Design, synthesis, and characterization of potent, slow-binding inhibitors that are selective for gelatinases

Gelatinases have been shown to play a key role in angiogenesis and tumor metastasis. Small molecular weight synthetic inhibitors for these enzymes are highly sought for potential use as anti-metastatic agents. Virtually all of the known inhibitors of matrix metalloproteinases (MMPs) are broad spectrum. We report herein the synthesis and kinetic characterization of two compounds, 4-(4-phenoxyphenylsulfonyl)butane-1,2-dithiol (compound 1) and 5-(4-phenoxyphenylsulfonyl)pentane-1,2-dithiol (compound 2), that are potent and selective gelatinase inhibitors. These compounds are slow, tight-binding inhibitors of gelatinases (MMP-2 and MMP-9) with K-i values in the nanomolar range. In contrast, competitive inhibition of the catalytic domain of membrane-type 1 metalloproteinase (MMP-14(cat)) with comparable K-i values (K-i similar to200 n(M)) was observed. Binding to stromelysin (MMP-3) was substantially weaker, with Ki values in the micromolar range (K-i similar to10 mu(M)). No binding to matrilysin (MMP-7) and collagenase 1 (MMP-1) was detected at inhibitor concentrations up to 60 mu(M). We have previously shown that synthetic MMP inhibitors work synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP in a process that depends on the affinity of the inhibitor toward MT1-MMP. It is shown herein that the dithiols are significantly less efficient (>100-fold) than marimastat, a broad-spectrum MMP inhibitor, in enhancing pro-MMP-2 activation in cells infected to express MT1-MMP, consistent with the lower affinity of the dithiols toward MT1-MMP. Thus, in contrast to broad-spectrum MMP inhibitors, the dithiols are less likely to promote MT1-MMP-dependent pro-MMP-2 activation in the presence of TIMP-2, while maintaining their ability to inhibit active MMP-2 effectively.