STROMELYSIN INHIBITORS DESIGNED FROM WEAKLY-BOUND FRAGMENTS - EFFECTSOF LINKING AND COOPERATIVITY

In the preceding paper,(1) we reported on the discovery of potent, non peptide inhibitors of the matrix metalloproteinase stromelysin that we re prepared by linking two ligands which bind weakly to adjacent sites on the protein. Here we describe the enthalpic and entropic contribut ions to the observed binding energy for both the linked and unlinked c ompounds using isothermal titration calorimetry. The results of the ca lorimetric experiments were interpreted on the basis of NMR-derived st ructures of stromelysin/inhibitor complexes. In addition, enzyme kinet ic assays were performed to measure the cooperative binding of the unt ethered ligands. For the untethered compounds, the presence of acetohy droxamic acid increases the binding energy of biaryl ligands by simila r to 1.3 kcal/mol. This gain in energy is enthalpic in nature and can be attributed, in part, to a direct dispersion interaction between the two ligands. For the linked compounds, enthalpic contributions to the binding energy depend critically on the linker length, whereas the en tropic contributions show virtually no dependence. The significant gai ns in enthalpy observed for a compound which linked the hydroxamate to the biaryl with a two methylene bridge was not observed for compounds with longer linkers due to a difference in the position of the biaryl moiety in the binding pocket. This difference disrupts key interactio ns between the ligand and the protein and highlights the importance of the linker in the design of tethered compounds.