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We report the development of compact and stabilized micelles incorporating a synthetic LXR agonist prodrug for the passive targeting of atherosclerotic lesions and therapeutic intervention. In vivo studies showed that the nanohybrid micelles exhibited favorable pharmacokinetics/biodistribution and were able to upregulate, to some extent, LXR target genes with no alteration of lipid metabolism.
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Aterosclerosis , Micelas , Humanos , Receptores X del Hígado/uso terapéutico , Distribución Tisular , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/patologíaRESUMEN
CD95L (also known as FasL or CD178) is a member of the tumor necrosis family (TNF) superfamily. Although this transmembrane ligand has been mainly considered as a potent apoptotic inducer in CD95 (Fas)-expressing cells, more recent studies pointed out its role in the implementation of non-apoptotic signals. Accordingly, this ligand has been associated with the aggravation of inflammation in different auto-immune disorders and in the metastatic occurrence in different cancers. Although it remains to decipher all key factors involved in the ambivalent role of this ligand, accumulating clues suggest that while the membrane bound CD95L triggers apoptosis, its soluble counterpart generated by metalloprotease-driven cleavage is responsible for its non-apoptotic functions. Nonetheless, the metalloproteases (MMPs and ADAMs) involved in the CD95L shedding, the cleavage sites and the different stoichiometries and functions of the soluble CD95L remain to be elucidated. To better understand how soluble CD95L triggers signaling pathways from apoptosis to inflammation or cell migration, we propose herein to summarize the different metalloproteases that have been described to be able to shed CD95L, their cleavage sites and the biological functions associated with the released ligands. Based on these new findings, the development of CD95/CD95L-targeting therapeutics is also discussed.
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Neoplasias , Receptor fas , Humanos , Proteína Ligando Fas , Ligandos , Metaloproteasas/metabolismo , Transducción de Señal , InflamaciónRESUMEN
The first matrix metalloproteinase (MMP) was discovered in 1962 from the tail of a tadpole by its ability to degrade collagen. As their name suggests, matrix metalloproteinases are proteases capable of remodeling the extracellular matrix. More recently, MMPs have been demonstrated to play numerous additional biologic roles in cell signaling, immune regulation, and transcriptional control, all of which are unrelated to the degradation of the extracellular matrix. In this review, we will present milestones and major discoveries of MMP research, including various clinical trials for the use of MMP inhibitors. We will discuss the reasons behind the failures of most MMP inhibitors for the treatment of cancer and inflammatory diseases. There are still misconceptions about the pathophysiological roles of MMPs and the best strategies to inhibit their detrimental functions. This review aims to discuss MMPs in preclinical models and human pathologies. We will discuss new biochemical tools to track their proteolytic activity in vivo and ex vivo, in addition to future pharmacological alternatives to inhibit their detrimental functions in diseases. SIGNIFICANCE STATEMENT: Matrix metalloproteinases (MMPs) have been implicated in most inflammatory, autoimmune, cancers, and pathogen-mediated diseases. Initially overlooked, MMP contributions can be both beneficial and detrimental in disease progression and resolution. Thousands of MMP substrates have been suggested, and a few hundred have been validated. After more than 60 years of MMP research, there remain intriguing enigmas to solve regarding their biological functions in diseases.
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Inhibidores de la Metaloproteinasa de la Matriz , Neoplasias , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz/uso terapéutico , Neoplasias/metabolismo , ProteolisisRESUMEN
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.
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Inmunoconjugados , Radioisótopos de CarbonoRESUMEN
In this report, a synthetic protocol for the preparation of phosphinic dipeptides of type 5 is presented. These compounds serve as valuable building blocks for the development of highly potent phosphinopeptidic inhibitors of medicinally relevant Zn-metalloproteases and aspartyl proteases. The proposed method is based on the tandem esterification of α-aminophosphinic and acrylic acids under silylating conditions in order to subsequently participate in a P-Michael reaction. The scope of the transformation was investigated by using a diverse set of readily available acrylic acids and (R)-α-aminophosphinic acids, and high yields were achieved in all cases. In most examples reported herein, the isolation of biologically relevant (R,S)-diastereoisomers became possible by simple crystallization from the crude products, thus enhancing the operational simplicity of the proposed method. Finally, functional groups corresponding to acidic or basic natural amino acids are also compatible with the reaction conditions. Based on the above, we expect that the practicality of the proposed protocol will facilitate the discovery of pharmacologically useful bioactive phosphinic peptides.
