CN114540376A - 一种抗肿瘤聚酮类化合物及其制备方法和用途 - Google Patents
一种抗肿瘤聚酮类化合物及其制备方法和用途 Download PDFInfo
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Abstract
本发明公开了化合物angumycinones C及其制备方法和应用。本发明构建了重组菌株A3(2)‑OE1(保藏编号:CCTCC M 20211134)生产包含聚酮类化合物的制备方法,同时涉及该类化合物在抗肿瘤领域的用途,angumycinones C结构式为:
Description
技术领域:
本发明涉及工业微生物领域,具体涉及Streptomyces coelicolor A3(2)-OE1(保藏编号: CCTCC M 20211134,保藏日期:2021年09月03日,保藏单位:中国典型培养物保藏中心, 保藏地址:湖北省武汉市武昌区八一路299号武汉大学生命科学学院,430072)生产聚酮类 化合物angumycinones C的方法;本发明还涉及该类化合物在抗肿瘤中的用途。
背景技术:
癌症是威胁人类身体健康的恶性肿瘤,是影响人们生活质量的主要难题之一。研究数据 表明,肺癌、前列腺癌以及乳腺癌等占据癌症死亡排行榜的首位。但是迄今为止仍没有一种 治疗显著的药物可以对癌症细胞进行彻底消除,以更好地对抗癌症这一顽疾。活性天然产物 因其结构多样和活性显著等特点,是当今发现药物靶点的一个主要来源,也是当今的一个研 究热点。聚酮类化合物结构复杂、活性多样,特别因其在抗肿瘤领域表现出来的良好应用前 景,颇受研究者们的青睐。
发明内容:
本发明旨在提供一种具有强烈的细胞毒抑制活性,同时对正常细胞无副作用的新化合物。
其结构式是
本发明的第一个目的是提供聚酮类化合物angumycinones C的生物合成基因簇spi,其 核苷酸序列如SEQ ID NO.1所示,包含25个基因,具体为:
负责角蒽醌骨架合成及合成过程中进行修饰的基因,即spi A、spi B、spi C、spiD、spi E、 spi F共6个基因;
spi A位于SEQ ID NO.1所示序列第23639-23944个碱基处,长度为306个碱基对,编码 酰基载体蛋白,101个氨基酸;
spi B位于SEQ ID NO.1所示序列第24020-25237个碱基处,长度为1218个碱基对,编码 聚酮链延长因子,405个氨基酸;
spi C位于SEQ ID NO.1所示序列第25234-26295个碱基处,长度为1062个碱基对,编码 Beta-酮基合酶,353个氨基酸;
spi D位于SEQ ID NO.1所示序列第26540-26869个碱基处,长度为330个碱基对,编码 环化酶,109个氨基酸;
spi E位于SEQ ID NO.1所示序列第21827-22762个碱基处,长度为936个碱基对,编码 环化酶,311个氨基酸;
spi F位于SEQ ID NO.1所示序列第22783-23565碱基处,长度为783个碱基对,编码酮 基还原酶,260个氨基酸;
编码与糖有关的基因,即spi P、spi Q、spi R、spi S、spi T、spi U、spi V、spi W共8个 基因:
spi P位于SEQ ID NO.1所示序列第6481-7314碱基处,长度为834个碱基对,编码N,N- 二甲基转移酶,277个氨基酸;
spi Q位于SEQ ID NO.1所示序列第7383-8540碱基处,长度为1158个碱基对,编码NDP- 己糖转氨酶,385个氨基酸;
spi R位于SEQ ID NO.1所示序列第8544-9134碱基处,长度为591个碱基对,编码NDP- 己糖-35-异构酶,196个氨基酸;
spi S位于SEQ ID NO.1所示序列第9147-10214碱基处,长度为1068个碱基对,编码葡 萄糖-1-磷酸胸腺嘧啶转移酶,355个氨基酸;
spi T位于SEQ ID NO.1所示序列第10211-11227碱基处,长度为1017个碱基对,编码 dTDP-葡萄糖-4,6-脱水酶,338个氨基酸;
spi U位于SEQ ID NO.1所示序列第11224-12528碱基处,长度为1305个碱基对,编码 NDP-己糖-3,4-脱水酶,434个氨基酸;
