RESUMO
TM9 family proteins (also named Phg1 proteins) have been previously shown to control cell adhesion by determining the cell surface localization of adhesion proteins such as the Dictyostelium SibA protein. Here, we show that the glycine-rich transmembrane domain (TMD) of SibA is sufficient to confer Phg1A-dependent surface targeting to a reporter protein. Accordingly, in Dictyostelium phg1A-knockout (KO) cells, proteins with glycine-rich TMDs were less efficiently transported out of the endoplasmic reticulum (ER) and to the cell surface. Phg1A, as well as its human ortholog TM9SF4 specifically associated with glycine-rich TMDs. In human cells, genetic inactivation of TM9SF4 resulted in an increased retention of glycine-rich TMDs in the endoplasmic reticulum, whereas TM9SF4 overexpression enhanced their surface localization. The bulk of the TM9SF4 protein was localized in the Golgi complex and a proximity-ligation assay suggested that it might interact with glycine-rich TMDs. Taken together, these results suggest that one of the main roles of TM9 proteins is to serve as intramembrane cargo receptors controlling exocytosis and surface localization of a subset of membrane proteins.
Assuntos
Membrana Celular/metabolismo , Glicina/metabolismo , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Animais , Dictyostelium , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Transporte ProteicoRESUMO
Legionella pneumophila is a facultative intracellular bacterium, which upon inhalation can cause a potentially fatal pneumonia termed Legionnaires' disease. The opportunistic pathogen grows in environmental amoebae and mammalian macrophages within a unique membrane-bound compartment, the 'Legionella-containing vacuole'. Bacteria are exposed to many environmental cues including small signalling molecules from eukaryotic cells. A number of pathogenic bacteria sense and respond to catecholamine hormones, such as adrenalin and noradrenalin, a process mediated via the QseBC two-component system in some bacteria. In this study, we examined the effect of adrenergic compounds on L. pneumophila, and discovered that the adrenergic receptor antagonists benoxathian, naftopidil, propranolol and labetalol, as well as the QseC sensor kinase inhibitor LED209, reduced the growth of L. pneumophila in broth or amoebae, while replication in macrophages was enhanced. Growth restriction was common to members of the genus Legionella and Mycobacterium, and was observed for L. pneumophila in the replicative but not stationary phase of the biphasic life cycle. Deletion of the L. pneumophila qseBC genes indicated that growth inhibition by adrenergics or LED209 is mediated only to a minor extent by this two-component system, implying the presence of other adrenergic sensing systems. This study identifies adrenergic molecules as novel inhibitors of extra- and intracellular growth of Legionella and reveals LED209 as a potential lead compound to combat infections with Legionella or Mycobacterium spp.
Assuntos
Antagonistas Adrenérgicos/metabolismo , Antibacterianos/metabolismo , Legionella pneumophila/efeitos dos fármacos , Legionella pneumophila/crescimento & desenvolvimento , Acanthamoeba castellanii/efeitos dos fármacos , Acanthamoeba castellanii/microbiologia , Animais , Linhagem Celular , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Mycobacterium/efeitos dos fármacos , Mycobacterium/crescimento & desenvolvimentoRESUMO
Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 µM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined.
Assuntos
Dictyostelium , Infecções por Klebsiella , Humanos , Klebsiella pneumoniae/genética , Dictyostelium/microbiologia , Fagócitos , Antibacterianos , Infecções por Klebsiella/microbiologiaRESUMO
Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.
