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1.
Microb Pathog ; 132: 230-242, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31082528

RESUMO

Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation to disrupt quorum-sensing in these bacteria could pave the way for the new development in decreasing resistance strains and are of significant interest for clinical, agricultural, and industrial applications. Isolated endophytic Bacillus thuringiensis strain KMCL07 showing quorum quenching activity on Pseudomonas aeruginosa PAO1 has been studied. AiiA lactonase KMMI17 identified belongs to metallo- ß-lactamase superfamily preserving conserved regions of 106HXDH-59 amino acids-H169-21 amino acids-D191 motif, significantly inhibits the biofilm formation and attenuates virulence factor pyocyanin production of PAO1. Insilico molecular docking analysis of lactonase KMMI17 using alternative catalytic site (PDB entry: 3DHA) with the AHL-based QS system regulators of PAO-1, C4 AHL, C6 AHL and 3-oxo-C12 AHL molecules showed good binding affinity between the protein and ligands, Phe111 and Tyr198 residues plays an important role in binding them. Crude enzyme extract was found to have Km value for C6-HSL: 134.2702 ±â€¯34.83 µM-1, C4-HSL: 308.217 ±â€¯139.9 µM-1 and 3-oxo-C12-HSL: 760.463 ±â€¯251.3 µM-1. LCMS analysis confirms the degradation activity of lactonase KMMI17 on AHL molecules and its hydrolytic process, which indicates the potential application of lactonase KMMI17 as a biocontrol agent or an anti-pathogenic drug.


Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Lactonas/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Percepção de Quorum/fisiologia , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Hidrolases de Éster Carboxílico/metabolismo , Índia , Madhuca/microbiologia , Metaloendopeptidases/metabolismo , Simulação de Acoplamento Molecular , Fenótipo , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Piocianina/metabolismo , Fatores de Virulência
2.
Toxicon ; 160: 38-46, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30802471

RESUMO

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in rural populations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000 to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced by hyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyper immune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebite envenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have produced recombinant single chain variable fragment scFv against the venom of the pit viper Bothrops asper at high levels expressed transiently and stably in transgenic plants and in vitro cultures that is reactive to BaP1 (a metalloproteinase from B. asper venom). The yield from stably transformed plants was significantly (p > 0.05) higher than the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stable transformation were: transgenic callus 62 µg/g (±2); biomass from cell suspension cultures 83 µg/g (±0.2); culture medium from suspensions 71.75 mg/L (±6.18). The activity of scFvBaP1 was confirmed by binding and neutralization of the fibrin degradation induced by BnP1 toxins from B. neuwiedi and by Atroxlysin Ia from B. atrox venoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to be used in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms to be used in human therapy against snakebites.


Assuntos
Metaloendopeptidases/antagonistas & inibidores , Planticorpos/farmacologia , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/farmacologia , Animais , Antivenenos/biossíntese , Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Testes de Neutralização , Planticorpos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Tabaco/genética , Tabaco/metabolismo
3.
Jpn J Infect Dis ; 71(4): 286-290, 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-29709984

RESUMO

Vaccinations with habu (Protobothrops flavoviridis) venom toxoid were administered to individuals living in Amami Oshima from 1965 to 2002, and its effectiveness was investigated in 1991. The results raised the possibility that normal human serum inherently contains an inhibitor of the hemorrhagic metalloproteinase HR2, considered to be one of the major components of habu venom. In this study, we investigated the interaction between the hemorrhagic metalloproteinases HR1 and HR2 from habu-venom and human alpha 2-macroglobulin (α2M). Hemorrhagic activity of HR2 was completely inhibited by human α2M. However, the hemorrhagic activity of the large molecule HR1a was not inhibited. Size exclusion chromatography revealed that human α2M captured the HR2 molecule and formed a complex with it, thus inhibiting hemorrhagic activity. These results suggest that human α2M plays an important role in the inhibition of hemorrhage induced by HR2 from habu venom.


