Natural as well as synthetic coumarins have recently drawn much attention due to its broad pharmacological activities. Many coumarins and their derivatives exert anti-coagulant, anti-tumor, anti-viral, anti-inflammatory and anti-oxidant effects, as well as anti-microbial and enzyme inhibition properties. The recognition of key structural features within coumarin family is crucial for the design and development of new analogues with improved activity and for the characterization of their mechanism of action and potential side effects. The different substituents in the coumarin nucleus strongly influence the biological activity of the resulting derivatives. Although some coumarins have been already characterized to evoke a particular biological activity, the challenge would be the design and synthesis of new derivatives with high specific activity for other pharmacological targets and define their mechanism of action to achieve new therapeutic drugs. The present review highlights the current progress in the development of coumarin scaffolds for drug discovery as novel anti-cancer agents. The major challenges about coumarins include the translation of current knowledge into new potential lead compounds and the repositioning of known compounds for the treatment of cancer.

Accurate characterization of the molecular mechanisms of the action of ligands is an extremely important issue for their appropriate research, pharmacological, and therapeutic uses. In view of this fact, the aim of the present work was to investigate the mechanisms involved in the actions of mepyramine at the guinea pig H 1 receptor stably expressed in Chinese hamster ovary cells. We found that mepyramine is able to decrease the basal constitutive activity of the guinea pig H 1 receptor, to bind with high affinity to a G q/11 protein-coupled form of the receptor and to promote a G protein-coupled inactive state of the H 1 receptor that interferes with the G q/11 -mediated signaling of the endogenously expressed ATP receptor, as predicted by the Cubic Ternary Complex Model of receptor occupancy. The effect of mepyramine on ATP-induced signaling was specifically neutralized by G␣ 11 overexpression, indicating that mepyramine is able to reduce G protein availability for other non-related receptors associated with the same signaling pathway. Finally, we found a loss of mepyramine efficacy in decreasing basal levels of intracellular calcium at high G␣ 11 expression levels, which can be theoretically explained in terms of high H 1 receptor constitutive activity. The whole of the present work sheds new light on H 1 receptor pharmacology and the mechanisms H 1 receptor inverse agonists could use to exert their observed negative efficacy.

The histamine H2 receptor (H2r) belongs to the heptahelical receptor family; upon agonist binding, members of this family activate a G protein and the downstream effector adenylyl cyclase. Like other G protein-coupled receptors, exposure of H2r to agonists produces a desensitization of the response. The present study focused on the desensitization mechanism of this receptor. Using transiently transfected COS-7 cells expressing tagged-H2r, the desensitization induced by amthamine, characterized by decreased cAMP production, was studied. Results show that the receptor was rapidly desensitized with a t(1/2) = 0.49 +/- 0.01 min. Because of the rapid nature of H2r desensitization, receptor phosphorylation was examined as a likely mechanism for signal attenuation. H2r desensitization was not affected by protein kinases A and C (PKA and PKC) inhibitors but was remarkably reduced by Zn(2+), an inhibitor of G protein-coupled receptor kinases (GRKs). Cotransfection experiments using tagged H2r and different GRKs (2, 3, 5, or 6), demonstrated that GRK2 and GRK3 were the most potent in augmenting desensitization, causing a reduction in the maximal response to amthamine and a decrease of the t(1/2) for desensitization, whereas GRK5 and GRK6 did not affect the signaling. Receptor phosphorylation correlates with desensitization for each GRK studied, whereas phosphorylation that is dependent on protein kinases A and C seemed irrelevant in receptor signal termination. These results indicate that in H2r-transfected COS-7 cells, exposure to an agonist caused desensitization controlled by H2r phosphorylation via GRK2 and GRK3.

H 1 and H 2 histamine receptor antagonists, although developed many decades ago, are still effective for the treatment of allergic and gastric acid-related conditions. This article focuses on novel aspects of the pharmacology and molecular mechanisms of histamine receptors that should be contemplated for optimizing current therapies, repositioning histaminergic ligands for new therapeutic uses, or even including agonists of the histaminergic system in the treatment of different pathologies such as leukemia or neurodegenerative disorders. In recent years, new signaling phenomena related to H 1 and H 2 receptors have been described that make them suitable for novel therapeutic approaches. Crosstalk between histamine receptors and other membrane or nuclear receptors can be envisaged as a way to modulate other signaling pathways and to potentiate the efficacy of drugs acting on different receptors. Likewise, biased signaling at histamine receptors seems to be a pharmacological feature that can be exploited to investigate nontraditional therapeutic uses for H 1 and H 2 biased agonists in malignancies such as acute myeloid leukemia and to avoid undesired side effects when used in standard treatments. It is hoped that the molecular mechanisms discussed in this review contribute to a better understanding of the different aspects involved in histamine receptor pharmacology, which in turn will contribute to increased drug efficacy, avoidance of adverse effects, or repositioning of histaminergic ligands.

