title: Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in 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 k(cat) and K(M), values of 1.02 +/- 0.06 s(-1) and 15 +/- 3 microM, 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.
authors.affiliation: Department of Radiology, Washington University School of Medicine, 4525 Scott Avenue, St. Louis, Missouri 63110, USA.
authors.forename: Zongren
authors.initials: Z
authors.lastname: Zhang
authors.forename: Jinda
authors.initials: J
authors.lastname: Fan
authors.forename: Philip P
authors.initials: PP
authors.lastname: Cheney
authors.forename: Mikhail Y
authors.initials: MY
authors.lastname: Berezin
authors.forename: W Barry
authors.initials: WB
authors.lastname: Edwards
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Akers
authors.forename: Duanwen
authors.initials: D
authors.lastname: Shen
authors.forename: Kexian
authors.initials: K
authors.lastname: Liang
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Culver
authors.forename: Samuel
authors.initials: S
authors.lastname: Achilefu
mesh_terms: D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism*; D004789:Enzyme Activation; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000201:enzymology; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D017239:Paclitaxel / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D005456:Fluorescent Dyes; D053148:Caspase 3; D017239:Paclitaxel