CN112457250A - 一种广谱荧光底物及其制备方法和应用 - Google Patents
一种广谱荧光底物及其制备方法和应用 Download PDFInfo
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- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
本发明提供了一种尿苷二磷酸葡萄糖醛酸转移酶的广谱荧光底物及其制备方法和应用,具体为卤代苯乙基‑4‑羟基‑1,8‑萘酰亚胺衍生物。该类底物可被多种UGT酶催化生成相应的单葡萄糖醛酸产物,且产物可在360nm荧光激发条件下产生明亮的荧光发射信号。以该类广谱荧光底物,以重组表达的UGT酶为酶源,可同步实现待测化合物对十种人源UGT酶的抑制/激活能力的筛选与评价。广谱荧光底物及其荧光产物的检测可借助液相‑荧光检测系统(LC‑FD)及荧光微孔板检测体系,具有检测通量高、操作简便、廉价高效等优点,具有良好的应用前景。
Description
技术领域
本发明属于医药技术领域,具体涉及一种广谱荧光底物,尤其涉及一种尿苷二磷酸葡萄糖醛酸转移酶(UGT)的广谱荧光底物及其制备方法,及其在UGT酶抑制剂/激活剂筛选与评价中的应用,以及在高通量筛选及评价UGT酶抑制剂/激活剂的应用。
背景技术
尿苷二磷酸葡萄糖醛酸转移酶(UGT)超家族是机体内重要的Ⅱ相代谢酶,其通过催化化合物与辅因子尿苷二磷酸葡萄糖醛酸(UDPGA)结合,从而增加底物的亲水性,使化合物能更有效地从尿液或胆汁中排出体外,这是机体的一个重要的解毒过程。哺乳动物的UGT可分为4个家族:UGT1,UGT2,UGT3和UGT8。其中参与药物结合代谢的多为UGT1A和UGT2B亚家族的成员。
作为机体最重要的II相代谢酶,UGT酶在各种异源生物(如治疗药物及其Ⅰ相代谢产物,食品化学成分或环境毒素)的代谢清除和解毒中发挥了关键作用。除异源物代谢外,UGT酶还参与许多内源性物质(如胆红素,类固醇和胆汁酸)的葡萄糖醛酸化代谢。当UGT酶出现功能障碍或被强效抑制后则可能会引发内源物代谢障碍及药物/草药相互作用(DDI/HDI),给人类健康带来不良影响。反之,部分UGT酶(如UGT1A1)的激活剂可加速胆红素等毒物的代谢清除,进而缓解疾病的发生发展进程。因此,业界一直期望能开发出UGT酶抑制剂/激活剂的高效筛选与评价方法。
由于UGT酶氨基酸序列的高度一致导致其底物谱高度重叠,因此UGT酶的特异性探针底物报道极少。到目前为止,仅有少数UGT亚型酶(包括UGT1A1,UGT1A4,UGT1A9和UGT2B7)具有公认的特异性底物。而对于其他UGT亚型酶的活性检测,只能依靠传统非荧光底物4-甲基伞形酮(4-MU),其检测通量低,样本前处理及检测过程操作繁琐复杂,无法同步开展目标物对多种UGT亚型酶抑制/激活能力的规模化筛选与评价。因此,设计研发UGT酶广谱型荧光探针底物及其匹配的UGT抑制剂/激活剂高通量筛选与评价方法对于UGT介导的药物相互作用评价具有重要意义。
发明内容
本发明属于医药技术领域,具体涉及一类尿苷二磷酸葡萄糖醛酸转移酶(UGTs)的广谱荧光底物及其制备方法,以及其在UGT酶抑制剂/激活剂筛选与评价中的应用。
本发明提出一种广谱荧光底物,该广谱荧光底物为具有如下结构通式(Ⅰ)的卤代苯乙基-4-羟基1,8-萘酰亚胺衍生物中的一种:
其中,R1选自F、Cl、Br或CF3中的一种。
本发明所述的广谱荧光底物的制备方法,步骤如下:
