RESUMO
Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.
Assuntos
Catepsinas/antagonistas & inibidores , Inibidores de Cisteína Proteinase/síntese química , Cisteína/química , Compostos de Epóxi/síntese química , Sulfonas/síntese química , Catepsinas/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacologia , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Humanos , Cinética , Simulação de Acoplamento Molecular , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologia , TermodinâmicaRESUMO
Protein kinase C (PKC) engenders motility through phosphorylation of α-tubulin at Ser-165 in nontransformed MCF-10A cells. Live cell imaging explored the impact of PKC-mediated phosphorylation on microtubule (MT) dynamics. MTs fluorescently labeled with GFP-α-tubulin were treated with diacylglycerol (DAG)-lactone (a membrane-permeable PKC activator), or cotransfected with a pseudophosphorylated S165D-α6-tubulin mutant. Each condition increased the dynamicity of MTs by stimulating the rate and duration of the growth phase and decreasing the frequency of catastrophe. In MDA-MB-231 metastatic breast cells where the intrinsic PKC activity is high, these MT growth parameters were also high but could be suppressed by expression of phosphorylation-resistant S165N-α6-tubulin or by treatment with a pan-PKC inhibitor (bis-indoleylmaleimide). Subcellular fractionation and immunofluorescence of MCF-10A cells showed that phosphorylation (via DAG-lactone) or pseudophosphorylation of α6-tubulin increased its partitioning into MTs as compared to controls, and produced longer, more stable MTs. Following expression of the plus-end binding protein GFP-EB1, DAG-lactone accelerated the formation and increased the number of nascent MTs. Expression of S165D-α6-tubulin promoted Rac1 activation and Rac1-dependent cell motility. These findings call attention to PKC-mediated phosphorylation of α-tubulin as a novel mechanism for controlling the dynamics of MTs that result in cell movement.
Assuntos
Mama/citologia , Microtúbulos/metabolismo , Proteína Quinase C/metabolismo , Tubulina (Proteína)/metabolismo , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Imunofluorescência , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/efeitos dos fármacos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Metástase Neoplásica , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Valeratos/farmacologia , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
A hybrid-design approach is undertaken to develop a highly potent and selective inhibitor of human cathepsin L. Studies involving human breast carcinoma MDA-MB-231 cells establish that this inhibitor can successfully block intracellular cathepsin L activity, and retard the cell-migratory potential of these highly metastatic cells.