spi V位于SEQ ID NO.1所示序列第12525-13907碱基处,长度为1383个碱基对,编码 NDP-己糖-2,3-脱水酶,460个氨基酸;
spi W位于SEQ ID NO.1所示序列第13907-14866碱基处,长度为960个碱基对,编码NDP- 己糖-3-酮基还原酶,319个氨基酸;
编码氧化还原酶的基因是spi H1,spi H2,spi H3:
spi H1位于SEQ ID NO.1所示序列第269-985碱基处,长度为717个碱基对,编码蒽酮单 加氧酶,238个氨基酸;
spi H2位于SEQ ID NO.1所示序列第19497-21824碱基处,长度为2328个碱基对,编码 FAD依赖的单加氧酶,775个氨基酸;
spi H3位于SEQ ID NO.1所示序列第27076-28551碱基处,长度为1476个碱基对,编码 FAD依赖的单加氧酶,491个氨基酸;
编码糖基转移酶的基因是spi L,spi N:
spi L位于SEQ ID NO.1所示序列第1030-2346碱基处,长度为1317个碱基对,编码假定 的糖基转移酶,438个氨基酸;
spi N位于SEQ ID NO.1所示序列第3212-4345碱基处,长度为1134个碱基对,编码糖基 转移酶,377个氨基酸;
编码与初级代谢相关的基因是spi I,spi J,spi K:
spi I位于SEQ ID NO.1所示序列第14925-16148碱基处,长度为1224个碱基对,编码甲 硫氨酸腺苷转移酶,407个氨基酸;
spi J位于SEQ ID NO.1所示序列第16150-17133碱基处,长度为984个碱基对,编码假定 的腺苷酸激酶,327个氨基酸;
spi K位于SEQ ID NO.1所示序列第17130-17999碱基处,长度为870个碱基对,编码亚 甲基四氢叶酸还原酶,289个氨基酸;
spi O位于SEQ ID NO.1所示序列第4421-6040碱基处,长度为1620个碱基对,编码自抗 性基因,539个氨基酸;
spi M位于SEQ ID NO.1所示序列第2343-3116碱基处,长度为774个碱基对,编码甲基 转移酶,257个氨基酸;
spi G位于SEQ ID NO.1所示序列第18378-19274碱基处,长度为897个碱基对,编码硫 酯酶,298个氨基酸;
SEQ ID NO.1的第1位到28551位的碱基序列的互补序列可根据DNA碱基互补原则随时 得到。SEQ ID NO.1的第1位到28551位的的核苷酸序列或部分核苷酸序列可以通过用合适 的限制性内切酶酶切得到。本发明提供了得到至少包含部分SEQ ID NO.1的第1位到28551 位中DNA序列的重组DNA载体的途径。
本发明的式Ⅰ化合物可通过微生物发酵培养来获取含有该类化合物的发酵物,然后对发 酵粗提物采用Sephadex LH20凝胶柱层析、中压MPLC和半制备HPLC等方法分离纯化得到。
本发明的第二个目的是提供新的聚酮类化合物angumycinones C,其结构式如式(I)所示:
本发明的三个目的是提供聚酮类化合物angumycinones C或其药用盐在制备抗肿瘤药物 中的应用
本发明的下述实施例中列举了利用S.coelicolor A3(2)-OE1制备本发明式Ⅰ化合物的实 例。
附图说明:
图1是重组菌株A3(2)-OE1粗提物的分析信息;
分析条件:色谱柱为Capcell Park C18柱:5μ,20mm×250mm,流动相包括A相和B相, 流动相A相:色谱甲醇+1‰(体积分数)的三氟乙酸;流动B相:水+1‰(体积分数)的三氟乙酸; 进样程序:0-60min,流动相比例为A相/B相(体积比):95:5-0:100,5-45min,检测波长190-600 nm,流速1mL/min,其中1代表化合物I。
图2是angumycinones C的关键的二维信号;
图3是angumycinones C的假定生合成路线;
具体实施方式:
在如下的实施例中所指的化合物Ⅰ的化学结构(结构式中的阿拉伯数字是化学结构中碳 原子的标位)是:
以下实施例是对本发明的进一步说明,而不是对本发明的限制。
实施例1聚酮类化合物angumycinones C的生物合成基因簇的异源表达
1.Streptomyces sp.HDN15129基因组序列扫描及angumycinones C的生物合成基因簇序列 分析和功能分析:
