CN115505030A - 用于tmprss2蛋白酶检测和抑制剂筛选的荧光探针、制备方法及其应用 - Google Patents
用于tmprss2蛋白酶检测和抑制剂筛选的荧光探针、制备方法及其应用 Download PDFInfo
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Abstract
Description
技术领域
本发明涉及荧光成像领域,具体涉及一种用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针、制备方法及筛选检测方法。
背景技术
跨膜丝氨酸蛋白酶2(TMPRSS2),属于II型跨膜丝氨酸蛋白酶(TTSPs),具有胰蛋白酶样内肽酶活性,在其催化活性位点有一个亲核的丝氨酸残基。TMPRSS2主要表达于前列腺、小肠、肺等多种组织的上皮细胞中。很多研究表明,TMPRSS2是包括SARS-COV-2、SARS-COV、MERS、CoV229E、OC43、NL63和HKU1等已知的7种冠状病毒蛋白水解激活的重要宿主细胞因子,抑制TMPRSS2可减少病毒对小鼠模型的感染。因此,抑制TMPRSS2蛋白酶活性的药物应该能够抑制SARS-CoV家族(包括突变的SARS-CoV-2)冠状病毒进入人细胞的感染。
因此需要一种荧光探针,旨在对TMPRSS2蛋白酶进行实时、快速、灵敏和特异的检测,同时对有潜力的抑制剂进行初步筛选。
发明内容
有鉴于此,本发明的目的在于提供一种用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针、制备方法及其应用,能够对TMPRSS2蛋白酶进行实时、快速、灵敏和特异的检测,同时对有潜力的抑制剂进行初步筛选。
本发明还公开一种用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针,所述荧光探针包含有多肽序列,所述多肽序列为X1-Arg-X2-nArg-X3-X4。
本发明还公开一种用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针,所述荧光探针由DQM-COOH荧光团与亲水的底物多肽偶联合成,所述荧光探针的化学结构式为:其中n=1、2、3、4,X1、X2为氨基酸,X3、X4为无或者是为氨基酸;
进一步,所述X1、X2为丙氨酸、谷酰氨、赖氨酸、丝氨酸、谷氨酸、苏氨酸中的一种,所述X3、X4为脯氨酸、谷酰氨、赖氨酸、丝氨酸、甘氨酸、苏氨酸中的一种;
进一步,所述DQM-COOH荧光团的合成路线为:
进一步,所述QM-COOH荧光团的制备方法包括以下步骤:
将化合物1和4-甲酰苯甲酸溶解在无水乙腈和哌啶中,在惰性气体保护下于85℃-90℃搅拌反应10-14h,冷却至室温并将粗产物纯化制得橙色DQM-COOH荧光团。
本发明的用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针的制备方法,包括以下步骤:
S1.将目的肽的第一个氨基酸C端通过共价键与高分子树脂连接,再脱去该氨基酸N端保护基团作为为合成起点,和过量的已活化的第二个氨基酸进行反应,接长肽链,重复操作,达到理想的合成肽链长度,最后将肽链从树脂上裂解下来,分离纯化,获得目标多肽;
S2.将目标多肽和荧光团DQM按照同样的操作用酰胺键连接。
本发明的利用荧光探针检测TMPRSS2的方法,包括以下步骤:
将荧光探针和TMPRSS2蛋白酶在缓冲液中孵育一定时间,然后采用380nm的波长激发,得到400-700nm的波长范围内的发射波长。
本发明的利用荧光探针筛选抑制剂的方法,包括以下步骤:
在缓冲液中加入TMPRSS2蛋白酶与待筛选的抑制剂分子并在室温下孵育,然后加入荧光探针充分混匀后继续孵育,并用荧光分析仪380nm的波长激发,得到400-700nm的波长范围内的发射光谱,然后对荧光发射光谱的面积积分,计算抑制剂对TMPRSS2蛋白酶活性的抑制率,完成荧光探针体外抑制剂筛选。
本发明的有益效果是:本发明公开的用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针、制备方法及其应用,该荧光探针能够对TMPRSS2蛋白酶进行实时、快速、灵敏和特异的检测,亲水的多肽增加了荧光团的水溶性,能够实现TMPRSS2蛋白酶在亲水环境中对探针的识别剪切,从而对TMPRSS2蛋白酶定量检测和抑制剂筛选。
