为蛋白质分子量标准（Thermo26614），1为未加IPTG诱导的大肠杆菌菌体对照，2为加入IPTG诱导MptpB表达的菌体，3为超声破碎上清液，4为穿流峰蛋白，5为杂蛋白，6是纯化后的MptpB。
图3为19-O-methyl-pyrrrocidineB对结核分枝杆菌酪氨酸磷酸酶B酶活性抑制的IC50曲线；横坐标是化合物浓度，纵坐标是抑制率，IC50值是39.1μM。
图4为PyrrocidineB对结核分枝杆菌酪氨酸磷酸酶B酶活性抑制的IC50曲线；横坐标是化合物浓度，纵坐标是抑制率，IC50值是75.5μM。
具体实施方式
下面结合说明书附图和具体实施例对本发明作进一步的解释说明，但具体实施例并不对本发明作任何限定。除非特别说明，实施例中所涉及的试剂、方法均为本领域常用的试剂和方法。本发明对化合物的来源不做任何形式的限定。
化合物19-O-methyl-pyrrrocidineB和PyrrocidineB用DMSO进行溶解，母液浓度为10mM，在室温避光下保存。
实施例1结核分枝杆菌酪氨酸磷酸酶B的融合表达
（1）结核分枝杆菌酪氨酸磷酸酶B的基因克隆，按照如下步骤进行：
根据抑制结核分枝杆菌酪氨酸磷酸酶B的DNA序列，设计并合成一对引物，在上、下游引物分别加上NdeⅠ、XhoⅠ的酶切位点，以结核分枝杆菌H37Ra的基因组DNA为模板，用上述的引物经PCR扩增出831bp长的结核分枝杆菌酪氨酸磷酸酶B的DNA片段。电泳并回收PCR扩增的DNA片段。
（2）表达结核分枝杆菌酪氨酸磷酸酶B载体的构建：
提取质粒pET28a(+)，用NdeⅠ、XhoⅠ双酶切质粒和结核分枝杆菌酪氨酸磷酸酶B的DNA片段，电泳后回收。回收后的产物，按质粒载体：基因片段为3:1的摩尔浓度比例混匀，加入T4DNA连接酶，室温连接2小时。
（3）重组质粒转化与筛选
上述步骤（2）的连接产物转化大肠杆菌DH5α，并涂布于含卡那霉素（50μg/mL）固体LB培养基上，置37°C培养过夜。次日挑取单菌落patch，置37°C培养过夜。对patch平板进行菌落PCR，鉴定阳性克隆子。经菌落PCR鉴定的阳性克隆接种到液体LB培养液，37°C震荡培养过夜。按质粒提取试剂盒提取阳性转化子的质粒，并转化到大肠杆菌BL21(DE3)，挑取阳性转化子进行后续实验。
（4）表达结核分枝杆菌酪氨酸磷酸酶B融合蛋白的表达与纯化
将上述的表达结核分枝杆菌酪氨酸磷酸酶B的大肠杆菌BL21(DE3)工程菌接种到含有卡那霉素（50μg/mL）的LB培养液，37°C震荡培养过夜获得培养物。按1:50（体积）的比例，将上述培养物接种于含50μg/mL卡那霉素的新鲜LB培养液中，37°C震荡培养至OD600=0.6，加入终浓度为0.1mM的IPTG，于20°C震荡培养过夜，诱导融合蛋白的表达。
（5）重组蛋白的提取与纯化：
将培养的表达结核分枝杆菌酪氨酸磷酸酶B的工程菌离心（5000×g，4°C，离心5min），收集菌体，并将菌体重悬于20mL裂解缓冲液（25mMTris，20mM咪唑，500mMNaCl，pH7.8），再次离心。重复上述步骤一次，以洗去培养基。使用5mL裂解缓冲液重悬菌体，并加入100μLTritonX-100，50μL1MDTT，5mL蛋白酶抑制剂，冰浴超声15min，离心（12000rpm，20min，4°C），收集上清液。利用镍螯合琼脂糖凝胶亲和层析纯化，将超声破碎离心的上清液加入镍层析柱，收集流出的穿流峰蛋白，然后用洗涤缓冲液（25mMTris，50mM咪唑，500mMNaCl，pH7.8）将非特异结合的杂蛋白洗去。最后，用洗脱缓冲液（25mMTris，350mM咪唑，500mMNaCl，pH7.8）洗脱目的蛋白。将蛋白洗脱液加入到孔径为10kDa的超滤管中，5000×g，4°C离心15min进行浓缩，弃去管底液体，加入适量蛋白保存缓冲液（25mMTris，100mMNaCl，pH7.8）5000×g，4°C离心15min，将洗脱蛋白的咪唑置换。离心直到滤膜上的液体为2mL，吸出于4°C保存。采用BCA蛋白浓度测定试剂盒（Pierce）测定MptpB浓度。
实施例2化合物Pyrrocidines对结核分枝杆菌酪氨酸磷酸酶B的抑制酶活性分析
