Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo.
Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo.
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DOI：
10.1021/mp800264k
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发表时间：
2009-03
影响因子：
4.9
通讯作者：
Achilefu S
中科院分区：
文献类型：
--
作者：
Zhang Z;Fan J;Cheney PP;Berezin MY;Edwards WB;Akers WJ;Shen D;Liang K;Culver JP;Achilefu S
我们已经开发了一种通用的方法来确定酶的活性在体外和监测其功能状态在体内。以七甲川菁染料为结构模板，建立了一种制备供体（CbOH）-受体（Me 2NCp）近红外荧光染料对的方法，用于制备酶可激活的分子体系。以caspase-3为模型酶，制备了两个新的caspase-3敏感性化合物：Me_2NCp-DEVD-K（CbOH）-OH（4）和AcGK（Me_2NCp）-DEVD-APK（CbOH）-NH_2（5）。猝灭机制是基于直接（经典）和反向荧光共振能量转移（FRET）的组合效应。胱天蛋白酶-3切割易断裂的DEVD酰胺键再生供体和受体染料的NIR荧光。虽然两种化合物都被半胱天冬酶-3裂解，但底物5比底物4更容易裂解，产生的kcat和KM值分别为1.02 ± 0.06 s-1和15 ± 3 μM。用紫杉醇处理A549肿瘤细胞导致通过NIR共聚焦显微镜观察的荧光强度增加>2倍，表明胱天蛋白酶原-3活化为胱天蛋白酶-3。在小鼠模型中观察到类似的趋势，其中相对于对照，荧光强度几乎是富含半胱天冬酶-3的组织中的值的两倍。这些结果表明，使用相同的NIR可活化的分子系统，用于监测酶的活性跨越广泛的空间尺度，从体外动力学测量到细胞内和体内半胱天冬酶-3活化的定位。近红外可激活的分子探针提供了一种有效的策略，以筛选新的药物在体外和监测治疗反应的活生物体。
We have developed a generic approach to determine enzyme activities in vitro and monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)-acceptor (Me2NCp) near infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me2NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me2NCp)-DEVD-APK(CbOH)-NH2 (5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5 was cleaved more readily than 4, yielding kcat and KM, values of 1.02 ± 0.06 s−1 and 15 ± 3 μM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in >2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitro kinetics measurements to in cellulo and in vivo localization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitro and monitor treatment response in living organisms.
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影响因子：
3.3
作者：
Berezin, Mikhail Y.;Lee, Hyeran;Achilefu, Samuel
通讯作者：
Achilefu, Samuel
影响因子：
4.9
作者：
Almutairi, Adah;Akers, Walter J.;Frechet, Jean M. J.
通讯作者：
Frechet, Jean M. J.
影响因子：
82.9
作者：
Bremer, C;Tung, CH;Weissleder, R
通讯作者：
Weissleder, R
影响因子：
46.9
作者：
Harpur, AG;Wouters, FS;Bastiaens, PIH
通讯作者：
Bastiaens, PIH
DOI：
10.1039/p29930000187
发表时间：
1993-02-01
期刊：
JOURNAL OF THE CHEMICAL SOCIETY-PERKIN TRANSACTIONS 2
影响因子：
--
作者：
GOTO, H;OSAWA, E
通讯作者：
OSAWA, E