Structure of malonic acid‐based inhibitors bound to human neutrophil collagenase. A new binding mode explains apparently anomalous data

Abstract

Matrix metalloproteinases (MMPs) are a family of zinc endopeptidases, which have been implicated in various disease processes. Various classes of MMP inhibitors, including hydroxamic acids, phosphinic acids, and thiols, have been previously described. Most of these mimic peptides, and most likely bind analogous to the corresponding peptide substrates. Among the hydroxamic acids, malonic acid derivatives have been used as MMP inhibitors, although optimization of their inhibition potency was not successful. Here we report the design of malonic acid-based inhibitors using the X-ray structure of a collagenase/inhibitor complex, which revealed a nonsubstrate-like binding mode. The proposed P-type turn-like conformation for the improved inhibitors was confirmed by X-ray crystallography. The observation of nonsubstrate-like binding confirms the original strategy for structure-based modeling of improved malonic acid inhibitors, and explains kinetic data that are inconsistent with substrate-like binding. Detailed interactions for the improved inhibitors seen in the crystal structure also suggest possibilities for further modifications in cycles of structure based drug design. Indeed, we have designed nonpeptidic inhibitors with approximately 500-fold improved inhibition based on these structures.

The 1.8-Å Crystal Structure of a Matrix Metalloproteinase 8-Barbiturate Inhibitor Complex Reveals a Previously Unobserved Mechanism for Collagenase Substrate Recognition

“…Intriguingly, Asn 188 -Tyr 189 , located at the corridor connecting the catalytic and the C-terminal domain, adopts a cis-peptide bond (Fig. 5). Although not yet recognized, this cis-peptide bond is not unique to the present crystal form; re-inspection confirmed its presence also in the alternative crystal form (26). This cis-peptide bond is located on the solvent-exposed loop preceding the "catalytic" ␣-helix…”

“…Inhibitor Conformation and Its Interaction with the Protein-Although identified as potent collagenase inhibitor by an independent screening program, the barbiturate-based inhibitor family exhibits striking similarities with well characterized classes of inhibitors, namely hydroxamic and malonic acidbased compounds (2,26). Fig.…”

“…ics, hydroxamic acid derivatives (2,14,21,22), phosphinamides and sulfodiimines (23,24), thiadiazole (25), and malonic acid derivatives (26,27).…”

Similarity of Binding Sites of Human Matrix Metalloproteinases

“…Subsite S1Ј exhibits the highest levels of similarity, followed by S2 and S3Ј subsites. The results add to multiple experimental proofs of flexibility of S1Ј subsite (12)(13)(14)(15)(16)(17)(18), help explain cases where desired specificity was not achieved upon optimization of groups fitting into S1Ј subsite (10,11), and contradict the view of S1Ј subsite as the specificity pocket of MMPs that is based on variability of rigid x-ray structures.…”

“…Traditionally, S1Ј subsite (5) has been labeled as the specificity pocket (6, 7) because of its significant size and shape differences among the rigid x-ray structures of various MMPs. However, modifications of P1Ј part of the ligands led to both increased selectivity (8,9) and failures (10,11) that were explained by observed changes in the structure of the pocket upon inhibitor binding (12)(13)(14)(15)(16)(17)(18). The importance of other parts of the binding site, especially the unprimed side (11, 19 -21), for the design of more potent and selective inhibitors has been demonstrated as well.…”