Ligand-Directed Modification of Active Matrix Metalloproteases: Activity-based Probes with no Photolabile Group.

Activity-based probes enable discrimination between the active enzyme and its inactive or inactivated counterparts. Since metalloproteases catalysis is non-covalent, activity-based probes targeting them have been systematically developed by decorating reversible inhibitors with photo-crosslinkers. By exploiting two types of ligand-guided chemistry, we identified novel activity-based probes capable of covalently modifying the active site of matrix metalloproteases (MMPs) without any external trigger. The ability of these probes to label recombinant MMPs was validated in vitro and the identity of the main labelling sites within their S3 ' region unambiguously assigned. We also demonstrated that our affinity probes can react with rhMMP12 at nanogram scale (that is, at 0.07 % (w/w)) in complex proteomes. Finally, this ligand-directed chemistry was successfully applied to label active MMP-12 secreted by eukaryote cells. We believe that this approach could be transferred more widely to many other metalloproteases, thus contributing to tackle their unresolved proteomic profiling in vivo.

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.

Biodistribution of Nanostructured Lipid Carriers in Mice Atherosclerotic Model.

Atherosclerosis is a major cardiovascular disease worldwide, that could benefit from innovative nanomedicine imaging tools and treatments. In this perspective, we here studied, by fluorescence imaging in ApoE-/- mice, the biodistribution of non-functionalized and RXP470.1-targeted nanostructured lipid carriers (NLC) loaded with DiD dye. RXP470.1 specifically binds to MMP12, a metalloprotease that is over-expressed by macrophages residing in atherosclerotic plaques. Physico-chemical characterizations showed that RXP-NLC (about 105 RXP470.1 moieties/particle) displayed similar features as non-functionalized NLC in terms of particle diameter (about 60-65 nm), surface charge (about -5 - -10 mV), and colloidal stability. In vitro inhibition assays demonstrated that RXP-NLC conserved a selectivity and affinity profile, which favored MMP-12. In vivo data indicated that NLC and RXP-NLC presented prolonged blood circulation and accumulation in atherosclerotic lesions in a few hours. Twenty-four hours after injection, particle uptake in atherosclerotic plaques of the brachiocephalic artery was similar for both nanoparticles, as assessed by ex vivo imaging. This suggests that the RXP470.1 coating did not significantly induce an active targeting of the nanoparticles within the plaques. Overall, NLCs appeared to be very promising nanovectors to efficiently and specifically deliver imaging agents or drugs in atherosclerotic lesions, opening avenues for new nanomedicine strategies for cardiovascular diseases.

Synthesis and in Vitro and in Vivo Evaluation of MMP-12 Selective Optical Probes.

In designing new tracers consisting of a small peptide conjugated to a reporter of comparable size, particular attention needs to be paid to the selection of the reporter group, which can dictate both the in vitro and the in vivo performances of the whole conjugate. In the case of fluorescent tracers, this is particularly true given the large numbers of available dye moieties differing in their structures and properties. Here, we have investigated the in vitro and in vivo properties of a novel series of MMP-12 selective probes composed of cyanine dyes varying in their structure, net charge, and hydrophilic character, tethered through a linker to a potent and specific MMP-12 phosphinic pseudopeptide inhibitor. The impact of linker length has been also explored. The crystallographic structure of one tracer in complex with MMP-12 has been obtained, providing the first crystal structure of a Cy5.5-derived probe and confirming that the binding of the targeting moiety is unaffected. MMP-12 remains the tracers' privileged target, as attested by their affinity selectivity profile evaluated in solution toward a panel of 12 metalloproteases. In vivo assessment of four selected probes has highlighted not only the impact of the dye structure but also that of the linker length on the probes' blood clearance rates and their biodistributions. These experiments have also provided valuable data on the stability of the dye moieties in vivo. This has permitted the identification of one probe, which combines favorable binding to MMP-12 in solution and on cells with optimized in vivo performance including blood clearance rate suitable for short-time imaging. Through this series of tracers, we have identified various critical factors modulating the tracers' in vivo behavior, which is both useful for the development and optimization of MMP-12 selective radiolabeled tracers and informative for the design of fluorescent probes in general.

