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By connection of Regasepin2, a heptapeptide inhibitor of matrix metalloproteinases (MMPs), to the N- or C-terminus of ES-2, an anti-angiogenic peptide of 11 residues, two designed peptides CPU1 and CPU2 were generated. Unexpectedly, CPU2 inhibited MMP-8 and MMP-9 activity in the nanomolar range, whereas CPU1 displayed a weaker inhibitory profile than Regasepin2 against TACE, MMP-8 and MMP-9. CPU1 showed a higher affinity than CPU2 with integrin α5β1 in a HUVEC adhesion assay. In an in vitro angiogenesis model of HUVEC migration, CPU1 showed higher inhibition potency than CPU2 (85% inhibition for CPU1 at a concentration of 0.8 μM versus 17% inhibition for CPU2 at the same concentration). In an in vivo angiogenesis model in chicken egg chorioallantoic membranes, 1.37 μM CPU1 showed a significant inhibition in the formation of new blood vessels, whereas CPU2 and Regasepin2 had no effect on angiogenesis. Furthermore, CPU1 significantly inhibited B16F10 melanoma growth in a syngeneic mouse model (inhibition rate of 56.91% by tumor weight analysis at a dose of 20 mg/kg/d), whereas CPU2 and Regasepin2 did not show any inhibitory effect. In view of recent findings that MMP-9 is pro-angiogenic, our results indicated that the combination of MMP inhibitors and anti-angiogenic peptides may generate novel molecules with potent in vivo anti-tumor effects.

The integrity of the vascular wall and its intrinsic basement membrane structures ensure that plasma and the corpuscular elements of the blood remain confined to the intravascular milieu and can enter into the extravascular compartment in a well controlled fashion in cases of tissue infection or inflammation. However, sometimes inflammatory stimuli act on blood leukocytes and on endothelial cells from within the blood vessels and in an overwhelming way, leading to inflammatory shock syndromes. These severe conditions with high mortality rates are characterized by intravascular neutrophil degranulation, permeability changes of endothelia and disintegration of basement membranes and lead to almost uncontrollable edema, coagulation changes and multi-organ failure. Matrix metalloproteinases (MMPs) have been functionally linked with septic and endotoxin shock, with cytokine release syndromes and with acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Here we review a number of association studies, compare inflammatory shock data from gene knockout studies in mice and provide some insights from recent investigations with inhibitors of MMPs. This evaluation strengthens the expectation that MMP inhibitors, in particular those blocking neutrophil proteases, may become useful in the early phase of acute inflammatory shock syndromes.

Matrix metalloproteinases (MMPs) are a large family of proteolytic enzymes involved in an array of physiological and pathological processes from development, morphogenesis, reproduction, wound healing, and aging to inflammation, angiogenesis, neurological disorders, and cancer cell invasion and metastasis. The imbalance between MMP activity and the inhibitory action of tissue inhibitors of metalloproteinases (TIMPs) are implicated in multiple diseases. Secreted in the body in a latent form, upon activation MMP-9 (gelatinase B) acts on many inflammatory substrates, and thus is suspected of contributing to the progression of cardiovascular disease, rheumatoid arthritis, and the subjects of this review, chronic obstructive pulmonary disease (COPD) and multiple sclerosis (MS). COPD is the fourth most common cause of death in the United States. In COPD, increased expression of MMP-9 by inflammatory cells e.g. neutrophils and macrophages is correlated with a variety of processes that cause lung damage. MMP-9 is also important in cytokine and protease modulation; it degrades the serine protease inhibitor alpha(1)-antitrypsin, which thus may lead to lung destruction. MS affects approximately 400,000 Americans and over a million people worldwide. Upregulation of MMP-9 increases the permeability of the blood brain barrier (BBB), facilitates the infiltration of leukocytes into the central nervous system, and causes myelin sheath degradation and neuronal damage. Early stage clinical trials have shown promising results when MMP-9 is inhibited in MS. These observations lead to the hypothesis that MMP-9 is a potential drug target for both COPD and MS and further development of highly potent and specific MMP-9 inhibitors is warranted.

Gelatinase B/MMP-9 fulfills crucial regulator or effector functions in disease states and may be pharmacologically targeted by specific inhibitors. The characteristics of cleavages of a natural gelatinase B substrate were simulated and amino acids with zinc-ion chelating side-groups were employed to design a library of peptide-based inhibitors. Here, we extend previous findings of combinatorial chemical synthesis and subsequent library deconvolution with a recently established high-throughput technology. This enabled us to study MMP inhibitors with two zinc-binding groups and to identify a new L-pyridylalanine-containing gelatinase B inhibitor. The peptide analog was found to inhibit, almost to the same degree, the neutrophil enzymes collagenase 2/ MMP-8 and MMP-9 and the monocytic tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) in vitro and to protect mice against lethal endotoxinemia in vivo. These data illustrate the usefulness of the screening platform for protective inhibitor discovery and complement recent insights in the pathogenesis and treatment of shock syndromes.