附图说明
下面结合附图和实施例对本发明作进一步描述:
图1为荧光团DQM-COOH核磁共振氢谱图
图2为采用水/DMSO混合物评价了蒸馏水(DW)的体积分数(fw)对DQM-COOH荧光的影响
图3为反应最优缓冲液筛选的荧光发射图
图4为反应最优缓冲液pH筛选的荧光发射图
图5为反应最优探针浓度筛选的荧光发射图
图6为反应最优蛋白酶浓度筛选的荧光发射图
图7为反应蛋白酶和多肽-荧光探针动力学荧光发射图
图8为反应最优温度筛选的荧光发射图
图9为特异性筛选的荧光发射图
图10为抑制剂筛选结果
图11为探针对细胞内TMPRSS2蛋白酶表达的验证图。
具体实施方式
荧光团DQM-COOH的合成:依次将化合物1(1g 4.25mmol)和4-甲酰苯甲酸(690mg4.6mmol)加入反应瓶中,在氩气保护下加入乙腈30ml和哌啶1ml室温下溶解,转至85℃-90℃度油浴反应12h。反应结束,冷却至室温,粗产物柱层析(二氯甲烷:甲醇=10:1)得到橙色化合物2,产率33%。
制备产物的核磁共振波谱如下:1H NMR(600MHz,DMSO-d6)δ8.92(d,J=8.4Hz,1H),8.09(d,J=8.9Hz,1H),7.99(d,J=8.2Hz,2H),7.95–7.91(m,3H),7.67(d,J=15.8Hz,1H),7.64–7.60(m,1H),7.49(d,J=15.8Hz,1H),7.01(s,1H),4.57(q,J=7.1Hz,2H),4.10(s,1H),1.39(t,J=7.1Hz,3H).(图1)
多肽-荧光探针(P-DQM)的合成:Fmoc固相合成法——将目的肽的第一个氨基酸C端通过共价键与高分子树脂连接,再脱去该氨基酸N端保护基团作为为合成起点,和过量的已活化的第二个氨基酸进行反应,接长肽链,重复操作,达到理想的合成肽链长度,最后将肽链从树脂上裂解下来,分离纯化,获得目标多肽。目标多肽和荧光团DQM按照同样的操作用酰胺键连接。所制得的荧光探针具体结构式为
荧光探针的体外性能验证:
(1)荧光性能验证:采用水/DMSO混合物评价了蒸馏水(DW)的体积分数(fw)对DQM-COOH荧光的影响(图2)。
(2)缓冲液筛选:选择了PBS(10mM)、PBST(10mM 0.2%Tween20)、HEPES(20mM)、HEPES(20mM 80mM KCI)、Tris-HCl(50mM,150mM NaCl)、Tris-HCl(50mM,150mM NaCl 0.2%Tween20)等6种缓冲液,pH7.2-7.4,加入TMPRSS2和P-DQM在缓冲液中反应后,用380nm波长光激发,得到400nm到700nm之间的发射光谱(图3)。
(3)缓冲液pH筛选:从上述筛选出的HEPES缓冲液调节pH为3-11,然后在不同pH的缓冲体系下加入探针P-DQM和TMPRSS2蛋白酶。用380nm波长光激发,得到400nm到700nm之间的发射光谱(图4)。
(4)固定酶量,探究合适探针浓度:将TMPRSS2蛋白酶溶液100nM与浓度分别30uM,60uM,90uM,120uM,150uM,200uM的探针溶液孵育,用380nm波长光激发,得到400nm到700nm之间的发射光谱(图5)。
(5)固定探针浓度,探究合适酶量:将探针溶液P-DQM(120uM)与浓度分别为200nM,100nM,80nM,60nM,40nM,20nM,0nM的蛋白溶液在HEPES孵育,用380nm波长光激发,得到400nm到700nm之间的发射光谱。(图6)
(6)动力学测定程序:在反应缓冲液HEPES中加入P-DQM(120uM)和TMPRSS2蛋白酶(100nM),在室温混合后加入比色皿,在荧光分析仪器上进行120min的动力学测试。Ex=380nm,Em=550nm,动力学间隔为1s(图7)。
(7)反应温度的探究:在反应缓冲液HEPES中加入P-DQM(120uM)和TMPRSS2蛋白酶(100nM),在4℃,25℃,37℃,40℃,45℃下孵育60分钟后,加入比色皿,在荧光分析仪器上进行荧光测试。用380nm波长光激发,得到400nm到700nm之间的发射光谱(图8)。
