Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity

J. Oliver McIntyre, Barbara Fingleton, K. Sam Wells, David W. Piston, Conor C. Lynch, Shiva Gautam, Lynn M. Matrisian

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Abstract

The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7VIS, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7VIS is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an F1 (fluorescein)-labelled peptide F1(AHX) RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7VIS is efficiently and selectively cleaved by MMP-7 with a k cat/Km value of 1.9 x 105 M-1 ·s-1 as measured by the rate of increase in F1 fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The Km value for PB-M7VIS is approx. 0.5 μM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k cat/Km value for PB-M7VIS is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7VIS, F1(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (F1/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced F1 fluorescence from MMP-7-positive tumours, but not from control tumours (P < 0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 {[4-(N-hydroxyamino)-2R-isobutyl-3S-(thienylthiomethyl)- succinyl]-L-phenylalanine-N-methylamide}, markedly decreased the F1 fluorescence over the MMP-7-positive tumour by approx. 60 %. Thus PB-M7VIS functions as a PB for in vivo detection of MMP-7 activity that serves to light this optical beacon and is, therefore, a selective in vivo optical molecular imaging contrast reagent.

Original languageEnglish
Pages (from-to)617-628
Number of pages12
JournalBiochemical Journal
Volume377
Issue number3
DOIs
StatePublished - Feb 1 2004

Keywords

  • Fluorescence assay
  • Fluorogenic dendrimer
  • Matrix metalloproteinase-7 (MMP-7)
  • Optical molecular imaging
  • Proteolytic beacon
  • SW480 xenograft

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