Tissue components hydrolyzing matrix metalloproteinases (MMPs) exhibit a high sequence similarity (56 -64% in catalytic domains) and yet a significant degree of functional specificity. The hexapeptide-binding sites of 24 known human MMPs were compared in terms of their force field interaction energies with five probes that are most frequently encountered in substrates and inhibitors. The probes moved along a grid enclosing partially flexible binding sites in rigid catalytic domains that were represented by published experimental structures and comparative models and new comparative models for nine most recently characterized MMPs. For individual MMPs, representative interaction energies were obtained as averages for all suitable experimental structures. Correlations of the representative energies for all MMP pairs were succinctly catalogued for individual probes, subsites, and correlation levels. Among the probes (neutral sp 3 carbon and sp 3 oxygen, positive sp 3 nitrogen and hydrogen, and negative carbonyl oxygen), the last probe is least distinctive. Similarities of subsites are decreasing as S1 > S2 > S3 > S1 ϳ S3 > S2 . Most interesting, occupancies of subsites in published structures of MMP-inhibitor complexes follow an almost parallel trend, alluding to overall low selectivity of known MMP inhibitors. Flexible subsite S1 that appears as the specificity pocket in rigid x-ray structures is actually very similar among individual MMPs. Several correlations indicated that MMPs 3, 8, and 12 have similar binding sites. Modeling results are corroborated with published experimental data on MMP inhibition and substrate specificities. The results provide numerous clues for development of specific inhibitors and substrates, as well as for selection of MMPs for testing that provides maximum information without redundant experiments.

Extracellular matrix (ECM) is a major tissue component that, besides its cell support function, is implicated in cell-cell signaling, wound repair, cell adhesion, and other cell and tissue functions. For small molecules acting in tissues, including chemicals, signaling peptides, effectors, inhibitors, and other man-made and physiological compounds, non-specific binding to ECM is a critical phenomenon affecting their disposition. We describe here a method for a quantitative characterization of the ECM binding, using a solidified ECM layer incubated with medium containing studied small molecules. Working conditions of Matrigel, a commercial basement membrane preparation, were optimized in terms of the protein concentration, surface area, gel layer thickness, solidification time, and mixing speed. The release of proteins from the solidified layer into the buffer was monitored and taken into account. Two major proteins, laminin and collagen IV, dissolve at different rates. The Matrigel stability data, obtained under varying incubation conditions and gentle mixing, can also be useful in other ECM-related research. The experimental binding data, averaged over all binding sites, were analyzed assuming a fast linear binding. The binding constants were determined for 10 small organic molecules for both dissolved proteins and the solidified layer. The binding constants tend to increase with lipophilicity of the compounds, as characterized by the 1-octanol/water partition coefficients.

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MMPs and TACE (ADAM-17) assume independent, parallel or opposite pathological roles in cancer, arthritis, and several other diseases. For therapeutic purposes, selective inhibition of individual MMPs and TACE is required in most cases due to distinct roles in diseases and the need to preserve activities in normal states. Toward this goal, we compared force-field interaction energies of five ubiquitous inhibitor atoms with flexible binding sites of 24 known human MMPs and TACE. The results indicate that MMPs 1−3, 10, 11, 13, 16, and 17 have at least one subsite very similar to TACE. S3 subsite is the best target for development of specific TACE inhibitors. Specific binding to TACE compared to most MMPs is promoted by placing a negatively charged ligand part at the bottom of S2 subsite, at the entrance of S1' subsite, or the part of S3' subsite that is close to catalytic zinc. Numerous other clues, consistent with available experimental data, are provided for design of selective inhibitors.

For small molecules acting in tissues, including signaling peptides, effectors, inhibitors, and other drug candidates, nonspecific binding to the extracellular matrix (ECM) is a critical phenomenon affecting their disposition, toxicity, and other effects. A commercially available ECM mimic, forming a solidified layer at the bottom of the vials, was used to measure the association constants of 25 simple aromatic compounds to two forms of ECM proteins, solidified (s-ECM) and dissolved (d-ECM) in the buffer during the incubation. Except for small homologous series, the binding data did not correlate with the lipophilicity and acidity of the compounds, contrary to a common expectation for nonspecific binding. To elucidate the putative structures of averaged binding sites of s-ECM and d-ECM, the Comparative Molecular Field Analysis (CoMFA) was applied in a modified version taking into consideration that multiple modes and multiple species may be involved. The method shapes a receptor site model from a set of grid points, in which the interaction energies between a probe atom and superimposed ligands are calculated. Electrostatic and steric energies in the grid points are characterized by regression coefficients. The forward-selection, nonlinear regression analysis was used to optimize the coefficients in the novel multi-species, multi-mode CoMFA models. These models showed satisfactory descriptive and predictive abilities for both s-ECM and d-ECM binding data, which were better than those obtained with the standard, one-mode CoMFA analysis. The calibrated models, defining the electrostatic and van der Waals regions of putative binding sites, are suitable for the prediction of ECM binding for untested chemicals.