Assuntos
Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Quimiotaxia/efeitos dos fármacos , Glucocorticoides , Humanos , Concentração Inibidora 50 , Fatores Inibidores da Migração de Macrófagos/genética , Mutação , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
Macrophage migration inhibitory factor (MIF) is a major proinflammatory cytokine that has been increasingly implicated in the pathogenesis of several inflammatory, autoimmune, infectious and oncogenic diseases. Accumulating evidence suggests that the tautomerase activity of MIF plays a role in modulating some of its intra- and extra-cellular activities. Therefore, the identification and development of small-molecule inhibitors targeting the catalytic activity of MIF has emerged as an attractive and viable therapeutic strategy to attenuate its function in health and disease. Herein we report a novel virtual screening protocol for the discovery of new inhibitors of MIF's tautomerase activity. Our protocol takes into account the flexibility and dynamics of the catalytic site by coupling molecular dynamics (MD) simulations aimed at modeling the protein's flexibility in solution to (i) docking with FlexX, or (ii) docking with FlexX and pharmacophoric filtering with Unity. In addition, we applied in parallel a standalone docking using the new version of Surflex software. The three approaches were used to screen the ChemBridge chemical library and the inhibitory activity of the top-ranked 333 compound obtained from each approach (1000 compound in total) was assessed in vitro using the tautomerase assay. This biochemical validation process resulted in the identification of 12 novel MIF inhibitors corresponding to a 1.2% hit rate. Six of these hits came from Surflex docking; two from FlexX docking with MD simulations and four hits were identified with MDS and pharmacophore filtering with minimal overlap between the hits from each approach. Six hits were identified with IC50 values lower than 10 microM (three hits with IC50 lower than 1 microM); four were shown to be suicide inhibitors and act via covalent modification of the N-terminal catalytic residues Pro1. One additional inhibitor, N-phenyl-N-1,3,4-thiadiazol-2-yl-thiourea, (IC50=300 nM) was obtained from FlexX docking combined to pharmacophoric filtering on one of the eight MD structures. These results demonstrate the power of integrative in silico approaches in the discovery of new modulator of MIF's tautomerase activity. The chemical diversity and mode of action of these compounds suggest that they could be used as molecular probes to elucidate the functions and biology of MIF and as lead candidates in drug developments of anti-MIF drugs.
Assuntos
Desenho de Fármacos , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Ligantes , Fatores Inibidores da Migração de Macrófagos/química , Simulação de Dinâmica MolecularRESUMO
Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.
Assuntos
Isotiocianatos/farmacologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/química , Doenças Autoimunes/fisiopatologia , Cromatografia em Gel , Citocinas/química , Glucocorticoides/farmacologia , Humanos , Inflamação/fisiopatologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/isolamento & purificação , Modelos Moleculares , Fosforilação , Conformação Proteica , Espalhamento de Radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The roles of NK cells in human melanoma remain only partially understood. We characterized NK cells from peripheral blood ex vivo by flow cytometry obtained from late stage (III/IV) melanoma patients. Interestingly, we found that the abundance of CD56bright NK cells negatively correlate with overall patient survival, together with distant metastases, in a multivariate cox regression analysis. The patients' CD56bright NK cells showed upregulation of CD11a, CD38 and CD95 as compared to healthy controls, pointing to an activated phenotype as well as a possible immune regulatory role in melanoma patients. After stimulation in vitro, CD56bright NK cells produced less TNFα and GMCSF in patients than controls. Furthermore, IFNγ production by the CD56bright NK cells correlated inversely with overall survival. Our results highlight that abundance and function of CD56bright NK cells are associated with melanoma patient survival, emphasizing the potential of NK cell subsets for biomarker discovery and future therapeutic targeting.
