RESUMO
Ubiquitin-specific proteases represent a family of enzymes that catalyze the cleavage of ubiquitin from specific substrate proteins to regulate their activity. USP48 is a rarely studied USP, which has recently been linked to inflammatory signaling via regulation of the transcription factor nuclear factor kappa B. Nonetheless, a crystal structure of USP48 has not yet been resolved and potent inhibitors are not known. We screened a set of 14 commercially available USP inhibitors for their activity against USP48 and identified the USP2 inhibitor "ML364" as a candidate for further optimization. Using a ligand-based approach, we derived and synthesized a series of ML364 analogs. The IC50 concentrations of the new compounds to inhibit USP48 were determined in a deubiquitinylase activity assay by measuring the fluorescence intensity using tetra-ubiquitin rhodamine110 as substrate. A compound containing a carboxylic acid functionalization (17e) inhibited USP48 activity toward tetra-ubiquitin rhodamine110 with an IC50 of 12.6 µM. Further structure-based refinements are required to improve the inhibition activity and specificity.
Assuntos
Transdução de Sinais , Proteases Específicas de Ubiquitina , Relação Estrutura-Atividade , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/metabolismo , Fatores de Transcrição , UbiquitinasRESUMO
Targeting metalloproteinases has been in the focus of drug design for a long time. However, meprin α and ß emerged as potential drug targets just recently and are linked to several diseases with different pathological background. Nevertheless, the validation of meprins as suitable drug targets still requires highly potent and selective inhibitors as chemical probes to elucidate their role in pathophysiology. Albeit highly selective inhibitors of meprin ß have already been reported, only inhibitors of meprin α with modest activity or selectivity are known. Starting from recently reported heteroaromatic scaffolds, the aim of this study was the optimisation of meprin α and/or meprin ß inhibition while keeping the favourable off-target inhibition profile over other metalloproteases. We report potent pan-meprin inhibitors as well as highly active inhibitors of meprin α with superior selectivity over meprin ß. The latter are suitable to serve as chemical probes and enable further target validation.
Assuntos
Metaloendopeptidases , Metaloproteases , Relação Estrutura-Atividade , Metaloproteases/metabolismo , Desenho de FármacosRESUMO
Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infections in infants, the elderly, and the immunocompromised, yet no licensed vaccine and only limited therapeutic options for prevention and treatment are available, which poses a global health challenge and emphasizes the urgent medical need for novel antiviral agents. In the current study, a novel potent small molecule inhibitor of RSV was identified by performing a screening and structure optimization campaign, wherein a naturally occurring dicaffeoylquinic acid (DCQA) compound served as a chemical starting point. The reported benzamide derivative inhibitor, designated as 2f, was selected for its improved stability and potent antiviral activity from a series of investigated structurally related compounds. 2f was well tolerated by cells and able to inhibit RSV infection with a half maximal inhibitory concentration (IC50) of 35 nM and a favorable selectivity index (SI) of 3742. Although the exact molecular target for 2f is not known, in vitro mechanism of action investigations revealed that the compound inhibits the early stage of infection by interacting with RSV virion and interferes primarily with the attachment and potentially with the virus-cell fusion step. Moreover, intranasal administration of 2f to mice simultaneously or prior to intranasal infection with RSV significantly decreased viral load in the lungs, pointing to the in vivo potential of the compound. Our results suggest that 2f is a viable candidate for further preclinical development and evaluation as an antiviral agent against RSV infections.