⑴将4-溴-1、8-萘酸酐和苯乙胺衍生物在乙醇中溶解混匀,反应并回流8小时后冷却至室温,倒入冰水中,分离出沉淀物,用水洗涤、干燥;
其中,4-溴-1、8-萘酸酐和苯乙胺衍生物的摩尔比为:1∶1.2;
其中,步骤⑴所述的苯乙胺衍生物为对氟苯乙胺、对溴苯乙胺、对氯苯乙胺和对三氟甲基苯乙胺中的一种;
⑵将步骤⑴所得产物和碳酸钾在甲醇中混合回流10小时,冷却至室温后,加入浓盐酸将pH值调节至1-2,过滤沉淀物,用水洗涤并干燥;
其中,步骤⑴所得产物和碳酸钾的摩尔比为:1∶9;
⑶将步骤⑵所得产物和吡啶盐酸盐溶解在环丁砜中,加热到200℃,反应8小时后冷却,将混合物倒入水中,分离出沉淀物,用水洗涤,干燥即得所述广谱荧光底物;
其中,步骤⑵所得产物和吡啶盐酸盐的摩尔比为:1∶20。
本发明的第二目的是提出广谱荧光底物的应用,步骤如下:
(1)将三羟甲基氨基甲烷-盐酸缓冲液(Tris-HCl缓冲液)与MgCl2,目标UGT酶,广谱荧光底物溶液、抑制剂混合,反应温度为20-60℃,pH值在5-10,预孵育3-5分钟;
(2)将不同浓度的抑制剂与步骤(1)所得的混合溶液混匀,设置空白对照组,在37℃孵育3-5分钟;
(3)在步骤(2)所得的混合溶液中加入尿苷二磷酸葡萄糖醛酸,反应10-60分钟;
(4)在步骤(3)所得的混合溶液中加入冰乙腈,在离心机上以20,000×g,4℃的条件下离心20分钟,反应液离心沉淀蛋白后,用过液相-荧光检测系统定量分析,荧光检测条件为:激发波长分别为450nm,360nm;发射波长分别为560nm,450nm;
(5)定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入待测抑制剂的反应速率相对于空白对照组反应速率的百分数作为抑制活性评估标准。
本发明中,步骤(1)中三羟甲基氨基甲烷-盐酸缓冲液的pH=7.4,目标UGT酶为人肝微粒体,步骤(2)中的抑制剂为尼罗替尼。
本发明中,步骤(1)中广谱荧光底物浓度为1/10-1Km,反应温度为37℃,pH为7.4。
本发明中,步骤(4)中检测条件以十八烷基甲硅烷基(ODS)柱为固定相,以0.2%甲酸水和乙腈为流动相,0-1min,80%甲酸水,1-2min,80%-60%甲酸水;2-6min,60%-20%甲酸水;6-6.5min,20%甲酸水;6.5-7.5min,20%-80%甲酸水。
本发明的第三目的是提出广谱荧光底物的另一种应用,步骤如下:
(1)将三羟甲基氨基甲烷-盐酸缓冲液(Tris-HCl缓冲液)与MgCl2,目标UGT酶,广谱荧光底物溶液以及抑制剂在96或384酶标仪孔板上混合,反应温度介于20-60℃之间,孵育体系pH介于5-10之间;
(2)预孵育,将不同浓度的抑制剂与步骤(1)所得混合溶液混匀,并设置空白对照组),37℃下孵育3-5min;
(3)在步骤(2)所得的混合溶液中加入尿苷二磷酸葡萄糖醛酸后放入酶标仪,反应10-60分钟;
(4)用酶标仪进行实时定量分析检测,荧光检测条件为:激发波长分别为450nm,360nm;发射波长分别为560nm,450nm;
(5)定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组反应速率的百分数作为抑制活性评估标准。
本发明中,步骤(1)中广谱荧光底物浓度为1/10-1Km,反应温度为37℃,pH为7.4。
本发明的广谱型底物是卤代苯乙基-4-羟基1,8-萘酰亚胺衍生物。与UGT传统底物4-MU相比,该类荧光底物的生成速率可直接用荧光微孔板/酶标仪进行检测,亦可借助液相-荧光系统来检测。上述检测方法具有检测灵敏、操作便捷、检测精密度和稳定性高、抗干扰能力强等特点,可用于UGT酶抑制剂或激活剂的高效筛选与评价。
