ABSTRACT
p21Cip1 (p21) is a universal cyclin-dependent kinase (CDK) inhibitor that halts cell proliferation and tumor growth by multiple mechanisms. The expression of p21 is often downregulated in cancer cells as a result of the loss of function of transcriptional activators, such as p53, or the increased degradation rate of the protein. To identify small molecules that block the ubiquitin-mediated degradation of p21 as a future avenue for cancer drug discovery, we have screened a compound library using a cell-based reporter assay of p21 degradation. This led to the identification of a benzodiazepine series of molecules that induce the accumulation of p21 in cells. Using a chemical proteomic strategy, we identified the ubiquitin-conjugating enzyme UBCH10 as a cellular target of this benzodiazepine series. We show that an optimized benzodiazepine analogue inhibits UBCH10 ubiquitin-conjugating activity and substrate proteolysis by the anaphase-promoting complex.
Subject(s)
Benzodiazepines , Ubiquitin-Conjugating Enzymes , Ubiquitin-Conjugating Enzymes/chemistry , Benzodiazepines/pharmacology , Proteomics , Ubiquitin/metabolism , Cell Nucleus/metabolismABSTRACT
First-generation RAF inhibitors paradoxically induce ERK signaling in normal and tumor cells exhibiting RAS activity. Compound-induced RAF dimerization through stabilization of the RAF ON/active state by inhibitors has emerged as a critical contributing factor. RAF inhibitors also enhance RAS-RAF association. Although this event is thought to play a key role in priming RAF activation, the underlying mechanism is not known. Here we report that RAF inhibitors induce the disruption of intramolecular interactions between the kinase domain and its N-terminal regulatory region independently of RAS activity. This provides a molecular basis to explain the induction of RAS-RAF association by RAF inhibitors, as well as the co-operativity observed between RAS activity and RAF kinase inhibitors in driving RAF activation. Profiling of second-generation RAF inhibitors confirmed their improved mode of action, but also revealed liabilities that allowed us to discern two properties of an ideal RAF inhibitor: high-binding affinity to all RAF paralogs and maintenance of the OFF/autoinhibited state of the enzyme.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-raf/antagonists & inhibitors , Allosteric Regulation/drug effects , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/metabolism , HEK293 Cells , Humans , Models, Biological , Mutation/genetics , Phosphorylation/drug effects , Protein Binding/drug effects , Protein Kinase Inhibitors/chemistry , Protein Multimerization , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins p21(ras)/geneticsABSTRACT
The discovery of a novel class of HCV NS5B polymerase inhibitors, 3-arylsulfonylamino-5-phenyl-thiophene-2-carboxylic acids is described. SAR studies have yielded several potent inhibitors of HCV polymerase as well as of HCV subgenomic RNA replication in Huh-7 cells.
Subject(s)
Enzyme Inhibitors/pharmacology , Hepacivirus/enzymology , RNA, Viral/metabolism , Sulfonamides/pharmacology , Thiophenes/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Carboxylic Acids , Carcinoma, Hepatocellular/chemistry , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/virology , Enzyme Inhibitors/chemistry , Genome, Viral , Humans , Liver Neoplasms/chemistry , Liver Neoplasms/enzymology , Liver Neoplasms/virology , RNA-Dependent RNA Polymerase/antagonists & inhibitors , Replicon/drug effects , Structure-Activity Relationship , Sulfonamides/chemistry , Thiophenes/chemistry , Virus Replication/drug effectsABSTRACT
Further SAR studies on the thiophene-2-carboxylic acids are reported. These studies led to the identification of a series of tertiary amides that show inhibition of both HCV NS5B polymerase in vitro and HCV subgenomic RNA replication in Huh-7 cells. Structural insights about the bioactive conformation of this class of molecules were deduced from a combination of modeling and transferred NOE (trNOE) studies.