Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.

The matrix metalloproteinases (MMPs)2 form a group of structurally related extracellular zinc endoproteases able to degrade at least one protein component of the extracellular matrix (1). Based on this property, MMPs are considered to be critical mediators of both normal and pathological tissue remodeling processes (2, 3). Their overexpression is observed in and associated with a variety of diseases, including cancer (4, 5), arthritis (6), multiple sclerosis (7,8), and atherosclerosis (9, 10). Therefore, there is substantial interest in developing MMP synthetic inhibitors for a variety of therapeutic indications (11)(12)(13)(14)(15). Results of the first clinical trials with broad spectrum MMP inhibitors in cancer therapy were disappointing, highlighting the need for better understanding of the exact role of each MMP during the different stages of tumor progression (16). Recent research in this field has focused on the development of inhibitors that fully differentiate one MMP from another (17). This is a particularly difficult task, since the topology and nature of the residues in the enzyme's active site are highly conserved among the different MMPs (18). Moreover, parts of the MMP catalytic domain, which play a critical role in enzyme specificity, seem to be highly flexible (19,20). This situation may explain why most previously reported MMP synthetic inhibitors preferentially inhibit some MMPs but are not exclusive inhibitors of a single MMP. A recent breakthrough in this field was achieved by identifying the first highly selective synthetic inhibitor of MMP-13 (21). Selective inhibitors for MMP-2 and MMP-9 have also been reported recently, but their degree of selectivity toward MMPs is less than that achieved for the MMP-13-selective inhibitor (22).To identify highly selective MMP inhibitors, libraries of phosphinic peptides were prepared. Phosphinic peptides are good transition state mimics and have been shown to behave as highly potent inhibitors of different zinc metalloproteinases (23). To probe the SЈ 1 cavity of MMPs, a chemical strategy that makes it possible to prepare phosphinic peptides harboring various substituents in their PЈ 1 position was used (see Scheme 1). This strategy relies on the use of a common precursor, which can be modified in one step, to prepare phosphinic peptides displaying substituted isoxazole side chains in their PЈ 1 position (24). In such phosphinic peptides, the isoxazole ring is used as a rigid scaffold to project in the right orientation various chemical groups able to interact with the SЈ 1 subsite of MMPs, which corresponds to a deep cavity. Based on this strategy, four libraries of phosphinic peptides, containing four different isoxazole side chains in their PЈ 1 position, were prepared by introducing additional chemical diversity in the PЈ 2 and PЈ 3 positions of the inhibitors (see Scheme 1). Screening of these libraries against 10 different MMPs allowed us to identify highly selective inhibitors of MMP-12. Based on the unique structure of these inhibitors, a highl...

Objective-Matrix metalloproteinase (MMP)-12 has been implicated in plaque progression and instability and is also amenable to selective inhibition. In this study, we investigated the influence of a greater than 10-fold selective synthetic MMP-12 inhibitor on plaque progression in the apolipoprotein E knockout mouse model of atherosclerosis. Methods and Results-A phosphinic peptide (RXP470.1) that is a potent, selective murine MMP-12 inhibitor significantly reduced atherosclerotic plaque cross-sectional area by approximately 50% at 4 different vascular sites in male and female apolipoprotein E knockout mice fed a Western diet. Furthermore, RXP470.1 treatment resulted in less complex plaques with increased smooth muscle cell:macrophage ratio, less macrophage apoptosis, increased cap thickness, smaller necrotic cores, and decreased incidence of calcification. Additional in vitro and in vivo findings indicate that attenuated monocyte/macrophage invasion and reduced macrophage apoptosis probably underlie the beneficial effects observed on atherosclerotic plaque progression with MMP-12 inhibitor treatment. However, recent findings reveal a more complex relationship between MMP activity and plaque stability. For example, we have demonstrated in hypercholesterolemic mice that MMP-3 and MMP-9 appear to have plaque-stabilizing roles, whereas MMP-12 is detrimental. 4 Other murine studies using either gene ablation or protein overexpression have also shown that some MMPs support the growth of stable plaques through smooth muscle cell migration or matrix deposition or have no effect, whereas others, as expected, promote an unstable plaque phenotype via several mechanisms, including ECM destruction (see review 5 ). Possibly because of the divergent roles MMPs play, broad-spectrum MMP inhibitors have failed to show clear benefits against atherosclerotic plaque growth or morphology in either animal or clinical studies. 6 -11 Manipulating expression of tissue inhibitors of MMPs in a mouse model of atherosclerosis has also yielded inconsistent results. 12 These findings suggest that selectively targeting MMPs with a clearly detrimental role could be more effective for inhibiting atherosclerotic plaque progression and instability. The close structural similarity of MMPs around their active sites has made developing highly selective MMP inhibitors a difficult task in general. 13 However, by exploiting phosphinic peptide chemistry, a strategy shown previously to provide more selective inhibitors of zinc-proteases, 14 has led to the identification of a novel potent and highly selective MMP-12 inhibitor, RXP470.1, which has a K i against human MMP-12 of 0.2 nmol/L but is 2 to 4 orders of magnitude less potent 18,19 we investigated the effect of RXP470.1 on plaque progression and morphology in apolipoprotein E (apoE) knockout mice. We treated mice with existing atherosclerosis with RXP470.1 for 4 weeks and tested its effects on plaques at 4 arterial sites. Conclusion-Our Materials and MethodsAn expanded Methods section is available...

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The molecular determinants responsible for the potency of the RXP470.1 phosphinic peptide inhibitor toward matrix metalloprotease-12 (MMP-12) remain elusive. To address this issue, structure-activity study, X-ray crystallography, and isothermal titration calorimetry (ITC) experiments were performed. The crystal structure of MMP-12/inhibitor complex (1.15 Å) reveals that the inhibitor establishes multiple interactions with the MMP-12 active site, with its long P(1)' side chain filling most of the S(1)' deep cavity. ITC experiments indicate that the binding of this inhibitor to MMP-12 is mostly entropy driven (ΔG° = -13.1 kcal/mol, ΔH° = -2.53 kcal/mol, and -TΔS° = -10.60 kcal/mol) and involves a proton uptake from the buffer. Comparing phosphinic versus hydroxamate inhibitors reveals that the chelation of the zinc ion is slightly different, leading the inhibitor backbone to adopt a position in which the hydrogen bonding with the MMP-12 active site is less favorable in phosphinic inhibitor while maintaining high affinity.