本发明所述的广谱荧光底物及基于该类底物所构建的UGT酶抑制剂/激活剂的筛选方法和高通量筛选及评价尿苷二磷酸葡萄糖醛酸转移酶抑制剂/激活剂中的方法具有以下优势:
(1)检测灵敏:该类荧光底物可被UGT酶催化代谢生成单一代谢产物,且该类底物的葡萄糖醛酸化产物具有极高的荧光量子产率。其荧光信号可通过酶标仪等常规分析手段获得,方便对其实时定量测定,在临床应用中,无需另行购买昂贵的检测设备。
(2)检测通量高:利用该广谱型荧光底物建立的抑制剂筛选方法可实现96、386孔板的高效快速实时检测,可实现每日2万个样品的高通量检测,具有单个测试成本低廉(<0.5元)的优势。
(3)操作简单、成本低廉:该类荧光底物经化学合成即可规模化制备,且合成工艺简单易行,原料廉价。UGT酶残余活性的检测及UGT酶抑制剂/激活剂的筛选方法也具有操作便捷、耗费低等特点,经济适用性较好。
申请人利用本发明披露的UGT广谱型荧光底物构建了UGT酶抑制剂筛选与评价的荧光检测阵列,并对多种天然产物和药物进行规模化筛选,证明该筛选体系及所用荧光底物具有极高的通量,具有非常良好的应用前景。
本发明披露了一类尿苷二磷酸葡萄糖醛酸转移酶(UGTs)的广谱荧光底物及其制备方法和应用。该类广谱荧光底物是根据人体多种UGT酶的底物偏好性,结合系列底物筛选发现的。该类底物具有代谢产物单一(仅生成一个单葡萄糖醛酸化产物)、能检测抑制剂存在条件下多种UGT亚型酶的残余活性。基于该类广谱底物开发的基于液相-荧光和荧光微孔板的UGT抑制剂/激活剂高通量筛选与评价方法具有灵敏度高、检测通量高、简单易操作等特点,具有良好的应用前景。
附图说明
图1.尿苷二磷酸葡萄糖醛酸转移酶广谱荧光底物的结构式。
图2.尿苷二磷酸葡萄糖醛酸转移酶广谱荧光底物的合成流程图。
图3.实施例2中四种荧光底物的代谢表型图。
图4.荧光底物4-HN-335和4-HN-395的核磁氢谱图,其中,上图是底物4-HN-335的氢谱,下图是底物4-HN-395的氢谱。
图5.实施例3中荧光底物4-HN-335及其产物的液相-荧光检测谱图。
图6.基于微孔板的UGT酶抑制剂高通量筛选示意图。
图7.实施例5中基于荧光检测阵列对UGT1A1抑制剂进行规模化筛选表。
具体实施方式
下面的实施例将对本发明予以进一步的说明,但并不因此而限制本发明。
实施例1.该类广谱荧光底物的制备与合成
(1)将4-溴-1、8-萘酸酐(1g,3.62mmol)和苯乙胺衍生物(0.482g,3.98mmol)溶于乙醇(50mL)。将反应混合物搅拌并回流8小时。冷却至室温后,将混合物倒入冰水中;分离出沉淀物,用水洗涤,干燥,得到1.21g淡黄色固体1(92.6%)。
(2)将化合物1(0.8g,2.22mmol)和K2CO3(2.54g,18.4mmol)在30mL CH3OH中的混合物回流10小时。冷却至室温后,通过加入浓HCl将pH值调节至2。过滤沉淀物,用水洗涤并干燥,得到黄色固体的化合物2(0.562g,收率:80.1%)
(3)将化合物2(0.3g,0.96mmol)和吡啶盐酸盐(2.31g,20mmol)溶解在环丁砜中,并加热到200℃8小时。冷却后,将混合物倒入水中。分离出沉淀物,用水洗涤,干燥,得到0.255的黄色针状固体(88.7%)。
尿苷二磷酸葡萄糖醛酸转移酶广谱荧光底物的结构式如图1所示。
尿苷二磷酸葡萄糖醛酸转移酶广谱荧光底物的合成流程如图2所示。
