Pharmacokinetics, oral bioavailability and metabolism of a novel isoquinolinone-based melatonin receptor agonist in rats.

7-Methoxy-6-(3-methoxy-benzyloxy)-2-methylisoquinolin-1(2H)-one (named as IS0042) is a newly identified melatoninergic agonist which exhibits selectivity to the type 2 melatonin receptor. Here, we examined the in vitro and in vivo pharmacokinetics properties of IS0042 in rats. IS0042 was considerably lipophilic with a modest aqueous solubility of 27.3 µg/mL. It was stable in simulated gastrointestinal fluid, and readily penetrated across differentiated Caco-2 cell model of intestinal barrier, suggesting good oral absorption. IS0042 underwent metabolism in rat intestinal and liver microsomes with an in vitro half-life of 367.5 ± 36.6 and 17.5 ± 2.7 min, respectively. Metabolite identification suggested that the major biotransformation pathways included the cleavage of ether bond, hydroxylation and demethylation. The same metabolites were also present in blood circulation following oral administration, indicating a good correlation between in vitro and in vivo metabolism. The pharmacokinetics parameters of IS0042 were evaluated after intravenous administration (10 or 25 mg/kg) and oral administration (100 mg/kg) of the drug to rats. IS0042 showed moderate clearance (0.73-1.02 L/h/kg), large volume of distribution (1.76-3.16 L/kg) and long elimination half-life (3.11-6.04 h) after intravenous administration. The absolute oral bioavailability of IS0042 was relatively low (9.8-18.6%). Overall, these results provide important parameters for the further development of this novel class of melatoninergic ligands.

Astragaloside IV and cycloastragenol stimulate the phosphorylation of extracellular signal-regulated protein kinase in multiple cell types.

Two Chinese herb-derived small molecule telomerase activators, astragaloside IV (AG-IV) and cycloastragenol (CAG), have recently been shown to improve the proliferative response of CD8+ T lymphocytes from HIV-infected patients by upregulating telomerase activity. Here, we examined the signaling mechanism of AG-IV and CAG. Telomerase activity in human embryonic kidney HEK293 fibroblasts was increased upon treatment with increasing concentrations of AG-IV or CAG. Both compounds induced the phosphorylation of extracellular signal-regulated protein kinase (ERK) in a time- and dose-dependent manner in HEK293 cells and HEK-neo keratinocytes. AG-IV and CAG also stimulated ERK phosphorylation in other cell lines of lung, brain, mammary, endothelial, and hematopoietic origins. Use of selective inhibitors and dominant negative mutants revealed the involvement of c-Src, MEK (ERK kinase), and epidermal growth factor receptor in CAG-induced ERK phosphorylation. Our data indicate that AG-IV and CAG may exert their cellular effects through the activation of the Src/MEK/ERK pathway.

3-Methoxylphenylpropyl amides as novel receptor subtype-selective melatoninergic ligands: characterization of physicochemical and pharmacokinetic properties.

Developing subtype-selective melatoninergic ligands has been a subject of considerable interest in drug discovery. A series of 3-methoxyphenylpropyl amide derivatives showing selective binding capacity to type 2 melatonin receptor with subnanomolar range of affinities has been identified recently by our laboratory. In the present study, their physicochemical properties, Caco-2 cell and mdr1-MDCK cell permeability, plasma protein binding, and metabolic stability were investigated. The selected compounds are lipophilic in nature, exhibiting aqueous solubility ranging from 40 to 200 microg/mL. Cell permeability studies on Caco-2 and mdr1-MDCK model revealed that they were readily transported through intestinal epithelium and possessed high penetration potential through blood-brain barrier, implying good oral absorption and central nervous system (CNS) distribution potential. They also showed substantial binding to human plasma protein ranging from 78.5% to 92.3%. These compounds were, however, subjected to rapid cytochrome P450-mediated degradation in rat and human liver microsomes with in vitro half-life of 9.5-31.9 min in rat and 5.5-66.7 min in human, which were much shorter than that of melatonin (approximately 73 min). Metabolite profiling unveiled that C6-ether linkage and methoxy substituents were likely the major metabolic soft spots in their structures, which provided important information for further improvement of their structural stability.

Synthesis of substituted N-[3-(3-methoxyphenyl)propyl] amides as highly potent MT(2)-selective melatonin ligands.

A series of substituted N-[3-(3-methoxyphenyl)propyl] amides were synthesized and their binding affinities towards human melatonin MT(1) and MT(2) receptors were evaluated. It was discovered that a benzyloxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT(2) binding affinity and at the same time decreased MT(1) binding affinity.

A hematopoietic perspective on the promiscuity and specificity of Galpha16 signaling.

