Fig 1.
Domain structure of MMPs (A) and schematic representation of galardin and compound 1b (B). All MMPs contain a signal peptide (cleaved off in the endoplasmatic reticulum), a pro-peptide domain and a catalytic domain. In addition, most MMPs contain a linker (hinge-region) and a hemopexin (HPX) like domain. The hinge region in MMP-9 differs from the other MMPs as it is longer and heavily O-glycosylated, and therefore also called the OG-domain. Three secreted (MMP-11, -21, -28) and all membrane-anchored MMPs have a basic RX[K/R]R motif at the C-terminal end of their pro-domain. This motif can be cleaved inside the cells by furin-like proteases. The two gelatinases (MMP-2, -9) contain three fibronectin II like repeats (FnII module) in their catalytic domain, located N-terminal to the catalytic Zinc-binding site. Four of the six membrane-type (MT)-MMPs are anchored to the cell membranes through a type I transmembrane domain and the other two through a glycosylphosphatidylinosityl (GPI) moiety.
Fig 2.
Purification and activation of proMMP-9.
(A) Imperial stained SDS-PAGE showing the purity of purified recombinant human full length proMMP-9 expressed in Sf9 cells (rproMMP-9) and of proMMP-9 purified from THP-1 cells (proMMP-9) as described in the Materials and Methods section. PT is the pass through fraction from Gelatin-Sepharose Chromatography of the recombinant enzyme, and 4 times more protein was loaded to the gel in the lanes labelled PT(2) compared to the lanes labelled PT(1). Std. 1 is the molecular size marker SpectraTM Mulitcolor High Range Protein Ladder and sb is sample buffer. Prior to electrophoresis, samples were either treated (+) or not treated (-) with DTT. Gelatin (B-D) and real-time gelatin (E) zymography of purified proMMP-9, trypsin activated (MMP-9) proMMP-9 from THP-1 cells, purified rproMMP-9, AMPA (rMMP-9(A)), trypsin (rMMP-9(T)) and MMP-3 (rMMP-9(M3)) activated recombinant proMMP-9. Std.2 in (B-E) is a mixture of proMMP-9 from THP-1 cells and proMMP-2 from human skin fibroblasts. Std. 3 is the 37 kDa catalytic domain of human MMP-9.
Table 1.
Inhibitory activity of galardin and compound 1b against human and bacterial metalloproteases.
Fig 3.
Inhibition of MMP-14 by galardin (A, B) and compound 1b (C, D). The inhibition constant Ki and [MMP-14] in assay were obtained from dose response plots vi/v0 vs [I] using the Morrison Eq (2) (A, C) and Henderson plots (B, D). In all plots, [MMP-14] was twice as high for experiments labelled (□) as for those labelled (●). The [S] is 5.0 μM except in the experiment in (B) labelled (□) where it is 10.0 μM. Shown in the figures is also the obtained Ki and [E] values (mean ± SD), in addition to the regression coefficient r2 and the number of individual assays (N) for each curve.
Fig 4.
Structural superimposition (backbone) of the 5cuh and the 1l6j x-ray structures of MMP-9.
The 1l6j structure contains the FnII domains (yellow) and the catalytic domain (green), while the 5cuh only contains the catalytic domain (blue). The pro-domain has been deleted from 1l6j, such that the sequence starts at F107. The co-crystallized hydroxamate inhibitor LT4 of the 5cuh in red, while the catalytic zinc and the coordinating histidines are in grey. The position of the co-crystallized inhibitor was used to define the docking grid during docking of galardin and compound 1b, and the figure shows that the docking into a structure lacking the FnII domains (5cuh) should not affect the docking results.
Fig 5.
Galardin and compound 1b docked into the catalytic site of MMP-9 and MMP-14.
The figure shows close ups of the active site region with the compound structures (xsticks), secondary structure elements and the most important amino acids for ligand binding (xsticks) indicated. Colour coding of atoms of amino acids and ligands: oxygen; red, nitrogen; blue, hydrogen; white, sulphur; yellow, carbon atoms of ligands; yellow, carbon atoms of amino acid side chains; pink, the zinc ion; light blue. The secondary structures elements are coloured from the N- to the C-terminal such that corresponding secondary elements of MMP-9 and MMP-14 obtain similar colour.
Fig 6.
Galardin and compound 1b docked into MMP-9 and galardin docked into pseudolysin.
Upper panel: The backbones of MMP-9 (5cuh) and pseudolysin (3dbk). The volume of the full binding pocket identified by the ICM Pocketfinder is displayed for both enzymes, with the S’1-subpocket indicated by an arrow. Middle panel: Galardin (red) and compound 1b (blue) docked into the binding pocket of MMP-9 and galardin (red) docked into pseudolysin. The panel shows that compounds may enter the S’1-subpocket of MMP-9 in a region between the side chains of Y423 and L188 on one side and the zinc. The corresponding entrance in pseudolysin is partly hindered by the side chain of R198. Lower panel: The complex from the middle section rotated 90 degrees and the ligands removed. The panel shows that the side chains of L188 and Y423 are located close to each other and hinder the entrance into the S’1-subpocket from the region above the zinc, while the corresponding region in pseudolysin is wider (side chains of N112 and L197).
Fig 7.
Galardin docked into the catalytic site of thermolysin, pseudolysin and auerolysin.
The figure shows close ups of the active site region with the compound structures (xsticks), secondary structure elements and the most important amino acids for ligand binding (xsticks) indicated. Colour coding of atoms of amino acids and ligands: oxygen; red, nitrogen; blue, hydrogen; white, carbon atoms of ligands; yellow, carbon atoms of amino acid side chains; grey, the zinc ion; light blue.