Effects of selective MMP-13 inhibition in squamous cell carcinoma depend on estrogen.

Matrix metalloproteinases like MMP-13 cleave and remodel the extracellular matrix and thereby play a crucial role in tumor progression in vivo. Using a highly selective inhibitor to block MMP-13 protein activity, we demonstrate a striking inhibitory effect on invasive tumor growth and vascularization in murine skin squamous cell carcinoma (SCC). Therapy outcome critically depends on animal age in C57Bl/6 mice and was successful in old female but not in young female mice. Treatment success was recovered by ovariectomy in young and abolished by 17ß-estradiol supplementation in old mice, suggesting a hormone dependent inhibitor effect. Responsiveness of the tumorigenic keratinocytes BDVII and fibroblasts to 17ß-estradiol was confirmed in vitro, where MMP-13 inhibitor treatment led to a reduction of cell invasion and vascular endothelial growth factor (VEGF) release. This correlated well with a less invasive and vascularized tumor in treated mice in vivo. 17ß-estradiol supplementation also reduced invasion and VEGF release in vitro with no additional reduction on MMP-13 inhibitor treatment. This suggests that low 17ß-estradiol levels in old mice in vivo lead to enhanced MMP-13 levels and VEGF release, allowing a more effective inhibitor treatment compared to young mice. In our study, we present a strong link between lower estrogen levels in old female mice, an elevated MMP-13 level, which results in a more effective MMP-13 inhibitor treatment in fibroblasts and SCC cells in vitro and in vivo.

Structural framework for covalent inhibition of Clostridium botulinum neurotoxin A by targeting Cys165.

Clostridium botulinum neurotoxin type A (BoNT/A) is one of the most potent toxins for humans and a major biothreat agent. Despite intense chemical efforts over the past 10 years to develop inhibitors of its catalytic domain (catBoNT/A), highly potent and selective inhibitors are still lacking. Recently, small inhibitors were reported to covalently modify catBoNT/A by targeting Cys(165), a residue located in the enzyme active site just above the catalytic zinc ion. However, no direct proof of Cys(165) modification was reported, and the poor accessibility of this residue in the x-ray structure of catBoNT/A raises concerns about this proposal. To clarify this issue, the functional role of Cys(165) was first assessed through a combination of site-directed mutagenesis and structural studies. These data suggested that Cys(165) is more involved in enzyme catalysis rather than in structural property. Then by peptide mass fingerprinting and x-ray crystallography, we demonstrated that a small compound containing a sulfonyl group acts as inhibitor of catBoNT/A through covalent modification of Cys(165). The crystal structure of this covalent complex offers a structural framework for developing more potent covalent inhibitors catBoNT/A. Other zinc metalloproteases can be founded in the protein database with a cysteine at a similar location, some expressed by major human pathogens; thus this work should find broader applications for developing covalent inhibitors.

Simple pseudo-dipeptides with a P2' glutamate: a novel inhibitor family of matrix metalloproteases and other metzincins.

A series of pseudo-peptides with general formula X-l-Glu-NH(2) (with X corresponding to an acyl moiety with a long aryl-alkyl side chain) have been synthesized, evaluated as inhibitors of matrix metalloproteases (MMPs), and found to display remarkable nanomolar affinity. The loss in potency associated with a substitution of the P(2)' l-glutamate by a l-glutamine corroborates the importance of a carboxylate at this position. The binding mode of some of these inhibitors was characterized in solution and by x-ray crystallography in complex with various MMPs. The x-ray crystal structures reveal an unusual binding mode with the glutamate side chain chelating the active site zinc ion. Competition experiments between these inhibitors and acetohydroxamic acid, a small zinc-binding molecule, are in accord with the crystallographic results. One of these pseudo-dipeptides displays potency and selectivity toward MMP-12 similar to the best MMP-12 inhibitors reported to date. This novel family of pseudo peptides opens new opportunities to develop potent and selective inhibitors for several metzincins.

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli.