Assuntos
Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Humano , Infecções Respiratórias , Lactente , Camundongos , Humanos , Animais , Idoso , Infecções por Vírus Respiratório Sincicial/tratamento farmacológico , Pulmão , Linhagem Celular , Infecções Respiratórias/tratamento farmacológico , Antivirais/farmacologiaRESUMO
Periodontitis is a severe yet underestimated oral disease. Since it is linked to several systemic diseases, such as diabetes, artheriosclerosis, and even Alzheimer's disease, growing interest in treating periodontitis has emerged recently. The major cause of periodontitis is a shift in the oral microbiome. A keystone pathogen that is associated with this shift is Porphyromonas gingivalis. Hence, targeting P. gingivalis came into focus of drug discovery for the development of novel antiinfective compounds. Among others, glutaminyl cyclases (QCs) of oral pathogens might be promising drug targets. Here, we report the discovery and structure-activity relationship of a novel class of P. gingivalis QC inhibitors according to a tetrahydroimidazo[4,5-c]pyridine scaffold. Some compounds exhibited activity in the lower nanomolar range and thus were further characterized with regard to their selectivity and toxicity.
RESUMO
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.
Assuntos
Aminoaciltransferases/química , Periodontite/microbiologia , Porphyromonas gingivalis/enzimologia , Prevotella intermedia/enzimologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/genética , Aminoaciltransferases/ultraestrutura , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Periodontite/tratamento farmacológico , Periodontite/genética , Porphyromonas gingivalis/patogenicidade , Prevotella intermedia/patogenicidade , Estrutura Terciária de Proteína/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/metabolismo , Tannerella forsythia/enzimologia , Tannerella forsythia/patogenicidadeRESUMO
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer Disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.
RESUMO
Astacin metalloproteinases, in particular meprins α and ß, as well as ovastacin, are emerging drug targets. Drug-discovery efforts have led to the development of the first potent and selective inhibitors in the last few years. However, the most recent compounds are based on a highly flexible tertiary amine scaffold that could cause metabolic liabilities or decreased potency due to the entropic penalty upon binding to the target. Thus, the aim of this study was to discover novel conformationally constrained scaffolds as starting points for further inhibitor optimization. Shifting from flexible tertiary amines to rigid heteroaromatic cores resulted in a boost in inhibitory activity. Moreover, some compounds already exhibited higher activity against individual astacin proteinases compared to recently reported inhibitors and also a favorable off-target selectivity profile, thus qualifying them as very suitable chemical probes for target validation.
Assuntos
Aminas/farmacologia , Antineoplásicos/farmacologia , Descoberta de Drogas , Hidrocarbonetos Aromáticos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Metaloproteases/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Aminas/síntese química , Aminas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Hidrocarbonetos Aromáticos/síntese química , Hidrocarbonetos Aromáticos/química , Metaloendopeptidases/metabolismo , Metaloproteases/metabolismo , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Despite huge progress in hormonal therapy and improved inâ vitro fertilization methods, the success rates in infertility treatment are still limited. A recently discovered mechanism revealed the interplay between the plasma protein fetuin-B and the cortical granule-based proteinase ovastacin to be a novel key mechanism in the regulation of fertilization. Upon sperm-egg fusion, cleavage of a distinct zona pellucida component by ovastacin destroys the sperm receptor, enhances zona robustness, and eventually provides a definitive block against polyspermy. An untimely onset of this zona hardening prior to fertilization would consequently result in infertility. Physiologically, this process is controlled by fetuin-B, an endogenous ovastacin inhibitor. Here we aimed to discover small-molecule inhibitors of ovastacin that could mimic the effect of fetuin-B. These compounds could be useful lead structures for the development of specific ovastacin inhibitors that can be used in infertility treatment or inâ vitro fertilization.
