Potent Mechanism-based Inhibitors for Matrix Metalloproteinases

Abstract

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that play important roles in physiological and pathological conditions. Both gelatinases (MMP-2 and -9) and membranetype 1 MMP (MMP-14) are important targets for inhibition, since their roles in various diseases, including cancer, have been well established. We describe herein a set of mechanism-based inhibitors that show high selectivity to gelatinases and MMP-14 (inhibitor 3) and to only MMP-2 (inhibitors 5 and 7). These molecules bind to the active sites of these enzymes, initiating a slow binding profile for the onset of inhibition, which leads to covalent enzyme modification. The full kinetic analysis for the inhibitors is reported. These are nanomolar inhibitors (K i ) for the formation of the noncovalent enzyme-inhibitor complexes. The onset of slow binding inhibition is rapid (k on of 10 2 to 10 4 M ؊1 s ؊1 ), and the reversal of the process is slow (k off of 10 ؊3 to 10 ؊4 s ؊1 ). However, with the onset of covalent chemistry with the best of these inhibitors (e.g. inhibitor 3), very little recovery of activity (<10%) was seen over 48 h of dialysis. We previously reported that broad spectrum MMP inhibitors like GM6001 enhance MT1-MMP-dependent activation of pro-MMP-2 in the presence of tissue inhibitor of metalloproteinases-2. Herein, we show that inhibitor 3, in contrast to GM6001, had no effect on pro-MMP-2 activation by MT1-MMP. Furthermore, inhibitor 3 reduced tumor cell migration and invasion in vitro. These results show that these new inhibitors are promising candidates for selective inhibition of MMPs in animal models of relevant human diseases.Extracellular proteolysis is an essential aspect of both physiological and pathological processes. Several enzyme families have been implicated in extracellular proteolysis, of which the matrix metalloproteinases (MMPs) 3 constitute an important group. The MMPs are zinc-dependent endopeptidases that play key roles in embryonic development, neurological processes, wound healing, angiogenesis, arthritis, cardiovascular diseases, and cancer, just to mention a few examples. In cancer, for instance, MMPs are implicated at all stages of tumor progression, including tumor growth, angiogenesis, and metastasis (1). Two MMPs, gelatinases A and B (MMP-2 and MMP-9, respectively), are highly expressed in human cancer, and a direct relationship between cancer progression and gelatinase expression and activity has been well established in many studies (2). As tumors manifest high levels of gelatinase activity, inhibitors specific for the gelatinases are highly sought.In the past 8 years, there have been numerous approaches aimed at targeting MMP activities in tumors, and several clinical trials were carried out to test the efficacy of various inhibitors. Unfortunately, the results of these trials were disappointing due to the lack of an objective clinical response and undesired side effects. Many reasons have been postulated for these effects, but at the core of the problem remains the issue of inhibitor ...

Distinct Functionality of Tumor Cell–Derived Gelatinases during Formation of Liver Metastases

“…In these approaches, a bipronged effect of the respective inhibitor could be observed, namely inhibition of the metastatic potential, as well as inhibition of metastatic foci outgrowth (26). Application of synthetic inhibitors did not allow analysis of whether host-derived or tumor cell -derived gelatinases are important for metastasis nor did it elucidate distinct functions of the two gelatinases (24). To date, distinction of the respective functions of MMP-2 and MMP-9 in metastasis could therefore only be investigated preferably in gene deficiency models.…”

“…The greatest progress has been made when inhibitors were synthesized which were relatively specific for gelatinases (22) or even displayed mechanism-based binding to gelatinases (23,24). Indeed, these gelatinase-specific inhibitors were efficient in inhibiting aggressive metastasis in the well-established syngeneic and immunocompetent L-CI.5s lymphoma liver metastasis model (25,26).…”

Reduced nonprotein thiols inhibit activation and function of MMP-9: Implications for chemoprevention

“…Because of the importance of MMP dysregulation in cancer development, identification and evaluation of MMP inhibitors as cancer chemotherapeutic agents are currently active areas of investigation [18][19][20]. Further, the use of MMP inhibitors in a cancer prevention ("chemoprevention") protocol is appealing due to the importance of basement membrane invasion in transformation of premalignant lesions, e.g., progression of oral epithelial dysplasia to HNSCC.…”

“…Okamoto et al determined that glutathione (GSH), but not N-acetylcysteine (NAC), complexed with MMP-9's auto-inhibitory domain [13]. Other investigations by Albini et al demonstrated that introduction of NAC to conditioned medium obtained from cultured Kaposi sarcoma cells inhibited MMP-2 and MMP-9 gelatinase activity and that treatment of cultured Kaposi sarcoma cells with NAC significantly suppressed tumor cell invasion [17].Because of the importance of MMP dysregulation in cancer development, identification and evaluation of MMP inhibitors as cancer chemotherapeutic agents are currently active areas of investigation [18][19][20]. Further, the use of MMP inhibitors in a cancer prevention ("chemoprevention") protocol is appealing due to the importance of basement membrane invasion in transformation of premalignant lesions, e.g., progression of oral epithelial dysplasia to HNSCC.…”

Sulfonate-Containing Thiiranes as Selective Gelatinase Inhibitors

“…18 We also investigated the type of inhibition that was observed. As reported earlier in our mechanistic studies of the thiirane-type gelatinase inhibitors, slow-binding behavior is a hallmark of the mechanism of action of this type of inhibitors.…”