A unique network of attack, defence and competence on the outer membrane of the periodontitis pathogen Tannerella forsythia.

Periodontopathogenic Tannerella forsythia uniquely secretes six peptidases of disparate catalytic classes and families that operate as virulence factors during infection of the gums, the KLIKK-peptidases. Their coding genes are immediately downstream of novel ORFs encoding the 98-132 residue potempins (Pot) A, B1, B2, C, D and E. These are outer-membrane-anchored lipoproteins that specifically and potently inhibit the respective downstream peptidase through stable complexes that protect the outer membrane of T. forsythia, as shown in vivo. Remarkably, PotA also contributes to bacterial fitness in vivo and specifically inhibits matrix metallopeptidase (MMP) 12, a major defence component of oral macrophages, thus featuring a novel and highly-specific physiological MMP inhibitor. Information from 11 structures and high-confidence homology models showed that the potempins are distinct ß-barrels with either a five-stranded OB-fold (PotA, PotC and PotD) or an eight-stranded up-and-down fold (PotE, PotB1 and PotB2), which are novel for peptidase inhibitors. Particular loops insert like wedges into the active-site cleft of the genetically-linked peptidases to specifically block them either via a new "bilobal" or the classic "standard" mechanism of inhibition. These results discover a unique, tightly-regulated proteolytic armamentarium for virulence and competence, the KLIKK-peptidase/potempin system.

Molecular interactions of PCSK9 with an inhibitory nanobody, CAP1 and HLA-C: Functional regulation of LDLR levels.

OBJECTIVE: The liver-derived circulating PCSK9 enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes. PCSK9 inhibition or silencing is presently used in clinics worldwide to reduce LDL-cholesterol, resulting in lower incidence of cardiovascular disease and possibly cancer/metastasis. The mechanism by which the PCSK9-LDLR complex is sorted to degradation compartments is not fully understood. We previously suggested that out of the three M1, M2 and M3 subdomains of the C-terminal Cys/His-rich-domain (CHRD) of PCSK9, only M2 is critical for the activity of extracellular of PCSK9 on cell surface LDLR. This likely implicates the binding of M2 to an unknown membrane-associated "protein X" that would escort the complex to endosomes/lysosomes for degradation. We reported that a nanobody P1.40 binds the M1 and M3 domains of the CHRD and inhibits the function of PCSK9. It was also reported that the cytosolic adenylyl cyclase-associated protein 1 (CAP1) could bind M1 and M3 subdomains and enhance the activity of PCSK9. In this study, we determined the 3-dimensional structure of the CHRD-P1.40 complex to understand the intricate interplay between P1.40, CAP1 and PCSK9 and how they regulate LDLR degradation. METHODS: X-ray diffraction of the CHRD-P1.40 complex was analyzed with a 2.2 Å resolution. The affinity and interaction of PCSK9 or CHRD with P1.40 or CAP1 was analyzed by atomic modeling, site-directed mutagenesis, bio-layer interferometry, expression in hepatic cell lines and immunocytochemistry to monitor LDLR degradation. The CHRD-P1.40 interaction was further analyzed by deep mutational scanning and binding assays to validate the role of predicted critical residues. Conformational changes and atomic models were obtained by small angle X-ray scattering (SAXS). RESULTS: We demonstrate that PCSK9 exists in a closed or open conformation and that P1.40 favors the latter by binding key residues in the M1 and M3 subdomains of the CHRD. Our data show that CAP1 is well secreted by hepatic cells and binds extracellular PCSK9 at distinct residues in the M1 and M3 modules and in the acidic prodomain. CAP1 stabilizes the closed conformation of PCSK9 and prevents P1.40 binding. However, CAP1 siRNA only partially inhibited PCSK9 activity on the LDLR. By modeling the previously reported interaction between M2 and an R-X-E motif in HLA-C, we identified Glu567 and Arg549 as critical M2 residues binding HLA-C. Amazingly, these two residues are also required for the PCSK9-induced LDLR degradation. CONCLUSIONS: The present study reveals that CAP1 enhances the function of PCSK9, likely by twisting the protein into a closed configuration that exposes the M2 subdomain needed for targeting the PCSK9-LDLR complex to degradation compartments. We hypothesize that "protein X", which is expected to guide the LDLR-PCSK9-CAP1 complex to these compartments after endocytosis into clathrin-coated vesicles, is HLA-C or a similar MHC-I family member. This conclusion is supported by the PCSK9 natural loss-of-function Q554E and gain-of-function H553R M2 variants, whose consequences are anticipated by our modeling.

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.

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.

Development of a Mass Spectrometry Imaging Method for Detecting and Mapping Graphene Oxide Nanoparticles in Rodent Tissues.

