Activatable Molecular Systems Using Homologous Near-Infrared Fluorescent Probes for Monitoring Enzyme Activitiesin Vitro,in Cellulo, andin Vivo

Abstract

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.

Biochemical Characterization of a Caspase-3 Far-red Fluorescent Probe for Non-invasive Optical Imaging of Neuronal Apoptosis

“…(Zhang et al 2009). Moreover, as the rate of the reaction is directly proportional to the concentration of recombinant caspase-3, QCASP3.2 represents a tool to quantify the amount of active enzyme.…”

“…More recently, other probes have been developed that directly target caspase-3 and become fluorescent only in the presence of active caspase-3, thus substantially increasing signal specificity (Bullok and Piwnica-Worms 2005;Lapeyre et al 2006). Functionality of these probes was demonstrated in human colon xenografts and in liver abscess mouse models (Bullok et al 2007), in isolated heart reperfusion injury (Pantos et al 2009) or after intra-dermal injection of caspase-3 (Zhang et al 2009). …”

Reporter nanoparticle that monitors its anticancer efficacy in real time

“…4E). In earlier studies, quenched fluorescent caspase substrate-based tracers have been used to detect apoptosis in vivo (37,51,52). As controls, we therefore included a separate group, where the animals were treated with a combination of free paclitaxel and the reporter element at a similar dose level to that in the reporter nanoparticles.…”

Optical Imaging in Cancer Research: Basic Principles, Tumor Detection, and Therapeutic Monitoring

“…These enzymes are up-regulated in cells undergoing apoptosis and studies have shown that disruption of apoptosis results in abnormal cell proliferation and tumor growth. The activities of these proteolytic enzymes have been recognized as important markers for developing imaging and therapeutic agents to noninvasively monitor tumor progress and therapy response [158,169,170] . For instance, Bullok et al [171,172] developed a FRET-based caspaseactivatable molecular probe, TcapQ647, composed of a fluorescent moiety in close proximity to a NIR fluorescent quencher linked with a caspase-cleavable peptide sequence.…”

“…They observed a greater increase in parasite-infected xenografts versus controls after TcapQ647 injection, thus demonstrating the potential of the compound for monitoring treatment response. An example of caspase-3-activatable probes that are based on FRET between two NIR cyanine dyes connected with a caspase-3-cleavable peptide substrate, Asp-Glu-Val-Asp (DEVD), was designed by Zhang et al [158] . Upon contact with caspase-3, whose overexpression was induced by the administration of the chemotherapeutic drug (paclitaxel) to A549 tumor-bearing mice, cleavage of the DEVD peptide sequence in the fluorescence-quenched probe resulted in fluorescence restoration.…”