Galpha(16), a member of G(q) subfamily, is expressed exclusively in hematopoietic cells, and its expression is highly modulated during lineage differentiation. Although functional redundancy within G(q) subclass members has been observed in many established models, Galpha(16)possesses unique structural and biochemical properties not shared by other family members. Its broad receptor-coupling capacity and unique downstream binding partners and effectors allow the occurrence of both inositol lipid-dependent and -independent signals. Apart from its recognized biological functions in hematopoietic cell responses, the enlistment of complicated signaling pathways further signifies the importance of Galpha(16)in signal integration. This review aims to provide an updated appreciation and rational discussion of Galpha(16) signaling with regard to its promiscuity and specificity.

Replacement of the alpha5 helix of Galpha16 with Galphas-specific sequences enhances promiscuity of Galpha16 toward Gs-coupled receptors.

G(16) can couple indiscriminately to a large number of G protein-coupled receptors (GPCRs), making it a prime candidate as a universal adaptor for GPCRs. In order to increase the promiscuity of Galpha(16), three chimeras incorporating increasing lengths of G(s)-specific residues (25, 44 or 81 residues) into the C-terminus of Galpha(16) were constructed and named 16s25, 16s44 and 16s81, respectively. The chimeras were examined for their ability to mediate receptor-induced stimulation of phospholipase C (PLC) and Ca(2+) mobilization. 16s25 was more effective than 16s44 and 16s81 at coupling to G(s)-linked receptors. 16s25 coupled productively to 10 different G(s)-coupled receptors examined and, for 50% of these receptors, 16s25-mediated PLC activities were higher than those mediated via Galpha(16). Similar results were observed for agonist-induced Ca(2+) mobilizations. These results show that incorporating the alpha5 helix of Galpha(s) into Galpha(16) can increase the promiscuity of 16s25 towards G(s)-coupled receptors.

Identification of a stretch of six divergent amino acids on the alpha5 helix of Galpha16 as a major determinant of the promiscuity and efficiency of receptor coupling.

A broad repertory of G-protein-coupled receptors shows effective coupling with the haematopoietic G16 protein. In the present study, individual residues along the C-terminal alpha5 helix of Galpha16 were examined for their contributions in defining receptor-coupling specificity. Residues that are relatively conserved within, but diverse between, the subfamilies of cloned Galpha subunits were mutated into the corresponding Galpha(z) residues. Six G(i)-linked receptors with different coupling efficiencies to Galpha16 were examined for their ability to utilize the various Galpha16 mutants to mediate agonist-induced inositol phosphate accumulation and Ca2+ mobilization. Co-operative enhancements of receptor coupling were observed with chimaeras harbouring multiple mutations at Glu350, Lys357 and Leu364 of Galpha16. Mutation of Leu364 into isoleucine appeared to be more efficient in enhancing receptor recognition compared with mutations at the other two sites. Mutation of a stretch of six consecutive residues (362-367) lying towards the end of the alpha5 helix was found to broaden significantly the receptor-coupling profile of Galpha16, and the effect was mediated partly through interactions with the beta2-beta3 loop. These results suggested that a stretch of six distinctive residues at the alpha5 helix of Galpha16 is particularly important, whereas other discrete residues spreading along the alpha5 helix function co-operatively for determining the specificity of receptor recognition.

Galpha(16/z) chimeras efficiently link a wide range of G protein-coupled receptors to calcium mobilization.

G protein-coupled receptors (GPCRs) represent a class of important therapeutic targets for drug discovery. The integration of GPCRs into contemporary high-throughput functional assays is critically dependent on the presence of appropriate G proteins. Given that different GPCRs can discriminate against distinct G proteins, a universal G protein adapter is extremely desirable. In this report, the authors evaluated two highly promiscuous Galpha(16/z) chimeras, 16z25 and 16z44, for their ability to translate GPCR activation into Ca(2+) mobilization using the fluorescence imaging plate reader (FLIPR) and aequorin. A panel of 24 G(s)- or G(i)-coupled receptors was examined for their functional association with the Galpha(16/z) chimeras. Although most of the GPCRs tested were incapable of inducing Ca(2+) mobilization upon their activation by specific agonists, the introduction of 16z25 or 16z44 allowed all of these GPCRs to mediate agonist-induced Ca(2+) mobilization. In contrast, only 16 of the GPCRs tested were capable of using Galpha(16) to mobilize intracellular Ca(2+). Analysis of dose-response curves obtained with the delta-opioid, dopamine D(1), and Xenopus melatonin Mel1c receptors revealed that the Galpha(16/z) chimeras possess better sensitivity than Galpha(16) in both the FLIPR and aequorin assays. Collectively, these studies help to validate the promiscuity of the Galpha(16/z) chimeras as well as their application in contemporary drug-screening assays that are based on ligand-induced Ca(2+) mobilization.

The beta6/alpha5 regions of Galphai2 and GalphaoA increase the promiscuity of Galpha16 but are insufficient for pertussis toxin-catalyzed ADP-ribosylation.

