RESUMO
Natural products represent compound classes with high chemical and structural diversity and various biological activities. Libraries based on natural products are valuable starting point in the search for novel biologically active substances. Here we report on the identification of the natural product podoverine A from the plant Podophyllum versipelle Hance as a novel tubulin-acting agent. A natural product compound collection was subjected to a high-content screen that monitors changes in cytoskeleton and DNA and podoverine A was identified as inhibitor of mitosis. This natural product causes mitotic arrest and inhibits microtubule polymerization in vitro and in cells by targeting the vinca binding site on tubulin.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Flavonas/farmacologia , Microtúbulos/efeitos dos fármacos , Podophyllum/química , Moduladores de Tubulina/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Flavonas/química , Flavonas/isolamento & purificação , Células HeLa , Humanos , Células MCF-7 , Microtúbulos/metabolismo , Estrutura Molecular , Polimerização/efeitos dos fármacos , Relação Estrutura-Atividade , Moduladores de Tubulina/química , Moduladores de Tubulina/isolamento & purificaçãoRESUMO
Chemical modulators are powerful tools to investigate biological processes. To identify circadian clock effectors, we screened a natural product library in the model plant Arabidopsis thaliana. Two compounds, prieurianin (Pri) and prieurianin acetate, were identified as causing a shorter circadian period. Recently, Pri was independently identified as a vesicle trafficking inhibitor and re-named endosidin 1 (ES1). Here we show that Pri primarily affects actin filament flexibility in vivo, later resulting in reduced severing and filament depolymerization. This stabilization of the actin cytoskeleton subsequently causes changes in vesicle trafficking. Pri also affected microfilaments in mammalian cells, indicating that its target is highly conserved; however, it did not alter actin dynamics in vitro, suggesting that its activity requires the presence of actin-associated proteins. Furthermore, well-characterized actin inhibitors shortened the period length of the Arabidopsis clock in a similar way to Pri, supporting the idea that Pri affects rhythms by altering the actin network. We conclude that actin-associated processes influence the circadian system in a light-dependent manner, but their disruption does not abolish rhythmicity. In summary, we propose that the primary effect of Pri is to stabilize the actin cytoskeleton system, thereby affecting endosome trafficking. Pri appears to stabilize actin filaments by a different mechanism from previously described inhibitors, and will be a useful tool to study actin-related cellular processes.
Assuntos
Citoesqueleto de Actina/efeitos dos fármacos , Actinas/efeitos dos fármacos , Arabidopsis/efeitos dos fármacos , Relógios Circadianos/efeitos dos fármacos , Limoninas/farmacologia , Vesículas Transportadoras/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Arabidopsis/ultraestrutura , Linhagem Celular , Chlorocebus aethiops , Testes Genéticos , Hipocótilo/efeitos dos fármacos , Hipocótilo/metabolismo , Hipocótilo/efeitos da radiação , Hipocótilo/ultraestrutura , Luz , Limoninas/química , Limoninas/isolamento & purificação , Microscopia Confocal , Microscopia Eletrônica de Varredura , Plantas Geneticamente Modificadas , Plântula/química , Plântula/efeitos dos fármacos , Plântula/metabolismo , Plântula/efeitos da radiação , Plântula/ultraestrutura , Bibliotecas de Moléculas Pequenas , Fatores de Tempo , Vesículas Transportadoras/metabolismoRESUMO
A Prins cyclization between a polymer-bound aldehyde and a homoallylic alcohol served as the key step in the synthesis of tetrahydropyran derivatives. A phenotypic screen led to the identification of compounds that inhibit mitosis (as seen by the accumulation of round cells with condensed DNA and membrane blebs). These compounds were termed tubulexins as they target the CSE1L protein and the vinca alkaloid binding site of tubulin.
Assuntos
Proteína de Suscetibilidade a Apoptose Celular/metabolismo , Piranos/síntese química , Piranos/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Sítios de Ligação , Proteína de Suscetibilidade a Apoptose Celular/química , Células HeLa , Humanos , Células MCF-7 , Mitose/efeitos dos fármacos , Moduladores de Mitose , Piranos/química , Tubulina (Proteína)/química , Moduladores de Tubulina/síntese química , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologiaRESUMO
A matter of common sense: a common recognition motif consisting of a negatively charged group five to six bonds away (red) from the (thio)ester functionality (green) and a positively charged tail group ten to twelve bonds away (blue) was identified in two native acyl protein thioesterase 1 (APT1) substrates. This similarity led to the design of potent inhibitors of the Ras-depalmitoylating enzyme APT1.
Assuntos
Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Lactonas/farmacologia , Tioléster Hidrolases/antagonistas & inibidores , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Animais , Linhagem Celular , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Lactonas/síntese química , Lactonas/química , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-Atividade , Especificidade por Substrato , Tioléster Hidrolases/metabolismoRESUMO
In biology-oriented synthesis the underlying scaffold classes of natural products selected in evolution are used to define biologically relevant starting points in chemical structure space for the synthesis of compound collections with focused structural diversity. Here we describe a highly enantioselective synthesis of natural-product-inspired 3,3'-pyrrolidinyl spirooxindoles--which contain an all-carbon quaternary centre and three tertiary stereocentres. This synthesis takes place by means of an asymmetric Lewis acid-catalysed 1,3-dipolar cycloaddition of an azomethine ylide to a substituted 3-methylene-2-oxindole using 1-3 mol% of a chiral catalyst formed from a N,P-ferrocenyl ligand and CuPF(6)(CH(3)CN)(4). Cellular evaluation has identified a molecule that arrests mitosis, induces multiple microtubule organizing centres and multipolar spindles, causes chromosome congression defects during mitosis and inhibits tubulin regrowth in cells. Our findings support the concept that compound collections based on natural-product-inspired scaffolds constructed with complex stereochemistry will be a rich source of compounds with diverse bioactivity.
Assuntos
Indóis/síntese química , Pirrolidinas/síntese química , Compostos de Espiro/síntese química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Indóis/farmacologia , Microtúbulos/efeitos dos fármacos , Pirrolidinas/farmacologia , Compostos de Espiro/farmacologia , EstereoisomerismoRESUMO
Most plasmalemmal proteins organize in submicrometer-sized clusters whose architecture and dynamics are still enigmatic. With syntaxin 1 as an example, we applied a combination of far-field optical nanoscopy, biochemistry, fluorescence recovery after photobleaching (FRAP) analysis, and simulations to show that clustering can be explained by self-organization based on simple physical principles. On average, the syntaxin clusters exhibit a diameter of 50 to 60 nanometers and contain 75 densely crowded syntaxins that dynamically exchange with freely diffusing molecules. Self-association depends on weak homophilic protein-protein interactions. Simulations suggest that clustering immobilizes and conformationally constrains the molecules. Moreover, a balance between self-association and crowding-induced steric repulsions is sufficient to explain both the size and dynamics of syntaxin clusters and likely of many oligomerizing membrane proteins that form supramolecular structures.