Abstract
The pathophysiological functions of the signaling molecules matrix metalloproteinase-14 (MMP-14) and integrin αvβ3 in various types of cancer are believed to derive from their collaborative activity in promoting invasion, metastasis, and angiogenesis, as shown in vitro and in vivo The two effectors act in concert in a cell-specific manner through the localization of pro-MMP-2 to the cell surface, where it is processed to intermediate and matured MMP-2. The matured MMP-2 product is localized to the cell surface via its binding to integrin αvβ3 The MMP-14/MMP-2/integrin αvβ3 axis thus constitutes an attractive putative target for therapeutic interventions, but the development of inhibitors that target this axis remains an unfulfilled task. To address the lack of such multitarget inhibitors, we have established a combinatorial approach that is based on flow cytometry screening of a yeast-displayed N-TIMP2 (N-terminal domain variant of tissue inhibitor of metalloproteinase-2) mutant library. On the basis of this screening, we generated protein monomers and a heterodimer that contain monovalent and bivalent binding epitopes to MMP-14 and integrin αvβ3 Among these proteins, the bi-specific heterodimer, which bound strongly to both MMP-14 and integrin αvβ3, exhibited superior ability to inhibit MMP-2 activation and displayed the highest inhibitory activity in cell-based models of a MMP-14-, MMP-2-, and integrin αvβ3-dependent glioblastoma and of endothelial cell invasiveness and endothelial capillary tube formation. These assays enabled us to show the superiority of the combined target effects of the inhibitors and to investigate separately the role each of the three signaling molecules in various malignant processes.
Highlights
6.3 a Tube formation is measured as number of segments. b Percent of inhibition was compared with the untreated control. c For untreated cells, the percentages of the different MMP-2 forms are as follows: 3.9% for MMP-2 intermediate; 3.5% for MMP-2 matured; and 7.4% for total active MMP-2. d Ϫ represents activation
The channel loaded with BSA served as a background control for integrin ␣v3 and MMP-14CAT binding
150 l of U87MG serum-free medium obtained after 48 h of cell growth, which contained all the MMP-2 forms, was added to each well, together with 2 M of either Ala–N-TIMP2WT, RGD or cRGD, and the plate was incubated overnight at 4 °C
Summary
An N-TIMP2RGD yeast surface display (YSD) loop library was designed in such a way that positions 51–59 at the apex of loop B–C in N-TIMP2, which naturally binds integrin ␣31, were replaced with a sequence comprising an RGD motif flanked on each side by three additional random amino acids (XXXRGDXXX) (Fig. 1A and Fig. S1). We engineered Ala–N-TIMP2WT, RGD, a protein variant having high affinity toward integrin ␣v3 and reduced inhibition activity toward MMP-14, by inserting an alanine residue before the cysteine residue at position 1 (Fig. 1C). The bi-specific monomer N-TIMP2WT, RGD was shown to bind both integrin ␣v3 and MMP-14CAT, with KD values of 64.8 nM (Fig. 2A) and 838 nM (Fig. 2C), respectively (Table 1). As expected, binding to integrin ␣v3 was not detected for the TIMP2WT and N-TIMP25M monospecific MMP-14 variants (Fig. S4A) In agreement with these results, the insertion of the five mutations into. N-TIMP25M, RGD significantly increased the affinity toward MMP-14CAT by ϳ400-fold (KD of 2 nM), the affinity toward integrin ␣v3 was decreased (KD of 531 nM) but not completely abolished (Table 1 and Fig. S4, C and D). Binding kinetics of N-TIMP2 variants ND means binding not detected at 1 M concentration; NA means not available; NT means not tested
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