Graphene-based nanoparticles are continuously being developed for biomedical applications, and their use raises concerns about their environmental and biological impact. In the literature, some imaging techniques based on fluorescence and radioimaging have been used to explore their fate in vivo. Here, we report on the use of label-free mass spectrometry and mass spectrometry imaging (MSI) for graphene oxide (GO) and reduced graphene oxide (rGO) analyses in rodent tissues. Thereby, we extend previous work by focusing on practical questions to obtain reliable and meaningful images. Specific radical anionic carbon clusters ranging from C2-• to C9-• were observed for both GO and rGO species, with a base peak at m/z 72 under negative laser desorption ionization mass spectrometry (LDI-MS) conditions. Extension to an LDI-MSI method was then performed, thus enabling the efficient detection of GO nanoparticles in lung tissue sections of previously exposed mice. The possibility of quantifying those nanoparticles on tissue sections has also been investigated. Two different ways of building calibration curves (i.e., GO suspensions spotted on tissue sections, or added to lung tissue homogenates) were evaluated and returned similar results, with linear dynamic concentration ranges over at least 2 orders of magnitude. Moreover, intra- and inter-day precision studies have been assessed, with relative standard deviation below 25% for each concentration point of a calibration curve. In conclusion, our study confirms that LDI-MSI is a relevant approach for biodistribution studies of carbon-based nanoparticles, as quantification can be achieved, provided that nanoparticle suspension and manufacturing are carefully controlled.

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.

Metalloprotease inhibitor TIMP proteins control FGF-2 bioavailability and regulate skeletal growth.

Regulated growth plate activity is essential for postnatal bone development and body stature, yet the systems regulating epiphyseal fusion are poorly understood. Here, we show that the tissue inhibitors of metalloprotease (TIMP) gene family is essential for normal bone growth after birth. Whole-body quadruple-knockout mice lacking all four TIMPs have growth plate closure in long bones, precipitating limb shortening, epiphyseal distortion, and widespread chondrodysplasia. We identify TIMP/FGF-2/IHH as a novel nexus underlying bone lengthening where TIMPs negatively regulate the release of FGF-2 from chondrocytes to allow IHH expression. Using a knock-in approach that combines MMP-resistant or ADAMTS-resistant aggrecans with TIMP deficiency, we uncouple growth plate activity in axial and appendicular bones. Thus, natural metalloprotease inhibitors are crucial regulators of chondrocyte maturation program, growth plate integrity, and skeletal proportionality. Furthermore, individual and combinatorial TIMP-deficient mice demonstrate the redundancy of metalloprotease inhibitor function in embryonic and postnatal development.

Synthesis and Structural/Functional Characterization of Selective M14 Metallocarboxypeptidase Inhibitors Based on Phosphinic Pseudopeptide Scaffold: Implications on the Design of Specific Optical Probes.

Metallocarboxypeptidases (MCPs) of the M14 family are Zn2+-dependent exoproteases present in almost every tissue or fluid in mammals. These enzymes perform a large variety of physiological functions and are involved in several pathologies, such as pancreatic diseases, inflammation, fibrinolysis, and cancer. Here, we describe the synthesis and functional/structural characterization of a series of reversible tight-binding phosphinic pseudopeptide inhibitors that show high specificity and potency toward these proteases. Characterization of their inhibitory potential against a large variety of MCPs, combined with high-resolution crystal structures of three selected candidates in complex with human carboxypeptidase A (CPA)1, allowed to decipher the structural determinants governing selectivity for type-A of the M14A MCP family. Further, the phosphinic pseudopeptide framework was exploited to generate an optical probe selectively targeting human CPAs. The phosphinic pseudopeptides presented here constitute the first example of chemical probes useful to selectively report on type-A MCPs activity in complex media.

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.

C-terminal truncation of IFN-γ inhibits proinflammatory macrophage responses and is deficient in autoimmune disease.

Controlled macrophage differentiation and activation in the initiation and resolution of inflammation is crucial for averting progression to chronic inflammatory and autoimmune diseases. Here we show a negative feedback mechanism for proinflammatory IFN-γ activation of macrophages driven by macrophage-associated matrix metalloproteinase 12 (MMP12). Through C-terminal truncation of IFN-γ at 135Glu↓Leu136 the IFN-γ receptor-binding site was efficiently removed thereby reducing JAK-STAT1 signaling and IFN-γ activation of proinflammatory macrophages. In acute peritonitis this signature was absent in Mmp12 -/- mice and recapitulated in Mmp12 +/+ mice treated with a MMP12-specific inhibitor. Similarly, loss-of-MMP12 increases IFN-γ-dependent proinflammatory markers and iNOS+/MHC class II+ macrophage accumulation with worse lymphadenopathy, arthritic synovitis and lupus glomerulonephritis. In active human systemic lupus erythematosus, MMP12 levels were lower and IFN-γ higher compared to treated patients or healthy individuals. Hence, macrophage proteolytic truncation of IFN-γ attenuates classical activation of macrophages as a prelude for resolving inflammation.