Assuntos
Biomarcadores Tumorais/sangue , Antígeno CD56/sangue , Células Matadoras Naturais/imunologia , Melanoma/mortalidade , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interferon gama/metabolismo , Masculino , Melanoma/imunologia , Melanoma/patologia , Metástase Neoplásica , Estadiamento de Neoplasias , Fenótipo , Análise de Regressão , Análise de SobrevidaRESUMO
Macrophage migration inhibitory factor (MIF) is a multifunctional protein and a major mediator of innate immunity. Although X-ray crystallography revealed that MIF exists as a homotrimer, its oligomerization state in vivo and the factors governing its oligomerization and stability remain poorly understood. The C-terminal region of MIF is highly conserved and participates in several intramolecular interactions that suggest a role in modulating the stability and biochemical activity of MIF. To determine the importance of these interactions, point mutations (A48P, L46A), insertions (P107) at the monomer-monomer interfaces, and C-terminal deletion (Delta 110-114NSTFA and Delta 105-114NVGWNNSTFA) variants were designed and their structural properties, thermodynamic stability, oligomerization state, catalytic activity and receptor binding were characterized using a battery of biophysical methods. The C-terminal deletion mutants DeltaC5 huMIF 1-109 and DeltaC10 huMIF 1-104 were enzymatically inactive and thermodynamically less stable than wild type MIF. Analytical ultracentrifugation studies demonstrate that both C-terminal mutants sediment as trimers and exhibit similar binding to CD74 as the wild type protein. Disrupting the conformation of the C-terminal region 105-114 and increasing its conformational flexibility through the insertion of a proline residue at position 107 was sufficient to reproduce the structural, biochemical and thermodynamic properties of the deletion mutants. P107 MIF forms an enzymatically inactive trimer and exhibits reduced thermodynamic stability relative to the wild type protein. To provide a rationale for the changes induced by these mutations at the molecular level, we also performed molecular dynamics simulations on these mutants in comparison to the wild type MIF. Together, our studies demonstrate that intersubunit interactions involving the C-terminal region 105-114, including a salt-bridge interaction between Arg73 of one monomer and the carboxy terminus of a neighboring monomer, play critical roles in modulating tertiary structure stabilization, enzymatic activity, and thermodynamic stability of MIF, but not its oligomerization state and receptor binding properties. Our results suggest that targeting the C-terminal region could provide new strategies for allosteric modulation of MIF enzymatic activity and the development of novel inhibitors of MIF tautomerase activity.
Assuntos
Fatores Inibidores da Migração de Macrófagos/química , Fatores Inibidores da Migração de Macrófagos/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Fatores Inibidores da Migração de Macrófagos/genética , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Secundária de Proteína , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , UltracentrifugaçãoRESUMO
Tuberculosis remains one of the major threats to public health worldwide. Given the prevalence of multi drug resistance (MDR) in Mycobacterium tuberculosis strains, there is a strong need to develop new anti-mycobacterial drugs with modes of action distinct from classical antibiotics. Inhibitors of mycobacterial virulence might target new molecular processes and may represent a potential new therapeutic alternative. In this study, we used a Dictyostelium discoideum host model to assess virulence of Mycobacterium marinum and to identify compounds inhibiting mycobacterial virulence. Among 9995 chemical compounds, we selected 12 inhibitors of mycobacterial virulence that do not inhibit mycobacterial growth in synthetic medium. Further analyses revealed that 8 of them perturbed functions requiring an intact mycobacterial cell wall such as sliding motility, bacterial aggregation or cell wall permeability. Chemical analogs of two compounds were analyzed. Chemical modifications altered concomitantly their effect on sliding motility and on mycobacterial virulence, suggesting that the alteration of the mycobacterial cell wall caused the loss of virulence. We characterized further one of the selected compounds and found that it inhibited the ability of mycobacteria to replicate in infected cells. Together these results identify new antimycobacterial compounds that represent new tools to unravel the molecular mechanisms controlling mycobacterial pathogenicity. The isolation of compounds with anti-virulence activity is the first step towards developing new antibacterial treatments.
