Structure–activity study of N-((trans)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide (SB269652), a bitopic ligand that acts as a negative allosteric modulator of the dopamine D2 receptor

We recently demonstrated that SB269652 (1) engages one protomer of a dopamine D2 receptor (D2R) dimer in a bitopic mode to allosterically inhibit the binding of dopamine at the other protomer. Herein, we investigate structural deter- minants for allostery, focusing on modifications to three moieties within 1. We find that orthosteric “head” groups with small 7-substituents were important to maintain the limited negative cooperativity of analogues of 1, and replacement of the tetrahydroisoquinoline head group with other D2R “privileged structures” generated orthosteric antagonists. Additionally, replacement of the cyclohexylene linker with polymethylene chains conferred linker length dependency in allosteric pharma- cology. We validated the importance of the indolic NH as a hydrogen bond donor moiety for maintaining allostery. Replacement of the indole ring with azaindole conferred a 30-fold increase in affinity while maintaining negative cooperativity. Combined, these results provide novel SAR insight for bitopic ligands that act as negative allosteric modulators of the D2R

The Energy Landscape Analysis of Cancer Mutations in Protein Kinases

The growing interest in quantifying the molecular basis of protein kinase activation and allosteric regulation by cancer mutations has fueled computational studies of allosteric signaling in protein kinases. In the present study, we combined computer simulations and the energy landscape analysis of protein kinases to characterize the interplay between oncogenic mutations and locally frustrated sites as important catalysts of allostetric kinase activation. While structurally rigid kinase core constitutes a minimally frustrated hub of the catalytic domain, locally frustrated residue clusters, whose interaction networks are not energetically optimized, are prone to dynamic modulation and could enable allosteric conformational transitions. The results of this study have shown that the energy landscape effect of oncogenic mutations may be allosteric eliciting global changes in the spatial distribution of highly frustrated residues. We have found that mutation-induced allosteric signaling may involve a dynamic coupling between structurally rigid (minimally frustrated) and plastic (locally frustrated) clusters of residues. The presented study has demonstrated that activation cancer mutations may affect the thermodynamic equilibrium between kinase states by allosterically altering the distribution of locally frustrated sites and increasing the local frustration in the inactive form, while eliminating locally frustrated sites and restoring structural rigidity of the active form. The energy landsape analysis of protein kinases and the proposed role of locally frustrated sites in activation mechanisms may have useful implications for bioinformatics-based screening and detection of functional sites critical for allosteric regulation in complex biomolecular systems

Coupling of β2-Adrenoceptors to XLαs and Gαs: A New Insight into Ligand-Induced G Protein ActivationS⃞

Gαs and extra-large Gαs (XLαs) can both transduce receptor activation into intracellular cAMP generation. It is unknown, however, whether these two GNAS-locus products display distinct properties with respect to receptor coupling. Here, we show that XLαs couples to the β2-adrenoceptor more efficiently than Gαs. In transfected human embryonic kidney 293 cells and mouse embryonic fibroblasts null for both Gαs and XLαs (2B2 cells), basal cAMP accumulation mediated by XLαs was higher than that mediated by Gαs. Inverse agonist treatment reduced Gαs-mediated basal activity, whereas its effect was markedly blunted on XLαs-mediated basal activity. Rank order of ligand efficacies regarding cAMP accumulation was the same when the receptor was coupled to XLαs or Gαs. However, ligand-induced and XLαs-mediated cAMP generation was higher than that mediated by Gαs. The relatively high efficiency of XLαs-mediated cAMP generation was conditional, disappearing with increased level of receptor expression or increased efficacy of ligand. Full-agonist responses in XLαs- and Gαs-expressing cells were comparable even at low receptor levels, whereas partial agonist responses became comparable only when the receptor expression was increased (>3 pmol/mg). Radioligand binding studies showed that the high-affinity component in agonist binding to β2-adrenoceptor was more pronounced in cells expressing XLαs than those expressing Gαs. We discuss these findings in the framework of current receptor-G protein activation models and offer an extended ternary complex model that can fully explain our observations

Ligands Raise the Constraint That Limits Constitutive Activation in G Protein-coupled Opioid Receptors

Using a cell-free bioluminescence resonance energy transfer strategy we compared the levels of spontaneous and ligand-induced receptor-G protein coupling in _ (DOP) and _ (MOP) opioid receptors. In this assay GDP can suppress spontaneous coupling, thus allowing its quantification. The level of constitutive activity was 4–5 times greater at the DOP than at the MOP receptor. A series of opioid analogues with a common peptidomimetic scaffold displayed remarkable inversions of efficacy in the two receptors. Agonists that enhanced coupling above the low intrinsic level of the MOP receptor were inverse agonists in reducing the greater level of constitutive coupling of the DOP receptor. Yet the intrinsic activities of such ligands are identical when scaled over the GDP base line of both receptors. This pattern is in conflict with the predictions of the ternary complex model and the “two state” extensions. According to this theory, the order of spontaneous and ligand-induced coupling cannot be reversed if a shift of the equilibrium between active and inactive forms raises constitutive activation in one receptor type.We propose that constitutive activation results from a lessened intrinsic barrier that restrains spontaneous coupling. Any ligand, regardless of its efficacy, must enhance this constraint to stabilize the ligand-bound complexed form

The effect of foreign affiliate employment on wages, employment, and the wage share in Austria

This paper estimates the effects of outward Foreign Direct Investment (employment in affiliates abroad) on employment, wages and the wage share in Austria using panel data for the period 1996–2005. There is evidence of significant negative effects of FDI on both employment and wages, and consequently on the wage share. The results are not limited to workers in low-skilled sectors. The negative employment effect is mainly due to the rise in employment in the foreign affiliates in Eastern Europe. The negative wage effects originate from affiliate employment in both Eastern Europe and the developed countries in the industrial sector, but FDI in Eastern Europe has positive wage effects in the services sector due to possible scope effects