Unregulated activities of the matrix metalloproteinase (MMP) family have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix components, such as collagen and laminin. However, clinical trials with small molecule MMP inhibitors have been largely unsuccessful, with a lack of selectivity considered particularly problematic. Enhanced selectivity could be achieved by taking advantage of differences in substrate secondary binding sites (exosites) within the MMP family. In this study, triple-helical substrates and triple-helical transition state analog inhibitors have been utilized to dissect the roles of potential exosites in MMP-9 collagenolytic behavior. Substrate and inhibitor sequences were based on either the ␣1(V)436 -450 collagen region, which is hydrolyzed at the Gly2Val bond selectively by MMP-2 and MMP-9, or the Gly2Leu cleavage site within the consensus interstitial collagen sequence ␣1(I-III)769 -783, which is hydrolyzed by MMP-1, MMP-2, MMP-8, MMP-9, MMP-13, and MT1-MMP. Exosites within the MMP-9 fibronectin II inserts were found to be critical for interactions with type V collagen model substrates and inhibitors and to participate in interactions with an interstitial (types I-III) collagen model inhibitor. A triple-helical peptide incorporating a fibronectin II insert-binding sequence was constructed and found to selectively inhibit MMP-9 type V collagen-based activities compared with interstitial collagen-based activities. This represents the first example of differential inhibition of collagenolytic activities and was achieved via an exosite-binding triple-helical peptide.Collagen catabolism (collagenolysis) is normally a well regulated physiological process critical to tissue and organ development, morphogenesis, and wound healing (1). Pathological conditions resulting from aberrant collagenolysis include primary and metastatic tumor growth, arthritis, arteriosclerosis, and periodontitis (1-4). A number of proteases have been described as exhibiting collagenolytic behavior, requiring that they catalyze the hydrolysis of the collagen triple helix (1). The most extensively studied are the matrix metalloproteinases (MMPs).2 MMPs that catalyze the hydrolysis of one or more of the interstitial collagens (types I-III) within their triple-helical domain include the secreted proteases MMP-1, MMP-2, MMP-8, MMP-9, and MMP-13 and the membrane-bound proteases MT1-MMP and MT2-MMP (5-8).Considerable work has been performed to define the MMP domains and regions that participate in collagenolysis. In the cases of MMP-1, MMP-8, MMP-13, MT1-MMP, and MT2-MMP, efficient collagenolytic activity in the isolated enzyme requires both the catalytic (CAT) and hemopexin (HPX)-like domains ( Fig. 1) (9 -14). The linker region between these domains also participates in collagenolysis, either by direct binding of substrate (15) or by allowing for the proper orientation of the CAT and HPX domains (16). The "gelatinase" members of the MMP family (MMP-2 and MMP-9)...

Alterations in activities of one family of proteases, the matrix metalloproteinases (MMPs), have been implicated in primary and metastatic tumor growth, angiogenesis, and pathological degradation of extracellular matrix (ECM) components, such as collagen and laminin. Since hydrolysis of the collagen triple-helix is one of the committed steps in ECM turnover, we envisioned modulation of collagenolytic activity as a strategy for creating selective MMP inhibitors. In the present study, a phosphinate transition state analogue has been incorporated within a triple-helical peptide template. The template sequence was based on the alpha1(V)436-450 collagen region, which is hydrolyzed at the Gly(439)-Val(440) bond selectively by MMP-2 and MMP-9. The phosphinate acts as a tetrahedral transition state analogue, which mimics the water-bound peptide bond of a protein substrate during hydrolysis. The phosphinate replaced the amide bond between Gly-Val in the P1-P1' subsites of the triple-helical peptide. Inhibition studies revealed Ki values in the low nanomolar range for MMP-2 and MMP-9 and low to middle micromolar range for MMP-8 and MMP-13. MMP-1, MMP-3, and MT1-MMP/MMP-14 were not inhibited effectively. Melting of the triple-helix resulted in a decrease in inhibitor affinity for MMP-2. The phosphinate triple-helical transition state analogue has high affinity and selectivity for the gelatinases (MMP-2 and MMP-9) and represents a new class of protease inhibitors that maximizes potential selectivity via interactions with both prime and nonprime active site subsites as well as with secondary binding sites (exosites).

A convenient and efficient method has been developed for the preparation of 9-fluorenylmethoxycarbonyl (Fmoc)-protected 1-aminoalkylphosphinic acids. Reproducible procedures for the synthesis and purification of free alpha-amino H-phosphinates are provided. Protection of free amino phosphinates as the N-Fmoc derivative was achieved by in situ trimethylsilylation of aminoalkylphosphinic acids, which then reacted with Fmoc-Cl to provide corresponding products in excellent yields and in high purity after simple extractive isolation. Mechanistic aspects of the silylation are discussed, and the application of the procedure to another class of amino phosphorus acids is presented.

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