Design and Synthesis of a Peptidyl-FRET Substrate for Tumor Marker Enzyme human Matrix Metalloprotease-2 (hMMP-2)

Matrix metalloproteases (MMPs) in particular MMP-2, have been associated with several pathological conditions such as ovarian, urothelial, cutaneous, gastric, breast, and cervical cancers, etc. Successful treatment of these pathological conditions requires sensitive, reliable, quick and effective diagnostic tools such as fluorescence resonance energy transfer (FRET) based assays in early stage of the disease. A peptidyl-FRET substrate having seven amino acid residues (PLGLKAR) with methoxycoumarin (Mca)/dinitrophenyl (Dnp) as fluorophore/quencher group has been synthesized using solid-phase fluorenylmethoxycarbonyl (Fmoc) peptide chemistry. The newly designed substrate is stable and shows a K (m) value of 15 mu M for hMMP-2. This K (m) value is the lowest compared with all other known hMMP-2 substrates having Mca/Dnp. Validation of the new FRET substrate in presence/absence of scorpion venom chlorotoxin, a known hMMP-2 inhibitor, shows an increase in detection efficiency of 6,250 times as compared to commonly used gelatin zymography. The new FRET substrate is much more cost effective and can be used for high throughput screening of hMMP-2 inhibitors in the laboratory for research and diagnostic purposes.