X-RAY STRUCTURES OF HUMAN NEUTROPHIL COLLAGENASE COMPLEXED WITH PEPTIDE HYDROXAMATE AND PEPTIDE THIOL INHIBITORS - IMPLICATIONS FOR SUBSTRATE-BINDING AND RATIONAL DRUG DESIGN

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases i nvolved in tissue remodeling. They have also been implicated in variou s disease processes including tumour invasion and joint destruction an d are therefore attractive targets for inhibitor design. For rational drug design, information of inhibitor binding at the atomic level is e ssential. Recently, we have published the refined high-resolution crys tal structure of the catalytic domain of human neutrophil collagenase (HNC) complexed with the inhibitor Pro-Leu-Gly-NHOH, which is a mimic for the unprimed (P3-P1) residues of a bound peptide substrate. We hav e now determined two additional HNC complexes formed with the thiol in hibitor HSCH2CH(CH(2)Ph)CO-L-Ala-Gly-NH2 and another hydroxamate inhib itor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2,, which were both refined to R-val ues of 0.183/0.198 at 0.240/0.225-nm resolution. The inhibitor thiol a nd hydroxamate groups ligand the catalytic zinc, giving rise to a slig htly distorted tetrahedral and trigonal-bipyramidal coordination spher e, respectively. The thiol inhibitor diastereomer with S-configuration at the P1' residue (corresponding to an L-amino acid analog) binds to HNC. Its peptidyl moiety mimics binding of primed (P1'-P3') residues of the substrate. In combination with our first structure a continuous hexapeptide corresponding to a peptide substrate productively bound t o HNC was constructed and energy-minimized. Proteolytic cleavage of th is Michaelis complex is probably general base-catalyzed as proposed fo r thermolysin, i.e. a glutamate assists nucleophilic attack of a water molecule. Although there are many structural and mechanistic similari ties to thermolysin, substrate binding to MMPs differs due to the inte ractions beyond S1'-P1' While thermolysin binds substrates with a kink at P1', substrates are bound in an extended conformation in the colla genases. This property explains the tolerance of thermolysin for D-ami no acid residues at the P1' position, in contrast to the collagenases. The third inhibitor, HONHCOCH(iBu)CO-L-Ala-Gly-NH2, unexpectedly bind s in a different manner than anticipated from its design and binding m ode in thermolysin. Its hydroxamate group obviously interacts with the catalytic zinc in a favourable bidentate manner, but in contrast its isobutyl (iBu) side chain remains outside of the S1' pocket, presumabl y due to severe constraints imposed by the adjacent planar hydroxamate group. Instead, the C-terminal Ala-Gly-NH2 tail adopts a bent conform ation and inserts into this S1' pocket, presumably in a non-optimized manner. Both the isobutyl side chain and the C-terminal peptide tail c ould be replaced by other, better fitting groups. Thus this inhibitor seems to represent a new lead structure suitable for designing better drugs.