ABSTRACT
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.
Subject(s)
Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Collagen Type I/metabolism , Cysteine Proteinase Inhibitors/chemistry , Cysteine Proteinase Inhibitors/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Drug Design , Humans , Kinetics , Protein BindingABSTRACT
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.
Subject(s)
Breast/cytology , Microtubules/metabolism , Protein Kinase C/metabolism , Tubulin/metabolism , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/pharmacology , Cell Line, Transformed , Cell Movement/drug effects , Cell Survival/drug effects , Enzyme Activation/drug effects , Female , Fluorescent Antibody Technique , Green Fluorescent Proteins/metabolism , Humans , Microtubule-Associated Proteins/metabolism , Microtubules/drug effects , Models, Biological , Mutant Proteins/metabolism , Neoplasm Metastasis , Phosphorylation/drug effects , Phosphoserine/metabolism , Recombinant Fusion Proteins/metabolism , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology , Valerates/pharmacology , rac1 GTP-Binding Protein/metabolismABSTRACT
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.
