Galphas-coupled receptor signaling actively down-regulates α4β1-integrin affinity: A possible mechanism for cell de-adhesion

<p>Abstract</p> <p>Background</p> <p>Activation of integrins in response to inside-out signaling serves as a basis for leukocyte arrest on endothelium, and migration of immune cells. Integrin-dependent adhesion is controlled by the conformational state of the molecule (i.e. change in the affinity for the ligand and molecular unbending (extension)), which is regulated by seven-transmembrane Guanine nucleotide binding Protein-Coupled Receptors (GPCRs). α₄β₁-integrin (CD49d/CD29, Very Late Antigen-4, VLA-4) is expressed on leukocytes, hematopoietic stem cells, hematopoietic cancer cells, and others. Affinity and extension of VLA-4 are both rapidly up-regulated by inside-out signaling through several Gα_i-coupled GPCRs. The goal of the current report was to study the effect of Gα_s-coupled GPCRs upon integrin activation.</p> <p>Results</p> <p>Using real-time fluorescent ligand binding to assess affinity and a FRET based assay to probe α₄β₁-integrin unbending, we show that two Gα_s-coupled GPCRs (H2-histamine receptor and β2-adrenergic receptor) as well as several cAMP agonists can rapidly down modulate the affinity of VLA-4 activated through two Gα_i-coupled receptors (CXCR4 and FPR) in U937 cells and primary human peripheral blood monocytes. This down-modulation can be blocked by receptor-specific antagonists. The Gα_s-induced responses were not associated with changes in the expression level of the Gα_i-coupled receptors. In contrast, the molecular unbending of VLA-4 was not significantly affected by Gα_s-coupled GPCR signaling. In a VLA-4/VCAM-1-specific myeloid cell adhesion system, inhibition of the VLA-4 affinity change by Gα_s-coupled GPCR had a statistically significant effect upon cell aggregation.</p> <p>Conclusion</p> <p>We conclude that Gα_s-coupled GPCRs can rapidly down modulate the affinity state of VLA-4 binding pocket through a cAMP dependent pathway. This plays an essential role in the regulation of cell adhesion. We discuss several possible implications of this described phenomenon.</p

The Mathematics of a Successful Deconvolution: A Quantitative Assessment of Mixture-Based Combinatorial Libraries Screened Against Two Formylpeptide Receptors

In the past 20 years, synthetic combinatorial methods have fundamentally advanced the ability to synthesize and screen large numbers of compounds for drug discovery and basic research. Mixture-based libraries and positional scanning deconvolution combine two approaches for the rapid identification of specific scaffolds and active ligands. Here we present a quantitative assessment of the screening of 32 positional scanning libraries in the identification of highly specific and selective ligands for two formylpeptide receptors. We also compare and contrast two mixture-based library approaches using a mathematical model to facilitate the selection of active scaffolds and libraries to be pursued for further evaluation. The flexibility demonstrated in the differently formatted mixture-based libraries allows for their screening in a wide range of assays

TIN-X:target importance and novelty explorer

Abstract Motivation The increasing amount of peer-reviewed manuscripts requires the development of specific mining tools to facilitate the visual exploration of evidence linking diseases and proteins. Results We developed TIN-X, the Target Importance and Novelty eXplorer, to visualize the association between proteins and diseases, based on text mining data processed from scientific literature. In the current implementation, TIN-X supports exploration of data for G-protein coupled receptors, kinases, ion channels, and nuclear receptors. TIN-X supports browsing and navigating across proteins and diseases based on ontology classes, and displays a scatter plot with two proposed new bibliometric statistics: Importance and Novelty. Availability and Implementation http://www.newdrugtargets.org </jats:sec

A Novel Flow Cytometric HTS Assay Reveals Functional Modulators of ATP Binding Cassette Transporter ABCB6

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6’s ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity

Pharmaceutical screen identifies novel target processes for activation of autophagy with a broad translational potential