通过对Streptomyces sp.HDN15129进行全基因组测序,并上传到antiSMASH上进行分析, 同时结合基因注释,在其中找到了29kb的与该化合物生物合成有关的基因簇,包含了20个 开放阅读框(open reading frames,ORFs)(表1)。根据生物信息学分析spi A、spi B、spi C、 spi D、spi E、spi F共6个基因负责聚酮链骨架合成及合成过程中的修饰;spi P、spi Q、spi R、spi S、spi T、spi U、spi V、spi W共8个基因负责编码与糖有关的基因;spi H1,spi H2, spi H3负责编码氧化还原酶的基因。该化合物生物合成途径初步推测如图3所示。
表1.angumycinones C生物合成基因簇的基因及其功能分析
实施例2化合物Ⅰ的重组菌株构建、发酵生产及分离精制
1.重组菌株的构建
(1)采用放线菌基因组提取的方法来提取Streptomyces sp.HDN15129放线菌基因组;
(2)使用MfeI/MseI限制性内切酶酶切基因组,来释放目的基因簇spi;同时将之前构 建好的引物来扩增克隆载体p15A-spi。
(3)使用Red/ET重组工程技术将胶回收后的克隆载体和酶切后的线性基因组构建目 的质粒,并通过转化子筛选得到正确的质粒。而后将正确的质粒重转化至GB05大肠中,以消除背景的影响。
(4)将重转化的正确质粒通过线环重组(linear plus circular homologousrecombination,LCHR)的方法添加强启动子kasOp*,以启动目的基因簇的表达。并通过该方法添加位点特异性重组元件pR6K-oriT-phiC31-kasOp*,最后将该质粒电转化入ET12567/PUZ8002中。
(5)链霉菌(S.coelicolor)A3(2)在MS[培养基组成(克/升):豆粉20g,甘露醇20g,琼脂粉20g,pH调节至7.2]平板中划线培养5-7天,长出的孢子用无菌棉签刮下孢子置 于50mL离心管中,50℃热激10min自然冷却的菌株作为接合转移的受体菌。供体菌E.coliET12567/pUZ8002/p15A-spi在100m L含50μg/mL卡那霉素、25μg/m L氯霉素和50μg/mL 阿泊拉霉素的LB液体培养基中于37℃生长至OD600值约为0.6-0.8时,离心收集菌体(9500rpm,1min),用无抗LB清洗菌体3遍,悬浮于1mL LB培养基中,作为接合转移 的供体菌。取上述受体菌400μL和供体菌200μL混合均匀,涂布于不含任何抗生素的 MS固体培养基上,吹干后,于30℃培养16-20h。然后将平板取出,用含有抗生素的水 覆盖平板,其终浓度为50μg/mL阿泊拉霉素和25μg/mL萘啶酮酸,吹干后,于30℃培养 箱中,培养10-20天后观察。
(6)当接合转移平板上长出小菌落后,用无菌牙签将其转接到含有25μg/mL阿泊拉霉素和 50μg/mL萘啶酮酸的MS平板上,30℃培养10-15天后,抽取各个突变株的基因组DNA,利用异源表达的检测引物spiF/R(引物序列见于表2)通过PCR检测获得阳性克隆,即获得angumycinones C生物合成基因簇异源表达菌株A3(2)-OE1。
表2.检测引物名称和序列
Primer | Sequences(5’-3’) |
spiF | GCAGGCTGCTGACGTGCGTG |
spiR | GTGTCCGAGGTGACCGGGAG |
2.发酵生产
生产菌的发酵培养:按培养微生物的常规方法,取S.coelicolor A3(2)-OE1适量,接种到 MS-Apra[添加阿泊拉霉素终浓度50μg/mL]的固体斜面培养基上,在30℃培养箱中培养10天。
取斜面培养10天的S.coelicolor A3(2)-OE1适量,接种到装有100mL培养基[培养基组 成(克/升):可溶性淀粉10g,蛋白胨2g,酵母提取物4g,pH调节至7.2]的500mL锥型 瓶中,在30℃,200rpm条件下摇床培养8天,获得发酵产物。
3.浸膏的获得
发酵液用纱布过滤得到上清液。用等量乙酸乙酯萃取三次,合并所有乙酸乙酯相,减压 浓缩得到粗浸膏,共12克。
4.化合物的分离精制
浸膏用甲醇溶解后,使用95%甲醇溶解,使用石油醚萃取,出去油脂成分,蒸干后以甲 醇-水为洗脱体系进行反相层析柱层析,分为7个流份。组分5先以甲醇为流动相进行sephadexG-200分子筛洗脱,后再经反相半制备高效液相色谱(甲醇:水=46:54)得化合物Ⅰ(4.6mg)。
