RESUMO
Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF) and p300 (1 to >50 micromol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 micromol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2-48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of alpha-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition.
Assuntos
Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Histona Acetiltransferases/antagonistas & inibidores , Tiazóis/farmacologia , Fatores de Transcrição/antagonistas & inibidores , Acetilação/efeitos dos fármacos , Animais , Antineoplásicos/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Células HCT116 , Células HT29 , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Relação Estrutura-Atividade , Tiazóis/química , Fatores de Transcrição/metabolismo , Fatores de Transcrição de p300-CBPAssuntos
Regulação Enzimológica da Expressão Gênica , Fosfolipases Tipo C/biossíntese , Fosfolipases Tipo C/genética , Animais , Inibidores Enzimáticos/farmacologia , Humanos , Hidrólise , Modelos Biológicos , Modelos Químicos , Fosfolipase C gama , Transdução de Sinais , Fatores de Tempo , Fosfolipases Tipo C/químicaRESUMO
Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca(2+) release from inositol 1,4,5-triphosphate-sensitive stores. U73122 (1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) has been extensively used as a pharmacological inhibitor of PLC to elucidate the importance of this enzyme family in signal transduction pathways. U73122 has an electrophilic maleimide group, which readily reacts with nucleophiles such as thiols and amines. In the current study the conjugation of U73122 to common components of cell culture medium, namely l-glutamine, glutathione, and bovine serum albumin (BSA), was demonstrated. The half-life of U73122 on incubation with phosphate-buffered saline (PBS), Hanks' buffered saline solution (with 2 mM glutamine), optimized basal nutrient medium (MCDB131, without BSA), complete medium, Dulbecco's modified Eagle's medium (with 2 mM l-glutamine) was approximately 150, 60, 32, 30, and 18 min, respectively. However, U73122 was not recoverable from medium supplemented with 0.5% BSA. U73122 underwent hydrolysis of the maleimide group when incubated with PBS. Glutamine conjugates of U73122 were identified in cell culture medium. Furthermore, the inhibition of epidermal growth factor-stimulated Ca(2+) release in a human epidermoid carcinoma cell line (A431) by U73122 was substantially reduced by the presence of BSA in a time-dependent manner. In complex cellular assays, the availability of U73122 to inhibit PLC may be limited by its chemical reactivity and lead to the misinterpretation of results in pharmacological assays.
Assuntos
Artefatos , Bioensaio , Sinalização do Cálcio/efeitos dos fármacos , Meios de Cultura/química , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Pirrolidinonas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Linhagem Celular Tumoral , Meios de Cultura/metabolismo , Estabilidade de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Estrenos/química , Estrenos/metabolismo , Glutamina/química , Glutationa/química , Meia-Vida , Humanos , Ligação Proteica , Pirrolidinonas/química , Pirrolidinonas/metabolismo , Soroalbumina Bovina/química , Fatores de TempoRESUMO
The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.