Objective— Matrix metalloproteinase (MMP)-12 has been implicated in plaque progression and instability and is also amenable to selective inhibition. In this study, we investigated the influence of a greater than 10-fold selective synthetic MMP-12 inhibitor on plaque progression in the apolipoprotein E knockout mouse model of atherosclerosis. Methods and Results— A phosphinic peptide (RXP470.1) that is a potent, selective murine MMP-12 inhibitor significantly reduced atherosclerotic plaque cross-sectional area by approximately 50% at 4 different vascular sites in male and female apolipoprotein E knockout mice fed a Western diet. Furthermore, RXP470.1 treatment resulted in less complex plaques with increased smooth muscle cell:macrophage ratio, less macrophage apoptosis, increased cap thickness, smaller necrotic cores, and decreased incidence of calcification. Additional in vitro and in vivo findings indicate that attenuated monocyte/macrophage invasion and reduced macrophage apoptosis probably underlie the beneficial effects observed on atherosclerotic plaque progression with MMP-12 inhibitor treatment. Conclusion— Our data demonstrate that a selective MMP-12 inhibitor retards atherosclerosis development and results in a more fibrous plaque phenotype in mice. Our study provides proof of principle to motivate translational work on MMP-12 inhibitor therapy in humans.

Urease inhibitors can be considered as a tool to control the damaging effect of ureolytic bacteria infections in humans which occur commonly in the developed countries. Computer-aided optimization of the aminomethylphosphinate structures by modifying both their N- and P-termini led to the invention of a novel group of inhibitors of bacterial ureases. Introduction of P-hydroxymethyl group into the molecule resulted in considerable increase of the inhibitory activity against enzymes purified from Bacillus pasteurii and Proteus vulgaris as compared with their P-methyl counterparts described previously. The designed compounds represent a competitive reversible class of urease inhibitors. The most potent, N-methyl-aminomethyl-P-hydroxymethylphosphinic acid, displayed K(i) = 360 nM against P. vulgaris enzyme.

A new series of phosphinic inhibitors able to interact with both angiotensin-converting enzyme (ACE) C-domain and endothelin-converting enzyme-1 (ECE-1), while sparing neprilysin (NEP), has been developed. The most potent and selective inhibitor in this series (compound 8(F2)) displays K(i) values of 0.65 nM, 150 nM, 14 nM and 6.7 microM toward somatic ACE C-domain, ACE N-domain, ECE-1, and NEP, respectively. Remarkably, in this series, the inhibitor's ability to discriminate between ECE-1 and NEP was observed to depend on the stereochemistry of the residue present in the inhibitor's P(1)' position. After iv administration, compound 8(F2) (10 mg/kg) lowered mean arterial blood pressure by 24 +/- 2 mmHg in spontaneously hypertensive rats, as compared with controls. Mixed ACE/ECE-1 inhibitor may lead to a new generation of vasopeptide inhibitors that should reduce the levels of angiotensin-II and endothelin-1, without interfering with bradykinin cleavage.

An unprecedented coupling of a P-C and a C-C bond-forming event in a practical operation was developed to access medicinally relevant phosphinic structures. The strategy relies on an Ireland-Claisen rearrangement triggered by the phospha-Michael addition of silyl phosphonites to allyl acrylates. This protocol was extended to a more versatile three-component variant that utilizes phosphinic acids, acryloyl chlorides, and allylic alcohols as starting materials.

The fusion of therapeutics and diagnostic medicine in an effort to provide individualized pharmacotherapy frequently requires the manipulation of drugs that target different enzymes and receptors. To this end, and as a strategy to increase the efficiency of drug development pipelines, new chemical entities are often developed that interact with more than one target. Angiotensin-converting enzyme (ACE), its homologue ACE2, neutral endopeptidase (NEP) and endothelin-converting enzyme (ECE-1) are metallopeptidases that are involved in the metabolism of biologically active peptides that impact on the regulation of the cardiovascular system. The benefit of the ACE/NEP; NEP/ECE and ACE/NEP/ECE dual and triple inhibitors is not only their possible increased efficacy with respect to blood pressure control, but also their other activities, such as antiproliferative, anti-fibrotic and anti-inflammatory, mediated by angiotensin II and atrial natriuretic peptide. Over the last few years a number of three-dimensional structures of these metallopeptidases have advanced our understanding of the mode of interaction between various ligands and their target binding sites. This information is invaluable in the rational design of new and improved drugs. Here we review the structural basis for the design of single and multiple metallopeptidase inhibitors for the treatment of cardiovascular disease. Moreover, we present recent advances in the development of ACE/ECE-1 inhibitors that are likely to have high potency and improved side effect profiles.

