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

Mol Pharm. Mar-Apr 2009;6(2):416-27. doi: 10.1021/mp800264k.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Antineoplastic Agents, Phytogenic / pharmacology
  • Caspase 3 / metabolism*
  • Enzyme Activation
  • Fluorescence
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Lung Neoplasms / drug therapy*
  • Lung Neoplasms / enzymology
  • Male
  • Mice
  • Microscopy, Fluorescence
  • Models, Molecular
  • Paclitaxel / pharmacology
  • Tumor Cells, Cultured

Substances

  • Antineoplastic Agents, Phytogenic
  • Fluorescent Dyes
  • Caspase 3
  • Paclitaxel