Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy.

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44-MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

Small-molecule anticancer compounds selectively target the hemopexin domain of matrix metalloproteinase-9.

Lack of target specificity by existing matrix metalloproteinase (MMP) inhibitors has hindered antimetastatic cancer drug discovery. Inhibitors that bind to noncatalytic sites of MMPs and disrupt protease signaling function have the potential to be more specific and selective. In this work, compounds that target the hemopexin (PEX) domain of MMP-9 were identified using an in silico docking approach and evaluated using biochemical and biological approaches. Two of the selected compounds interfere with MMP-9-mediated cancer cell migration and proliferation in cells expressing exogenous or endogenous MMP-9. Furthermore, these inhibitors do not modulate MMP-9 catalytic activity. The lead compound, N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide, specifically binds to the PEX domain of MMP-9, but not other MMPs. This interaction between the compound and the PEX domain results in the abrogation of MMP-9 homodimerization and leads to blockage of a downstream signaling pathway required for MMP-9-mediated cell migration. In a tumor xenograft model, this pyrimidinone retarded MDA-MB-435 tumor growth and inhibited lung metastasis. Thus, we have shown for the first time that a novel small-molecule interacts specifically with the PEX domain of MMP-9 and inhibits tumor growth and metastasis by reducing cell migration and proliferation.

Regulation of plasma hemopexin activity by stimulated endothelial or mesangial cells.

The pathogenesis of glomerular alterations and proteinuria in corticosteroid-responsive nephrotic syndrome (CRNS) is unknown. As an isoform of plasma hemopexin (Hx) with protease activity may be implicated in this disease, we have studied the inhibition of Hx by ADP and reactivation to its active form by endothelial or mesangial cells in vitro. We hypothesized that these cells might potentially be able to convert the inactivated form of Hx (Hxi) to active Hx (Hxa) in vitro, mediated by cellular ecto-ADPase. Since ecto-ADPase (CD39) is upregulated after stimulation of these cells with lipopolysaccharide (LPS) or certain cytokines, we postulated that this conversion might occur specifically after inflammatory stimulation of these cells. Human endothelial or mesangial cell cultures were incubated overnight with or without LPS (10.0 ng/ml) or TNFalpha (10.0 ng/ml), washed and subsequently incubated with Hxi (1.5 mg/ml) in serum-free conditions (Hxi was prepared by treatment of Hxa with ADP or ADP-beta-S). After 60 min, supernatants were tested for their capacity to alter glomerular extracellular matrix molecules (i.e. ecto-apyrase) in vitro using standard methods, and compared with Hxi that had not been incubated with cells. Supernatants containing Hxa (1.5 mg/ml) served as positive control. The results show significant activity in supernatants with Hxi (prepared using native ADP). However, Hxi inactivated by ADP-beta-S (which is non-hydrolyzable) could not be reactivated after contact with LPS-stimulated or unstimulated cells in vitro. As ecto-ADPase of these cells is upregulated by LPS, we believe that reactivation of Hxi to Hxa is mediated by cellular ecto-ADPase. Although the relevance of this inflammation-mediated activation mechanism of Hx in patients with CRNS requires further experimentation, our preliminary observations suggesting that this mechanism is corticosteroid dependent may support a potential role of Hxa in CRNS.