The MMP-2 Fluorometric (also known as fluorimetric) Drug Discovery Kit, RED is a complete assay system designed to screen MMP-2 inhibitors using a quenched fluorogenic substrate OMNIMMP® RED: TQ3-GABA-Pro-Cha-Abu-Smc-His-Ala-Dab(6-TAMRA)-Ala-Lys-NH2 [TQ3=quencher; GABA=4-aminobutyric acid; Cha=L-cyclohexylalanine; Abu=2-aminobutyric acid; Smc=S-methyl-L-cysteine; Dab=2,4-diaminobutyric acid; 6-TAMRA=6-tetramethylrhodamine]. TAMRA fluorescence is thoroughly quenched by the TQ3 group until cleavage by MMPs separates the two moieties.
The assays are performed in a convenient 96-well microplate format. The kit is useful to screen inhibitors of MMP-2, a potential therapeutic target. The compound NNGH is also included as a prototypic control inhibitor.
|Alternative Name:||Gelatinase A, 72 kDa Type IV collagenase|
|Applications:||Fluorescent detection, HTS|
|Application Notes:||Designed to screen MMP-2 inhibitors using a quenched fluorogenic peptide.|
|Handling:||Avoid freeze/thaw cycles.|
|Shipping:||Shipped on Dry Ice|
|Long Term Storage:||-80°C|
|Kit/Set Contains:||1 vial MMP-2 enzyme|
1 vial substrate (OMNIMMP® RED)
1 vial 6'-TAMRA calibration standard
1 vial control inhibitor (NNGH)
1 bottle (20 ml) assay buffer
1 black 96-well microplate
|Scientific Background:||Matrix metalloproteinase-2 (MMP-2, gelatinase A, 72kDa type IV collagenase) is a member of the MMP family of extracellular proteases. These enzymes play a role in many normal and disease states by virtue of their broad substrate specificities. Targets of MMP-2 include native and denatured collagens, fibronectin, elastin, laminin-5, pro-TNF-α, and neurocan. MMP-2 is secreted as a 72kDa proenzyme (as measured by SDS-PAGE), and activated by cleavage to 62 and 59kDa. MMP-2 is an important target for inhibitor screening due to its involvement in diseases such as atherosclerosis, and cancer growth, angiogenesis, and metastasis.|
|Technical Info/Product Notes:||NCBI Reference Sequence: NM_004530|
The OMNIMMP® RED substrate offers key advantages over other MMP substrates.
- Emission at the red end of the spectrum (576 nm after excitation at 545 nm) avoids the interference at lower wavelengths often exhibited by screening compounds, and by substances commonly found in biological samples and tissue culture medium.
- MMP substrate peptides display poor aqueous solubility, often with Kms near their limits of solubility, making enzyme and inhibitor kinetics difficult. MMP Kms for OMNIMMP® RED substrate are below its solubility limit.In addition to the efficient binding as exhibited by low Kms, OMNIMMP® RED is avidly cleaved by MMPs, with kcat/Kms in the range of 104-106 M-1sec-1.
- The ultra-strong fluorescence of OMNIMMP® RED allows for substrate concentrations much lower than the Km, a condition generally desirable in inhibitor screening assays.
General Literature References
Matrix metalloproteinases: regulators of the tumor microenvironment
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Updated biological roles for matrix metalloproteinases and new "intracellular" substrates revealed by degradomics
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Determination of protease cleavage site motifs using mixture-based oriented peptide libraries
: B.E. Turk et al.; Nat. Biotechnol. 19
, 661 (2001), Abstract
Matrix metalloproteinases: they're not just for matrix anymore!: L.J. McCawley & L.M. Matrisian; Curr. Opin. Cell Biol. 13, 534 (2001),
Therapeutic potential of matrix metalloproteinase inhibitors in atherosclerosis
: S.J. George; Expert Opin. Investig. Drugs 9
, 993 (2000), Abstract
Discovery of CGS 27023A, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits
: L.J. MacPherson, et al.; J. Med. Chem. 40
, 2525 (1997), Abstract
Induction of cell migration by matric metalloproteinase -2 cleavage of laminin-5
: G. Giannelli, et al.; Science 277
, 225 (1997), Abstract
The hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2) requires Ca2+ for fibronectin and heparin binding. Binding properties of recombinant gelatinase A C domain to extracellular matrix and basement membrane components
: U.M. Wallon & C.M. Overall; J. Biol. Chem. 272
, 7473 (1997), Abstract
; Full Text
Intermolecular autolytic cleavage can contribute to the activation of progelatinase A by cell membranes
: S.J. Atkinson et al.; J. Biol. Chem. 270
, 30479 (1995), Abstract
; Full Text