化合物Ⅰ为亮黄色针状固体,分子式C19H16O7,HR-ESI-MS m/z:355.0824[M-H]-,计算 值355.0823);IR(KBr)νmax 3399,1699,1683,1653,1577,1541,1488,1207,1136,1027cm-1。 1H and 13C NMR核磁数据归属见表2。
表2化合物Ⅰ的1H和13C NMR数据(600和150MHz,in DMSO-d6)a
a)本表信号归属基于DEPT、HMQC及HMBC图谱解析结果。碳信号的多重度利用DEPT方法确定并分别用s(单重峰)、d(二重峰)、t(三重峰)和q(四重峰)、m(多重峰) 表示。
b)此栏中的数字和代号分别代表在1H-1H COSY谱中与相应行中的1H给出偶合相关信号的 1H核。
c)此栏中的数字和代号分别代表在HMBC谱中与相应行中的1H给出偶合相关信号的13C 核。
实施例2化合物的细胞毒活性测试
1实验样品及实验方法
被测样品溶液的配制:测试样品为上述实施例1中分离精制的化合物Ⅰ纯品。精密称取 适量样品,用DMSO配置成30μM的母液,然后采用二倍稀释法稀释成15,7.5,3.75,1.875,0.9375μM的待测溶液,阳性药阿霉素(ADM)配置成1μM,供测活性。
供测细胞:L-02、H69AR(SRB法)。
测试方法:SRB实验方法如下检测及数据分析
(1)细胞处理:贴壁细胞,胰酶消化对数期细胞,制成细胞悬液。
(2)细胞计数:使用计数板和计数器,吸取10-20μL,计四个角上16个方格细胞的平均数x(x 大致20-50比较好),40÷x=y,y为一个板需要加的细胞的毫升数,每个板一共需要6mL,6 -y=z,z为需要加的培养基的体积(计数原则,计上不计下,计左不计右)。(每个96孔板的 细胞数量在38-41万)
(3)细胞铺板:96孔板中每孔加入90μL细胞悬液
(4)加样品:细胞贴壁生长24h后,每孔加入10μL不同浓度的化合物溶液。
(5)细胞放入37℃培养箱培养
(6)72h后,去掉培养基,每孔加入100μL TCA溶液,4℃固定1h以上,自来水冲洗5-6遍,自然晾干,每孔加入100μL SRB溶液进行染色,染色5-10min后,去除SRB,1%的 冰醋酸溶液冲洗5-6遍,自然晾干,干燥之后加入150μLTris溶液,震荡摇匀,用酶标仪在 515nm测吸光度。
2实验结果
在细胞毒活性测试中,不同浓度的化合物I对所测试的肿瘤细胞的实验结果见表3。
表3不同浓度的化合物I对H69AR细胞的抑制率
3结论
化合物Ⅰ具有强烈的肿瘤细胞增殖抑制活性,作为新型抗肿瘤化合物结构在癌症治疗领 域具有良好的应用前景。
Claims (6)
1.一种聚酮类化合物angumycinones C的生物合成基因簇,其特征在于,其核苷酸序列如SEQ ID NO.1所示。
3.一种重组菌株streptomyces coelicolor A3(2)-OE1,其特征在于,是用南海海泥来源的放线菌Streptomyces sp.HDN15129进行全基因组测序后,经过生物信息学分析,使用Red/ET重组的方法将其中的typeII PKS导入到异源宿主S.coelicolor A3(2)中,得到重组菌株A3(2)-OE1,保藏编号:CCTCC M 20211134。
4.权利要求2所述化合物的制备方法,其特征在于包括以下步骤:天蓝色链霉菌Streptomyces coelicolor A3(2)-OE1先在含有抗生素Apra 50μg/ml的MS固体培养基上于30℃培养箱中培养10天,再次接种于液体发酵培养基中对该菌株进行30℃,200rpm摇床条件发酵培养8天;发酵液过滤得到发酵液,用等量乙酸乙酯萃取3次,合并所有乙酸乙酯相,减压浓缩得到粗浸膏。
5.权利要求2所述化合物的精制纯化方法,将权利要求4所述粗浸膏通过C-18ODS反相柱梯度分离,以甲醇/水为流动相进行梯度洗脱;再经sephadexG-200分子筛洗脱,以甲醇为流动相进行洗脱;最后经反相半制备高效液相色谱分离纯化得权利要求1所述化合物,制备梯度为甲醇:水=46:54,其中所述发酵培养基成分为可溶性淀粉10g,蛋白胨2g,酵母提取物4g,水1L,pH 7.2-7.4。
6.权利要求2所述化合物在制备抗肿瘤药物中的用途。
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