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Acrilatos/química , Dipéptidos , Ácidos Fosfínicos/química , Dipéptidos/síntesis química , Dipéptidos/química , EsterificaciónRESUMEN
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 inâ 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 inâ vivo.
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Understanding pharmacokinetics and biodistribution of antibody-drug conjugates (ADCs) is a one of the critical steps enabling their successful development and optimization. Their complex structure combining large and small molecule characteristics brought out multiple bioanalytical methods to decipher the behavior and fate of both components in vivo. In this respect, these methods must provide insights into different key elements including half-life and blood stability of the construct, premature release of the drug, whole-body biodistribution, and amount of the drug accumulated within the targeted pathological tissues, all of them being directly related to efficacy and safety of the ADC. In this review, we will focus on the main strategies enabling to quantify and characterize ADCs in biological matrices and discuss their associated technical challenges and current limitations.
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Macrophage elastase [matrix metalloproteinase (MMP)-12] is the most upregulated MMP in abdominal aortic aneurysm (AAA) and, hence, MMP-12-targeted imaging may predict AAA progression and rupture risk. Here, we report the design, synthesis, and evaluation of three novel hydroxamate-based selective MMP-12 inhibitors (CGA, CGA-1, and AGA) and the methodology to obtain MMP-12 selectivity from hydroxamate-based panMMP inhibitors. Also, we report two 99mTc-radiotracers, 99mTc-AGA-1 and 99mTc-AGA-2, derived from AGA. 99mTc-AGA-2 displayed faster blood clearance in mice and better radiochemical stability compared to 99mTc-AGA-1. Based on this, 99mTc-AGA-2 was chosen as the lead tracer and tested in murine AAA. 99mTc-AGA-2 uptake detected by autoradiography was significantly higher in AAA compared to normal aortic regions. Specific binding of the tracer to MMP-12 was demonstrated through ex vivo competition. Accordingly, this study introduces a novel family of selective MMP-12 inhibitors and tracers, paving the way for further development of these agents as therapeutic and imaging agents.
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Ácidos Hidroxámicos/farmacología , Metaloproteinasa 12 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Oligopéptidos/farmacología , Compuestos de Organotecnecio/farmacología , Radiofármacos/farmacología , Animales , Aneurisma de la Aorta Abdominal/enzimología , Aneurisma de la Aorta Abdominal/metabolismo , Diseño de Fármacos , Humanos , Ácidos Hidroxámicos/síntesis química , Inhibidores de la Metaloproteinasa de la Matriz/síntesis química , Ratones Endogámicos C57BL , Imagen Molecular/métodos , Estructura Molecular , Oligopéptidos/síntesis química , Compuestos de Organotecnecio/síntesis química , Radiofármacos/síntesis química , Relación Estructura-ActividadRESUMEN
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.
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Aorta/diagnóstico por imagen , Metaloproteinasa 12 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/química , Imagen Molecular/métodos , Compuestos de Organotecnecio/química , Animales , Aneurisma de la Aorta Abdominal/diagnóstico por imagen , Humanos , Masculino , Inhibidores de la Metaloproteinasa de la Matriz/farmacocinética , Ratones , Ratones Endogámicos C57BL , Trazadores Radiactivos , Radioquímica , Distribución Tisular , Regulación hacia ArribaRESUMEN
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.
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Portadores de Fármacos/química , Lípidos/química , Nanomedicina , Nanoestructuras/química , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/metabolismo , Técnicas de Química Sintética , Modelos Animales de Enfermedad , Portadores de Fármacos/síntesis química , Humanos , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Noqueados , Nanomedicina/métodos , Nanopartículas/química , Nanoestructuras/ultraestructura , Distribución TisularRESUMEN
A straightforward, late-stage diversification strategy for the installation of side chains on readily accessible unsaturated phosphinopeptidic scaffolds based on a Giese-type addition of alkyl radicals has been investigated. Among different alternatives, the preferred methodology is operationally simple as it can be carried out in an open flask with no need for protection of acidic moieties. Direct application to the synthesis of SPPS-compatible building blocks or to longer peptides is also reported.
