Benzothiophene containing Rho kinase inhibitors: Efficacy in an animal model of glaucoma.

We identified a new benzothiophene containing Rho kinase inhibitor scaffold in an ultra high-throughput enzymatic activity screen. SAR studies, driven by a novel label-free cellular impedance assay, were used to derive 39, which substantially reduced intraocular pressure in a monkey model of glaucoma-associated ocular hypertension.

Rac1 links leading edge and uropod events through Rho and myosin activation during chemotaxis.

Chemotactic responsiveness is crucial to neutrophil recruitment to sites of infection. During chemotaxis, highly divergent cytoskeletal programs are executed at the leading and trailing edge of motile neutrophils. The Rho family of small GTPases plays a critical role in cell migration, and recent work has focused on elucidating the specific roles played by Rac1, Rac2, Cdc42, and Rho during cellular chemotaxis. Rac GTPases regulate actin polymerization and extension of the leading edge, whereas Rho GTPases control myosin-based contraction of the trailing edge. Rac and Rho signaling are thought to crosstalk with one another, and previous research has focused on mutual inhibition of Rac and Rho signaling during chemotaxis. Indeed, polarization of neutrophils has been proposed to involve the activity of a negative feedback system where Rac activation at the front of the cell inhibits local Rho activation, and vice versa. Using primary human neutrophils and neutrophils derived from a Rac1/Rac2-null transgenic mouse model, we demonstrate here that Rac1 (and not Rac2) is essential for Rho and myosin activation at the trailing edge to regulate uropod function. We conclude that Rac plays both positive and negative roles in the organization of the Rhomyosin "backness" program, thereby promoting stable polarity in chemotaxing neutrophils.

Constitutive p21-activated kinase (PAK) activation in breast cancer cells as a result of mislocalization of PAK to focal adhesions.

Cytoskeletal remodeling is critical for cell adhesion, spreading, and motility. p21-activated kinase (PAK), an effector molecule of the Rho GTPases Rac and Cdc42, has been implicated in cytoskeletal remodeling and cell motility. PAK kinase activity and subcellular distribution are tightly regulated by rapid and transient localized Rac and Cdc42 activation, and by interactions mediated by adapter proteins. Here, we show that endogenous PAK is constitutively activated in certain breast cancer cell lines and that this active PAK is mislocalized to atypical focal adhesions in the absence of high levels of activated Rho GTPases. PAK localization to focal adhesions in these cells is independent of PAK kinase activity, NCK binding, or GTPase binding, but requires the association of PAK with PIX. Disruption of the PAK-PIX interaction with competitive peptides displaces PAK from focal adhesions and results in a substantial reduction in PAK hyperactivity. Moreover, disruption of the PAK-PIX interaction is associated with a dramatic decrease of PIX and paxillin in focal adhesions, indicating that PAK localization to these structures via PIX is required for the maintenance of paxillin- and PIX-containing focal adhesions. Abnormal regulation of PAK localization and activity may contribute to the tumorigenic properties of certain breast cancer cells.

Spatial and temporal analysis of Rac activation during live neutrophil chemotaxis.

The ability of cells to recognize and respond with directed motility to chemoattractant agents is critical to normal physiological function. Neutrophils represent the prototypic chemotactic cell in that they respond to signals initiated through the binding of bacterial peptides and other chemokines to G protein-coupled receptors with speeds of up to 30 microm/min. It has been hypothesized that localized regulation of cytoskeletal dynamics by Rho GTPases is critical to orchestrating cell movement. Using a FRET-based biosensor approach, we investigated the dynamics of Rac GTPase activation during chemotaxis of live primary human neutrophils. Rac has been implicated in establishing and maintaining the leading edge of motile cells, and we show that Rac is dynamically activated at specific locations in the extending leading edge. However, we also demonstrate activated Rac in the retracting tail of motile neutrophils. Rac activation is both stimulus and adhesion dependent. Expression of a dominant-negative Rac mutant confirms that Rac is functionally required both for tail retraction and for formation of the leading edge during chemotaxis. These data establish that Rac GTPase is spatially and temporally regulated to coordinate leading-edge extension and tail retraction during a complex motile response, the chemotaxis of human neutrophils.