SPELUNKING STROMELYSIN: A CASE HISTORY OF STRUCTURE BASED DRUG DESIGN Jens J. Birktoft, R. Crowther, U. Kammlott, B. Graves, D. Waugh, W. Levin, N. Fotouhi, K. Hull, A. Hanglow, S. Pietranico, C. Michoud and M. Visnick. Roche Research Center. Hoffmann La-Roche Inc., Nutley, NJ,. USA.

Stromelysin-1 (MMP-3) is a member of a group of zinc containing proteolytic enzymes named the metalloproteinases (MMPs). Stromelysin has been implicated in rheumatoid arthritis and osteoarthritis as well as in the metastatic phase of cancer. A characteristic feature of these diseases is the irreversible erosion of connective tissue. The established roles of MMPs in these demographically important diseases make these enzymes ideal targets for pharmacological intervention. Two routes have been taken in the development of MMP inhibitors, one starting from the structure of putative substrate (and product) - enzyme complexes, while the other approach, to be discussed here, is rooted in the perceived organization of the pro-form of the MMPs. Specifically, the interactions of the so-called switch region (#73-PRCGVP-#78) in the pro-segment with the protease domain featuring ligation of zinc by cysteine formed the starting point for the design of inhibitors targeted toward stromelysin (Fotouhi et al, JBC, 269, 30227, 1994). Comparison of high resolution crystal structures of a truncated form of prostromelysin and of a complex formed between active stromelysin and a tight binding cyclic peptide demonstrated that the switch region peptide binds to stromelysin in nearly identical manner in prostromelysin and the cyclic peptide. In both pro-stromelysin and the inhibitor complex a mixed beta-sheet structure is formed between the RCGV peptide and the protein. Furthermore, the orientation of this peptide is opposite to that observed with peptides bound in a substrate-like mode. Additionally, analysis of these two crystal structures revealed that the zinc-bound cysteine side chain only partially fills the S1' substrate binding site. The remainder of this large cavity, which extends through the protein core, is occupied by solvent molecules. Additional, but smaller binding cavities form the S1-S3 and S2'-S4' subsites. The binding properties of the large S1' binding site have been exploited in the development of inhibitors derived from a cysteine or iso-cysteine building block and incorporating aromatic moieties. The analysis of several crystal structures will be described, and will include descriptions of enzyme conformational changes induced by different ligands and as well as changes in ligand interactions resulting from ligand modifications.