Structure-activity relationship studies, regarding the influence of side chains of phosphinic pseudotripeptidic inhibitors on matrix metalloproteinases (MMPs), provided potent and selective inhibitors for this family of structurally and functionally related proteases. Among them, phosphinic pseudopeptide CbzPhepsi[P(O)(OH)CH(2)] phenylpropyl TrpNH(2), known as RXP03, has been extensively used for in vivo and in vitro studies so far. The large quantities of RXP03 required for in vivo studies, as well as the necessity for diastereoisomeric purity, motivated us to further explore and develop an efficient synthetic methodology, which allows separation of the four diastereoisomers of RXP03 based on the astonishing observed differences in solubility of the four isomers in various solvents. This fact prompted us to examine theoretically the conformational differences of these four isomers via computer simulations in the solvents used experimentally. Given the fact that the four examined diastereoisomeric forms of the phosphinic peptides exhibit different behavior in terms of potency and selectivity profiles toward zinc-metalloproteases, this theoretical study provides valuable information on the conformation of phosphinic inhibitors and therefore improves the design and synthesis of active structures. The differences in solubility of RXP03 diastereoisomers in the used solvents were examined in terms of intra- and intermolecular structure. It is found that the different solubility of the RRS and RSS diastereoisomers in EtOH is a result of the different number of hydrogen bonds formed by each isomer with EtOH molecules. In the case of SRS and SSS in Et(2)O, their different solubility might be attributed to the different intramolecular hydrogen bonds formed on these diastereoisomers.

Various attempts to detect matrix metalloproteinase (MMP) active forms from complex proteomes, based on the use of specific photoactivatable affinity probes, have up to now failed. To overcome this failure, an affinity approach has been evaluated as an alternative to the photoaffinity one. For this purpose, two probes were synthesized to interact specifically with the active site of MMPs and allow isolation of MMP/probe complexes on magnetic beads through a biotin linker. Using phosphinic peptide chemistry, we prepared an affinity probe displaying picomolar potency toward several MMPs, and a related photoaffinity probe incorporating a photoactivatable azido group exhibiting subnanomolar affinity toward these targets. By a combination of silver-staining detection and MALDI peptide mass fingerprints, a systematic comparison was made of both strategies in terms of hMMP-12 and hMMP-8 recovery and identification when present in mixtures of different complexity. The results obtained show that the affinity protocol is superior to the photoaffinity strategy in terms of quantity of captured MMPs and number of MMP tryptic fragments detected in MALDI-MS. The specificity and efficiency of the affinity capture protocol developed in this study allowed easy, fast, and unambiguous detection by MALDI-MS of three hMMPs (2, 8, and 12), from a single affinity capture experiment, when added (10-36 ng of MMPs) to a tumor extract (10 microg). Thus, the tools and approaches reported should enable us to progress in the detection of endogenous active forms of MMPs in complex proteomes, an important objective with many diagnostic applications.

The synthesis of the phosphinic analogue of thyrotropin releasing hormone (TRH) GlpPsi[P(O)(OH)]HisProNH2, where the scissile peptide bond of TRH has been replaced by the hydrolytically stable phosphinic bond, has been achieved by a multistep synthetic strategy, providing thus one of the most potent synthetic inhibitors of pyroglutamyl peptidase II (PPII) reported to date (170 nM). The key synthetic step, an Ugi-type condensation reaction, produced directly the suitably protected for solid-phase peptide synthesis pseudodipeptidic block FmocGlu(OMe)Psi[P(O)(OH)]His(Tr)OH. Formation of the pyroglutamic ring was performed on solid phase, providing thus a general method for synthesizing pyroglutamyl phosphinic peptides on solid phase. Using this strategy, the phosphinic analogue of TRH has been synthesized for the first time.

Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia–warm reperfusion (CI-WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after 2 conditions of CI (4°C for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI-WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, whereas gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10 μM) was added to the University of Wisconsin solution, and livers were preserved for 42 hours (4°C), then reperfused for 1 hour in Krebs solution (37°C), containing 20% erythrocytes. Liver viability parameters were recorded, and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2%–60% of liver biopsies; mean, 26% ± 9%) to isolated necrotic hepatocytes as found in treated livers (0.2%–12%; mean, 3% ± 2%) ( P < 0.05). Conclusion: These data demonstrate that MMPs, by altering the ECM, play a major role in liver CI-WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions. (Hepatology 2007.)

Angiotensin-converting enzyme 2 (ACE2), a recently identified human homologue of angiotensin-converting enzyme, is a zinc metallocarboxypeptidase which may play a unique role in cardiovascular and renal function. Here we report the discovery of potent and selective inhibitors of ACE2, which have been identified by evaluating a series of phosphinic di- and tripeptides of the general formula: Z-Xaa(PO 2-CH 2)YaaOH and Ac-Zaa-Xaa(PO 2-CH 2)YaaOH. The most potent inhibitor in this series is a tripeptide that displays a K i value of 0.4 nM toward ACE2 and is 3 orders of magnitude less potent toward carboxypeptidase A. Phosphinic tripeptides exhibit high potency exclusively when the Xaa position is occupied by a pseudoproline. A model of interaction between one inhibitor of this series and ACE2 suggests that the critical role played by a proline in inhibitors, but also for substrates hydrolysis, may rely on the presence of Tyr (510) in the ACE2 active site.