(8)特异性的测定程序:在反应缓冲液HEPES中加入P-DQM(120uM)和其他蛋白、离子、葡萄糖等,在37℃下孵育80分钟,加入比色皿,在荧光分析仪器上进行荧光测试。用380nm波长光激发,得到400nm到700nm之间的发射光谱。其他蛋白酶或者其他物质分别为:ALP、BSA、D+葡萄糖、L-谷氨酰胺、Mg2+、Fe3+(图9)。
(9)抑制剂筛选:按照以下步骤进行抑制剂筛选:在缓冲液中加入TMPRSS2蛋白酶与待筛选的抑制剂分子在室温下孵育30分钟,再加入上述荧光探针充分混匀后继续孵育60分钟,然后在荧光分析仪上采用380nm的波长激发,得到400nm到700nm的波长范围内的发射光谱,然后对不同组的荧光发射光谱计算积分面积,计算抑制剂对TMPRSS2蛋白酶活性的抑制率,完成荧光探针体外抑制剂筛选(图10)。
(10)细胞培养:将细胞在含5%CO2,37℃的潮湿环境中培养。Vero E6细胞在补充了10%胎牛血清,1%青霉素-链霉素的Dulbecco改良Eagle培养基(DMEM)中生长。
(11)TMPRSS2过表达:培养的Vero E6细胞在密度为60%~70%时进行转染,用培养基稀释Lipofectamine 3000试剂,再用P3000试剂制备DNA预混液。两种试剂混合孵育15分钟后直接加入培养基中。
(12)TMPRSS2蛋白酶活性验证:不同浓度探针和对照探针P’-DQM溶解在培养基中,然后孵育细胞,在荧光显微镜下观察(图11)。
本实施例中,当n=为2、3、4时,以及X1、X2,X3、X4为其他氨基酸时,或者X3、X4为无时,以及X1、X2与X3、X4的氨基酸种类不同时,所制得的荧光探针性能差异性不大,均能实现本发明的目的。
上述实施例中,可采用荧光团DQM-COOH与现有技术中的一般多肽合成荧光探针,或者采用现有技术中荧光团与本发明的多肽合成用于荧光探针,索合成的荧光探针均能用于TMPRSS2蛋白酶检测和抑制剂筛选。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (9)
2.一种用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针,其特征在于:所述荧光探针包含有多肽序列,所述多肽序列为X1-Arg-X2-nArg-X3-X4。
4.根据权利要求3所述的用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针,其特征在于:所述X1、X2为丙氨酸、谷酰氨、赖氨酸、丝氨酸、谷氨酸、苏氨酸中的一种,所述X3、X4为脯氨酸、谷酰氨、赖氨酸、丝氨酸、甘氨酸、苏氨酸中的一种。
6.根据权利要求5所述的用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针,其特征在于:所述QM-COOH荧光团的制备方法包括以下步骤:
将化合物1和4-甲酰苯甲酸溶解在无水乙腈和哌啶中,在惰性气体保护下于85℃-90℃搅拌反应10-14h,冷却至室温并将粗产物纯化制得橙色DQM-COOH荧光团。
7.根据权利要求1所述的用于TMPRSS2蛋白酶检测和抑制剂筛选的荧光探针的制备方法,其特征在于:包括以下步骤:
S1.将目的肽的第一个氨基酸C端通过共价键与高分子树脂连接,再脱去该氨基酸N端保护基团作为为合成起点,和过量的已活化的第二个氨基酸进行反应,接长肽链,重复操作,达到理想的合成肽链长度,最后将肽链从树脂上裂解下来,分离纯化,获得目标多肽;
S2.将目标多肽和荧光团DQM按照同样的操作用酰胺键连接。
8.一种利用权利要求1所述的荧光探针检测TMPRSS2的方法,其特征在于:包括以下步骤:
将荧光探针和TMPRSS2蛋白酶在缓冲液中孵育一定时间,然后采用380nm的波长激发,得到400-700nm的波长范围内的发射波长。
9.一种利用权利要求1所述的荧光探针筛选抑制剂的方法,其特征在于:包括以下步骤:
在缓冲液中加入TMPRSS2蛋白酶与待筛选的抑制剂分子并在室温下孵育,然后加入荧光探针充分混匀后继续孵育,并用荧光分析仪380nm的波长激发,得到400-700nm的波长范围内的发射光谱,然后对荧光发射光谱的面积积分,计算抑制剂对TMPRSS2蛋白酶活性的抑制率,完成荧光探针体外抑制剂筛选。
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