Bifunctional Inhibitors as a New Tool To Reduce Cancer Cell Invasion by Impairing MMP-9 Homodimerization

Abstract

Protein homodimers play important roles in physiological and pathological processes, including cancer invasion and metastasis. Recently, MMP-9 natural homodimerization via the PEX domain has been correlated with high migration rates of aggressive cancer cells. Here we propose that bifunctional MMP-9 inhibitors designed to impair natural MMP-9 homodimerization promoted by PEX-PEX interactions might be an effective tool to fight cancer cell invasion. Elaborating a previously described dimeric hydroxamate inhibitor 1, new ligands were synthesized with different linker lengths and branch points. Evaluation of the modified bifunctional ligands by X-ray crystallography and biological assays showed that 7 and 8 could reduce invasion in three glioma cell lines expressing MMP-9 at different levels. To rationalize these results, we present a theoretical model of full-length MMP-9 in complex with 7. This pioneering study suggests that a new approach using MMP-9 selective bifunctional inhibitors might lead to an effective therapy to reduce cancer cell invasion.

Keywords: Bifunctional inhibitors; MMP-9 homodimerization; X-ray crystallography; glioblastoma multiforme.

Figure 1

New bifunctional inhibitors design from the crystallographic structure of the complex 1-MMP-9 catalytic domain. (A) Crystal structure of MMP-9 (PDB 4H2E) in complex with 1 (green) showing how the long linker spans the distance between the two MMP-9 catalytic domains present in the crystal lattice. (B) Superimposition of the SC-74020 (magenta) complex with-MMP-2 from the NMR structure (PDB 1HOV) on each of the two crystallographic cat-MMP-9. The shortest distance between the two SC-74020 is only 3.6 Å (red box). Given the similarity of the central cores around the ZBG of 1 and SC-74020, changing the linkage point from P1 to P2′ should yield a shorter DEI linker that might induce the same MMP-9 dimer observed in the crystal.

Chart 1. Chemical Structures of New Compounds 5–10 and the Known Compounds 2–4

Scheme 1. Synthesis of P2′ Dimers 7–10

Reagents and conditions: (a) N-hydroxysuccinimide, Et₃N, THF, rt, 2 h; (b) DIPEA, DMF, rt, 16 h; (c) TFA, CH₂Cl₂, 0 °C, 5 h; (d) TBDMSiONH₂, EDC, CH₂Cl₂, rt, 16 h; (e) TFA, CH₂Cl₂, 0 °C, 5 h.

Figure 2

Superimposition of bifunctional ligands 1, 7, and 8. (A) The shortening and repositioning of the branch point results in reduced geometrical freedom (1, PDB 4H2E, green; 8 PDB 4H82, cyan). (B) Comparison of the dimerization mode of MMP-9 (gray ribbons) and MMP-12 (PDB 4H49, yellow) to highlight that it is unlikely that different MMPs will share dimerization modes. (C) Superposition on Cα atoms of residues from the cat-MMP-9: molecule B of PDB 4HMA, orange cartoon with ligand 7 (yellow sticks) was superposed with molecule B of PDB 4H82, gray cartoon with ligand 8 in cyan sticks. Changing the ZBG from the hydroxamate of 8 to carboxylate of 7 in the MMP-9 complex alters only slightly the overall linker-induced trimer. (D) Zoom on the MMP-9 zinc atom to highlight the subtle changes induced by changing the ZBG. The position of the zinc in the hydroxamate complex, 8 (gray) differs slightly from that in the MMP-9 carboxylate complex 7 (black). The difference in the zinc position and in the chelation mode of the hydroxamate moiety compared to the carboxylate alters only slightly the positioning of the linker, which at long distance changes the dimerization. Such subtle differences impact strongly on affinity and selectivity.

Figure 3

MMP inhibitors effects on cell invasiveness. The invasiveness of treated and untreated (DMSO, control) cells (U87MG, U343MG, and T98G) were assessed in matrigel basement membrane matrix. Cells able to invade were visualized by crystal violet probe and counted by the means of ImageJ software. Data are presented as the mean ± SEM, and derived from at least three independent experiments done in duplicate. *p < 0.05; **p < 0.01; ***p < 0.005; *Significance vs control using ANOVA with post hoc Newman–Keuls test. ^§p < 0.05; ^§§p < 0.01; ^§significance vs CGS27023A, using ANOVA with post hoc Newman–Keuls test.

Figure 4

Proposed trimeric adduct MMP-9-7-MMP-9. Surface visualization (A) and ribbon representation (B) (domains colors: pink, catalytic; orange, fibronectin; blue, hemopexin). Compound 7 is colored green.