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 Fl (fluorescein)-labelled peptide Fl(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 1.9x10(5) M(-1).s(-1) as measured by the rate of increase in Fl 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 microM, 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, Fl(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 (Fl/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced Fl 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)-2 R -isobutyl-3 S -(thienylthiomethyl)-succinyl]-L-phenylalanine- N -methylamide), markedly decreased the Fl 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.
McIntyre
J Oliver
JO
Department of Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232-6840, U.S.A. oliver.mcintyre@vanderbilt.edu
Fingleton
Barbara
B
Wells
K Sam
KS
Piston
David W
DW
Lynch
Conor C
CC
Gautam
Shiva
S
Matrisian
Lynn M
LM
D000595:Amino Acid Sequence; D000818:Animals; D002138:Calibration; D050091:Dendrimers; D019793:Fluorescein / Q000138:chemical synthesis / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000378:metabolism; D007090:Image Interpretation, Computer-Assisted; D020783:Matrix Metalloproteinase 7 / Q000138:chemical synthesis / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000138:chemical synthesis / Q000378:metabolism; D009368:Neoplasm Transplantation; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011073:Polyamines / Q000138:chemical synthesis / Q000378:metabolism; D012878:Skin Neoplasms / Q000201:enzymology*; D013379:Substrate Specificity; D014183:Transplantation, Heterologous
D050091:Dendrimers; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins; C104700:PAMAM Starburst; D010455:Peptides; D011073:Polyamines; D020783:Matrix Metalloproteinase 7; D019793:Fluorescein