Functionalized Phenylbenzamides Inhibit Aquaporin-4 Reducing Cerebral Edema and Improving Outcome in Two Models of CNS Injury.

Cerebral edema in ischemic stroke can lead to increased intracranial pressure, reduced cerebral blood flow and neuronal death. Unfortunately, current therapies for cerebral edema are either ineffective or highly invasive. During the development of cytotoxic and subsequent ionic cerebral edema water enters the brain by moving across an intact blood brain barrier and through aquaporin-4 (AQP4) at astrocyte endfeet. Using AQP4-expressing cells, we screened small molecule libraries for inhibitors that reduce AQP4-mediated water permeability. Additional functional assays were used to validate AQP4 inhibition and identified a promising structural series for medicinal chemistry. These efforts improved potency and revealed a compound we designated AER-270, N-[3,5-bis (trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide. AER-270 and a prodrug with enhanced solubility, AER-271 2-{[3,5-Bis(trifluoromethyl) phenyl]carbamoyl}-4-chlorophenyl dihydrogen phosphate, improved neurological outcome and reduced swelling in two models of CNS injury complicated by cerebral edema: water intoxication and ischemic stroke modeled by middle cerebral artery occlusion.

Interactions between E6, FAK, and GIT1 at paxillin LD4 are necessary for transformation by bovine papillomavirus 1 E6.

UNLABELLED: Bovine papillomavirus 1 E6 interacts with two similar proteins that regulate cell attachment and cell migration called paxillin (PXN) and HIC-5 (also known as HIC5, ARA55, HIC-5, TSC-5, and TGFB1I1). Despite the similarity between HIC-5 and paxillin, paxillin is required for E6 to transform mouse embryo fibroblasts while HIC-5 is not. Using mutants of paxillin, we found that dynamic competitive interactions between E6, focal adhesion kinase, and the GIT1 ARF-GAP protein for binding to paxillin are required but not sufficient for transformation by E6. Using mutants of paxillin and chimeric proteins between HIC-5 and paxillin, we demonstrate that a critical difference between HIC-5 and paxillin is within the LIM domains of paxillin that do not directly interact with E6. Mutational analysis indicates that at least six distinct domains of paxillin are required for E6 transformation. IMPORTANCE: Papillomaviruses cause epitheliomas in vertebrates through the actions of virus-encoded oncoproteins. Despite the immense diversity of papillomavirus types, our understanding of the mechanisms by which the virus-encoded E6 oncoproteins contribute to cell transformation is restricted to human papillomavirus types that are associated with cancer. Bovine papillomavirus 1 (BPV-1) E6 has served as a model system for studies of E6 structure and function. This study examines the mechanisms by which BPV-1 E6 association with the cellular focal adhesion adapter protein paxillin contributes to cell transformation and extends our knowledge of the diverse mechanisms by which papillomaviruses transform host cells.

Paxillin enables attachment-independent tyrosine phosphorylation of focal adhesion kinase and transformation by RAS.

Paxillin and HIC5 are closely related adapter proteins that regulate cell migration and are tyrosine-phosphorylated by focal adhesion kinase (FAK). Paxillin, HIC5, and FAK tyrosine phosphorylation increase upon cell attachment and decrease upon detachment from extracellular matrix. Unexpectedly, we found that although FAK tyrosine phosphorylation in attached cells did not require paxillin, in detached fibroblasts there was remaining FAK tyrosine phosphorylation that required expression of paxillin and was not supported by HIC5. The support of attachment-independent FAK tyrosine phosphorylation required the paxillin LIM domains and suggested that paxillin might facilitate oncogenic transformation. Paxillin but not HIC5 augmented anchorage-independent cell proliferation induced by RAS. Both anchorage-independent FAK tyrosine phosphorylation and RAS-induced colony formation required multiple docking sites on paxillin, including LD4 (docking sites for FAK-Src and GIT1/2-PIX-NCK-PAK complex), LD5, and all four carboxyl-terminal LIM domains (that bind tubulin and PTP-PEST). Analysis using paxillin mutants dissociated domains of paxillin that are required for regulation of cell migration from domains that are required for anchorage-independent cell proliferation and demonstrated essential functions of the paxillin LIM domains that are not found in HIC5 LIM domains. These results highlight the role of paxillin in facilitating attachment-independent signal transduction implicated in cancer.