苯乙胺衍生物分别使用对氟苯乙胺、对氯苯乙胺、对溴苯乙胺、和对三氟甲基苯乙胺合成底物4-HN-335、4-HN--351、4-HN--395、4-HN—385分别对应R1为F、Cl、Br和CF3。运用核磁共振技术,分别获得了四种荧光底物的1H谱和13C谱图,见图4(4-HN-335为例)。
实施例2.人源UGT酶参与荧光底物结合代谢的测试
参与UGT酶广谱型荧光底物葡萄糖结合代谢的UGT酶表型筛选。采用13种商业化的重组人源UGT单酶对荧光底物进行代谢表型筛选。具体如下:
(1)将Tris-HCl(三羟甲基氨基甲烷-盐酸)缓冲液(50mM,pH=7.4)与MgCl2(5mM),目标UGT酶(浓度统一为0.05mg/ml),荧光底物分别使用4-HN-335、4-HN--351、4-HN--395、4-HN—385(底物浓度统一为10μM)在EP管中混合,于37℃条件下预孵育3-5分钟。反应温度介于20~60℃之间,优选37℃为最佳反应温度;孵育体系pH介于5~10之间,优选pH7.4为最佳反应pH值。
(2)加入UDPGA反应60分钟,在酶标仪上检测其葡萄糖醛酸产物荧光,比较其荧光值大小判断各个底物与不同UGT亚型酶的反应速率。结果发现四种荧光底物均能被10种UGT酶代谢。(如图3所示)
结果表明其能被至少十种人UGT亚型酶代谢。
实施例3.基于LC-FD法筛选UGT酶抑制剂方法的构建及其应用
(1)在生理条件下将Tris-HCl缓冲液(50mM,pH=7.4)与MgCl2(5mM),以人肝微粒体为酶源,荧光底物分别使用4-HN-335、4-HN--351、4-HN--395、4-HN—385(以图1所示一类萘酰亚胺类化合物作为广谱型荧光底物,浓度选择1/10~1Km)以及尼罗替尼在EP管中混合,反应温度介于20~60℃之间,优选37℃为最佳反应温度;孵育体系pH介于5~10之间,优选pH7.4为最佳反应pH值。
(2)预孵育,将不同浓度的抑制剂尼罗替尼与Buffer混匀(设置空白对照组),加入底物,37℃孵育3~5min。
(3)加入UDPGA反应时间10-60分钟,确保所述产物生成率或底物转化率低于20%。
(4)加入等体积200微升的冰乙腈终止反应,在离心机上以20,000×g,4℃的条件下离心20分钟。反应液离心沉淀蛋白后借助过液相-荧光检测系统(LC-FD)对底物和产物进行定量分析。检测条件以ODS(十八烷基甲硅烷基)柱为固定相,以0.2%甲酸水(B)和乙腈(A)为流动相,0-1min,80%B,1-2min,80%-60%B;2-6min,60%-20%B;6-6.5min,20%B;6.5-7.5min,20%-80%B。底物及产物的荧光检测条件为:激发波长分别为450nm,360nm;发射波长分别为450nm,560nm。(图5)
(5)测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组反应速率的百分数作为抑制活性评估标准。
检测后发现了尼罗替尼对UGT1A1有强效抑制作用,不同底物4-HN-335、-351、-395、-385对应的IC50分别为19.63、18.69、14.33、17.64μM。
实施例4.UGT酶抑制剂的高通量筛选阵列的构建及其应用
从广谱底物的筛选发现,到代谢表型评价,接着利用酶标仪建立了UGT酶抑制剂的高通量筛选荧光阵列方法,并开展了部分化合物对多种UGT酶抑制作用的评价,得到相应的剂量-抑制曲线和IC50数据。如图6所示,具体步骤为:
