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

The Expanding Role of MT1-MMP in Cancer Progression

For over 20 years, membrane type 1 matrix metalloproteinase (MT1-MMP) has been recognized as a key component in cancer progression. Initially, the primary roles assigned to MT1-MMP were the activation of proMMP-2 and degradation of fibrillar collagen. Proteomics has revealed a great array of MT1-MMP substrates, and MT1-MMP selective inhibitors have allowed for a more complete mapping of MT1-MMP biological functions. MT1-MMP has extensive sheddase activities, is both a positive and negative regulator of angiogenesis, can act intracellularly and as a transcription factor, and modulates immune responses. We presently examine the multi-faceted role of MT1-MMP in cancer, with a consideration of how the diversity of MT1-MMP behaviors impacts the application of MT1-MMP inhibitors

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

Phosphorylation of PEA-15 switches its binding specificity from ERK/MAPK to FADD

Cell signalling pathways that regulate proliferation and those that regulate programmed cell death (apoptosis) are co-ordinated. The proteins and mechanisms that mediate the integration of these pathways are not yet fully described. The phosphoprotein PEA-15 (phosphoprotein enriched in astrocytes) can regulate both the ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) pathway and the death receptor-initiated apoptosis pathway. This is the result of PEA-15 binding to the ERK/MAPK or the proapoptotic protein FADD (Fas-activated death domain protein) respectively. The mechanism by which binding of PEA-15 to these proteins is controlled has not been elucidated. PEA-15 is a phosphoprotein containing a Ser-104 phosphorylated by protein kinase C and a Ser-116 phosphorylated by CamKII (calcium/calmodulin-dependent protein kinase II) or AKT. Phosphorylation of Ser-104 is implicated in the regulation of glucose metabolism, while phosphorylation at Ser-116 is required for PEA-15 recruitment to the DISC (death-initiation signalling complex). Moreover, PEA-15 must be phosphorylated at Ser-116 to inhibit apoptosis. In the present study, we report that phosphorylation at Ser-104 blocks ERK binding to PEA-15 in vitro and in vivo, whereas phosphorylation at Ser-116 promotes its binding to FADD. We further characterize phospho-epitope-binding antibodies to these sites. We report that phosphorylation does not influence the distribution of PEA-15 between the cytoplasm and nucleus of the cell since all phosphorylated states are found predominantly in the cytoplasm. We propose that phosphorylation of PEA-15 acts as the switch that controls whether PEA-15 influences proliferation or apoptosis

Synchrotron-microtomographic studies of normal and pathological cranial sutures - a further insight

ADAM17 is implicated in several debilitating diseases. However, drug discovery efforts targeting ADAM17 have failed due to the utilization of zinc-binding inhibitors. We previously reported discovery of highly selective nonzinc-binding exosite-targeting inhibitors of ADAM17 that exhibited not only enzyme isoform selectivity but synthetic substrate selectivity as well ( J. Biol. Chem. 2013, 288, 22871). As a result of SAR studies presented herein, we obtained several highly selective ADAM17 inhibitors, six of which were further characterized in biochemical and cell-based assays. Lead compounds exhibited low cellular toxicity and high potency and selectivity for ADAM17. In addition, several of the leads inhibited ADAM17 in a substrate-selective manner, which has not been previously documented for inhibitors of the ADAM family. These findings suggest that targeting exosites of ADAM17 can be used to obtain highly desirable substrate-selective inhibitors. Additionally, current inhibitors can be used as probes of biological activity of ADAM17 in various in vitro and, potentially, in vivo systems

Structure-Based Design and Synthesis of Potent and Selective Matrix Metalloproteinase 13 Inhibitors

We describe the use of comparative structural analysis and structure-guided molecular design to develop potent and selective inhibitors (<b>10d</b> and (<i>S</i>)-<b>17b</b>) of matrix metalloproteinase 13 (MMP-13). We applied a three-step process, starting with a comparative analysis of the X-ray crystallographic structure of compound <b>5</b> in complex with MMP-13 with published structures of known MMP-13·inhibitor complexes followed by molecular design and synthesis of potent but nonselective zinc-chelating MMP inhibitors (e.g., <b>10a</b> and <b>10b</b>). After demonstrating that the pharmacophores of the chelating inhibitors (<i>S</i>)-<b>10a</b>, (<i>R</i>)-<b>10a</b>, and <b>10b</b> were binding within the MMP-13 active site, the Zn²⁺ chelating unit was replaced with nonchelating polar residues that bridged over the Zn²⁺ binding site and reached into a solvent accessible area. After two rounds of structural optimization, these design approaches led to small molecule MMP-13 inhibitors <b>10d</b> and (<i>S</i>)-<b>17b</b>, which bind within the substrate-binding site of MMP-13 and surround the catalytically active Zn²⁺ ion without chelating to the metal. These compounds exhibit at least 500-fold selectivity versus other MMPs