The actin cytoskeleton supports diverse cellular processes such as endocytosis, oriented growth, adhesion and migration. The dynamic nature of the cytoskeleton, however, has made it difficult to define the roles of the many accessory molecules that modulate actin organization, especially the multifunctional adapter protein annexin II. We now report that the compound withaferin A (1) can alter cytoskeletal architecture in a previously unknown manner by covalently binding annexin II and stimulating its basal F-actin cross-linking activity. Drug-mediated disruption of F-actin organization is dependent on annexin II expression by cells and markedly limits their migratory and invasive capabilities at subcytotoxic concentrations. Given the extensive ethnobotanical history of withaferin-containing plant preparations in the treatment of cancer and inflammatory and neurological disorders, we suggest that annexin II represents a feasible, previously unexploited target for therapeutic intervention by small-molecule drugs.

AKT, a phospholipid-binding serine/threonine kinase, is a key component of the phosphoinositide 3-kinase cell survival signaling pathway that is aberrantly activated in many human cancers. Many attempts have been made to inhibit AKT; however, selectivity remains to be achieved. We have developed a novel strategy to inhibit AKT by targeting the pleckstrin homology (PH) domain. Using in silico library screening and interactive molecular docking, we have identified a novel class of non -lipid-based compounds that bind selectively to the PH domain of AKT, with ''in silico'' calculated K D values ranging from 0.8 to 3.0 Mmol/L. In order to determine the selectivity of these compounds for AKT, we used surface plasmon resonance to measure the binding characteristics of the compounds to the PH domains of AKT1, insulin receptor substrate-1, and 3-phosphoinositide -dependent protein kinase 1. There was excellent correlation between predicted in silico and measured in vitro K D s for binding to the PH domain of AKT, which were in the range 0.4 to 3.6 Mmol/L. Some of the compounds exhibited PH domain -binding selectivity for AKT compared with insulin receptor substrate-1 and 3-phosphoinositide -dependent protein kinase 1. The compounds also inhibited AKT in cells, induced apoptosis, and inhibited cancer cell proliferation. In vivo, the lead compound failed to achieve the blood concentrations required to inhibit AKT in cells, most likely due to rapid metabolism and elimination, and did not show antitumor activity. These results show that these compounds are the first small molecules selectively targeting the PH domain of AKT. [Mol Cancer Ther 2008; 7(9):2621 -32]

OCT1 and OCT2 are involved in renal secretion of cationic drugs. Although they have similar selectivity for some substrates (e.g. tetraethylammonium (TEA)), they have distinct selectivities for others (e.g. cimetidine). We postulated that "homolog-specific residues," i.e. the 24 residues that are conserved in OCT1 orthologs as one amino acid and in OCT2 as a different one, influence homologspecific selectivity and examined the influence on substrate binding of three of these conserved residues that are found in the C-terminal half of the rabbit orthologs of OCT1/2. The N353L and R403I substitutions (OCT2 to OCT1) did not significantly change the properties of OCT2. However, the E447Q replacement shifted substrate selectivity toward an OCT1-like phenotype. Substitution of glutamate with cationic amino acids (E447K and E447R) abolished transport activity, and the E447L mutant displayed markedly reduced transport of TEA and cimetidine while retaining transport of 1-methyl-4-phenylpyridinium. In a novel homology model of the three-dimensional structure of OCT2, Glu 447 was found in a putative docking region within a hydrophilic cleft of the protein. Renal excretion is the principal pathway for elimination of many clinically used drugs and is the exclusive pathway for eliminating many end products of drug-metabolizing enzymes (1-3). Transporters in the renal tubule epithelium mediate secretion and thus play a critical role in detoxification (4). In addition, transporters control the exposure of renal cells to nephrotoxic drugs and environmental toxins and thereby influence xenobiotic-induced nephrotoxicity. A large fraction of these agents fall into the chemical class commonly referred to as "organic cations" (OCs), 2 i.e. a diverse array of primary, secondary, tertiary, or quaternary amines that have a net positive charge on the amine nitrogen at physiological pH. The proximal tubule is the primary site of renal OC secretion (3,5), and the first step in the process is OC entry from the blood into proximal cells across the peritubular (i.e. basolateral) membrane. Three homologous transporters (OCT1, OCT2, and OCT3) have been cloned and subsequently shown to be expressed in the basolateral membrane of proximal cells (1,6). In all species examined, including the human (7), the kinetic and selectivity profile of OCT3 and the comparatively low levels of mRNA and protein expression of this homolog in the kidney suggest that renal secretion is dominated by some combination of OCT1 and OCT2 activity. Perhaps the most convincing evidence of the significance of OCT1 and OCT2 in renal OC secretion is the observation that secretion of the prototypic OC, tetraethylammonium (TEA), is completely eliminated in OCT1/2 Ϫ/Ϫ mice (8).In human kidney, OCT2 appears to be the predominant OC transporter. Expression of mRNA for OCT2 far exceeds that for OCT1, and immunocytochemistry clearly shows a basolateral expression for OCT2 and little or no presence of OCT1 (9). There are, however, clear species differences in this profile, with subst...