Development of Thioaryl-Based Matrix Metalloproteinase-12 Inhibitors with Alternative Zinc-Binding Groups: Synthesis, Potentiometric, NMR, and Crystallographic Studies.

Matrix metalloproteinase-12 (MMP-12) selective inhibitors could play a role in the treatment of lung inflammatory and cardiovascular diseases. In the present study, the previously reported 4-methoxybiphenylsulfonyl hydroxamate and carboxylate based inhibitors (1b and 2b) were modified to enhance their selectivity for MMP-12. In the newly synthesized thioaryl derivatives, the nature of the zinc binding group (ZBG) and the sulfur oxidation state were changed. Biological assays carried out in vitro on human MMPs with the resulting compounds led to identification of a sulfide, 4a, bearing an N-1-hydroxypiperidine-2,6-dione (HPD) group as new ZBG. Compound 4a is a promising hit compound since it displayed a nanomolar affinity for MMP-12 with a marked selectivity over MMP-9, MMP-1, and MMP-14. Solution complexation studies with Zn2+ were performed to characterize the chelating abilities of the new compounds and confirmed the bidentate binding mode of HPD derivatives. X-ray crystallography studies using MMP-12 and MMP-9 catalytic domains were carried out to rationalize the biological results.

Bifunctional Inhibitors as a New Tool To Reduce Cancer Cell Invasion by Impairing MMP-9 Homodimerization.

Protein homodimers play important roles in physiological and pathological processes, including cancer invasion and metastasis. Recently, MMP-9 natural homodimerization via the PEX domain has been correlated with high migration rates of aggressive cancer cells. Here we propose that bifunctional MMP-9 inhibitors designed to impair natural MMP-9 homodimerization promoted by PEX-PEX interactions might be an effective tool to fight cancer cell invasion. Elaborating a previously described dimeric hydroxamate inhibitor 1, new ligands were synthesized with different linker lengths and branch points. Evaluation of the modified bifunctional ligands by X-ray crystallography and biological assays showed that 7 and 8 could reduce invasion in three glioma cell lines expressing MMP-9 at different levels. To rationalize these results, we present a theoretical model of full-length MMP-9 in complex with 7. This pioneering study suggests that a new approach using MMP-9 selective bifunctional inhibitors might lead to an effective therapy to reduce cancer cell invasion.

Cyanobacteria: photosynthetic factories combining biodiversity, radiation resistance, and genetics to facilitate drug discovery.

Cyanobacteria are ancient, abundant, and widely diverse photosynthetic prokaryotes, which are viewed as promising cell factories for the ecologically responsible production of chemicals. Natural cyanobacteria synthesize a vast array of biologically active (secondary) metabolites with great potential for human health, while a few genetic models can be engineered for the (low level) production of biofuels. Recently, genome sequencing and mining has revealed that natural cyanobacteria have the capacity to produce many more secondary metabolites than have been characterized. The corresponding panoply of enzymes (polyketide synthases and non-ribosomal peptide synthases) of interest for synthetic biology can still be increased through gene manipulations with the tools available for the few genetically manipulable strains. In this review, we propose to exploit the metabolic diversity and radiation resistance of cyanobacteria, and when required the genetics of model strains, for the production and radioactive (14C) labeling of bioactive products, in order to facilitate the screening for new drugs.

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.

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.

Optimization and in Vivo Validation of Peptide Vectors Targeting the LDL Receptor.

Active targeting and delivery to pathophysiological organs of interest is of paramount importance to increase specific accumulation of therapeutic drugs or imaging agents while avoiding systemic side effects. We recently developed a family of new peptide ligands of the human and rodent LDL receptor (LDLR), an attractive cell-surface receptor with high uptake activity and local enrichment in several normal or pathological tissues (Malcor et al., J. Med. Chem. 2012, 55 (5), 2227). Initial chemical optimization of the 15-mer, all natural amino acid compound 1/VH411 (DSGL[CMPRLRGC]cDPR) and structure-activity relationship (SAR) investigation led to the cyclic 8 amino acid analogue compound 22/VH445 ([cMPRLRGC]c) which specifically binds hLDLR with a KD of 76 nM and has an in vitro blood half-life of ∼3 h. Further introduction of non-natural amino acids led to the identification of compound 60/VH4106 ([(d)-"Pen"M"Thz"RLRGC]c), which showed the highest KD value of 9 nM. However, this latter analogue displayed the lowest in vitro blood half-life (∼1.9 h). In the present study, we designed a new set of peptide analogues, namely, VH4127 to VH4131, with further improved biological properties. Detailed analysis of the hLDLR-binding kinetics of previous and new analogues showed that the latter all displayed very high on-rates, in the 106 s-1.M-1 range, and off-rates varying from the low 10-2 s-1 to the 10-1 s-1 range. Furthermore, all these new analogues showed increased blood half-lives in vitro, reaching ∼7 and 10 h for VH4129 and VH4131, respectively. Interestingly, we demonstrate in cell-based assays using both VH445 and the most balanced optimized analogue VH4127 ([cM"Thz"RLRG"Pen"]c), showing a KD of 18 nM and a blood half-life of ∼4.3 h, that its higher on-rate correlated with a significant increase in both the extent of cell-surface binding to hLDLR and the endocytosis potential. Finally, intravenous injection of tritium-radiolabeled 3H-VH4127 in wild-type or ldlr -/- mice confirmed their active LDLR targeting in vivo. Overall, this study extends our previous work toward a diversified portfolio of LDLR-targeted peptide vectors with validated LDLR-targeting potential in vivo.