(1)设置200μL的反应体系,将Tris-HCl缓冲液(50mM,pH=7.4),MgCl2(5mM),重组人源UGT1A1(0.03mg/ml),荧光底物分别使用4-HN-335、4-HN--351、4-HN--395、4-HN—385(浓度选择1/10~1Km),天然产物抑制剂(1μM,10μM,100μM)等溶液加入96孔板,混匀,于37℃条件下预孵3分钟。
(2)向反应体系中加入10μL UDPGA(终浓度2mM)起始反应后,将酶标板放入酶标仪连续实时检测30分钟(底物及产物的荧光检测条件为:激发波长分别为450nm,360nm;发射波长分别为560nm,450nm);并确保所述产物生成率或底物转化率低于20%。定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组产物生成速率的百分数作为抑制活性评估标准,发现了尼罗替尼对UGT1A1有强效抑制作用,不同底物4-HN-335、-351、-395、-385对应的IC50分别为17.58、19.32、15.22、16.95μM。
实施例5.运用荧光检测阵列对UGT1A1抑制剂进行规模化筛选
(1)设置200μL的反应体系,将Tris-HCl缓冲液(50mM,pH=7.4),MgCl2(5mM),重组人源UGT1A1(0.03mg/ml),荧光底物4-HN-335溶液浓度选择1/10~1Km(1.5μM),待测化合物穗花杉双黄酮和甘草查尔酮(1μM,10μM,100μM)等溶液加入96孔板,混匀,于37℃条件下预孵3分钟。
(2)向反应体系中加入10μL UDPGA(终浓度2mM)起始反应后,将酶标板放入酶标仪连续实时检测30分钟(底物及产物的荧光检测条件为:激发波长分别为450nm,360nm;发射波长分别为560nm,450nm);并确保所述产物生成率或底物转化率低于20%。定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组产物生成速率的百分数作为抑制活性评估标准,发现了穗花杉双黄酮和甘草查尔酮A有最强的抑制效果。(图7)
实施例6.穗花杉双黄酮对十种UGT酶抑制作用的评价
设置200μL的反应体系,将Tris-HCl缓冲液(50mM,pH=7.4),MgCl2(5mM),重组人源UGT1A1(0.03mg/ml),荧光底物4-HN-335溶液浓度选择1/10~1Km(1.5μM),待测化合物穗花杉双黄酮(1μM-100μM)等溶液加入96孔板,混匀,于37℃条件下预孵3分钟。
向反应体系中加入10μL UDPGA(终浓度2mM)起始反应后,将酶标板放入酶标仪连续实时检测30分钟(底物及产物的荧光检测条件为:激发波长分别为450nm,360nm;发射波长分别为560nm,450nm);并确保所述产物生成率或底物转化率低于20%。定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组产物生成速率的百分数作为抑制活性评估标准,发现了穗花杉双黄酮对十种UGT亚型酶都有强效的抑制效果,IC50分别为0.09±0.01μM,0.10±0.01μM,1.01±0.16μM,0.39±0.02μM,0.35±0.03μM,2.45±0.16μM,0.53±0.03μM,3.19±0.50μM,3.10±0.45μM and 6.46±0.37μM。
Claims (8)
2.一种如权利要求1所述的广谱荧光底物的制备方法,其特征在于步骤如下:
⑴将4-溴-1、8-萘酸酐和苯乙胺衍生物在乙醇中溶解混匀,反应并回流8小时后冷却至室温,倒入冰水中,分离出沉淀物,用水洗涤、干燥;