Assuntos
Aminas/farmacologia , Ácidos Hidroxâmicos/farmacologia , Infertilidade Feminina/tratamento farmacológico , Metaloproteases/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Aminas/química , Animais , Biocatálise , Relação Dose-Resposta a Droga , Feminino , Ácidos Hidroxâmicos/química , Infertilidade Feminina/metabolismo , Metaloproteases/metabolismo , Camundongos , Modelos Moleculares , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
We describe the development of flexible minocycline-lipid-complex extrudates with optimized mechanical and drug release properties. These extrudates contain a minocycline - magnesium stearate chelate complex with a higher stability in aqueous media, which has now been incorporated in a PEG-PLGA (polyethylene glycol - poly(lactic-co-glycolic acid)) matrix. PEG 1500 has been utilized in different concentrations to serve as plasticizer. The novel formulations have been characterized by texture analysis, X-Ray powder diffraction (XRPD) and differential scanning calorimetry (DSC). Extrudates with a reduced diameter of 300⯵m (previously 600⯵m) were introduced, and a more sensitive quantification method with a tandem-mass spectrometry detector was developed. From all tested formulations, the extrudates consisting of Expansorb DLG 50 - 6P (PEG-PLGA, molar weight 30-60â¯kDa) paired with 10% PEG 1500 emerged as best formulation. These extrudates feature a drug content of 11.5% and a controlled release over at least 42â¯days. The release profile is without a lag time and shows initially a slightly higher release rate, which is desired. Compared to previous developments, the extrudates now offer a high flexibility combined with a large mechanical resilience, which will ease the handling and administration.
Assuntos
Antibacterianos/administração & dosagem , Lipídeos/química , Minociclina/administração & dosagem , Poliésteres/química , Polietilenoglicóis/química , Antibacterianos/química , Química Farmacêutica , Preparações de Ação Retardada , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Minociclina/química , Periodontite/tratamento farmacológico , Plastificantes/química , Ácidos Esteáricos/químicaRESUMO
Amyloidogenic plaques are hallmarks of Alzheimer's disease (AD) and typically consist of high percentages of modified Aß peptides bearing N-terminally cyclized glutamate residues. The human zinc(II) enzyme glutaminyl cyclase (QC) was shown in vivo to catalyze the cyclization of N-terminal glutamates of Aß peptides in a pathophysiological side reaction establishing QC as a druggable target for therapeutic treatment of AD. Here, we report crystallographic snapshots of human QC catalysis acting on the neurohormone neurotensin that delineate the stereochemical course of catalysis and suggest that hydrazides could mimic the transition state of peptide cyclization and deamidation. This hypothesis is validated by a sparse-matrix inhibitor screening campaign that identifies hydrazides as the most potent metal-binding group compared to classic Zn binders. The structural basis of hydrazide inhibition is illuminated by X-ray structure analysis of human QC in complex with a hydrazide-bearing peptide inhibitor and reveals a pentacoordinated Zn complex. Our findings inform novel strategies in the design of potent and highly selective QC inhibitors by employing hydrazides as the metal-binding warhead.
Assuntos
Doença de Alzheimer/enzimologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/metabolismo , Inibidores Enzimáticos/química , Hidrazinas/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Aminoaciltransferases/química , Cristalografia por Raios X , Ciclização/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Hidrazinas/farmacologia , Modelos Moleculares , Terapia de Alvo Molecular , Neurotensina/metabolismo , Conformação Proteica/efeitos dos fármacosRESUMO
Periodontal therapy using antimicrobials that are topically applied requires slow or controlled release devices. The in vitro antimicrobial activity of biodegradable polymer formulations that contain a new minocycline lipid complex (P-MLC) was evaluated. The new P-MLC formulations that contained 11.5% minocycline were compared with pure minocycline or an existing commercial formulation, which included determination of minimal inhibitory concentration (MIC) values against two oral bacteria and activity on six-species periodontal biofilm. Moreover, the flow of gingival crevicular fluid (GCF) was modeled up to 42 d and the obtained eluates were tested both for MIC values and inhibiting biofilm formation. In general, MICs of the P-MLC formulations were slightly increased as compared with pure minocycline. Biofilm formation was clearly inhibited by all tested formulations containing minocycline with no clear difference between them. In 3.5 d old biofilms, all formulations with 250 µg/mL minocycline decreased bacterial counts by 3 log10 and metabolic activity with no difference to pure antimicrobials. Eluates of experimental formulations showed superiority in antimicrobial activity. Eluates of one experimental formulation (P503-MLC) still inhibited biofilm formation at 28 d, with a reduction by 1.87 log10 colony forming units (CFU) vs. the untreated control. The new experimental formulations can easily be instilled in periodontal pockets and represent alternatives in local antimicrobials, and thus warrant further testing.