Transformation by bovine papillomavirus type 1 E6 requires paxillin.

Papillomavirus E6 proteins are adapters that change the function of cellular regulatory proteins. The bovine papillomavirus type 1 E6 (BE6) binds to LXXLL peptide sequences termed LD motifs (consensus sequence LDXLLXXL) on the cellular protein paxillin that is a substrate of Src and focal adhesion kinases. Anchorage-independent transformation induced by BE6 required both paxillin and BE6-binding LD motifs on paxillin but was independent of the major tyrosine phosphorylation sites of paxillin. The essential role of paxillin in transformation by BE6 highlights the role of paxillin in the transduction of cellular signals that result in anchorage-independent cell proliferation.

Minimal features of paxillin that are required for the tyrosine phosphorylation of focal adhesion kinase.

Tyrosine phosphorylation of FAK (focal adhesion kinase) regulates signalling that results from the interaction of integrins with extracellular matrix and growth factor receptors. A critical step in this process is the phosphorylation of Tyr397 of FAK, which creates a binding site for Src family kinases, PI3K (phosphoinositide 3-kinase) and Shc (Src homology and collagen homology). An intact Tyr397 site is required for FAK-mediated regulation of cell migration, survival signals and full responsiveness to soluble growth factors. We showed previously that the adaptor protein paxillin is required for the overall tyrosine phosphorylation of FAK in embryonic stem cells [Wade, Bohl and Vande Pol (2002) Oncogene 21, 96-107]. In the present paper, we identify the minimal structural features of paxillin that are required to support overall FAK tyrosine phosphorylation and Tyr397 phosphorylation. Paxillin contains N-terminal leucine-rich LD motifs that bind directly to FAK and four LIM (Lin-11, Isl-1 and Mec-3) domains in the C-terminus. We show that paxillin LIM domains 1, 2 and 3 are each required for FAK tyrosine phosphorylation, while LIM4 is dispensable. In addition to paxillin LIM domains 1, 2 and 3, a single LD motif on paxillin is required to support FAK tyrosine phosphorylation in embryonic stem cells. Both sequence and spatial requirements exist for LD motifs to support FAK tyrosine phosphorylation. Interestingly, synthetic LD motifs that fail to bind FAK in vitro are able to fully support FAK tyrosine phosphorylation, indicating that minimal interactions of LD motifs with FAK suffice. Our results demonstrate at least four distinct structural domains of paxillin support at least three distinct functions that are each required for FAK tyrosine phosphorylation.

Paxillin null embryonic stem cells are impaired in cell spreading and tyrosine phosphorylation of focal adhesion kinase.

Paxillin is a focal-adhesion associated protein implicated in the regulation of integrin signaling and organization of the actin cytoskeleton. Paxillin associates with numerous signaling molecules including adaptor molecules (p130Cas, CRK), kinases (FAK, Pyk2, PAK and SRC), tyrosine phosphatases (PTP-PEST), ARF-GAP proteins (p95pkl, PAG3) and papillomavirus E6 oncoproteins. Although paxillin is tyrosine phosphorylated in cellular processes such as cell attachment and spreading, little direct evidence is available about paxillin's role in these events. Targeted gene disruption was used to generate paxillin null mouse embryonic stem (ES) cells and paxillin null differentiated cells. Paxillin null ES cells exhibit delayed spreading on integrin binding substrates fibronectin and laminin, and there is reduced tyrosine phosphorylation of Focal Adhesion Kinase (FAK). Both of these phenotypes are recovered in paxillin knockout cells upon exogenous re-expression of paxillin. The individual LD motifs of paxillin that are binding sites for FAK, vinculin and ARF-GAP proteins, as well as tyrosine residues that when phosphorylated create binding sites for CRK family members, are dispensable for FAK phosphorylation and early cell spreading. These results demonstrate that paxillin contributes to attachment-dependent tyrosine phosphorylation of FAK and early cell spreading in ES cells.