RESUMO
Traditional herbal medicines, which emphasize a holistic, patient-centric view of disease treatment, provide an exciting starting point for discovery of new immunomodulatory drugs. Progress on identification of herbal molecules with proven single agent activity has been slow, in part because of insufficient consideration of pharmacology fundamentals. Many molecules derived from medicinal plants exhibit low oral bioavailability and rapid clearance, leading to low systemic exposure. Recent research suggests that such molecules can act locally in the gut or liver to activate xenobiotic defense pathways that trigger beneficial systemic effects on the immune system. We discuss this hypothesis in the context of four plant-derived molecules with immunomodulatory activity: indigo, polysaccharides, colchicine, and ginsenosides. We end by proposing research strategies for identification of novel immunomodulatory drugs from herbal medicine sources that are informed by the possibility of local action in the gut or liver, leading to generation of systemic immune mediators.
Assuntos
Descoberta de Drogas/métodos , Imunomodulação , Plantas Medicinais/química , Animais , Colchicina/farmacologia , Ginsenosídeos/farmacologia , Índigo Carmim/farmacologia , Fitoterapia , Polissacarídeos/farmacologia , XenobióticosRESUMO
Colchicine has served as a traditional medicine for millennia and remains widely used to treat inflammatory and other disorders. Colchicine binds tubulin and depolymerizes microtubules, but it remains unclear how this mechanism blocks myeloid cell recruitment to inflamed tissues. Here we show that colchicine inhibits myeloid cell activation via an indirect mechanism involving the release of hepatokines. We find that a safe dose of colchicine depolymerizes microtubules selectively in hepatocytes but not in circulating myeloid cells. Mechanistically, colchicine triggers Nrf2 activation in hepatocytes, leading to secretion of anti-inflammatory hepatokines, including growth differentiation factor 15 (GDF15). Nrf2 and GDF15 are required for the anti-inflammatory action of colchicine in vivo. Plasma from colchicine-treated mice inhibits inflammatory signalling in myeloid cells in a GDF15-dependent manner, by positive regulation of SHP-1 (PTPN6) phosphatase, although the precise molecular identities of colchicine-induced GDF15 and its receptor require further characterization. Our work shows that the efficacy and safety of colchicine depend on its selective action on hepatocytes, and reveals a new axis of liver-myeloid cell communication. Plasma GDF15 levels and myeloid cell SHP-1 activity may be useful pharmacodynamic biomarkers of colchicine action.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Colchicina/farmacologia , Citocinas/fisiologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Células Mieloides/efeitos dos fármacos , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Antioxidantes/farmacologia , Colchicina/farmacocinética , Simulação por Computador , Citocinas/biossíntese , Fator 15 de Diferenciação de Crescimento/genética , Hepatócitos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Peritonite/induzido quimicamente , Peritonite/prevenção & controle , Proteína Tirosina Fosfatase não Receptora Tipo 6/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Useful small molecule tools can be discovered in imaging screens that measure phenotype in single cells or small organisms. Recent examples include identification of small molecule inhibitors of processes such as cell migration, cytokinesis, mitotic spindle length determination, melanogenesis, aggresome formation, membrane transport and nuclear export. Imaging screens are currently limited by challenges in the areas of image analysis and target identification. We discuss the use of model organisms such as zebrafish in screens and review different methods of target identification. The emerging field of automated image analysis is also introduced.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Processamento de Imagem Assistida por Computador , Animais , HumanosRESUMO
Automated fluorescence microscopy provides a powerful tool for analyzing the physiological state of single cells with high throughput and high information content. Here I discuss two types of experiments in which this technology was used to discover and characterize bioactive small molecules. In phenotypic-screening experiments, the goal is to find "hits" with specific effects on cells by screening large libraries of small molecules. An example is screening for small molecules that perturb mitosis by novel mechanisms. In cytological-profiling experiments, the goal is to characterize the bioactivity of a limited number of small molecules in considerable depth, and thus understand their mechanism and toxicities at the cellular level. I discuss an example in which 100 small molecules with known bioactivity were profiled by using multiple fluorescent probes, and clustered into mechanistic classes by automated statistical analysis.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Microscopia de Fluorescência/métodos , Algoritmos , Automação , Células Cultivadas , Relação Dose-Resposta a Droga , HumanosRESUMO
Maintenance of centrosome number is essential for cell-cycle progression and genomic stability, but investigation of this regulation has been limited by assay difficulty. We present a fully automated image-based centrosome-duplication assay that is accurate and robust enough for both careful cell-biology studies and high-throughput screening, and employ this assay in a series of chemical-genetic studies. We observe that a simple cytometric profiling strategy, which is based on organelle size, groups compounds with similar mechanisms of action; this suggests a simple strategy for excluding compounds that undesirably target such activities as protein synthesis and microtubule dynamics. Screening a library of compounds of known activity, we found unexpected effects on centrosome duplication by a number of drugs, most notably isoform-specific protein kinase C inhibitors and retinoic acid receptor agonists. From a 16 320-member library of uncharacterized small molecules, we identified five potent centrosome-duplication inhibitors that do not target microtubule dynamics or protein synthesis. The analysis methodology reported here is directly relevant to studies of centrosome regulation in a variety of systems and is adaptable to a wide range of other biological problems.
Assuntos
Centrossomo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Animais , Bioensaio/métodos , Células Cultivadas , Centrossomo/metabolismo , Biologia ComputacionalRESUMO
We present a method for high-throughput cytological profiling by microscopy. Our system provides quantitative multidimensional measures of individual cell states over wide ranges of perturbations. We profile dose-dependent phenotypic effects of drugs in human cell culture with a titration-invariant similarity score (TISS). This method successfully categorized blinded drugs and suggested targets for drugs of uncertain mechanism. Multivariate single-cell analysis is a starting point for identifying relationships among drug effects at a systems level and a step toward phenotypic profiling at the single-cell level. Our methods will be useful for discovering the mechanism and predicting the toxicity of new drugs.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Microscopia de Fluorescência , Farmacologia/métodos , Testes de Toxicidade/métodos , Automação , Ciclo Celular/efeitos dos fármacos , Análise por Conglomerados , DNA/análise , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Células HeLa , Humanos , Processamento de Imagem Assistida por Computador , Fenótipo , Estatística como AssuntoRESUMO
FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target.