Assuntos
Venenos de Crotalídeos/enzimologia , Metaloendopeptidases/antagonistas & inibidores , alfa 2-Macroglobulinas Associadas à Gravidez/metabolismo , Trimeresurus , Animais , Humanos , Ligação Proteica
4.
Int J Biol Macromol ; 115: 940-954, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29680505

RESUMO

Methionine aminopeptidase 2 (MAP2) is a principal regulator of apoptosis for Leishmania donovani and a potential candidate for the design and synthesis of novel antileishmanials. The LdMAP2 gene was cloned in pET28a(+)-SUMO vector, expressed in E. coli and then purified by chromatographic methods. It was found to be a monomer and required divalent metal ion for its activity against synthetic substrates with Co(II), Mg(II), Mn(II) and Ni(II) being the major activators. Moreover, Ca(II) showed the tightest binding with Km value of 124.7 ±â€¯9.2 µM, while Co(II) proved most efficient for catalysis with kcat value of 128.1 ±â€¯4 min-1. The naturally occurring aminopeptidase B inhibitor bestatin was found to be a potent inhibitor of LdMAP2 with a Ki value of 0.86 µM. Further, structural studies with circular dichroism (CD) showed an increase in the α-helical and ß-sheet contents and a decrease in random coils in LdMAP2 upon interactions with both bestatin and fluorogenic substrates. Finally, structural studies pointed out key differences in the structure of LdMAP2 and HsMAP2 and their interactions with inhibitor bestatin, Ala-AMC, Leu-AMC and Met-AMC. The structural differences of two orthologs and different binding modes with bestatin can be crucial for the development of novel and specific inhibitor against leishmaniasis.


Assuntos
Aminopeptidases/química , Aminopeptidases/metabolismo , Leishmania donovani/enzimologia , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Sequência de Aminoácidos , Aminopeptidases/antagonistas & inibidores , Estabilidade Enzimática , Humanos , Cinética , Leucina/análogos & derivados , Leucina/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , Especificidade por Substrato , Temperatura Ambiente
5.
PLoS One ; 13(4): e0196474, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29698456

RESUMO

Upon their translocation into the mitochondrial matrix, the N-terminal pre-sequence of nuclear-encoded proteins undergoes cleavage by mitochondrial processing peptidases. Some proteins require more than a single processing step, which involves several peptidases. Down-regulation of the putative Trypanosoma brucei mitochondrial intermediate peptidase (MIP) homolog by RNAi renders the cells unable to grow after 48 hours of induction. Ablation of MIP results in the accumulation of the precursor of the trypanosomatid-specific trCOIV protein, the largest nuclear-encoded subunit of the cytochrome c oxidase complex in this flagellate. However, the trCOIV precursor of the same size accumulates also in trypanosomes in which either alpha or beta subunits of the mitochondrial processing peptidase (MPP) have been depleted. Using a chimeric protein that consists of the N-terminal sequence of a putative subunit of respiratory complex I fused to a yellow fluorescent protein, we assessed the accumulation of the precursor protein in trypanosomes, in which RNAi was induced against the alpha or beta subunits of MPP or MIP. The observed accumulation of precursors indicates MIP depletion affects the activity of the cannonical MPP, or at least one of its subunits.


Assuntos
Metaloendopeptidases/metabolismo , Mitocôndrias/enzimologia , Trypanosoma brucei brucei/metabolismo , Sequência de Aminoácidos , Regulação para Baixo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/classificação , Metaloendopeptidases/genética , Microscopia de Fluorescência , Filogenia , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Especificidade por Substrato
6.
Cell Physiol Biochem ; 42(4): 1657-1669, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28738346

RESUMO

BACKGROUND/AIMS: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. METHODS: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. RESULTS: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. CONCLUSION: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.


Assuntos
Antibacterianos/farmacologia , Azitromicina/farmacologia , Berberina/farmacologia , Biofilmes/efeitos dos fármacos , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Alginatos , Animais , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Ciclofosfamida , Fibrose Cística/microbiologia , DNA Bacteriano/antagonistas & inibidores , DNA Bacteriano/biossíntese , Combinação de Medicamentos , Sinergismo Farmacológico , Ácido Glucurônico/antagonistas & inibidores , Ácido Glucurônico/biossíntese , Ácidos Hexurônicos/antagonistas & inibidores , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/microbiologia , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Metaloproteases/antagonistas & inibidores , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Neutropenia/induzido quimicamente , Neutropenia/tratamento farmacológico , Neutropenia/genética , Neutropenia/patologia , Oligopeptídeos/antagonistas & inibidores , Oligopeptídeos/biossíntese , Infecções por Pseudomonas/induzido quimicamente , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Piocianina/antagonistas & inibidores , Piocianina/biossíntese , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
7.
Bioorg Med Chem Lett ; 27(11): 2428-2431, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28408220

