Activatable molecular systems using homologous near-infrared fluorescent Probes for monitoring enzyme activities in vitro, in pCellulo, and in vivo

Zongren Zhang, Jinda Fan, Philip P. Cheney, Mikhail Y. Berezin, W. Barry Edwards, Walter J. Akers, Duanwen Shen, Kexian Liang, Joseph P. Culver, Samuel Achilefu

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

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 (Me 2NCp) 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)-NH 2 (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 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.

Original languageEnglish
Pages (from-to)416-427
Number of pages12
JournalMolecular Pharmaceutics
Volume6
Issue number2
DOIs
StatePublished - Apr 6 2009

Keywords

  • Activatable probes
  • Caspase
  • Enzyme
  • FRET
  • Kinetics
  • Near-infrared fluorescence
  • Quenching mechanism

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