BACKGROUND: Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited. RESULTS: Here we report a high throughput screening approach that allowed the systematic investigation of the solubilizing and folding influence of twelve cytoplasmic partners on 28 DRPs in the strains BL21 (DE3) pLysS, Origami B (DE3) pLysS and SHuffle® T7 Express lysY (1008 conditions). The screening identified the conditions leading to the successful soluble expression of the 28 DRPs selected for the study. Amongst 336 conditions tested per bacterial strain, soluble expression was detected in 196 conditions using the strain BL21 (DE3) pLysS, whereas only 44 and 50 conditions for soluble expression were identified for the strains Origami B (DE3) pLysS and SHuffle® T7 Express lysY respectively. To assess the redox states of the DRPs, the solubility screen was coupled with mass spectrometry (MS) to determine the exact masses of the produced DRPs or fusion proteins. To validate the results obtained at analytical scale, several examples of proteins expressed and purified to a larger scale are presented along with their MS and functional characterization. CONCLUSIONS: Our results show that the production of soluble and functional DRPs with cytoplasmic partners is possible in E. coli. In spite of its reducing cytoplasm, BL21 (DE3) pLysS is more efficient than the Origami B (DE3) pLysS and SHuffle® T7 Express lysY trxB(-)/gor(-) strains for the production of DRPs in fusion with solubilizing partners. However, our data suggest that oxidation of the proteins occurs ex vivo. Our protocols allow the production of a large diversity of DRPs using DsbC as a fusion partner, leading to pure active DRPs at milligram scale in many cases. These results open up new possibilities for the study and development of DRPs with therapeutic or biotechnological interest whose production was previously a limitation.

Correlative radioimaging and mass spectrometry imaging: a powerful combination to study 14C-graphene oxide in vivo biodistribution.

Research on graphene based nanomaterials has flourished in the last decade due their unique properties and emerging socio-economic impact. In the context of their potential exploitation for biomedical applications, there is a growing need for the development of more efficient imaging techniques to track the fate of these materials. Herein we propose the first correlative imaging approach based on the combination of radioimaging and mass spectrometry imaging for the detection of Graphene Oxide (GO) labelled with carbon-14 in mice. In this study, 14C-graphene oxide nanoribbons were produced from the oxidative opening of 14C-carbon nanotubes, and were then intensively sonicated to provide nano-size 14C-GO flakes. After Intravenous administration in mice, 14C-GO distribution was quantified by radioimaging performed on tissue slices. On the same slices, MS-imaging provided a highly resolved distribution map of the nanomaterial based on the detection of specific radical anionic carbon clusters ranging from C2˙- to C9˙- with a base peak at m/z 72 (12C) and 74 (14C) under negative laser desorption ionization mass spectrometry (LDI-MS) conditions. This proof of concept approach synergizes the strength of each technique and could be advantageous in the pre-clinical development of future Graphene-based biomedical applications.

A selective matrix metalloproteinase-12 inhibitor retards atherosclerotic plaque development in apolipoprotein E-knockout mice.

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. CONCLUSIONS: 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.

Novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphinic tripeptide.

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97Å (1 Å=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15Å) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P(1)' residue with the unusual R configuration which, surprisingly, is accommodated by the large S(2)' pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi-pi stacking interactions with the amino benzoyl group of the first molecule locking them in a 'hand-shake' conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.

Monitoring In Vivo Performances of Protein-Drug Conjugates Using Site-Selective Dual Radiolabeling and Ex Vivo Digital Imaging.

In preclinical models, the development and optimization of protein-drug conjugates require accurate determination of the plasma and tissue profiles of both the protein and its conjugated drug. To this aim, we developed a bioanalytical strategy based on dual radiolabeling and ex vivo digital imaging. By combining enzymatic and chemical reactions, we obtained homogeneous dual-labeled anti-MMP-14 Fabs (antigen-binding fragments) conjugated to monomethyl auristatin E where the protein scaffold was labeled with carbon-14 (14C) and the conjugated drug with tritium (3H). These antibody-drug conjugates with either a noncleavable or a cleavable linker were then evaluated in vivo. By combining liquid scintillation counting and ex vivo dual-isotope radio-imaging, it was possible not only to monitor both components simultaneously during their circulation phase but also to quantify accurately their amount accumulated within the different organs.