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Alanina/análogos & derivados , Péptidos/síntesis química , Ácidos Fosfínicos/síntesis química , Alanina/síntesis química , Alanina/química , Alquilación , Radicales Libres/química , Estructura Molecular , Péptidos/química , Ácidos Fosfínicos/químicaRESUMEN
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.
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Carboxipeptidasas A/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Oligopéptidos/farmacología , Ácidos Fosfínicos/farmacología , Carboxipeptidasas A/química , Carboxipeptidasas A/metabolismo , Dominio Catalítico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Células HEK293 , Células HeLa , Humanos , Indoles/síntesis química , Indoles/farmacología , Cinética , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Ácidos Fosfínicos/síntesis química , Ácidos Fosfínicos/metabolismo , Unión ProteicaRESUMEN
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.
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Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Zinc/metabolismo , Sitios de Unión , Calorimetría , Cristalización , Inhibidores de la Metaloproteinasa de la Matriz/química , Inhibidores de la Metaloproteinasa de la Matriz/metabolismo , Relación Estructura-ActividadRESUMEN
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.
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Aneurisma/diagnóstico por imagen , Arterias Carótidas/diagnóstico por imagen , Macrófagos/metabolismo , Metaloproteinasa 12 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/metabolismo , Imagen Óptica/métodos , Aneurisma/inmunología , Aneurisma/metabolismo , Aneurisma/patología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Arterias Carótidas/inmunología , Arterias Carótidas/metabolismo , Arterias Carótidas/patología , Modelos Animales de Enfermedad , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Expresión Génica , Humanos , Inflamación , Macrófagos/inmunología , Macrófagos/patología , Metaloproteinasa 12 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/síntesis química , Ratones , Ratones Endogámicos C57BL , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Compuestos de Amonio Cuaternario/química , Compuestos de Amonio Cuaternario/metabolismo , Ácidos Sulfónicos/química , Ácidos Sulfónicos/metabolismoRESUMEN
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.
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Metaloproteinasa 12 de la Matriz/análisis , Imagen Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Animales , Carbocianinas , Técnicas de Química Sintética , Cristalografía por Rayos X , Células HeLa , Humanos , Metaloproteinasa 12 de la Matriz/química , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones Endogámicos C57BL , Sondas Moleculares/farmacocinética , Óptica y Fotónica/métodos , Péptidos/química , Distribución TisularRESUMEN
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.
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Radioisótopos de Cobre/química , Dendrímeros/síntesis química , Dendrímeros/farmacocinética , Glicerol/química , Polímeros/química , Sulfatos/química , Tritio/química , Animales , Compuestos Aza/química , Quelantes/química , Técnicas de Química Sintética , Dendrímeros/química , Estabilidad de Medicamentos , Femenino , Marcaje Isotópico , Ratones , Piperidinas/química , Tomografía de Emisión de Positrones , Radioquímica , Ratas , Distribución TisularRESUMEN
A P-C bond-forming reaction between silyl phosphonites and Morita-Baylis-Hillman acetates (MBHAs) is explored as a general alternative towards medicinally relevant ß-carboxyphosphinic structural motifs. Conversion rates of diversely substituted MBHAs to phosphinic acids 9 or 14 that were recorded by using (31) Pâ NMR spectroscopy revealed unexpected reactivity differences between ester and nitrile derivatives. These kinetic profiles and DFT calculations support a mechanistic scenario in which observed differences can be explained from the "lateness" of transition states. In addition, we provide experimental evidence suggesting that enolates due to initial P-Michael addition are not formed. Based on the proposed mechanistic scenario in conjunction with DFT calculations, an interpretation of the E/Z stereoselectivity differences between ester and nitriles is proposed. Synthetic opportunities stemming from this transformation are presented, which deal with the preparation of several synthetically capricious phosphinic building blocks, whose access through the classical P-Michael synthetic route is not straightforward.
