Prodigiosins (Ps) represent a family of naturally occurring red pigments characterized by a common pyrrolylpyrromethene skeleton. Some members of this family have been shown to possess interesting immunosuppressive properties exerted with a novel mechanism of action, different from that of currently used drugs. In fact, Ps inhibit phosphorylation and activation of JAK-3, a cytoplasmic tyrosine kinase associated with a cell surface receptor component called common gamma-chain, which is exclusive of all IL-2 cytokine family receptors. Blocking common gamma-chain transduction activity results in a potent and specific immunosuppressive activity. With respect to the interesting and unexploited immunomodulating properties of this family of compounds we initiated a medicinal chemistry program aimed at finding novel prodigiosin derivatives with improved immunosuppressive activity and lower toxicity. Utilizing an unprecedented and flexible way of assembling the prodigiosin frame, a number of new derivatives have been prepared and tested leading to the choice of 4-benzyloxy-5-[(5-undecyl-2H-pyrrol-2-ylidene)methyl]-2, 2'-bi-1H-pyrrole (PNU-156804, 16) as a lead immunosuppressant.

No abstract

β-arrestins are critical signalling molecules that regulate many fundamental physiological functions including the maintenance of euglycemia and peripheral insulin sensitivity. Here we show that inactivation of the β-arrestin-2 gene, barr2, in β-cells of adult mice greatly impairs insulin release and glucose tolerance in mice fed with a calorie-rich diet. Both glucose and KCl-induced insulin secretion and calcium responses were profoundly reduced in β-arrestin-2 (barr2) deficient β-cells. In human β-cells, barr2 knockdown abolished glucose-induced insulin secretion. We also show that the presence of barr2 is essential for proper CAMKII function in β-cells. Importantly, overexpression of barr2 in β-cells greatly ameliorates the metabolic deficits displayed by mice consuming a high-fat diet. Thus, our data identify barr2 as an important regulator of β-cell function, which may serve as a new target to improve β-cell function.

ABSTRACT3 H]spiperone to Chinese hamster ovary-transfected D 3 receptors when radioligands were used at 0.2 and 0.5 nM, respectively. However, even at high concentrations (5 M), SB269,652 only submaximally inhibited the specific binding of these radioligands when they were employed at 10-fold higher concentrations. By analogy, although SB269,652 potently blocked D 3 receptor-mediated activation of G␣ i3 and phosphorylation of extracellular-signal-regulated kinase (ERK)1/2, when concentrations of dopamine were increased by 10-fold, from 1 M to 10 M, SB269,652 only submaximally inhibited dopamine-induced stimulation of G␣ i3 . SB269,652 (up to 10 M) only weakly and partially (by approximately 20 -30%) inhibited radioligand binding to D 2 receptors. Likewise, SB269,652 only submaximally suppressed D 2 receptor-mediated stimulation of G␣ i3 and G␣ qi5 (detected with the aequorin assay) and phosphorylation of ERK1/2 and Akt. Furthermore, SB269,652 only partially (35%) inhibited the dopamine-induced recruitment of ␤-arrestin2 to D 2 receptors. Finally, Schild analysis using G␣ i3 assays, and studies of radioligand association and dissociation kinetics, supported allosteric actions of SB269,652 at D 3 and D 2 receptors.

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternative therapeutic target; however, drug discovery programmes focused on this G protein-coupled receptor (GPCR) have failed largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviours and hyper-locomotion. By mapping the upstream signalling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signalling in driving clinically relevant outcomes and in controlling adverse effects including "epileptic-like" seizures. We conclude that M1 mAChR ligands that promote receptor-phosphorylation dependent signalling would protect against cholinergic-adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behaviour relevant for the treatment of AD.

G protein-coupled receptors (GPCRs) regulate virtually all aspects of human physiology and represent an important class of therapeutic drug targets. Many GPCR-targeted drugs resemble endogenous agonists, often resulting in poor selectivity among receptor subtypes and restricted pharmacologic profiles. The muscarinic acetylcholine receptor family exemplifies these problems; thousands of ligands are known, but few are receptor subtype-selective and nearly all are cationic in nature. Using structure-based docking against the M 2 and M 3 muscarinic receptors, we screened 3.1 million molecules for ligands with new physical properties, chemotypes, and receptor subtype selectivities. Of 19 docking-prioritized molecules tested against the M 2 subtype, 11 had substantial activity and 8 represented new chemotypes. Intriguingly, two were uncharged ligands with low micromolar to high nanomolar K i values, an observation with few precedents among aminergic GPCRs. To exploit a single amino-acid substitution among the binding pockets between the M 2 and M 3 receptors, we selected molecules predicted by docking to bind to the M 3 and but not the M 2 receptor. Of 16 molecules tested, 8 bound to the M 3 receptor. Whereas selectivity remained modest for most of these, one was a partial agonist at the M 3 receptor without measurable M 2 agonism. Consistent with this activity, this compound stimulated insulin release from a mouse b-cell line. These results support the ability of structure-based discovery to identify new ligands with unexplored chemotypes and physical properties, leading to new biologic functions, even in an area as heavily explored as muscarinic pharmacology.

No abstract

D and D dopamine receptors belong to the largest family of cell surface proteins in eukaryotes, the G protein-coupled receptors (GPCRs). Considering their crucial physiologic functions and their relatively accessible cellular locations, GPCRs represent one of the most important classes of therapeutic targets. Until recently, the only strategy to develop drugs regulating GPCR activity was through the identification of compounds that directly acted on the orthosteric sites for endogenous ligands. However, many efforts have recently been made to identify small molecules that are able to interact with allosteric sites. These sites are less well-conserved, therefore allosteric ligands have greater selectivity on the specific receptor. Strikingly, the use of allosteric modulators can provide specific advantages, such as an increased selectivity for GPCR subunits and the ability to introduce specific beneficial therapeutic effects without disrupting the integrity of complex physiologically regulated networks. In 2010, our group unexpectedly found that -[(1r,4r)-4-[2-(7-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]-1H-indole-2-carboxamide (SB269652), a compound supposed to interact with the orthosteric binding site of dopamine receptors, was actually a negative allosteric modulator of D- and D-receptor dimers, thus identifying the first allosteric small molecule acting on these important therapeutic targets. This review addresses the progress in understanding the molecular mechanisms of interaction between the negative modulator SB269652 and D and D dopamine receptor monomers and dimers, and surveys the prospects for developing new dopamine receptor allosteric drugs with SB269652 as the leading compound.