Insights from selective non-phosphinic inhibitors of MMP-12 tailored to fit with an S1' loop canonical conformation.

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.

First evidence that emerging pinnatoxin-G, a contaminant of shellfish, reaches the brain and crosses the placental barrier.

Massive proliferation of some toxic marine dinoflagellates is responsible for the occurrence of harmful algal blooms and the contamination of fish and shellfish worldwide. Pinnatoxins (PnTx) (A-H) comprise an emerging phycotoxin family belonging to the cyclic imine toxin group. Interest has been focused on these lipophilic, fast-acting and highly potent toxins because they are widely found in contaminated shellfish, and can represent a risk for seafood consumers. PnTx display a potent antagonist effect on nicotinic acetylcholine receptors (nAChR), and in this study we assessed in vivo the ability of PnTx-G to cross physiological barriers to reach its molecular target. Radiolabeled [3H]-PnTx-G synthesized with good radiochemical purity and yield retained the high affinity of the natural toxin. Oral gavage or intravenous administration to adult rats and digital autoradiographic analyses revealed the biodistribution and toxicokinetics of [3H]-PnTx-G, which is rapidly cleared from blood, and accumulates in the liver and small intestine. The labeling of peripheral and brain adult/embryo rat tissues highlights its ability to cross the intestinal, blood-brain and placental barriers. High-resolution 3D-imaging and in vitro competition studies on rat embryo sections revealed the specificity of [3H]-PnTx-G binding and its selectivity for muscle and neuronal nAChR subtypes (such as α7 subtype). The use of a human perfused cotyledon model and mass spectrometry analyses disclosed that PnTx-G crosses the human placental barrier. The increasing worldwide occurrence of both the dinoflagellate Vulcanodinium rugosum and PnTx-contaminated shellfish, due to climate warming, raises concerns about the potential adverse impact that exposure to pinnatoxins may have for human health.

Novel Matrix Metalloproteinase 12 Selective Radiotracers for Vascular Molecular Imaging.

Matrix metalloproteinase-12 (MMP-12) is highly upregulated in several inflammatory diseases, including abdominal aortic aneurysm (AAA). Here we report four novel 99mTc-labeled radiotracers derived from a highly selective competitive MMP-12 inhibitor. These tracers in their 99gTc version were assessed in vitro on a set of human metalloproteases and displayed high affinity and selectivity toward MMP-12. Their radiolabeling with 99mTc was shown to be efficient and stable in both buffer and mouse blood. The tracers showed major differences in their biodistribution and blood clearance. On the basis of its in vivo performance, [99mTc]-1 was selected for evaluation in murine AAA, where MMP-12 gene expression is upregulated. Autoradiography of aortae at 2 h postinjection revealed high uptake of [99mTc]-1 in AAA relative to adjacent aorta. Tracer uptake specificity was demonstrated through in vivo competition. This study paves the way for further evaluation of [99mTc]-1 for imaging AAA and other MMP-12-associated diseases.

Development of potent and selective phosphinic peptide inhibitors of angiotensin-converting enzyme 2.

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.

Inhibitors of BMP-1/tolloid-like proteinases: efficacy, selectivity and cellular toxicity.

BMP-1/tolloid-like proteinases belong to the astacin family of human metalloproteinases, together with meprins and ovastacin. They represent promising targets to treat or prevent a wide range of diseases such as fibrotic disorders or cancer. However, the study of their pathophysiological roles is still impaired by the lack of well-characterized inhibitors and the questions that remain regarding their selectivity and in vivo efficiency. As a first step towards the identification of suitable tools to be used in functional studies, we have undertaken a systematic comparison of seven molecules known to affect the proteolytic activity of human astacins including three hydroxamates (FG-2575, UK383,367, S33A), the protein sizzled, a new phosphinic inhibitor (RXP-1001) and broad-spectrum protease inhibitors (GM6001, actinonin). Their efficacy in vitro, their cellular toxicity and efficacy in cell cultures were thoroughly characterized. We found that these molecules display very different potency and selectivity profiles, with hydroxamate FG-2575 and the protein sizzled being very powerful and selective inhibitors of BMP-1, whereas phosphinic peptide RXP-1001 behaves as a broad-spectrum inhibitor of astacins. Their use should therefore be carefully considered in agreement with the aim of the study to avoid result misinterpretation.