RESUMO

The astacin proteases meprin α and ß are emerging drug targets for treatment of disorders such as kidney failure, fibrosis or inflammatory bowel disease. However, there are only few inhibitors of both proteases reported to date. Starting from NNGH as lead structure, a detailed elaboration of the structure-activity relationship of meprin ß inhibitors was performed, leading to compounds with activities in the lower nanomolar range. Considering the preference of meprin ß for acidic residues in the P1' position, the compounds were optimized. Acidic modifications induced potent inhibition and >100-fold selectivity over other structurally related metalloproteases such as MMP-2 or ADAM10.


Assuntos
Ácidos Hidroxâmicos/química , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/química , Sulfonamidas/química , Ácidos Hidroxâmicos/síntese química , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Proteases/síntese química , Relação Estrutura-Atividade , Sulfonamidas/síntese química
8.
Bioorg Med Chem Lett ; 27(9): 2018-2022, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28347665

RESUMO

Snakebites represent an important public health problem, with a great number of victims with permanent sequelae or fatal outcomes, particularly in rural, agriculturally active areas. The snake venom metalloproteases (SVMPs) are the principal proteins responsible for some clinically-relevant effects, such as local and systemic hemorrhage, dermonecrosis, and myonecrosis. Because of the difficulties in neutralizing them rapidly and locally by antivenoms, the search and design of small molecules as inhibitors of SVMPs are proposed. The Bothrops asper metalloprotease P1 (BaP1) is hereby used as a target protein and by High Throughput Virtual Screening (HTVS) approach, the free access virtual libraries: ZINC, PubChem and ChEMBL, were searched for potent small molecule inhibitors. Results from the aforementioned approaches provided strong evidences on the structural requirements for the efficient BaP1 inhibition such as the presence of the pyrimidine-2,4,6-trione moiety. The two proposed compounds have also shown excellent results in performed in vitro interaction studies against BaP1.


Assuntos
Antídotos/química , Antídotos/farmacologia , Bothrops/metabolismo , Metaloendopeptidases/antagonistas & inibidores , Pirimidinonas/química , Pirimidinonas/farmacologia , Venenos de Serpentes/antagonistas & inibidores , Animais , Simulação por Computador , Descoberta de Drogas , Metaloendopeptidases/metabolismo , Simulação de Acoplamento Molecular , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia
9.
Int J Biol Macromol ; 98: 436-446, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28163123

RESUMO

Resistance of snakes and some other animals to snake envenomation has been attributed to soluble factors present in their tissues. Here we report the isolation of a novel metalloprotease inhibitor from Bothrops alternatus snake serum (named BaltMPI) with high purity, using a four-step chromatographic method. BaltMPI has molecular weights of 60.5 and 42.4kDa, as determined by SDS-PAGE and mass spectrometry, respectively, and pI=5.27. The first 60 amino acids from the N-terminal region of BaltMPI, determined by Edman's degradation, showed high homology (97%) with the snake venom metalloprotease inhibitor (SVMPI) BJ46a and other SVMPIs (78-82%). The chromatographic fractions and purified BaltMPI exhibited anti-hemorrhagic activity against Batroxase and BjussuMP-I. BaltMPI was stable over wide ranges of pH (1, 5, 8, and 9) and temperature (-80, -20, 4, 60, and 100°C), and suppressed the fibrinogenolytic, fibrinolytic, and azocaseinolytic activities of Batroxase. BaltMPI specifically inhibited the activity of metalloproteases, without affecting the activity of serine proteases. Together, our results suggest that BaltMPI and other SVMPIs are promising molecules for the treatment of snake envenomation, in particular that caused by Bothrops sp.


