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

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)/K-m value of 1.9 x 10(5) 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 K-m value for PB-M7vis is approx. 0.5 muM, 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)/K-m 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-phenylatanine-N-methylamide} markedly decreased the F1 fluorescence over the MMP-7-positive turnout 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.