The idea of G protein-coupled receptors (GPCRs) coupling to G protein solely in their active form was abolished when it was found that certain ligands induce a G protein-coupled but inactive receptor form. This receptor form interferes with signaling of other receptors by sequestering G protein. However, the spontaneous existence of this receptor species has never been established. The aim of the present work was to evaluate the existence of the spontaneous conformation of the receptor inactively coupled to G protein able to interfere with the response of other GPCRs. According to the law of mass action, receptor overexpression should lead to increased amounts of all spontaneously occurring species. Based on this, we generated Chinese hamster ovary (CHO-K1)-derived cell lines expressing various amounts of the human histamine H2 receptor. In these systems, the signaling of other endogenously and transiently expressed GPCRs was attenuated proportionally to human H2 receptor expression levels. G protein transfection specifically reverted this attenuation, strongly suggesting hijacking of the G protein from a common pool. Similar attenuation effects were observed when the ␤ 2 -adrenergic receptor was overexpressed, suggesting that this is a more general phenomenon. Moreover, in human mammary MDA-MB-231 cells, a consistent increase in the response of other GPCRs was observed when endogenous expression of ␤ 2 -adrenergic receptor was knocked down using specific small interfering RNAs. Our findings show that GPCRs may interact with the signaling of other receptors by modulating the availability of the G protein and suggest the existence of GPCR spontaneous coupling to G proteins in an inactive form. G protein-coupled receptors (GPCRs)2 form a large and functionally diverse superfamily of proteins that transduce signals across cell membranes. Although much is known about structural features of GPCRs involved in ligand recognition and G protein binding, the actual mechanism underlying GPCR signaling remains unclear.Traditionally, agonist occupancy of GPCRs is believed to result in a conformational change in the receptor, leading to activation of G proteins (1). However, in genetically engineered systems where receptors can be expressed at high densities, Costa and Herz (2) noted that high levels of receptor expression uncovered the existence of a population of spontaneously (unliganded) active receptors, resulting in an elevated basal response in the system.The histamine H2 receptor (H2R) is an extensively characterized member of the GPCR family, which in most systems couples to G s proteins to activate adenylyl cyclase (3-6). Compared with other GPCRs, the H2R is unique in that the wildtype receptor possesses a remarkably high degree of constitutive activity. With a receptor density of 300 fmol/mg protein, constitutive H2 receptor activity could be detected in Chinese hamster ovary (CHO-K1) cells (7).The notion that GPCRs also signal without an external chemical trigger, i.e. in a constitutive or spontaneous manner, res...

It is widely assumed that G protein-coupled receptor kinase 2 (GRK2)-mediated specific inhibition of G protein-coupled receptors (GPCRs) response involves GRK-mediated receptor phosphorylation followed by ␤-arrestin binding and subsequent uncoupling from the heterotrimeric G protein. It has recently become evident that GRK2-mediated GPCRs regulation also involves phosphorylation-independent mechanisms. In the present study we investigated whether the histamine H2 receptor (H2R), a G␣ s -coupled GPCR known to be desensitized by GRK2, needs to be phosphorylated for its desensitization and/or internalization and resensitization. For this purpose we evaluated the effect of the phosphorylating-deficient GRK2K220R mutant on H2R signaling in U937, COS7, and HEK293T cells. We found that although this mutant functioned as dominant negative concerning receptor internalization and resensitization, it desensitized H2R signaling in the same degree as the GRK2 wild type. To identify the domains responsible for the kinase-independent receptor desensitization, we co-transfected the receptor with constructions encoding the GRK2 RGS-homology domain (RH) and the RH or the kinase domain fused to the pleckstrinhomology domain. Results demonstrated that the RH domain of GRK2 was sufficient to desensitize the H2R. Moreover, disruption of RGS functions by the use of GRK2D110A/ K220R double mutant, although coimmunoprecipitating with the H2R, reversed GRK2K220R-mediated H2R desensitization. Overall, these results indicate that GRK2 induces desensitization of H2R through a phosphorylation-independent and RGS-dependent mechanism and extends the GRK2 RH domainmediated regulation of GPCRs beyond G␣ q -coupled receptors. On the other hand, GRK2 kinase activity proved to be necessary for receptor internalization and the resulting resensitization.It has been classically described that G protein-coupled receptor (GPCR) 2 desensitization involves rapid receptor phosphorylation by second messenger-dependent kinases and/or G protein-coupled receptor kinases (GRKs). GRK-mediated receptor phosphorylation promotes arrestin binding to the receptor followed by GPCR internalization in clathrin-coated pits. Therefore, the paradigmatic GRK major function is to enable arrestin binding to the receptor and uncoupling from the heterotrimeric G protein (1). However, GPCR signaling may also be specifically attenuated not only at the receptor level but also at the G protein level (2). In recent years, the intracellular regulator of G protein signaling (RGS) proteins have been discovered to serve additional, mostly negative modulatory roles, in G protein-mediated signal transduction (3). More than 30 proteins containing RGS or RGS homology (RH) domains have been described so far (4). Most of these proteins interact with G␣ i or G␣ q subunits, but there is also evidence for the interaction of RGS2 and the truncated form of RGS3 with the G␣ s subunit (5, 6). The seven GRKs proteins described so far contain an N-terminal RGS-homology domain (RH), a catalytic central...

