Summary The NADPH oxidase enzyme complex, NOX2, is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator of inflammation. Here, we have performed rational design and in silico screen to identify a small molecule inhibitor, Phox-I1, targeting the interactive site of p67phox with Rac GTPase that is a necessary step of the signaling leading to NOX2 activation. Phox-I1 binds to p67phox with a submicromolar affinity and abrogates Rac1 binding, and is effective in inhibiting NOX2-mediated superoxide production dose-dependently in human and murine neutrophils without detectable toxicity. Medicinal chemistry characterizations have yielded promising analogs and initial information of the structure-activity relationship of Phox-I1. Our studies suggest the potential utility of Phox-I class inhibitors in NOX2 oxidase inhibition and present the first application of rational targeting of a small GTPase - effector interface.

Rho GTPases represent a family of small GTP-binding proteins that are involved in many important cellular functions relevant to cancer including cell cytoskeleton organization, migration, transcription, and proliferation. Since deregulation of members of Rho GTPase family is often found associated with many disease states, targeting of Rho GTPases and related signaling pathways for potential therapeutic benefits has been extensively pursued. Recent progress in this field of studies by peptide and peptidomemic inhibitors has provided important validations to this principle. The possibility to design and synthesize specific peptides that can bind to specific surface of the targeting proteins to elicit transient and specific blockade of the signal flows that require defined protein-protein interactions makes peptide inhibitors an attractive approach. In this review we summarize the recent advances in the design and application of a number of polypeptide and peptidomimetic structures that specifically target individual members of Rho GTPases and their up- or down-stream signaling regulators/effectors with an emphasis on cancer, inflammation and neurodegenerative diseases. The principle derived from the peptidic inhibitors has led to discoveries of the first generation of small molecule inhibitors of Rac GTPase of the Rho family. The implication of these studies in the pathobiology of various human diseases makes targeting Rho GTPases a valid strategy for future therapies.