Insights from Selective Non-phosphinic Inhibitors of MMP-12 Tailored to Fit with an S1′ Loop Canonical Conformation

Abstract

After the disappointment of clinical trials with early broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs), the field is now resurging with a new focus on the development of selective inhibitors that fully discriminate between different members of the MMP family with several therapeutic applications in perspective. Here, we report a novel class of highly selective MMP-12 inhibitors, without a phosphinic zinc-binding group, designed to plunge deeper into the S 1 cavity of the enzyme. The best inhibitor from this series, identified through a systematic chemical exploration, displays nanomolar potency toward MMP-12 and selectivity factors that range between 2 and 4 orders of magnitude toward a large set of MMPs. Comparison of the high resolution x-ray structures of MMP-12 in free state or bound to this new MMP-12 selective inhibitor reveals that this compound fits deeply within the S 1 specificity cavity, maximizing surface/volume ratios, without perturbing the S 1 loop conformation. This is in contrast with highly selective MMP-13 inhibitors that were shown to select a particular S 1 loop conformation. The search for such compounds that fit precisely to preponderant S 1 loop conformation of a particular MMP may prove to be an alternative effective strategy for developing selective inhibitors of MMPs. The association of matrix metalloproteinases (MMPs)2 with a variety of pathological states has stimulated impressive efforts over the past 20 years to develop synthetic compounds able to block efficiently (1-7) and selectively the uncontrolled activity of these enzymes (8). Extremely potent inhibitors of MMPs have been developed, but in most cases these compounds act as broad spectrum inhibitors of MMPs (9). The arguments that have been proposed to explain the difficulties in identifying inhibitors able to differentiate one MMP from another include: (a) marked sequence similarities between the catalytic domains of MMPs, (b) a well conserved enzyme active site topology (backbone RMSD between the MMP catalytic domains is 0.7-0.8 Å), and (c) the mobility of residues in the so-called S 1 Ј specificity loop (10, 11).MMPs form a group of 23 proteins in humans, all of which contain a catalytic domain belonging to the zinc metalloproteinase family (12, 13). Retrospective analysis suggests that the incorporation of strong zinc-binding groups, such as hydroxamate functions, potentiates MMP inhibition but unfortunately in an indiscriminate manner affecting most members of the MMP family (7), as well as other unrelated zinc-proteinases (14). The use of a less avid zinc-binding group, like the phosphoryl group present in phosphinic peptide transition state analogs, has led to a second generation of more selective MMP inhibitors, like the selective inhibitors reported for MMP-12 (macrophage-metalloelastase) (15). The third generation MMP inhibitors possess no zinc-binding group and exploit mainly the depth of the S 1 Ј cavity present in most MMPs (16,17). This strategy has led to the discovery of the first extre...

Simple Pseudo-dipeptides with a P2′ Glutamate

“…The crystal structure of the complex between MMP-12 and compound 3 in presence of AHA does not support this suggestion (39). AHA is a small zinc-binding group that acts as a weak MMP inhibitor, classically used to prevent enzyme autolysis during protein preparation and crystallization (40) (57).…”

“…Data reduction for MMP-12 with compound 8 was carried out with MOSFLM (47) and scaled with SCALA from the CCP4 suite of programs (48). Data reduction for MMP-12 with compound 3 made use of the script "xdsme" and structure solution by molecular replacement using MOLREP (50) with model coordinates from PDB code 3LIK (39). Rigid body refinement with REFMAC (51) was sufficient for MMP-12 with compound 8 as the lattice parameters did not vary substantially from those of compound 3 (supplemental Table S1).…”

“…The crystal structure of this inhibitor bound to MMP-12 in presence of acetohydroxamic acid (AHA) showed that this inhibitor adopts a "standard" binding mode with the two P 2 Ј and P 3 Ј glutamate side chains pointing toward the S 2 Ј and S 3 Ј subsites within the active site (39). Subsequent studies of this inhibitor and analogues possessing the general formula X-Glu-Glu-NH 2 , X-Gln-Glu-NH 2 , or X-Glu-NH 2 , where X corresponds to the long P 1 Ј aryl-alkyl side chain, have produced data that were not explained by MMP-12⅐AHA⅐3 complex crystal structure.…”

“…With the aim to develop a new generation of inhibitors of lower molecular weight than 1, compound 3, the most selective MMP-12 inhibitor to date, was designed (38,39). The crystal structure of this inhibitor bound to MMP-12 in presence of acetohydroxamic acid (AHA) showed that this inhibitor adopts a "standard" binding mode with the two P 2 Ј and P 3 Ј glutamate side chains pointing toward the S 2 Ј and S 3 Ј subsites within the active site (39).…”

Discovery of a New Class of Potent MMP Inhibitors by Structure-Based Optimization of the Arylsulfonamide Scaffold

“…22−25 However, the presented scaffold easily allows for further chemical modifications aimed at improving the selectivity toward a specific MMP. In this respect, the availability of several X-ray structures of MMPs, as well as structural hints provided by selective MMP inhibitors developed so far, such as those presented by Dive, 22 can drive the lead optimization strategy.…”