Screening MT1-MMP Activity and Inhibition in Three-Dimensional Tumor Spheroids.

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for tumor angiogenesis, invasion, and metastasis, and thus MT1-MMP is a high priority target for potential cancer therapies. To properly evaluate MT1-MMP inhibitors, a screening protocol is desired by which enzyme activity can be quantified in a tumor microenvironment-like model system. In the present study, we applied a fluorogenic, collagen model triple-helical substrate to quantify MT1-MMP activity for tumor spheroids embedded in a collagen hydrogel. The substrate was designed to be MT1-MMP selective and to possess fluorescent properties compatible with cell-based assays. The proteolysis of the substrate correlated to glioma spheroid invasion. In turn, the application of either small molecule or protein-based MMP inhibitors reduced proteolytic activity and glioma spheroid invasion. The presence of MT1-MMP in glioma spheroids was confirmed by western blotting. Thus, spheroid invasion was dependent on MT1-MMP activity, and inhibitors of MT1-MMP and invasion could be conveniently screened in a high-throughput format. The combination of the fluorogenic, triple-helical substrate, the three-dimensional tumor spheroids embedded in collagen, and Hit-Pick software resulted in an easily adaptable in vivo-like tumor microenvironment for rapidly processing inhibitor potential for anti-cancer use.

Matrix Metalloproteinase 13 Inhibitors for Modulation of Osteoclastogenesis: Enhancement of Solubility and Stability.

Matrix metalloproteinase 13 (MMP-13) activity has been correlated to breast cancer bone metastasis. It has been proposed that MMP-13 contributes to bone metastasis through the promotion of osteoclastogenesis. To explore the mechanisms of MMP-13 action, we previously described a highly efficacious and selective MMP-13 inhibitor, RF036. Unfortunately, further pursuit of RF036 as a probe of MMP-13 in vitro and in vivo activities was not practical due to the limited solubility and stability of the inhibitor. Our new study has explored replacing the RF036 backbone sulfur atom and terminal methyl group to create inhibitors with more favorable pharmacokinetic properties. One compound, designated inhibitor 3, in which the backbone sulfur and terminal methyl group of RF036 were replaced by nitrogen and oxetane, respectively, had comparable activity, selectivity, and membrane permeability to RF036, while exhibiting greatly enhanced solubility and stability. Inhibitor 3 effectively inhibited MMP-13-mediated osteoclastogenesis but spared collagenolysis, and thus represents a next-generation MMP-13 probe applicable for in vivo studies of breast cancer metastasis.

Matrix Metalloproteinase Triple-Helical Peptide Inhibitors: Potential Cross-Reactivity with Caspase-11.

Triple-helical peptide inhibitors (THPIs) of matrix metalloproteinases (MMPs) have recently been demonstrated to be effective in a variety of animal models of disease, coincidental with knockout studies. However, passenger mutations have been described in MMP knockout mice that impact the activity of other proteins, including caspase-11. Thus, it is possible that the results observed with THPIs may be based on inhibition of caspase-11, not MMPs. The present study evaluated whether THPIs were cross-reactive with caspase-11. Two different THPIs were tested, one that is known to inhibit MMP-1 and MMP-8 (GlyΨ{PO2H-CH2}Ile-His-Lys-Gln THPI) and one that is selective for MMP-2 and MMP-9 (α1(V)GlyΨ{PO2H-CH2}Val [mep14,32,Flp15,33] THPI). No inhibition of caspase-11 was observed with GlyΨ{PO2H-CH2}Ile-His-Lys-Gln THPI, even at an inhibitor concentration of 5 µM, while 5 µM α1(V)GlyΨ{PO2H-CH2}Val [mep14,32,Flp15,33] THPI exhibited 40% inhibition of caspase-11. Further testing of GlyΨ{PO2H-CH2}Ile-His-Lys-Gln THPI revealed nM inhibition of MMP-2, MMP-9, and MMP-13. Thus, the effectiveness of GlyΨ{PO2H-CH2}Ile-His-Lys-Gln THPI observed in a sepsis animal model may not be due to caspase-11 inhibition, but may be due to broader MMP inhibition than previously thought.