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.

Sugar-Based Arylsulfonamide Carboxylates as Selective and Water-Soluble Matrix Metalloproteinase-12 Inhibitors.

Matrix metalloproteinase-12 (MMP-12) can be considered an attractive target to study selective inhibitors useful in the development of new therapies for lung and cardiovascular diseases. In this study, a new series of arylsulfonamide carboxylates, with increased hydrophilicity resulting from conjugation with a ß-N-acetyl-d-glucosamine moiety, were designed and synthesized as MMP-12 selective inhibitors. Their inhibitory activity was evaluated on human MMPs by using the fluorimetric assay, and a crystallographic analysis was performed to characterize their binding mode. Among these glycoconjugates, a nanomolar MMP-12 inhibitor with improved water solubility, compound 3 [(R)-2-(N-(2-(3-(2-acetamido-2-deoxy-ß-d-glucopyranosyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid], was identified.

N-O-Isopropyl Sulfonamido-Based Hydroxamates as Matrix Metalloproteinase Inhibitors: Hit Selection and in Vivo Antiangiogenic Activity.

Matrix metalloproteinases (MMPs) have been shown to be involved in tumor-induced angiogenesis. In particular, MMP-2, MMP-9, and MMP-14 have been reported to be crucial for tumor angiogenesis and the formation of metastasis, thus becoming attractive targets in cancer therapy. Here, we report our optimization effort to identify novel N-isopropoxy-arylsulfonamide hydroxamates with improved inhibitory activity toward MMP-2, MMP-9, and MMP-14 with respect to the previously discovered compound 1. A new series of hydroxamates was designed, synthesized, and tested for their antiangiogenic activity using in vitro assays with human umbilical vein endothelial cells (HUVECs). A nanomolar MMP-2, MMP-9, and MMP-14 inhibitor was identified, compound 3, able to potently inhibit angiogenesis in vitro and also in vivo in the matrigel sponge assay in mice. Finally, X-ray crystallographic and docking studies were conducted for compound 3 in order to investigate its binding mode to MMP-9 and MMP-14.

Synthesis and biodistribution studies of (3)H- and (64)Cu-labeled dendritic polyglycerol and dendritic polyglycerol sulfate.

Dendritic polyglycerol sulfate (dPGS) is a biocompatible, bioactive polymer which exhibits anti-inflammatory activity in vivo and thus represents a promising candidate for therapeutic and diagnostic applications. To investigate the in vivo pharmacokinetics in detail, dPGS with a molecular weight of approx. 10 kDa was radiolabeled with (3)H and (64)Cu, and evaluated by performing biodistribution studies and small animal positron emission tomography (PET). (3)H-labeling was accomplished by an oxidation-reduction process with sodium periodate and [(3)H]-borohydride. (64)Cu-labeling was achieved by conjugation of isothiocyanate- or maleimide-functionalized copper(II)-chelating ligands based on 1,4-bis(2-pyridinylmethyl)-1,4,7-triazacyclononane (DMPTACN) to an amino functionalized dPGS scaffold, followed by reaction with an aqueous solution containing (64)CuCl2. Independent biodistribution by radioimaging and PET imaging studies with healthy mice and rats showed that the neutral dPG was quantitatively renally eliminated, whereas the polysulfated analogues accumulated mainly in the liver and spleen. Small amounts of the dPGS derivatives were slowly excreted via the kidneys. The degree of uptake by the reticuloendothelial system (RES) was similar for dPGS with 40% or 85% sulfation, and surface modification of the scaffold with the DMPTACN chelator did not appear to significantly affect the biodistribution profile. On the basis of our data, the applicability of bioactive dPGS as a therapeutic agent might be limited due to organ accumulation even after 3 weeks. The inert characteristics and clearance of the neutral polymer, however, emphasizes the potential of dPG as a multifunctional scaffold for various nanomedical applications.