Assuntos
Bothrops/sangue , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Animais , Caseínas/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Hemorragia/tratamento farmacológico , Metaloendopeptidases/metabolismo , Camundongos , Inibidores de Proteases/sangue , Inibidores de Proteases/química , Proteólise/efeitos dos fármacos
10.
Endocrinology ; 158(1): 41-55, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27849360

RESUMO

Hypothalamic lesions or deficient melanocortin (MC) signaling via MC4 receptor (MC4r) mutations often lead to hyperphagia and severe treatment-resistant obesity. We tested the methionine aminopeptidase 2-inhibitor beloranib (ZGN-440) in 2 male rat models of obesity, one modeling hypothalamic obesity with a combined medial hypothalamic lesion (CMHL) and the other modeling a monogenic form of obesity with MC4r mutations (MC4r knockout [MC4rKO]). In CMHL rats (age 3 months), postsurgery excess weight gain was significantly inhibited (ZGN-440, 0.2 ± 0.7 g/d; vehicle, 3.8 ± 0.6 g/d; P < 0.001) during 12 days of ZGN-440 treatment (0.1 mg/kg daily subcutaneously) together with a 30% reduction of daily food intake vs vehicle injection. In addition, ZGN-440 treatment improved glucose tolerance and reduced plasma insulin, and circulating levels of α-melanocyte stimulating hormone were increased. Serum lipid levels did not differ significantly in ZGN-440-treated vs vehicle-treated rats. Similar results were found in MC4rKO rats: ZGN-440 treatment (14-21 d) was associated with significant reductions of body weight gain (MC4rKO, -1.7 ± 0.6 vs 2.8 ± 0.4 g/d; lean wild-type controls, -0.7 ± 0.2 vs 1.7 ± 0.7 g/d; ZGN-440 vs vehicle, respectively), reduction of food intake (MC4rKO, -28%; lean controls, -7.5%), and insulin resistance, whereas circulating levels of interleukin-1ß did not change. In both obesity models, body temperature and locomotor activity were not affected by ZGN-440 treatment. In conclusion, the robust reduction of body weight in response to ZGN-440 observed in rats with severe obesity is related to a strong reduction of food intake that is likely related to changes in the central regulation of feeding.


Assuntos
Aminopeptidases/antagonistas & inibidores , Cinamatos/uso terapêutico , Cicloexanos/uso terapêutico , Compostos de Epóxi/uso terapêutico , Hipotálamo Médio/lesões , Metaloendopeptidases/antagonistas & inibidores , Obesidade/tratamento farmacológico , Receptor Tipo 4 de Melanocortina/genética , Sesquiterpenos/uso terapêutico , Animais , Temperatura Corporal , Peso Corporal , Cinamatos/farmacologia , Cicloexanos/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Ingestão de Alimentos , Compostos de Epóxi/farmacologia , Expressão Gênica , Teste de Tolerância a Glucose , Hiperfagia/complicações , Resistência à Insulina , Leptina/sangue , Metabolismo dos Lipídeos , Fígado/enzimologia , Masculino , Obesidade/sangue , Obesidade/etiologia , Ratos Sprague-Dawley , Ratos Transgênicos , Sesquiterpenos/farmacologia
11.
Protein Sci ; 26(2): 242-257, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27774687

RESUMO

The function and localization of proteins and peptides containing C-terminal "CaaX" (Cys-aliphatic-aliphatic-anything) sequence motifs are modulated by post-translational attachment of isoprenyl groups to the cysteine sulfhydryl, followed by proteolytic cleavage of the aaX amino acids. The zinc metalloprotease ZMPSTE24 is one of two enzymes known to catalyze this cleavage. The only identified target of mammalian ZMPSTE24 is prelamin A, the precursor to the nuclear scaffold protein lamin A. ZMPSTE24 also cleaves prelamin A at a second site 15 residues upstream from the CaaX site. Mutations in ZMPSTE24 result in premature-aging diseases and inhibition of ZMPSTE24 activity has been reported to be an off-target effect of HIV protease inhibitors. We report here the expression (in yeast), purification, and crystallization of human ZMPSTE24 allowing determination of the structure to 2.0 Å resolution. Compared to previous lower resolution structures, the enhanced resolution provides: (1) a detailed view of the active site of ZMPSTE24, including water coordinating the catalytic zinc; (2) enhanced visualization of fenestrations providing access from the exterior to the interior cavity of the protein; (3) a view of the C-terminus extending away from the main body of the protein; (4) localization of ordered lipid and detergent molecules at internal and external surfaces and also projecting through fenestrations; (5) identification of water molecules associated with the surface of the internal cavity. We also used a fluorogenic assay of the activity of purified ZMPSTE24 to demonstrate that HIV protease inhibitors directly inhibit the human enzyme in a manner indicative of a competitive mechanism.