Autophagy is a conserved homeostatic process active in all human cells and affecting a spectrum of diseases. Here we use a pharmaceutical screen to discover new mechanisms for activation of autophagy. We identify a subset of pharmaceuticals inducing autophagic flux with effects in diverse cellular systems modelling specific stages of several human diseases such as HIV transmission and hyperphosphorylated tau accumulation in Alzheimer’s disease. One drug, flubendazole, is a potent inducer of autophagy initiation and flux by affecting acetylated and dynamic microtubules in a reciprocal way. Disruption of dynamic microtubules by flubendazole results in mTOR deactivation and dissociation from lysosomes leading to TFEB (transcription factor EB) nuclear translocation and activation of autophagy. By inducing microtubule acetylation, flubendazole activates JNK1 leading to Bcl-2 phosphorylation, causing release of Beclin1 from Bcl-2-Beclin1 complexes for autophagy induction, thus uncovering a new approach to inducing autophagic flux that may be applicable in disease treatment

A Potent and Selective Inhibitor of Cdc42 GTPase

Cdc42, a member of the Rho family of GTPases, has been shown to play a role in cell adhesion, cytoskeletal arrangement, phagocytosis and cell motility and migration, in addition to a host of other diverse biological processes. The function of Rho-family GTPases in disease pathogenesis has been well established and identification of small, cell permeable molecules that selectively and reversibly regulate Rho GTPases is of high scientific and potentially therapeutic interest. There has been limited success in identifying inhibitors that specifically interact with small Rho family GTPases. The identified probe, ML141 (CID-2950007), is demonstrated to be a potent, selective and reversible non-competitive inhibitor of Cdc42 GTPase suitable for in vitro assays, with low micromolar potency and selectivity against other members of the Rho family of GTPases (Rac1, Rab2, Rab7). Given the highly complementary nature of the function of the Rho family GTPases, Cdc42 selective inhibitors such as those reported here should help untangle the roles of the proteins in this family

Characterization of a Cdc42 Protein Inhibitor and Its Use as a Molecular Probe

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.This work was supported by National Science Foundation (NSF) Grant MCB0956027 and National Institutes of Health Grant R03 MH081231-01 from the Molecular Libraries Program (to A. W. N.); University of New Mexico Center for Molecular Discovery Molecular Libraries Probe Production Centers (UNMCMD MLPCN) National Institutes of Health Grants U54MH084690 and R01HL081062 (to L. A. S.); UNM National Center for Research Resources (NCRR) Grant 5P20RR016480 (to L. G. H.); National Institutes of Health Grant R21 CA170375-01 through the NCI (to A. W. N., L. G. H., and J. E. G.); National Institutes of Health Grants NS066429 and AI092130 (to T. B.); and University of Kansas Specialized Chemistry Center (KUSCC) MLPCN National Institutes of Health Grant U54HG005031 (to J. A.)

Ligand-receptor-G-protein molecular assemblies on beads for mechanistic studies and screening by flow cytometry.

ABSTRACT G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gi␣2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein. Streptavidin-coated polystyrene beads used biotinylated anti-FLAG antibodies to bind FLAG-tagged G proteins for ternary complex assembly. Validation was achieved by showing time and concentration dependence of ternary complex formation. Affinity measurements of ligand for receptor on particles, of the ligand-receptor complex for G protein on the particles, and receptor-Gi␣2 fusion protein for G␤␥, were consistent with comparable assemblies in detergent suspension. Performance was assessed in applications representing the potential of these assemblies for ternary complex mechanisms. We showed the relationship for a family of ligands between LR and LRG affinity and characterized the affinity of both the wild-type and GFP fusion receptors with G protein. We also showed the potential of kinetic measurements to allow observation of individual steps of GTP-induced ternary complex disassembly and discriminated a fast step caused by RG disassembly compared with the slower step of G␣␤␥ disassembly. GPCRs interact with extracellular stimuli, such as photons, hormones, neurotransmitters, and odorants The formyl peptide receptor (FPR) responds to the presence of N-formyl methionine-containing peptides resulting from bacterial and mitochondrial protein synthesis, as well as other hydrophobic peptide