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1.
Nanoscale ; 15(11): 5510-5518, 2023 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-36853236

RESUMO

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.


Assuntos
Grafite , Nanotubos de Carbono , Animais , Camundongos , Grafite/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Distribuição Tecidual , Radioisótopos de Carbono
2.
J Med Chem ; 65(9): 6953-6968, 2022 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-35500280

RESUMO

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.


Assuntos
Imunoconjugados , Radioisótopos de Carbono
3.
Sci Total Environ ; 790: 148125, 2021 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-34380275

RESUMO

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.


Assuntos
Placenta , Frutos do Mar , Animais , Encéfalo , Feminino , Humanos , Gravidez , Ratos , Alimentos Marinhos , Distribuição Tecidual
4.
Angew Chem Int Ed Engl ; 60(33): 18272-18279, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34096148

RESUMO

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.

5.
J Med Chem ; 62(21): 9743-9752, 2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31603669

RESUMO

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.


Assuntos
Aorta/diagnóstico por imagem , Metaloproteinase 12 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/química , Imagem Molecular/métodos , Compostos de Organotecnécio/química , Animais , Aneurisma da Aorta Abdominal/diagnóstico por imagem , Humanos , Masculino , Inibidores de Metaloproteinases de Matriz/farmacocinética , Camundongos , Camundongos Endogâmicos C57BL , Traçadores Radioativos , Radioquímica , Distribuição Tecidual , Regulação para Cima
6.
Molecules ; 24(19)2019 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-31561608

RESUMO

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.


Assuntos
Portadores de Fármacos/química , Lipídeos/química , Nanomedicina , Nanoestruturas/química , Animais , Apolipoproteínas E/deficiência , Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Aterosclerose/metabolismo , Técnicas de Química Sintética , Modelos Animais de Doenças , Portadores de Fármacos/síntese química , Humanos , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Knockout , Nanomedicina/métodos , Nanopartículas/química , Nanoestruturas/ultraestrutura , Distribuição Tecidual
7.
FEBS Open Bio ; 8(12): 2011-2021, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30524951

RESUMO

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.

8.
Proc Natl Acad Sci U S A ; 114(27): 7154-7159, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28630289

RESUMO

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.


Assuntos
Antagonistas dos Receptores de Hormônios Antidiuréticos/farmacologia , Dendroaspis , Peptídeos Natriuréticos/farmacologia , Peptídeos/farmacologia , Doenças Renais Policísticas/tratamento farmacológico , Receptores de Vasopressinas/genética , Venenos de Serpentes/farmacologia , Animais , Benzazepinas/farmacologia , Células CHO , Cricetinae , Cricetulus , Cristalografia por Raios X , AMP Cíclico/metabolismo , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Doenças Renais Policísticas/metabolismo , Transdução de Sinais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Tolvaptan , Tripsina/química
9.
J Med Chem ; 60(1): 403-414, 2017 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-27996256

RESUMO

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.


Assuntos
Inibidores de Metaloproteinases de Matriz/farmacologia , Zinco/metabolismo , Sítios de Ligação , Calorimetria , Cristalização , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/metabolismo , Relação Estrutura-Atividade
10.
Sci Rep ; 6: 38345, 2016 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-27917892

RESUMO

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.


Assuntos
Aneurisma/diagnóstico por imagem , Artérias Carótidas/diagnóstico por imagem , Macrófagos/metabolismo , Metaloproteinase 12 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz/metabolismo , Imagem Óptica/métodos , Aneurisma/imunologia , Aneurisma/metabolismo , Aneurisma/patologia , Animais , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/metabolismo , Artérias Carótidas/imunologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Modelos Animais de Doenças , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Expressão Gênica , Humanos , Inflamação , Macrófagos/imunologia , Macrófagos/patologia , Metaloproteinase 12 da Matriz/genética , Inibidores de Metaloproteinases de Matriz/síntese química , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Compostos de Amônio Quaternário/química , Compostos de Amônio Quaternário/metabolismo , Ácidos Sulfônicos/química , Ácidos Sulfônicos/metabolismo
11.
Bioconjug Chem ; 27(10): 2407-2417, 2016 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-27564088

RESUMO

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.


Assuntos
Metaloproteinase 12 da Matriz/análise , Imagem Molecular/métodos , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Animais , Carbocianinas , Técnicas de Química Sintética , Cristalografia por Raios X , Células HeLa , Humanos , Metaloproteinase 12 da Matriz/química , Metaloproteinase 12 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Sondas Moleculares/farmacocinética , Óptica e Fotônica/métodos , Peptídeos/química , Distribuição Tecidual
12.
Chem Biol ; 21(3): 408-13, 2014 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-24583051

RESUMO

Matrix metalloproteinases (MMPs) are a large family of zinc-dependent endoproteases that catalyze cleavage of extracellular matrix and nonmatrix proteins. MMPs play a role in tissue remodeling, and their uncontrolled activity is associated with number of diseases, including tumor metastasis. Thus, there is a need to develop methods to monitor MMP activity, and number of probes has been previously described. The key problem many probes encounter is the issue of selectivity, since 23 human MMPs, despite playing different physiological roles, have structurally similar active sites. Here, we introduce the halogen bonding concept into the probe design and show that the probe containing iodine exhibits an unprecedented selectivity for MMP-9. We provide structure-based explanation for the selectivity, confirming that it is due to formation of the halogen bond that supports catalysis, and we highlight the value of exploring halogen bonding in the context of selective probe design.