Assuntos
Inibidores da Protease de HIV/química , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Domínio Catalítico , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
12.
Eur J Clin Pharmacol ; 73(1): 15-28, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27742998

RESUMO

INTRODUCTION: EP217609 is a representative of a new class of synthetic parenteral anticoagulants with a dual mechanism of action. It combines in a single molecule a direct thrombin inhibitor and an indirect factor Xa inhibitor. EP217609 can be neutralized by a specific antidote avidin, which binds to the biotin moiety of EP217609. PURPOSE: The primary objective was to assess the neutralization of EP217609 by avidin in healthy subjects. Secondary objectives were to define the optimal avidin monomer/EP217609 molar ratio to achieve an adequate neutralization of EP217609 and to assess the safety and tolerability of EP217609 and avidin. METHODS: Healthy subjects (n = 36) were randomized to a 3 by 3 replicated Latin square design between 3 EP217609 doses (4, 8, 12 mg) and 3 avidin monomer/EP217609 molar ratios (1:1; 2:1; 3:1). EP217609 was administered as a single intravenous bolus, and avidin as a 30-min intravenous infusion, starting 90 min after EP217609 administration. RESULTS: Overall, EP217609 and avidin were well tolerated. One subject experienced a benign and transient typical pseudo-allergic reaction. The administration of EP217609 resulted in dose-dependent increases in pharmacodynamic markers. Avidin triggered a rapid and irreversible neutralization of EP217609 without rebound effect. Adequate neutralization of the anticoagulant activity was achieved with both 2:1 and 3:1 avidin monomer/EP217609 molar ratios. All safety parameters did not show any treatment-emergent clinically relevant changes or abnormalities in any dose group. CONCLUSIONS: These results will allow further investigation in patients requiring a neutralizable anticoagulant as those undergoing cardiac surgery. STUDY REGISTRATION: EudraCT number 2010-020216-10.


Assuntos
Anticoagulantes/farmacologia , Antídotos/farmacologia , Avidina/farmacologia , Biotina/análogos & derivados , Oligossacarídeos/farmacologia , Adulto , Anticoagulantes/efeitos adversos , Anticoagulantes/sangue , Anticoagulantes/farmacocinética , Antídotos/efeitos adversos , Antídotos/farmacocinética , Avidina/efeitos adversos , Avidina/sangue , Avidina/farmacocinética , Biotina/efeitos adversos , Biotina/sangue , Biotina/farmacocinética , Biotina/farmacologia , Testes de Coagulação Sanguínea , Venenos de Crotalídeos/antagonistas & inibidores , Fator Xa , Humanos , Masculino , Metaloendopeptidases/antagonistas & inibidores , Oligossacarídeos/efeitos adversos , Oligossacarídeos/sangue , Oligossacarídeos/farmacocinética , Adulto Jovem
13.
Virulence ; 8(6): 938-958, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-27763824

RESUMO

Pseudomonas aeruginosa is among the most common pathogens responsible for both acute and chronic infections of high incidence and severity. Additionally, P. aeruginosa resistance to conventional antimicrobials has increased rapidly over the past decade. Therefore, it is crucial to explore new therapeutic options, particularly options that specifically target the pathogenic mechanisms of this microbe. The ability of a pathogenic bacterium to cause disease is dependent upon the production of agents termed 'virulence factors', and approaches to mitigate these agents have gained increasing attention as new antibacterial strategies. Although blue light irradiation is a promising alternative approach, only limited and preliminary studies have described its effect on virulence factors. The current study aimed to investigate the effects of lethal and sub-lethal doses of blue light treatment (BLT) on P. aeruginosa virulence factors. We analyzed the inhibitory effects of blue light irradiation on the production/activity of several virulence factors. Lethal BLT inhibited the activity of pyocyanin, staphylolysin, pseudolysin and other proteases, but sub-lethal BLT did not affect the production/expression of proteases, phospholipases, and flagella- or type IV pili-associated motility. Moreover, a eukaryotic cytotoxicity test confirmed the decreased toxicity of blue light-treated extracellular P. aeruginosa fractions. Finally, the increased antimicrobial susceptibility of P. aeruginosa treated with sequential doses of sub-lethal BLT was demonstrated with a checkerboard test. Thus, this work provides evidence-based proof of the susceptibility of drug-resistant P. aeruginosa to BLT-mediated killing, accompanied by virulence factor reduction, and describes the synergy between antibiotics and sub-lethal BLT.


