Analysis of the binding of hydroxamic acid and carboxylic acid inhibitors to the stromelysin-1 (matrix metalloproteinase-3) catalytic domain by isothermal titration calorimetry

Matrix metalloproteinases (MMPs) are implicated in diseases such as arthrit is and cancer. Among these enzymes, stromelysin-1 can also activate the pro enzymes of other MMPs, making it an attractive target for pharmaceutical de sign. Isothermal titration calorimetry (ITC) was used to analyze the bindin g of three inhibitors to the stromelysin catalytic domain (SCD). One inhibi tor (Galardin) uses a hydroxamic acid group (pK(a) congruent to 8.7) to bin d the active site zinc; the others (PD180557 and PD166793) use a carboxylic acid group (pK(a) congruent to 4.7). Binding affinity increased dramatical ly as the pH was decreased over the range 5.5-7.5. Experiments carried out at pH 6.7 in several different buffers revealed that approximately one and two protons are transferred to the enzyme-inhibitor complexes for the hydro xamic and carboxylic acid inhibitors, respectively. This suggests that both classes of inhibitors bind in the protonated state, and that one amino aci d residue of the enzyme also becomes protonated upon binding. Similar exper iments carried out with the H224N mutant gave strong evidence that this res idue is histidine 224. Delta G, Delta H, Delta S, and Delta C-p were determ ined for the three inhibitors at pH 6.7, and Delta C-p was used to obtain e stimates of the solvational, translational, and conformational components o f the entropy term. The results suggest that: (1) a polar group at the P1 p osition can contribute a large favorable enthalpy, (2) a hydrophobic group at P2' can contribute a favorable entropy of desolvation, and (3) P1' subst ituents of certain sizes may trigger an entropically unfavorable conformati onal change in the enzyme upon binding. These findings illustrate the value of complete thermodynamic profiles generated by ITC in discovering binding interactions that might go undetected when relying on binding affinities a lone.