RESUMO
The objective of this study was to develop an improved drug delivery system for the local antimicrobial treatment of periodontitis, that offers enhanced drug stability, easy application and controlled release over several weeks. Chelate complexes consisting of a tetracycline antibiotic and a fatty acid salt were developed. Minocycline and doxycycline were paired with magnesium- and calcium stearate in different molar ratios. These chelate complexes stabilize the active pharmaceutical ingredient and enable the incorporation into a PLGA (poly(lactic-co-glycolic acid)) polymer matrix via hot melt extrusion. The chelate complexes were characterized via UV/Vis- and IR-spectroscopy. A high antibiotic activity of the complex was observed in a disc diffusion test. The drug complex was mixed with different PLGA-polymers and cryomilled in advance of the extrusion. The hot melt extrusion yielded homogeneous extrudates with a diameter from 600 to 900⯵m. They contain 11.5% of minocycline, are adjustable in length and are easy to handle. In vitro release studies revealed a controlled release of the drug over 42â¯days. In conclusion, the developed extrudates are promising systems to improve the treatment of periodontitis.
Assuntos
Antibacterianos/química , Doxiciclina/química , Portadores de Fármacos , Minociclina/química , Periodontite/tratamento farmacológico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Ácidos Esteáricos/química , Antibacterianos/administração & dosagem , Preparações de Ação Retardada , Doxiciclina/administração & dosagem , Composição de Medicamentos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Cinética , Minociclina/administração & dosagem , Tamanho da Partícula , Periodontite/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimentoRESUMO
Thia analogs of fosmidomycin are potent inhibitors of the non-mevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (IspC, Dxr) of Plasmodium falciparum. Several new thioethers displayed antiplasmodial in vitro activity in the low nanomolar range, without apparent cytotoxic effects in HeLa cells. The (S)-(+)-enantiomer of a typical representative selectively inhibited IspC and the growth of P. falciparum in continuous culture. The inhibitor was stable at pH 7.6 and room temperature, and no racemization was observed under these conditions during a period of up to two days. Oxidation of selected thioethers to sulfones reduced antiplasmodial activity and the inhibitory activity against Escherichia coli, Mycobacterium tuberculosis and P. falciparum IspC orthologs.
Assuntos
Antibacterianos/farmacologia , Antiprotozoários/farmacologia , Escherichia coli/efeitos dos fármacos , Fosfomicina/análogos & derivados , Mycobacterium tuberculosis/efeitos dos fármacos , Plasmodium falciparum/efeitos dos fármacos , Antibacterianos/síntese química , Antibacterianos/química , Antiprotozoários/síntese química , Antiprotozoários/química , Células CACO-2 , Relação Dose-Resposta a Droga , Escherichia coli/crescimento & desenvolvimento , Fosfomicina/síntese química , Fosfomicina/química , Fosfomicina/farmacologia , Células HeLa , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/crescimento & desenvolvimento , Testes de Sensibilidade Parasitária , Plasmodium falciparum/crescimento & desenvolvimento , Relação Estrutura-AtividadeRESUMO
OBJECTIVES: Human glutaminyl cyclases (QC and isoQC) play an important role in maintaining inflammatory conditions. Meanwhile a glutaminyl cyclase synthesized by Porphyromonas gingivalis (PgQC), a key pathogen in developing periodontitis and a potential link of periodontitis with rheumatoid arthritis (RA), was discovered. This study was aimed to determine the expression of QC, isoQC and PgQC in patients with chronic periodontitis (CP) and RA. DESIGN: Thirty volunteers were enrolled in a pilot study and divided into 3 groups (healthy, CP and RA individuals). Blood samples, biofilm and gingival crevicular fluid (GCF) were analysed for mRNA expression of QC, isoQC and P. gingivalis QC. Major bacteria being associated with periodontal disease were quantified in subgingival biofilm and protein levels for monocyte chemoattractant protein (MCP)-1, MCP-3 and interleukin (IL)-1ß) were determined in the GCF. Expression of PgQC on the mRNA and protein levels was assessed in two P. gingivalis strains. RESULTS: PgQC is expressed in P. gingivalis strains and the protein seems to be located mainly in peri-plasmatic space. mRNA expression of QC was significantly increased in the peripheral blood from RA patients vs. healthy subjects and CP patients (p = 0.013 and p = 0.003, respectively). In GCF of RA patients, QC mRNA was detected more frequently than in healthy controls (p = 0.043). In these samples IL-1ß levels were also elevated compared to GCF from periodontally healthy individuals (p = 0.003). PgQC was detected in eight out of the 13 P. gingivalis positive biofilm samples. CONCLUSION: Activity of QC may play a supportive role in maintaining chronic periodontal inflammation and destruction in RA. PgQC is expressed in vivo but further research is needed to evaluate biological importance of this enzyme and if it constitutes a potential target in periodontal antimicrobial therapy.