Assuntos
Antibacterianos/farmacologia , Luz , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos da radiação , Fatores de Virulência/efeitos da radiação , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/efeitos da radiação , Cor , Humanos , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/efeitos da radiação , Testes de Sensibilidade Microbiana , Elastase Pancreática/efeitos da radiação , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/terapia , Virulência/efeitos da radiação , Fatores de Virulência/antagonistas & inibidores
14.
Nat Chem ; 8(12): 1152-1158, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27874871

RESUMO

Off-target binding of hydrophobic drugs can lead to unwanted side effects, either through specific or non-specific binding to unintended membrane protein targets. However, distinguishing the binding of drugs to membrane proteins from that of detergents, lipids and cofactors is challenging. Here, we use high-resolution mass spectrometry to study the effects of HIV protease inhibitors on the human zinc metalloprotease ZMPSTE24. This intramembrane protease plays a major role in converting prelamin A to mature lamin A. We monitored the proteolysis of farnesylated prelamin A peptide by ZMPSTE24 and unexpectedly found retention of the C-terminal peptide product with the enzyme. We also resolved binding of zinc, lipids and HIV protease inhibitors and showed that drug binding blocked prelamin A peptide cleavage and conferred stability to ZMPSTE24. Our results not only have relevance for the progeria-like side effects of certain HIV protease inhibitor drugs, but also highlight new approaches for documenting off-target drug binding.


Assuntos
Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Preparações Farmacêuticas/metabolismo , Inibidores da Protease de HIV/química , Inibidores da Protease de HIV/metabolismo , Humanos , Lamina Tipo A/metabolismo , Lipídeos/química , Espectrometria de Massas , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/química , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Preparações Farmacêuticas/química , Ligação Proteica , Prenilação de Proteína , Especificidade por Substrato , Zinco/química , Zinco/metabolismo
15.
Bioorg Med Chem Lett ; 26(21): 5254-5259, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27692546

RESUMO

A collection of twenty-six organoselenium compounds, ebselen and its structural analogues, provided a novel approach for inhibiting the activity of human methionine aminopeptidase 2 (MetAP2). This metalloprotease, being responsible for the removal of the amino-terminal methionine from newly synthesized proteins, plays a key role in angiogenesis, which is essential for the progression of diseases, including solid tumor cancers. In this work, we discovered that ebselen, a synthetic organoselenium drug molecule with anti-inflammatory, anti-oxidant and cytoprotective activity, inhibits one of the main enzymes in the tumor progression pathway. Using three-step synthesis, we obtained twenty-five ebselen derivatives/analogues, ten of which are new, and tested their inhibitory activity toward three neutral aminopeptidases (MetAP2, alanine and leucine aminopeptidases). All of the tested compounds proved to be selective, slow-binding inhibitors of MetAP2. Similarly to ebselen, most of its analogues exhibited a moderate potency (IC50=1-12µM). Moreover, we identified three strong inhibitors that bind favorably to the enzyme with the half maximal inhibitory concentration in the submicromolar range.