Zinc-Metalloproteinase Inhibitors: Evaluation of the Complex Role Played by the Zinc-Binding Group on Potency and Selectivity.

The most exploited strategy to develop potent zinc-metalloprotease inhibitors relies on a core zinc chelator and a peptidic or nonpeptidic scaffold that provides supplementary interactions for optimized potency and selectivity. Applied to matrix metalloproteases (MMPs) with highly conserved catalytic domains, this strategy failed to identify inhibitors with the desired selectivity profiles. To question the precise role of the zinc-binding group (ZBG), we have carried out a study on MMP-12 inhibitors with a common peptidic core but different ZBGs. We find that exchanging the ZBG modifies inhibitor positioning and affects its dynamics and selectivity. The binding properties of these compounds were compared through biochemical, structural, and calorimetric studies, showing a complex interplay between cooperative interactions and dynamics dictated by the ZBG. Improving selectivity will require expanding the ZBG repertoire within inhibitor libraries, since relying on a single ZBG significantly decreases our chance to identify effective inhibitors.

Roles of the two active sites of somatic angiotensin-converting enzyme in the cleavage of angiotensin I and bradykinin: insights from selective inhibitors.

Somatic angiotensin-converting enzyme (ACE) contains two homologous domains, each bearing a functional active site. The in vivo contribution of each active site to the release of angiotensin II (Ang II) and the inactivation of bradykinin (BK) is still unknown. To gain insights into the functional roles of these two active sites, the in vitro and in vivo effects of compounds able to selectively inhibit only one active site of ACE were determined, using radiolabeled Ang I or BK, as physiological substrates of ACE. In vitro studies indicated that a full inhibition of the Ang I and BK cleavage requires a blockade of the two ACE active sites. In contrast, in vivo experiments in mice demonstrated that the selective inhibition of either the N-domain or the C-domain of ACE by these inhibitors prevents the conversion of Ang I to Ang II, while BK protection requires the inhibition of the two ACE active sites. Thus, in vivo, the cleavage of Ang I and BK by ACE appears to obey to different mechanisms. Remarkably, in vivo the conversion of Ang I seems to involve the two active sites of ACE, free of inhibitor. Based on these findings, it might be suggested that the gene duplication of ACE in vertebrates may represent a means for regulating the cleavage of Ang I differently from that of BK.

Optical imaging of MMP-12 active form in inflammation and aneurysm.

Matrix metalloproteinase (MMP)-12 plays a key role in the development of aneurysm. Like other members of MMP family, MMP-12 is produced as a proenzyme, mainly by macrophages, and undergoes proteolytic activation to generate an active form. Accordingly, molecular imaging of the MMP-12 active form can inform of the pathogenic process in aneurysm. Here, we developed a novel family of fluorescent probes based on a selective MMP-12 inhibitor, RXP470.1 to target the active form of MMP-12. These probes were stable in complex media and retained the high affinity and selectivity of RXP470.1 for MMP-12. Amongst these, probe 3 containing a zwitterionic fluorophore, ZW800-1, combined a favorable affinity profile toward MMP-12 and faster blood clearance. In vivo binding of probe 3 was observed in murine models of sterile inflammation and carotid aneurysm. Binding specificity was demonstrated using a non-binding homolog. Co-immunostaining localized MMP-12 probe binding to MMP-12 positive areas and F4/80 positive macrophages in aneurysm. In conclusion, the active form of MMP-12 can be detected by optical imaging using RXP470.1-based probes. This is a valuable adjunct for pathophysiology research, drug development, and potentially clinical applications.