Knowing the importance for research and pharmacological uses of proper ligand classification into agonists, inverse agonists, and antagonists, the aim of this work was to study the behavior of tiotidine, a controversial histamine H2 receptor ligand. We found that tiotidine, described previously as an H2 antagonist, actually behaves as an inverse agonist in U-937 cells, diminishing basal cAMP levels. [3 H]Tiotidine showed two binding sites, one with high affinity and low capacity and the other with low affinity and high capacity. The former site disappeared in the presence of guanosine 5Ј-O-(3-thio)triphosphate, indicating that it belongs to a subset of receptors coupled to G-protein, showing the classic binding profile for an agonist. Considering the occupancy models developed up to now, the only one that explains tiotidine dual behavior is the cubic ternary complex (CTC) model. This model allows Gprotein to interact with the receptor even in the inactive state. We showed by theoretical simulations based on the CTC model of dose-response and binding experiments that tiotidine biases the system to a G-protein-coupled form of the receptor that is unable to evoke a response. This theoretical approach was supported by experimental results in which an unrelated Gprotein-coupled receptor that also signals through G␣ s -protein (␤ 2 -adrenoreceptor) was impeded by tiotidine. This interference clearly implies that tiotidine biases the system to G␣ s -coupled form of the H2 receptor and turns G␣ s -protein less available to interact with ␤ 2 -adrenoreceptor. These findings not only show that tiotidine is an H2 inverse agonist in U-937 cells but also provide experimental support for the CTC model.Receptors coupled to G-proteins (GPCRs) play a major role in signal transduction and are the targets for a large number of therapeutic drugs. Although much is known about signal transduction pathways, the mechanism by which ligands bind to and activate GPCRs remains unclear. In an attempt to understand such mechanisms, several occupancy models have been developed. The first theoretical model of receptor function that included G-proteins was the ternary complex model of De Léan et al. (1980) in which the receptor possesses two binding sites, one for the ligand on the extracellular side and other for the G-protein in the intracellular side. This model is a generalization of former models, in which only the free receptor and the receptor ligand complex existed, allowing

Histamine mediates numerous functions acting through its four receptor subtypes all belonging to the large family of seven transmembrane G-protein coupled receptors. In particular, histamine H2 receptor (H2R) is mainly involved in gastric acid production, becoming a classic pharmacological target to treat Zollinger–Ellison disease and gastric and duodenal ulcers. H2 ligands rank among the most widely prescribed and over the counter-sold drugs in the world. Recent evidence indicate that some H2R ligands display biased agonism, selecting and triggering some, but not all, of the signaling pathways associated to the H2R. The aim of the present work is to study whether famotidine, clinically widespread used ligand acting at H2R, exerts biased signaling. Our findings indicate that while famotidine acts as inverse agonist diminishing cAMP basal levels, it mimics the effects of histamine and the agonist amthamine concerning receptor desensitization and internalization. Moreover, the treatment of HEK293T transfected cells with any of the three ligands lead to a concentration dependent pERK increment. Similarly in AGS gastric epithelial cells, famotidine treatment led to both, the reduction in cAMP levels as well as the increment in ERK phosphorylation, suggesting that this behavior could have pharmacological relevant implications. Based on that, histidine decarboxylase expression was studied by quantitative PCR in AGS cells and its levels were increased by famotidine as well as by histamine and amthamine. In all cases, the positive regulation was impeded by the MEK inhibitor PD98059, indicating that biased signaling toward ERK1/2 pathway is the responsible of such enzyme regulation. These results support that ligand bias is not only a pharmacological curiosity but has physiological and pharmacological implications on cell metabolism.