Assuntos
Aminopeptidases/antagonistas & inibidores , Azóis/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Compostos Organosselênicos/farmacologia , Inibidores de Proteases/farmacologia , Azóis/química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , Compostos Organosselênicos/química , Relação Estrutura-Atividade
16.
Toxins (Basel) ; 8(9)2016 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-27571103

RESUMO

The research on natural snake venom metalloendopeptidase inhibitors (SVMPIs) began in the 18th century with the pioneering work of Fontana on the resistance that vipers exhibited to their own venom. During the past 40 years, SVMPIs have been isolated mainly from the sera of resistant animals, and characterized to different extents. They are acidic oligomeric glycoproteins that remain biologically active over a wide range of pH and temperature values. Based on primary structure determination, mammalian plasmatic SVMPIs are classified as members of the immunoglobulin (Ig) supergene protein family, while the one isolated from muscle belongs to the ficolin/opsonin P35 family. On the other hand, SVMPIs from snake plasma have been placed in the cystatin superfamily. These natural antitoxins constitute the first line of defense against snake venoms, inhibiting the catalytic activities of snake venom metalloendopeptidases through the establishment of high-affinity, non-covalent interactions. This review presents a historical account of the field of natural resistance, summarizing its main discoveries and current challenges, which are mostly related to the limitations that preclude three-dimensional structural determinations of these inhibitors using "gold-standard" methods; perspectives on how to circumvent such limitations are presented. Potential applications of these SVMPIs in medicine are also highlighted.


Assuntos
Antídotos/uso terapêutico , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/uso terapêutico , Proteínas de Répteis/antagonistas & inibidores , Mordeduras de Serpentes/tratamento farmacológico , Venenos de Serpentes/antagonistas & inibidores , Animais , Antídotos/história , História do Século XVIII , História do Século XIX , História do Século XX , História do Século XXI , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/história , Metaloendopeptidases/metabolismo , Inibidores de Proteases/história , Conformação Proteica , Proteínas de Répteis/química , Proteínas de Répteis/história , Proteínas de Répteis/metabolismo , Mordeduras de Serpentes/enzimologia , Mordeduras de Serpentes/história , Venenos de Serpentes/química , Venenos de Serpentes/enzimologia , Venenos de Serpentes/história , Relação Estrutura-Atividade
17.
Molecules ; 21(5)2016 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-27164068

RESUMO

Coronary artery disease (CAD) is the most common cause of heart attack and the leading cause of mortality in the world. It is associated with mitochondrial dysfunction and increased level of reactive oxygen species production. According to the Ottawa Heart Genomics Study genome-wide association study, a recent research identified that Q688 spastic paraplegia 7 (SPG7) variant is associated with CAD as it bypasses the regulation of tyrosine phosphorylation of AFG3L2 and enhances the processing and maturation of SPG7 protein. This study aims to identify potential compounds isolated from Traditional Chinese Medicines (TCMs) as potential lead compounds for paraplegin (SPG7) inhibitors. For the crystallographic structure of paraplegin, the disordered disposition of key amino acids in the binding site was predicted using the PONDR-Fit protocol before virtual screening. The TCM compounds saussureamine C and 3-(2-carboxyphenyl)-4(3H)-quinazolinone, have potential binding affinities with stable H-bonds and hydrophobic contacts with key residues of paraplegin. A molecular dynamics simulation was performed to validate the stability of the interactions between each candidate and paraplegin under dynamic conditions. Hence, we propose these compounds as potential candidates as lead drug from the compounds isolated from TCM for further study in drug development process with paraplegin protein for coronary artery disease.


Assuntos
Asparagina/análogos & derivados , Doença da Artéria Coronariana/genética , Medicamentos de Ervas Chinesas/química , Inibidores Enzimáticos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Quinazolinonas/farmacologia , ATPases Associadas a Diversas Atividades Celulares , Asparagina/química , Asparagina/farmacologia , Sítios de Ligação , Simulação por Computador , Doença da Artéria Coronariana/enzimologia , Cristalografia por Raios X , Medicamentos de Ervas Chinesas/farmacologia , Inibidores Enzimáticos/química , Humanos , Metaloendopeptidases/química , Metaloendopeptidases/genética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Quinazolinonas/química , Relação Estrutura-Atividade
18.
Nucleic Acid Ther ; 26(4): 250-6, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27228357

