Synthesis and evaluation of a novel fluorescent photoprobe for imaging matrix metalloproteinases.
The measurement of matrix metalloproteinase (MMP) activity in diseases like inflammation, oncogenesis, or atherosclerosis in vivo is highly desirable. Fine-tuned pyrimidine-2,4,6-triones (barbiturates) offer nonpeptidyl lead structures for developing imaging agents for specifically visualization of activated MMPs in vivo. The aim of this study was to modify a C-5-disubstituted barbiturate and thus design a highly affine, nonpeptidic, optical MMP inhibitor (MMPI)-ligand for imaging of activated MMPs in vivo. A convergent 10 step synthesis was developed, starting with a malonic ester and (4-bromophenoxy)benzene to generate 5-bromo-pyrimidine-2,4,6-trione as the key intermediate. To minimize the interactions between activated MMPs and the dye of the conjugate 6, a PEGylated piperazine derivative was used as a spacer and an azide as a protected amino function. After linking both building blocks, reducing the azide ( Staudinger reaction) and labeling with Cy 5.5, we obtained the nonhydroxamate MMP inhibitor 6 with high affinity (IC 50-value: 48 nM for MMP-2) measured in a fluorogenic assay using commercially available MMP-substrates and the purified enzyme. Zymography revealed an efficient blocking of enzyme activity of purified MMP-2 and MMP-9 and of MMP-containing cell supernatants (HT-1080), (A-673) using the PEGylated barbiturate 5. Fluorescence microscopy studies using a highly (A-673) and a moderate (HT-1080) MMP-2 secreting cell line showed efficient binding of the Cy 5.5 labeled tracer 6 to the MMP-2 positive cells while MMP-2 negative cells (MCF-7) did not bind. Therefore, this new barbiturate-based MMP-probe has a high affinity and specificity toward MMP-2 and -9 and is thus a promising candidate for sensitive MMP detection in vivo.
Faust
Andreas
A
Department of Clinical Radiology and Department of Nuclear Medicine, University Hospital Münster, Albert-Schweitzer-Str. 33, 48149 Münster, Germany. faustan@uni-muenster.de
Waschkau
Bianca
B
Waldeck
Jens
J
Höltke
Carsten
C
Breyholz
Hans-Jörg
HJ
Wagner
Stefan
S
Kopka
Klaus
K
Heindel
Walter
W
Schäfers
Michael
M
Bremer
Christoph
C
D001463:Barbiturates / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D003952:Diagnostic Imaging; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000737:chemistry* / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D008856:Microscopy, Fluorescence / Q000379:methods; D015394:Molecular Structure; D016133:Polymerase Chain Reaction / Q000379:methods; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D012680:Sensitivity and Specificity; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D001463:Barbiturates; D005456:Fluorescent Dyes; D008024:Ligands; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9