Assuntos
Halogênios/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Halogênios/química , Humanos , Metaloproteinase 9 da Matriz/química , Simulação de Dinâmica Molecular , Especificidade por Substrato
13.
Int J Cancer ; 135(12): 2749-59, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-24676718

RESUMO

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.


Assuntos
Carcinoma de Células Escamosas/metabolismo , Estrogênios/metabolismo , Metaloproteinase 13 da Matriz/fisiologia , Inibidores de Metaloproteinases de Matriz/farmacologia , Neoplasias Cutâneas/metabolismo , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Estradiol/metabolismo , Matriz Extracelular/enzimologia , Feminino , Fibroblastos/citologia , Queratinócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Transplante de Neoplasias , Neovascularização Patológica , Neoplasias Cutâneas/tratamento farmacológico , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
FEBS J ; 281(3): 943-56, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24289879

RESUMO

Human somatic angiotensin-I converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase and a central component of the renin angiotensin aldosterone system (RAAS). Its involvement in the modulation of physiological actions of peptide hormones has positioned ACE as an important therapeutic target for the treatment of hypertension and cardiovascular disorders. Here, we report the crystal structures of the two catalytic domains of human ACE (N- and C-) in complex with FI, the S enantiomer of the phosphinic ACE/ECE-1 (endothelin converting enzyme) dual inhibitor FII, to a resolution of 1.91 and 1.85 Å, respectively. In addition, we have determined the structure of AnCE (an ACE homologue from Drosophila melanogaster) in complex with both isomers. The inhibitor FI (S configuration) can adapt to the active site of ACE catalytic domains and shows key differences in its binding mechanism mostly through the reorientation of the isoxazole phenyl side group at the P1' position compared with FII (R configuration). Differences in binding are also observed between FI and FII in complex with AnCE. Thus, the new structures of the ACE-inhibitor complexes presented here provide useful information for further exploration of ACE inhibitor pharmacophores involving phosphinic peptides and illustrate the role of chirality in enhancing drug specificity.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/química , Modelos Moleculares , Oligopeptídeos/química , Peptidil Dipeptidase A/química , Ácidos Fosfínicos/química , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Domínio Catalítico , Proteínas de Drosophila , Drosophila melanogaster/enzimologia , Humanos , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Ligantes , Mutagênese Sítio-Dirigida , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/genética , Peptidil Dipeptidase A/metabolismo , Ácidos Fosfínicos/metabolismo , Ácidos Fosfínicos/farmacologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Especificidade por Substrato , Difração de Raios X
15.
Microb Cell Fact ; 12: 37, 2013 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-23607455

RESUMO

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.


Assuntos
Escherichia coli/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Citoplasma/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Oxirredução , Isomerases de Dissulfetos de Proteínas/genética , Dobramento de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
16.
J Med Chem ; 56(3): 1149-59, 2013 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-23343195

RESUMO

The molecular determinants responsible for the potency of the RXP470.1 phosphinic peptide inhibitor toward matrix metalloprotease-12 (MMP-12) remain elusive. To address this issue, structure-activity study, X-ray crystallography, and isothermal titration calorimetry (ITC) experiments were performed. The crystal structure of MMP-12/inhibitor complex (1.15 Å) reveals that the inhibitor establishes multiple interactions with the MMP-12 active site, with its long P(1)' side chain filling most of the S(1)' deep cavity. ITC experiments indicate that the binding of this inhibitor to MMP-12 is mostly entropy driven (ΔG° = -13.1 kcal/mol, ΔH° = -2.53 kcal/mol, and -TΔS° = -10.60 kcal/mol) and involves a proton uptake from the buffer. Comparing phosphinic versus hydroxamate inhibitors reveals that the chelation of the zinc ion is slightly different, leading the inhibitor backbone to adopt a position in which the hydrogen bonding with the MMP-12 active site is less favorable in phosphinic inhibitor while maintaining high affinity.