Assuntos
Aminoaciltransferases/metabolismo , Artrite Reumatoide/metabolismo , Periodontite Crônica/metabolismo , Adulto , Idoso , Western Blotting , Quimiocina CCL2/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Líquido do Sulco Gengival/química , Humanos , Interleucina-1beta/metabolismo , Masculino , Pessoa de Meia-Idade , Periodonto/metabolismo , Projetos Piloto , Porphyromonas gingivalis/enzimologia , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The formation of amyloid-ß (Aß) peptides is causally involved in the development of Alzheimer's disease (AD). A significant proportion of deposited Aß is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-Aß). These forms show enhanced neurotoxicity compared to full-length Aß. Although the truncation may occur by aminopeptidases after formation of Aß, recently discovered processing pathways of amyloid-ß protein precursor (AßPP) by proteases such as meprin ß may also be involved. Here, we assessed a role of meprin ß in forming Aß3-40/42, which is the precursor of pGlu-Aß3-40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin ß mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin ß in neurons and astrocytes in the vicinity of pGlu-Aß containing deposits. Cleavage of AßPP-derived peptides by meprin ß in vitro results in peptides Aß1-x, Aß2-x, and Aß3-x. The formation of N-truncated Aß by meprin ß was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-Aß3-40 by an addition of glutaminyl cyclase, supporting the preceding formation of Aß3-40. Further analysis of the meprin ß cleavage revealed a yet unknown dipeptidyl-peptidase-like activity specific for the N-terminus of Aß1-x. Thus, our data suggest that meprin ß contributes to the formation of N-truncated Aß by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-Aß formation.
Assuntos
Aminoaciltransferases/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Metaloendopeptidases/metabolismo , Fragmentos de Peptídeos/metabolismo , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Aminoaciltransferases/genética , Peptídeos beta-Amiloides/genética , Animais , Encéfalo/patologia , Células CHO , Cricetinae , Cricetulus , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Ativação Enzimática/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Metaloendopeptidases/genética , Fragmentos de Peptídeos/genéticaRESUMO
Metalloproteinases of the astacin family are drawing ever increasing attention as potential drug targets. However, knowledge regarding inhibitors thereof is limited in most cases. Crucial for the development of metalloprotease inhibitors is high selectivity, to avoid side effects brought about by inhibition of off-target proteases and interference with physiological pathways. In this study we aimed at the design of novel selective inhibitors for the astacin proteinase meprinâ α. Based on a recently identified tertiary amine scaffold, a series of compounds was synthesized and evaluated. The compounds exhibit reasonable inhibitory activity with high selectivity over other metalloproteases. The isoenzyme meprinâ ß is only slightly inhibited. Hence, the present study revealed a novel class of selective meprinâ α inhibitors with improved selectivity over known compounds.