Assuntos
Dictyostelium/microbiologia , Mycobacterium marinum/efeitos dos fármacos , Virulência/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Mycobacterium marinum/patogenicidade , Mycobacterium marinum/fisiologia , Mycobacterium marinum/ultraestrutura , Bibliotecas de Moléculas PequenasRESUMO
Here, we report on the design, synthesis, and biological evaluation of 4-thiazolidinone (rhodanine) derivatives targeting Mycobacterial tuberculosis (Mtb) trans-2-enoyl-acyl carrier protein reductase (InhA). Compounds having bulky aromatic substituents at position 5 and a tryptophan residue at position N-3 of the rhodanine ring were the most active against InhA, with IC50 values ranging from 2.7 to 30 µM. The experimental data showed consistent correlations with computational studies. Their antimicrobial activity was assessed against Mycobacterium marinum (Mm) (a model for Mtb), Pseudomonas aeruginosa (Pa), Legionella pneumophila (Lp), and Enterococcus faecalis (Ef) by using anti-infective, antivirulence, and antibiotic assays. Nineteen out of 34 compounds reduced Mm virulence at 10 µM. 33 exhibited promising antibiotic activity against Mm with a MIC of 0.21 µM and showed up to 89% reduction of Lp growth in an anti-infective assay at 30 µM. 32 showed high antibiotic activity against Ef, with a MIC of 0.57 µM.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Oxirredutases/antagonistas & inibidores , Rodanina/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Enterococcus faecalis/efeitos dos fármacos , Legionella pneumophila/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Mycobacterium marinum/efeitos dos fármacos , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Rodanina/síntese química , Rodanina/química , Relação Estrutura-AtividadeRESUMO
The causative agent of Legionnaires' disease, Legionella pneumophila, grows in environmental amoebae and mammalian macrophages within a distinct compartment, the 'Legionella-containing vacuole' (LCV). Intracellular bacteria are protected from many antibiotics, and thus are notoriously difficult to eradicate. To identify novel compounds that restrict intracellular bacterial replication, we previously developed an assay based on a coculture of amoebae and GFP-producing L. pneumophila. This assay was used to screen a pathway-based, highly diverse chemical library, referred to as the Sinergia library. In this work, we chose to focus on a group of 11 hit compounds, the majority of which originated from the query molecule CN585, a compound that targets the protein phosphatase calcineurin. Further studies on 78 related compound variants revealed crucial structural attributes, namely a triple-ring scaffold with a central triazine moiety, substituted in positions 3 and 5 by two piperidine or pyrrolidine rings, and in position 1 by an amine group bearing a single aliphatic chain moiety. The most effective compound, ZINC00615682, inhibited intracellular replication of L. pneumophila with an IC50 of approximately 20 nM in Acanthamoeba castellanii and slightly less efficiently in Dictyostelium discoideum or macrophages. Pharmacological and genetic attempts to implicate calcineurin in the intracellular replication of L. pneumophila failed. Taken together, these results show that the amoebae-based screen and structure-activity relationship analysis is suitable for the identification of novel inhibitors of the intracellular replication of L. pneumophila. The most potent compound identified in this study targets (an) as yet unidentified host factor(s).
RESUMO
In Pseudomonas aeruginosa, the production of many secreted virulence factors is controlled by a quorum-sensing (QS) circuit, constituted of transcriptional activators (LasR, RhlR, PqsR) and their cognate signaling molecules (3-oxo-C12-HSL, C4-HSL, PQS). QS is a cooperative behavior that is beneficial to a population but can be exploited by "QS-cheaters", individuals which do not respond to the QS-signal, but can use public goods produced by QS-cooperators. In order to identify QS-deficient clones we designed a genetic screening based on a lasB-lacZ fusion. We isolated one clone (PT1617) deficient in QS-dependent gene expression and virulence factor production despite wild type lasR, rhlR and pqsR alleles. Whole genome sequencing of PT1617 revealed a 3,552 bp deletion encompassing ORFs PA2228-PA2229-PA2230 and the pslA gene. However, complementation of PT1617 by plasmid-encoded copies of these ORFs, did not restore QS. Unexpectedly, gene expression levels of ORFs PA2228, PA2227 (vqsM) and PA2222, located adjacent to the deletion, were 10 to 100 fold higher in mutant PT1617 than in PAO1. When expressed from a constitutive promoter on a plasmid, PA2226, alone was found to be sufficient to confer a QS-negative phenotype on PAO1 as well as on PA14. Co-expression of PA2226 and PA2225 in PAO1 further prevented induction of the type III secretion system. In summary, we have identified a novel genetic locus including ORF2226 termed qsrO (QS-repressing ORF), capable of down-regulating all three known QS-systems in P. aeruginosa.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Fatores de Virulência/genética , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/genética , Sequência de Bases , Teste de Complementação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Dados de Sequência Molecular , Fases de Leitura Aberta , Plasmídeos , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Deleção de Sequência , Transdução de Sinais , Virulência , Fatores de Virulência/metabolismoRESUMO
Tuberculosis is considered to be one of the world's deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches.
