ABSTRACT
Cdc42 plays important roles in cytoskeleton organization, cell cycle progression, signal transduction, and vesicle trafficking. Overactive Cdc42 has been implicated in the pathology of cancers, immune diseases, and neuronal disorders. Therefore, Cdc42 inhibitors would be useful in probing molecular pathways and could have therapeutic potential. Previous inhibitors have lacked selectivity and trended toward toxicity. We report here the characterization of a Cdc42-selective guanine nucleotide binding lead inhibitor that was identified by high throughput screening. A second active analog was identified via structure-activity relationship studies. The compounds demonstrated excellent selectivity with no inhibition toward Rho and Rac in the same GTPase family. Biochemical characterization showed that the compounds act as noncompetitive allosteric inhibitors. When tested in cellular assays, the lead compound inhibited Cdc42-related filopodia formation and cell migration. The lead compound was also used to clarify the involvement of Cdc42 in the Sin Nombre virus internalization and the signaling pathway of integrin VLA-4. Together, these data present the characterization of a novel Cdc42-selective allosteric inhibitor and a related analog, the use of which will facilitate drug development targeting Cdc42-related diseases and molecular pathway studies that involve GTPases.
Subject(s)
Enzyme Inhibitors/pharmacology , Molecular Probes/pharmacology , Pyrazoles/pharmacology , Sulfonamides/pharmacology , cdc42 GTP-Binding Protein/antagonists & inhibitors , 3T3 Cells , Allosteric Regulation , Animals , Antiviral Agents/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Enzyme Activation/drug effects , Humans , Integrin alpha4beta1/antagonists & inhibitors , Integrin alpha4beta1/physiology , Mice , Oligopeptides/metabolism , Phenylurea Compounds/metabolism , Protein Binding , Pseudopodia/drug effects , Sin Nombre virus/physiology , Structure-Activity Relationship , Virus Internalization/drug effects , cdc42 GTP-Binding Protein/chemistry , cdc42 GTP-Binding Protein/metabolism , rac1 GTP-Binding Protein/metabolism , rhoA GTP-Binding Protein/metabolismABSTRACT
A three-component method for the synthesis of highly substituted gamma-lactams from readily available maleimides, aldehydes, and amines is described. A new reductive amination/intramolecular lactamization sequence provides a straightforward route to the lactam products in a single manipulation. The general utility of this method is demonstrated by the parallel synthesis of a gamma-lactam library.