Assuntos
Ácidos Hidroxâmicos/química , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/química , Domínio Catalítico , Desenho de Fármacos , Ensaios Enzimáticos , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteases/síntese química , Inibidores de Proteases/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The metalloproteinase meprin ß emerged as a current drug target for the treatment of a number of disorders, among those fibrosis, inflammatory bowel disease and Morbus Alzheimer. A major obstacle in the development of metalloprotease inhibitors is target selectivity to avoid side effects by blocking related enzymes with physiological functions. Here, we describe the structure-guided design of a novel series of compounds, based on previously reported highly active meprin ß inhibitors. The bioisosteric replacement of the sulfonamide scaffold gave rise to a next generation of meprin inhibitors. Selected compounds based on this novel amine scaffold exhibit high activity against meprin ß and also remarkable selectivity over related metalloproteases, i.e., matrix metalloproteases and A disintegrin and metalloproteinases.
Assuntos
Cristalografia por Raios X , Desenho de Fármacos , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Metaloproteinases de Matriz/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Domínio Catalítico , Sobrevivência Celular , Células Hep G2 , Humanos , Metaloendopeptidases/metabolismo , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Colicins are natural non-antibiotic bacterial proteins with a narrow spectrum but an extremely high antibacterial activity. These proteins are promising food additives for the control of major pathogenic Shiga toxin-producing E. coli serovars in meats and produce. In the USA, colicins produced in edible plants such as spinach and leafy beets have already been accepted by the U. S. Food and Drug Administration (FDA) and U. S. Department of Agriculture (USDA) as food-processing antibacterials through the GRAS (generally recognized as safe) regulatory review process. Nicotiana benthamiana, a wild relative of tobacco, N. tabacum, has become the preferred production host plant for manufacturing recombinant proteins-including biopharmaceuticals, vaccines, and biomaterials-but the purification procedures that have been employed thus far are highly complex and costly. We describe a simple and inexpensive purification method based on specific acidic extraction followed by one chromatography step. The method provides for a high recovery yield of purified colicins, as well as a drastic reduction of nicotine to levels that could enable the final products to be used on food. The described purification method allows production of the colicin products at a commercially viable cost of goods and might be broadly applicable to other cost-sensitive proteins.
Assuntos
Antibacterianos/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Colicinas/isolamento & purificação , Aditivos Alimentares/isolamento & purificação , Carne/microbiologia , Nicotiana/genética , Sequência de Aminoácidos , Animais , Antibacterianos/química , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/farmacologia , Bovinos , Colicinas/biossíntese , Colicinas/química , Colicinas/farmacologia , Aditivos Alimentares/química , Aditivos Alimentares/metabolismo , Aditivos Alimentares/farmacologia , Testes de Sensibilidade Microbiana , Nicotina/antagonistas & inibidores , Nicotina/biossíntese , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/crescimento & desenvolvimento , Nicotiana/química , Nicotiana/metabolismoRESUMO
Structural optimization of 3-hydroxy-N'-arylidenepropanehydrazonamides provided new analogs with nanomolar to subnanomolar antiplasmodial activity against asexual blood stages of Plasmodium falciparum, excellent parasite selectivity, and nanomolar activity against the earliest forms of gametocyte development. Particularly, derivatives with a 1,3-dihalo-6-trifluoromethylphenanthrene moiety showed outstanding in vivo properties and demonstrated in part curative activity in the Plasmodium berghei mouse model when administered perorally.
Assuntos
Amidas/química , Antimaláricos/química , Hidrazonas/química , Malária/tratamento farmacológico , Fenantrenos/química , Plasmodium berghei/efeitos dos fármacos , Amidas/síntese química , Amidas/farmacologia , Animais , Antimaláricos/síntese química , Antimaláricos/farmacologia , Células Hep G2 , Humanos , Hidrazonas/síntese química , Hidrazonas/farmacologia , Malária/parasitologia , Camundongos , Fenantrenos/síntese química , Fenantrenos/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
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.