Synthesis and evaluation of a novel hydroxamate based fluorescent photoprobe for imaging of matrix metalloproteinases.
The assessment of matrix metalloproteinase (MMP) activity in vivo is highly desirable in various human diseases such as cancers. Hydroxamic acids based on CGS27023A or CGS25966 are nonpeptidyl lead structures that specifically target activated MMPs in vivo. The aim of this study was the modification and fluorescent labeling of these lead structures to develop a highly affine, nonpeptide MMP inhibitor (MMPI)-ligand for molecular optical imaging of activated MMPs. An 11 step synthesis was developed involving a PEGylated benzyl derivative as a spacer to minimize the interactions between the activated MMP and the dye of conjugate 11 with an azide as a protected amino function. After reducing the azide (Staudinger reaction) and labeling with Cy5.5, we obtained a CGS-based MMP inhibitor 11 with a fluorescent signaling flag. To evaluate the biological properties of this photoprobe, three human cancer cell lines (A-673, HT-1080 and BT-20) were characterized with respect to their MMP-2 and -9 (gelatinases) expression levels (real-time PCR) and protein levels (Western blotting). Initially, fluorogenic inhibition assays were used to assess the MMP inhibition potential. The PEGylated CGS 10 showed complete inhibition of MMP-2 and MMP-9 activities in vitro both for purified MMP-2/-9 (active and pro-forms) and MMP-2/-9 containing cell culture supernatants. To test the imaging potential in biological tissues, gelatinase activity was measured on tumor cryostat sections of the above-mentioned tumor cells using FITC-labeled dye-quenched gelatin. Gelatinase positive tumors revealed strong binding of CGS-Cy5.5 11, while gelatinase negative tumors were not targeted. In conclusion, this new CGS-based MMP photoprobe has a high affinity for MMP-2 and -9 and is thus a promising candidate for sensitive imaging of MMP activity in various diseases in patients.
Department of Clinical Radiology, Albert-Schweitzer-Strasse 33, University Hospital Munster, Munster, Germany. faustan@uni-muenster.de
Andreas
A
Faust
Bianca
B
Waschkau
Jens
J
Waldeck
Carsten
C
Höltke
Hans-Jörg
HJ
Breyholz
Stefan
S
Wagner
Klaus
K
Kopka
Otmar
O
Schober
Walter
W
Heindel
Michael
M
Schäfers
Christoph
C
Bremer
D002232:Carbocyanines / Q000737:chemistry; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D006877:Hydroxamic Acids / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008024:Ligands; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D057054:Molecular Imaging / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry; D014176:Protein Biosynthesis; D011719:Pyrazines / Q000378:metabolism / Q000494:pharmacology; D013449:Sulfonamides / Q000378:metabolism / Q000494:pharmacology; D014158:Transcription, Genetic
C097658:CGS 27023A; C098793:CY5.5 cyanine dye; D002232:Carbocyanines; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006877:Hydroxamic Acids; D008024:Ligands; D061965:Matrix Metalloproteinase Inhibitors; C490459:N-hydroxy-2-(((4'-methoxyphenyl)sulfonyl)benzylamino)-3-methylbutanamide; D011719:Pyrazines; D013449:Sulfonamides; D011092:Polyethylene Glycols; D020782:Matrix Metalloproteinases