Assuntos
Acanthamoeba castellanii/microbiologia , Antibacterianos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Mycobacterium marinum/fisiologia , Acanthamoeba castellanii/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , FluorescênciaRESUMO
Legionella pneumophila is a ubiquitous fresh-water bacterium which reproduces within its erstwhile predators, environmental amoeba, by subverting the normal pathway of phagocytosis and degradation. The molecular mechanisms which confer resistance to amoeba are apparently conserved and also allow replication within macrophages. Thus, L. pneumophila can act as an 'accidental' human pathogen and cause a severe pneumonia known as Legionnaires' disease. The intracellular localisation of L. pneumophila protects it from some antibiotics, and this fact must be taken into account to develop new anti-bacterial compounds. In addition, the intracellular lifestyle of L. pneumophila may render the bacteria susceptible to compounds diminishing bacterial virulence and decreasing intracellular survival and replication of this pathogen. The development of a single infection cycle intracellular replication assay using GFP-producing L. pneumophila and Acanthamoebacastellanii amoeba is reported here. This fluorescence-based assay allows for continuous monitoring of intracellular replication rates, revealing the effect of bacterial gene deletions or drug treatment. To examine how perturbations of the host cell affect L. pneumophila replication, several known host-targeting compounds were tested, including modulators of cytoskeletal dynamics, vesicle scission and Ras GTPase localisation. Our results reveal a hitherto unrealized potential antibiotic property of the ß-lactone-based Ras depalmitoylation inhibitor palmostatin M, but not the closely related inhibitor palmostatin B. Further characterisation indicated that this compound caused specific growth inhibition of Legionella and Mycobacterium species, suggesting that it may act on a common bacterial target.
Assuntos
Antibacterianos/farmacologia , Espaço Intracelular/microbiologia , Legionella pneumophila/efeitos dos fármacos , Animais , Linhagem Celular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Concentração Inibidora 50 , Espaço Intracelular/efeitos dos fármacos , Legionella pneumophila/crescimento & desenvolvimento , Camundongos , Testes de Sensibilidade Microbiana , Peso Molecular , Mycobacterium/efeitos dos fármacos , Mycobacterium/crescimento & desenvolvimento , Propiolactona/análogos & derivados , Propiolactona/farmacologia , Especificidade da Espécie , Sulfonas/farmacologiaRESUMO
Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the ß-strand ß3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF's trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state.
Assuntos
Interações Hidrofóbicas e Hidrofílicas , Oxirredutases Intramoleculares/química , Leucina/química , Fatores Inibidores da Migração de Macrófagos/química , Sequência de Aminoácidos , Sequência Conservada , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Proteínas Mutantes/química , Mutação/genética , Ligação Proteica/genética , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Macrophage migration inhibitory factor (MIF) is a major mediator of innate immunity and inflammation and presents a potential therapeutic target for various inflammatory, infectious, and autoimmune diseases, including cancer. Although a number of inhibitors have been identified and designed based on the modification of known nonphysiological substrates, the lack of a suitable high-throughput assay has hindered the screening of chemical libraries and the discovery of more diverse inhibitors. Herein the authors report the development and optimization of a robust high-throughput kinetic-based activity assay for the identification of new MIF inhibitors. Using this assay, they screened 80,000 small molecules and identified and validated 13 novel inhibitors of MIF catalytic activity. These small molecules demonstrated inhibition constant (K(i,app)) values ranging from 0.5 to 13 microM.