The human somatic angiotensin converting enzyme (ACE) contains two homologous domains, each bearing a zinc-dependent active site. All of the synthetic inhibitors of this enzyme used in clinical applications interact with these two active sites to a similar extent. Recently, several lines of evidence have suggested that the N-terminal active site of ACE might be involved in specific hydrolysis of some important physiological substrates, like Acetyl-Seryl-Aspartyl-LysylProline, a negative regulator of hematopoietic stem cell differentiation and proliferation. These findings have stimulated studies aimed at identifying new ACE inhibitors able to block only one of the two active sites of this enzyme. By screening phosphinic peptide libraries, we discovered a phosphinic peptide Ac-Asp-(L) Phe(PO 2 -CH 2 ) (L) Ala-Ala-NH 2 , called RXP 407, which is able to differentiate the two ACE active sites, with a dissociation constant three orders of magnitude lower for the N-domain of the enzyme. The usefulness of a combinatorial chemistry approach to develop new lead structures is underscored by the unusual chemical structure of RXP 407, as compared with classical ACE inhibitors. As a highly potent and selective inhibitor of the N-terminal active site of wild ACE (K i ‫؍‬ 12 nM), RXP 407, which is metabolically stable in vivo, may lead to a new generation of ACE inhibitors able to block in vivo only a subset of the different functions regulated by ACE.

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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...