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Interferon-α (IFN-α) is essential for antiviral immunity, but in the absence of matrix metalloproteinase-12 (MMP-12) or IκBα (encoded by NFKBIA) we show that IFN-α is retained in the cytosol of virus-infected cells and is not secreted. Our findings suggest that activated IκBα mediates the export of IFN-α from virus-infected cells and that the inability of cells in Mmp12(-/-) but not wild-type mice to express IκBα and thus export IFN-α makes coxsackievirus type B3 infection lethal and renders respiratory syncytial virus more pathogenic. We show here that after macrophage secretion, MMP-12 is transported into virus-infected cells. In HeLa cells MMP-12 is also translocated to the nucleus, where it binds to the NFKBIA promoter, driving transcription. We also identified dual-regulated substrates that are repressed both by MMP-12 binding to the substrate's gene exons and by MMP-12-mediated cleavage of the substrate protein itself. Whereas intracellular MMP-12 mediates NFKBIA transcription, leading to IFN-α secretion and host protection, extracellular MMP-12 cleaves off the IFN-α receptor 2 binding site of systemic IFN-α, preventing an unchecked immune response. Consistent with an unexpected role for MMP-12 in clearing systemic IFN-α, treatment of coxsackievirus type B3-infected wild-type mice with a membrane-impermeable MMP-12 inhibitor elevates systemic IFN-α levels and reduces viral replication in pancreas while sparing intracellular MMP-12. These findings suggest that inhibiting extracellular MMP-12 could be a new avenue for the development of antiviral treatments.
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Núcleo Celular/genética , Inmunidad/genética , Interferón-alfa/genética , Metaloproteinasa 12 de la Matriz/genética , Animales , Sitios de Unión , Núcleo Celular/inmunología , Núcleo Celular/metabolismo , Citosol/metabolismo , Citosol/virología , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Metaloproteinasa 12 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Inhibidor NF-kappaB alfa , Páncreas/inmunología , Páncreas/virología , Virus del Sarcoma de Rous/genética , Virus del Sarcoma de Rous/patogenicidad , Replicación Viral/efectos de los fármacosRESUMEN
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.
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Carcinoma de Células Escamosas/metabolismo , Estrógenos/metabolismo , Metaloproteinasa 13 de la Matriz/fisiología , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Neoplasias Cutáneas/metabolismo , Animales , Carcinoma de Células Escamosas/tratamiento farmacológico , Estradiol/metabolismo , Matriz Extracelular/enzimología , Femenino , Fibroblastos/citología , Queratinocitos/citología , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Trasplante de Neoplasias , Neovascularización Patológica , Neoplasias Cutáneas/tratamiento farmacológico , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To develop enzyme-activatable Förster resonance energy transfer (FRET) substrate probes to detect matrix metalloproteinase 12 (MMP-12) and MMP-13 activities in vivo in mouse models of inflammatory arthritis. METHODS: Peptidic FRET probes activated by MMP-12 and MMP-13 were reverse designed from inhibitors selected from a phosphinic peptide inhibitor library. Selectivity of the probes was demonstrated in vitro using MMP-1, MMP-2, MMP-3, MMP-12, and MMP-13. In vivo activation of the probes was tested in the zymosan-induced mouse model of inflammation, and probe specificity was evaluated by the MMP inhibitor GM6001 and specific synthetic inhibitors of MMP-12 and MMP-13. The probes were used to monitor these enzyme activities in the collagen-induced arthritis (CIA) model in vivo. RESULTS: The MMP-12 and MMP-13 activity probes (MMP12ap and MMP13ap, respectively) discriminated between the activities of the 2 enzymes. The in vivo activation of these probes was inhibited by GM6001 and by their respective specific inhibitors. In the CIA model, MMP12ap activation peaked 5 days after disease onset and showed strong correlation with disease severity during this time (r = 0.85, P < 0.0001). MMP13ap activation increased gradually after disease onset and correlated with disease severity over a longer period of 15 days (r = 0.58, P < 0.0001). CONCLUSION: We generated two selective FRET probes that can be used to monitor MMP-12 and MMP-13 activities in live animals. MMP12ap follows the initial stage of inflammation in CIA, while MMP13ap follows the progression of the disease. The specificity of these probes is useful in monitoring the efficacy of MMP inhibitors.