We developed a ligand-mimetic antibody Fab fragment specific for Drosophila ␣PS2␤PS integrins to probe the ligand binding affinities of these invertebrate receptors. TWOW-1 was constructed by inserting a fragment of the extracellular matrix protein Tiggrin into the H-CDR3 of the ␣v␤3 ligand-mimetic antibody WOW-1. The specificity of ␣PS2␤PS binding to TWOW-1 was demonstrated by numerous tests used for other integrin-ligand interactions. Binding was decreased in the presence of EDTA or RGD peptides and by mutation of the TWOW-1 RGD sequence or the ␤PS metal iondependent adhesion site (MIDAS) motif. TWOW-1 binding was increased by mutations in the ␣PS2 membrane-proximal cytoplasmic GFFNR sequence or by exposure to Mn 2؉ . Although Mn 2؉ is sometimes assumed to promote maximal integrin activity, TWOW-1 binding in Mn 2؉ could be increased further by the ␣PS2 GFFNR 3 GFANA mutation. A mutation in the ␤PS I domain (␤PS-b58; V409D) greatly increased ligand binding affinity, explaining the increased cell spreading mediated by ␣PS2␤PS-b58. Further mutagenesis of this residue suggested that Val-409 normally stabilizes the closed head conformation. Mutations that potentially reduce interaction of the integrin ␤ subunit plexin-semaphorinintegrin (PSI) and stalk domains have been shown to have activating properties. We found that complete deletion of the ␤PS PSI domain enhanced TWOW-1 binding. Moreover the PSI domain is dispensable for at least some other integrin functions because ␤PS-⌬PSI displayed an enhanced ability to mediate cell spreading. These studies establish a means to evaluate mechanisms and consequences of integrin affinity modulation in a tractable model genetic system.Integrins are the primary family of receptors that connects cells to the extracellular matrix (ECM) 2 (1). The cytoplasmic tails of integrin subunits associate with multiple intracellular components, which mediate both signaling functions and ECM-cytoskeleton connections (2). Cells can regulate the functions of integrins at least in part by inducing conformational changes in integrin structure that alter the affinity of the integrin heterodimer for ECM ligands. Similarly integrin binding to ligands can lead to outside-in signal propagation, which can regulate cellular behaviors such as growth, differentiation, and survival (3-7).The integrin heterodimer is composed of ␣ and ␤ subunits that are nonhomologous to one another but strongly conserved structurally across the animal kingdom (8, 9). A long history of studies with conformation-sensitive antibodies indicates that integrins undergo large and concerted conformational changes as a result of cellular activation, and recent studies have begun to provide details of this structural switching (10 -16). Inactive integrins are likely to be bent in the middle so that the headpiece faces in toward the membrane-proximal part of the extracellular stalks. As a result of cellular activation, the integrin adopts an extended conformation with the headpiece facing away from the cell in optimal position to en...

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