The involvement of many zinc metalloproteinases belonging to the metzincin family with a variety of pathological states raises the possibility of therapeutic intervention using synthetic inhibitors with appropriate selectivity. Knowledge of the catalytic domain 3D-structures for various members of the metzincin family has been successfully exploited by chemists to develop potent synthetic inhibitors. However, despite intense efforts, very few highly selective inhibitors of metzincins have been discovered up to now. A survey of the literature suggests that the over-exploitation of the hydroxamate function as a zinc-binding group to develop inhibitors might be responsible for this situation. The use of alternative zinc-binding groups has led to more selective inhibitors, but the most encouraging results have been obtained for MMP-13 with compounds that do not incorporate zinc-binding groups in their structure. This new family of inhibitors exploits the presence of a deep S(1)(') cavity in the protease active site, a specific trait shared by many members of the metzincin family. However, to be successfully transposed to the metzincin members, this strategy will not only be able to exploit the structural detail of these S(1)(') cavities, but probably also subtle difference in their dynamics.

A new group of organophosphorus inhibitors of urease, P-methyl phosphinic acids was discovered by using the structure based inhibitor design approach. Several derivatives of the lead compound, aminomethyl(P-methyl)phosphinic acid, were synthesized successfully. Their potency was evaluated in vitro against urease from Bacillus pasteurii and Proteus vulgaris. The studied compounds constitute a group of competitive, reversible inhibitors of bacterial ureases. Obtained thiophosphinic analogues of the most effective structures exhibited kinetic characteristics of potent, slow binding urease inhibitors, with K-i = 170 nM (against B. pasteurii enzyme) for the most active N-(N'-benzyloxycarbonylglycyl)aminomethyl(P-methyl)phosphinothioic acid.

Zn-metalloproteinases are an important class of hydrolytic enzymes that are characterized by the presence of a catalytic zinc(II) atom in their active center which is fundamental for proteolytic activity. Metzincins, a superfamily of Zn-metalloproteinases with many structural and functional commonalities among its members, are responsible for the fine tuning of key physiological functions in mammals and the deregulation of their activity is directly connected to numerous inflammatory and degenerative diseases such as arthritis or cancer. Development of small-molecule exogenous inhibitors of metzincins able to re-establish normal proteolytic activity in pathological conditions has been a field of intense research effort for many years but applications in the clinic were not always successful. One of the main reasons for this failure is the uncontrolled action of these inhibitors on target as well as anti-target metzincin family members. Current medicinal efforts have been shifted to the discovery of target-specific inhibitors that will help to improve our understanding of metzincins biological function and provide the basis for the development of safer pharmaceutical agents. This review focuses on the cases of certain medicinally important metzincins [matrix metalloproteinases (MMPs), a disintegrin and metalloproteinases (ADAMs), ADAMs with thrombospondin motifs (ADAMTSs), and procollagen C-proteinase (PCP)] and summarizes the latest advances on the discovery of inhibitors of these enzymes that display improved selectivity profiles.

The association of matrix metalloproteinases (MMPs) with a variety of pathological states has stimulated impressive efforts over the past 20 years to develop synthetic compounds able to block potently and selectively the uncontrolled activity of these enzymes. Extremely potent inhibitors of MMPs have been developed, but in most cases these compounds act as broad-spectrum inhibitors of MMPs. Retrospective analysis suggests that the use of strong zinc-binding groups, like the hydroxamate function, to achieve potent MMP inhibition is responsible not only for the development of inhibitors displaying poor selectivity towards MMP members but also in their ability to potently block other unrelated zinc proteinases. The use of less avid zinc-binding group, like the phosphoryl group present in phosphinic peptide transition-state analogues, has led to a second generation of highly selective MMP inhibitors (MMP-12 selective inhibitors). The third generation of highly selective MMP inhibitors (MMP-13 selective inhibitors) possess no zinc-binding group and exploit the deep S1’ cavity present in some MMPs. Past research on the development of MMP inhibitors has probably underestimated the role of flexibility in the MMP active site and its impact in accommodating different inhibitor structures. Combined use of several biophysical techniques, like nuclear magnetic resonance, X-ray crystallography and isothermal titration experiments, should greatly improve our understanding of the specific structural and dynamic features that can be exploited to obtain series of inhibitors able to specifically block each MMP validated as a therapeutic target.