以对硝基苯磷酸二钠（pNPP）为底物，对重组结核分枝杆菌酪氨酸磷酸酶B进行酶活性分析，所用的结核分枝杆菌酪氨酸磷酸酶B是实施例1纯化得到的重组结核分枝杆菌酪氨酸磷酸酶B融合蛋白。储存于-20°C的500mMpNPP母液置于冰上溶解，用反应缓冲液（50mMTris，100mMNaCl，pH7.8）对pNPP和MptpB进行适量稀释。在96孔板中依次加入终浓度为6μg/mL的MptpB，浓度梯度的化合物（0μM，3.9μM，7.8μM，15.6μM，31.25μM，62.5μM，125μM，250μM），1.5mM的pNPP，反应总体积为200μL。37°C反应5min，通过光吸收酶标仪读取405nm下各孔的吸收值。根据吸光值按照以下计算方法算出化合物对结核分枝杆菌酪氨酸磷酸酶B酶活性的抑制率：抑制率（%）=（实验组-空白组）/（阴性对照-空白组）。以化合物浓度的对数为横坐标，酶活性抑制率为纵坐标作图，由OriginPro8软件拟合曲线和计算IC50，测出化合物19-O-methyl-pyrrrocidineB和PyrrocidineB对结核分枝杆菌酪氨酸磷酸酶B抑制的IC50分别为39.1μM，75.5μM。
对比例1
实验方法同实施例2，唯一不同的是，所用的化合物为下列结构的化合物（式III），结果表明：100μM浓度的A240-A对MptpB的抑制率只有8%，即式III所述化合物对MptpB酶活性没有抑制作用。
式（III）。
Claims (5)
1.化合物Pyrrocidines或其衍生物或其药学上可接受的盐作为酪氨酸磷酸酶抑制剂的应用，其特征在于，所述化合物Pyrrocidines的结构式为式（I）或式（II），
。
2.化合物Pyrrocidines或其衍生物或其药学上可接受的盐在制备酪氨酸磷酸酶抑制剂的应用，其特征在于，所述化合物Pyrrocidines的结构式为式（I）或式（II），
。
3.根据权利要求1或2所述的应用，其特征在于，所述酪氨酸磷酸酶为结核分枝杆菌酪氨酸磷酸酶B。
4.化合物Pyrrocidines或其衍生物或其药学上可接受的盐在制备抗结核病的药物中的应用，其特征在于，所述化合物Pyrrocidines的结构式为式（I）或式（II），
。
5.根据权利要求1或2或4所述的应用，其特征在于，所述式（I）的浓度为39.1uM~81.8uM，式（II）的浓度为75.5~181.2uM。
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| CN109836396A (zh) * | 2017-11-29 | 2019-06-04 | 扬州蓝色生物医药科技有限公司 | 一种新型噻唑磺酰胺类化合物及其作为抗结核药物的应用 |
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| CN112843027A (zh) * | 2021-02-03 | 2021-05-28 | 中山大学 | 2-氯-6-甲氧基间苯二酚在抑制结核分枝杆菌酪氨酸磷酸酶a中的用途 |
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| CN106701594A (zh) * | 2016-11-23 | 2017-05-24 | 西藏自治区高原生物研究所 | 一株大花绿绒蒿内生真菌DH24及其生产Pyrrocidines类化合物的应用 |
| CN109836396A (zh) * | 2017-11-29 | 2019-06-04 | 扬州蓝色生物医药科技有限公司 | 一种新型噻唑磺酰胺类化合物及其作为抗结核药物的应用 |
| CN109836395A (zh) * | 2017-11-29 | 2019-06-04 | 扬州蓝色生物医药科技有限公司 | 一种邻三氟甲基苯氧乙酰胺基噻唑磺酰胺MptpB抑制剂 |
| CN109836396B (zh) * | 2017-11-29 | 2020-08-04 | 扬州蓝色生物医药科技有限公司 | 一种新型噻唑磺酰胺类化合物及其作为抗结核药物的应用 |
| CN109836395B (zh) * | 2017-11-29 | 2020-08-04 | 扬州蓝色生物医药科技有限公司 | 一种邻三氟甲基苯氧乙酰胺基噻唑磺酰胺MptpB抑制剂 |
| CN111658629A (zh) * | 2020-07-07 | 2020-09-15 | 中山大学 | Fusarielin M及其衍生物、药学上可接受的盐的应用 |
| CN112843027A (zh) * | 2021-02-03 | 2021-05-28 | 中山大学 | 2-氯-6-甲氧基间苯二酚在抑制结核分枝杆菌酪氨酸磷酸酶a中的用途 |
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