Selective Modulation of Matrix Metalloproteinase 9 (MMP-9) Functions via Exosite Inhibition

Abstract

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)...

Exosite Interactions Impact Matrix Metalloproteinase Collagen Specificities

“…Enzyme aliquots were kept on wet ice and used the same day. MMP activity was initially evaluated using the Knight single-stranded peptide and compared with prior data (17,18). In this way, Knight singlestranded peptide activity was used as an indicator of enzyme integrity rather than tissue inhibitor of metalloproteinase titration as performed previously (8).…”

“…fTHP-15, fTHP-16, and fTHP-17 (see Table 1 for sequences) were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase chemistry as described previously (8,17,18,30). Peptide synthesis was carried out on a Protein Technologies PS3 peptide synthesizer (Tucson, AZ).…”

“…For the aforementioned collagenases, efficient catalytic activities toward collagens require both the CAT and HPX domains (11)(12)(13)(14)(15)(16)(17). Taking into account the importance of the CAT and HPX domains, the collagenolytic process has been proposed to involve discrete, sequential steps (17). First, an MMP binds a triple helix utilizing secondary binding sites (exosites) with prominent exosites identified in the HPX domain (18).…”

“…THP substrates have been powerful tools for elucidating MMP kinetic behavior and substrate cleavage selectivity (17,18,29,30). In a prior study, we recently have found that by varying the composition of collagen model sequences within THPs we can explore unique binding interactions in the CAT and HPX domains of MMP-1 (18).…”

The Collagen Binding Domain of Gelatinase A Modulates Degradation of Collagen IV by Gelatinase B

“…[24][25][26] Collagenolytic activities and MMP inhibition have been largely studied on triple-helical synthetic substrates. 27,28 Also, the role of the CBD exosite in modulating type I collagen degradation has been extensively documented. [29][30][31][32][33][34] Nevertheless, despite the importance of type IV collagen degradation, it has been little studied.…”