Assuntos
Metaloproteinase 12 da Matriz/efeitos dos fármacos , Inibidores de Proteases/farmacologia , Cristalografia por Raios X , Modelos Moleculares , Inibidores de Proteases/química , Termodinâmica
17.
FEBS J ; 280(1): 139-59, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23121732

RESUMO

Artificial miniproteins that are able to target catalytic sites of matrix metalloproteinases (MMPs) were designed using a functional motif-grafting approach. The motif corresponded to the four N-terminal residues of TIMP-2, a broad-spectrum protein inhibitor of MMPs. Scaffolds that are able to reproduce the functional topology of this motif were obtained by exhaustive screening of the Protein Data Bank (PDB) using STAMPS software (search for three-dimensional atom motifs in protein structures). Ten artificial protein binders were produced. The designed proteins bind catalytic sites of MMPs with affinities ranging from 450 nm to 450 µm prior to optimization. The crystal structure of one artificial binder in complex with the catalytic domain of MMP-12 showed that the inter-molecular interactions established by the functional motif in the artificial binder corresponded to those found in the MMP-14-TIMP-2 complex, albeit with some differences in geometry. Molecular dynamics simulations of the ten binders in complex with MMP-14 suggested that these scaffolds may allow partial reproduction of native inter-molecular interactions, but differences in geometry and stability may contribute to the lower affinity of the artificial protein binders compared to the natural protein binder. Nevertheless, these results show that the in silico design method used provides sets of protein binders that target a specific binding site with a good rate of success. This approach may constitute the first step of an efficient hybrid computational/experimental approach to protein binder design.


Assuntos
Inibidores de Metaloproteinases de Matriz/química , Simulação de Dinâmica Molecular , Proteínas Recombinantes de Fusão/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Bases de Dados de Proteínas , Humanos , Ligação de Hidrogênio , Metaloproteinase 12 da Matriz/química , Metaloproteinase 14 da Matriz/química , Inibidores de Metaloproteinases de Matriz/síntese química , Dados de Sequência Molecular , Ligação Proteica , Engenharia de Proteínas , Estabilidade Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/síntese química , Inibidor Tecidual de Metaloproteinase-2/química
18.
J Biol Chem ; 287(40): 33607-14, 2012 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-22869371

RESUMO

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.


Assuntos
Toxinas Botulínicas Tipo A/antagonistas & inibidores , Clostridium botulinum/metabolismo , Cisteína/química , Domínio Catalítico , Química Farmacêutica/métodos , Cristalografia por Raios X/métodos , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Mutagênese Sítio-Dirigida , Peptídeo Hidrolases/química , Peptídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteína 25 Associada a Sinaptossoma/química , Zinco/química
19.
J Biol Chem ; 287(32): 26647-56, 2012 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-22689580

RESUMO

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.


Assuntos
Dipeptídeos/farmacologia , Ácido Glutâmico/química , Inibidores de Metaloproteinases de Matriz , Inibidores de Proteases/farmacologia , Domínio Catalítico , Cristalografia por Raios X , Dipeptídeos/química , Metaloproteinases da Matriz/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/química , Especificidade por Substrato
20.
Biochimie ; 94(2): 461-70, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21889567

RESUMO

Long-sarafotoxins (l-SRTXs) have recently been identified in both the venom of Atractaspis microlepidota and that of Atractaspis irregularis. They are characterized by different C-terminus extensions that follow the invariant Trp21, which plays a crucial role in endothelin-receptor binding. We initially determined the toxicity and three-dimensional structures of two chemically synthesized l-SRTXs that have different C-terminus extensions, namely SRTX-m (24 aa, including extension "D-E-P") and SRTX-i3 (25 aa, including extension "V-N-R-N"). Both peptides were shown to be highly toxic in mice and displayed the cysteine-stabilized α-helical motif that characterizes endothelins and short-SRTXs, to which a longer C-terminus with variable flexibility is added. To discern the functional and pharmacological consequences of the supplementary amino acids, different chimerical as well as truncated forms of SRTX were designed and synthesized. Thus, we either removed the extra-C-terminal residues of SRTX-m or i3, or grafted the latter onto the C-terminal extremity of a short-SRTX (s-SRTX) (ie. SRTX-b). Our competitive binding assays where SRTXs competed for iodinated endothelin-1 binding to cloned ET(A) and ET(B) receptor subtypes over-expressed in CHO cells, revealed the essential role of the C-terminus extensions for ET-receptor recognition. Indeed, l-SRTXs displayed an affinity three to four orders of magnitude lower as compared to SRTX-b for the two receptor subtypes. Moreover, grafting the C-terminus extension to SRTX-b induced a drastic decrease in affinity, while its removal (truncated l-SRTXs) yielded an affinity for ET-receptors similar to that of s-SRTXs. Furthermore, we established by intracellular Ca(2+) measurements that l-SRTXs, as well as s-SRTXs, display agonistic activities. We thus confirmed in these functional assays the major difference in potency for these two SRTX families as well as the crucial role of the C-terminus extension in their various pharmacological profiles. Finally, one of the chimeric toxin synthesized in this study appears to be one of the most potent and selective ligand of the ET(B) receptor known to date.


Assuntos
Endotelina-1/metabolismo , Peptídeos/síntese química , Receptores de Endotelina/agonistas , Venenos de Víboras , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células CHO , Cálcio/metabolismo , Cricetinae , Injeções Intravenosas , Transporte de Íons/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peptídeos/toxicidade , Ligação Proteica , Engenharia de Proteínas , Estrutura Secundária de Proteína , Receptores de Endotelina/metabolismo , Relação Estrutura-Atividade , Taxa de Sobrevida , Transfecção , Vasoconstritores/síntese química , Vasoconstritores/toxicidade , Venenos de Víboras/síntese química , Venenos de Víboras/toxicidade
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