RESUMO

Heterosporis saurida, a microsporidian parasite of lizardfish, Saurida undosquamis, causes severe economic losses in marine aquaculture. Among the novel approaches being explored for treatment of parasitic infections in aquaculture is small interfering RNA molecules. The aim of the present study was to investigate the efficiency of using siRNA to knock down expression of specific genes of H. saurida in vitro. For this purpose, siRNAs specific for ATP/ADP antiporter 1 and methionine aminopeptidase II genes were designed and tested using a previously developed in vitro cultivation model. Silencing of H. saurida target genes was assessed and the efficacy of using siRNA for inhibition of gene expression was measured by quantitative real-time polymerase chain reaction (PCR). Silencing of ATP/ADP antiporter 1 or methionine aminopeptidase II by siRNA reduced H. saurida infection levels in EK-1 cells 40% and 60%, respectively, as measured by qRT-PCR and spore counts. Combined siRNA treatment of both ATP/ADP antiporter 1 and methionine aminopeptidase II siRNAs was more effective against H. saurida infection as seen by the 16S rRNA level and spore counts. Our study concluded that siRNA could be used to advance development of novel approaches to inhibit H. saurida and provide an alternative approach to combat microsporidia.


Assuntos
Peixes/genética , Inativação Gênica , Interferência de RNA , RNA Interferente Pequeno/genética , Aminopeptidases/antagonistas & inibidores , Aminopeptidases/genética , Animais , Antiporters/antagonistas & inibidores , Antiporters/genética , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Doenças dos Peixes/prevenção & controle , Peixes/microbiologia , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/genética , Microsporídios/patogenicidade , RNA Interferente Pequeno/farmacologia
19.
J Enzyme Inhib Med Chem ; 31(6): 1632-7, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27028668

RESUMO

The CSN complex plays a key role in various cellular pathways: through a metalloprotease activity of its Csn5 deneddylating enzyme, it regulates the activity of Cullin-RING ligases (CRLs). Indeed, Csn5 has been found amplified in many tumors, but, due to its pleiotropic effects, it is difficult to dissect its function and the involvement in cancer progression. Moreover, while growing evidences point to the neddylation function as a good target for drug development; specific inhibitors have not yet been developed for the CSN. Here, we propose the yeast Saccharomyces cerevisiae as a model system to screen libraries of small molecules as inhibitors of cullins deneddylation, taking advantage of the unique feature of this organism to survive without a functional CSN5 gene and to accumulate a fully neddylated cullin substrate. By combining molecular modeling and simple genetic tools, we were able to identify two small molecular fragments as selective inhibitors of Csn5 deneddylation function.


Assuntos
Metaloendopeptidases/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/metabolismo , Complexo do Signalossomo COP9 , Simulação de Dinâmica Molecular
20.
Comb Chem High Throughput Screen ; 19(3): 246-58, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26875789

RESUMO

Human meprin-α and-ß are important regulators of angiogenesis, cancer, inflammation, fibrosis, and neurodegenerative diseases and hence important therapeutic targets. Meprins are the only astacin proteases that are expressed in membrane-bound and secreted form. The cleavage specificity of human meprins is similar in certain cases but differs markedly in others. The inhibitor selectivity of human meprins is controlled by the specific residues involved in binding at the active-site cleft of the proteases. Meprins are inhibited by various small molecular inhibitors as well as macromolecular endogenous inhibitors, making them good drug targets. In the current study, molecular dynamics simulation was performed for 10 ns on ten systems consisting of two apoenzymes of meprin -α/ß and eight complexes of human meprin-α and -ß complexed to four inhibitors with different metal binding moieties and comparable Ki values. These simulation studies helped to elucidate the molecular details of how several parameters influence protein-inhibitor binding affinity. Analysis of the interaction energies of the protein-inhibitor complexes revealed the diverse binding nature of this series of inhibitors. Several structural segments of human meprins exhibited certain conformational changes during the simulation time course. Among the inhibitors studied captopril had a different disposition in the meprin-bound complexes compared to the other three inhibitors, namely Pro- Leu-Gly-hydroxamate, galardin and EDTA. Comparison of the interaction energies for each system helped us to conclude that the hydroxamic acid-based inhibitors are the most potent inhibitors of meprins.


Assuntos
Metaloendopeptidases/antagonistas & inibidores , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacologia , Sítios de Ligação/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Simulação de Acoplamento Molecular , Estrutura Molecular , Inibidores de Proteases/química , Relação Estrutura-Atividade
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