其中,4-溴-1、8-萘酸酐和苯乙胺衍生物的摩尔比为:1∶1.2;
其中,步骤⑴所述的苯乙胺衍生物为对氟苯乙胺、对溴苯乙胺、对氯苯乙胺和对三氟甲基苯乙胺中的一种;
⑵将步骤⑴所得产物和碳酸钾在甲醇中混合回流10小时,冷却至室温后,加入浓盐酸将pH值调节至1-2,过滤沉淀物,用水洗涤并干燥;
其中,步骤⑴所得产物和碳酸钾的摩尔比为:1∶9;
⑶将步骤⑵所得产物和吡啶盐酸盐溶解在环丁砜中,加热到200℃,反应8小时后冷却,将混合物倒入水中,分离出沉淀物,用水洗涤,干燥即得所述广谱荧光底物;
其中,步骤⑵所得产物和吡啶盐酸盐的摩尔比为:1∶20。
3.一种如权利要求1所述的广谱荧光底物的应用,其特征在于步骤如下:
(1)将三羟甲基氨基甲烷-盐酸缓冲液与MgCl2,目标UGT酶,广谱荧光底物溶液、抑制剂混合,反应温度为20-60℃,pH值在5-10,预孵育3-5分钟;
(2)将不同浓度的抑制剂与步骤(1)所得的混合溶液混匀,设置空白对照组,在37℃孵育3-5分钟;
(3)在步骤(2)所得的混合溶液中加入尿苷二磷酸葡萄糖醛酸,反应10-60分钟;
(4)在步骤(3)所得的混合溶液中加入冰乙腈,在离心机上以20,000×g,4℃的条件下离心20分钟,反应液离心沉淀蛋白后,用过液相-荧光检测系统定量分析,荧光检测条件为:激发波长分别为450nm,360nm;发射波长分别为560nm,450nm;
(5)定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入待测抑制剂的反应速率相对于空白对照组反应速率的百分数作为抑制活性评估标准。
4.一种如权利要求3所述的广谱荧光底物的应用,其特征在于步骤(1)中三羟甲基氨基甲烷-盐酸缓冲液的pH=7.4,目标UGT酶为人肝微粒体,步骤(2)中的抑制剂为尼罗替尼。
5.一种如权利要求4所述的广谱荧光底物的应用,其特征在于步骤(1)中广谱荧光底物浓度为1/10-1Km,反应温度为37℃,pH为7.4。
6.一种如权利要求3所述的广谱荧光底物的应用,其特征在于步骤(4)中检测条件以十八烷基甲硅烷基柱为固定相,以0.2%甲酸水和乙腈为流动相,0-1min,80%甲酸水,1-2min,80%-60%甲酸水;2-6min,60%-20%甲酸水;6-6.5min,20%甲酸水;6.5-7.5min,20%-80%甲酸水。
7.一种如权利要求1所述的广谱荧光底物的应用,其特征在于步骤如下:
(1)将三羟甲基氨基甲烷-盐酸缓冲液与MgCl2,目标UGT酶,广谱荧光底物溶液以及抑制剂在96或384酶标仪孔板上混合,反应温度介于20-60℃之间,孵育体系pH介于5-10之间;
(2)预孵育,将不同浓度的抑制剂与步骤(1)所得混合溶液混匀,并设置空白对照组),37℃下孵育3-5min;
(3)在步骤(2)所得的混合溶液中加入尿苷二磷酸葡萄糖醛酸后放入酶标仪,反应10-60分钟;
(4)用酶标仪进行实时定量分析检测,荧光检测条件为:激发波长分别为450nm,360nm;发射波长分别为560nm,450nm;
(5)定量测定反应时间内葡萄糖醛酸产物的生成速率并计算加入抑制剂组的反应速率相对于空白对照组反应速率的百分数作为抑制活性评估标准。
8.一种如权利要求7所述的广谱荧光底物的应用,其特征在于步骤(1)中广谱荧光底物浓度为1/10-1Km,抑制剂为穗花杉双黄酮,反应温度为37℃,pH为7.4。
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