Replacement of beta6/alpha5 region at the C-terminus on Galpha16 with Galphaz-specific residues has been shown to broaden the promiscuity of Galpha16. Here, we substituted the last 44 residues of Galpha16 with the corresponding region from either Galphai2 or GalphaoA (16i44 and 16o44). 16i44 and 16o44 chimeras were more effective than Galpha16 at coupling to Gi-linked delta-opioid, mu-opioid, and Xenopus melatonin MT1c receptors when coexpressed in green monkey fibroblast (COS-7) cells. 16i44, but not 16o44, also enhanced the formyl peptide-induced stimulation of phospholipase C activity. Both chimeras were resistant to pertussis toxin-catalyzed [32P]ADP-ribosylation, despite the fact that pertussis toxin partially inhibited the chimera-mediated stimulation of phospholipase Cbeta. The use of Galphat1 as a Gbetagamma scavenger revealed that the pertussis toxin-sensitivity can be attributed to endogenous Gbetagamma subunits released from G(i/o). Although incorporation of a Galphai-like beta6/alpha5 region into the C-terminus of Galpha16 increases its promiscuity, this region is not sufficient to support recognition by pertussis toxin.

Synthesis and functional characterization of substituted isoquinolinones as MT2-selective melatoninergic ligands.

A series of substituted isoquinolinones were synthesized and their binding affinities and functional activities towards human melatonin MT1 and MT2 receptors were evaluated. Structure-activity relationship analysis revealed that substituted isoquinolinones bearing a 3-methoxybenzyloxyl group at C5, C6 or C7 position respectively (C5>C6>C7 in terms of their potency) conferred effective binding and selectivity toward the MT2 receptor, with 15b as the most potent compound. Most of the tested compounds were MT2-selective agonists as revealed in receptor-mediated cAMP inhibition, intracellular Ca2+ mobilization and phosphorylation of extracellular signal-regulated protein kinases. Intriguingly, compounds 7e and 7f bearing a 4-methoxybenzyloxyl group or 4-methylbenzyloxyl at C6 behaved as weak MT2-selective antagonists. These results suggest that substituted isoquinolinones represent a novel family of MT2-selective melatonin ligands. The position of the substituted benzyloxyl group, and the substituents on the benzyl ring appeared to dictate the functional characteristics of these compounds.

Characterization of substituted phenylpropylamides as highly selective agonists at the melatonin MT2 receptor.

Melatonin is a widely distributed hormone that regulates several major physiological processes, including the circadian rhythm and seasonal adaptation. The two subtypes of mammalian G protein-coupled melatonin receptors are primarily responsible for mediating the actions of melatonin. Because synthetic melatonin agonists have considerable therapeutic potentials in modulating insomnia and circadian- related sleep disorders, it is highly desirable to develop subtype-selective melatoninergic compounds. The pharmacological potencies of a series of substituted N-[3-(3-methoxyphenyl)propyl] amides towards human melatonin MT(1) and MT(2) receptors were evaluated by the FLIPR high-throughput screening assay, whilst their subtype-selectivity was subsequently verified with ERK phosphorylation and cAMP assays. Structure-activity relationship analysis of highly potent subtype-selective ligands (MT(2) EC(50) 10-90 pM) revealed that a benzyloxyl substituent incorporated at C6 position of the 3-methoxyphenyl ring dramatically enhanced the MT(2) potency and at the same time decreased MT(1) potency. Incorporation of structural moieties conferring the subtype selectivity produced several extremely potent MT(2)-selective ligands. The most potent subtype-selective ligand, 2q had a substantially higher potency for MT(2) receptor than melatonin for elevation of [Ca(2+)]i and inhibition of forskolin-elevated cAMP. Representative MT(2)-selective ligands also induced ERK phosphorylation in both recombinant and native cell lines, and no cross-reactivity to 17 other GPCRs could be detected. These ligands represent invaluable tools for delineating the functional roles of distinct melatonin receptor subtypes and are viable candidates for drug development.

Expression of Gα(z) in C2C12 cells restrains myogenic differentiation.

The recent identification of Gα(z) expression in C2C12 myoblasts and its demonstrated interaction with the transcription factor Eya2 inferred an unanticipated role of Gα(z) in muscle development. In the present study, endogenous Gα(z) mRNA and protein expressions in C2C12 cells increased upon commencement of myogenesis and peaked at around 4-6days after induction but were undetectable in adult skeletal muscle. Surprisingly, stable expression of recombinant Gα(z) in C2C12 myoblasts strongly suppressed myotube formation upon serum deprivation, and the constitutively active mutant Gα(z)QL exerted more pronounced effects. Transcriptional activities of reporter genes responsive to early (MyoD, MEF2 and myogenin) and late (muscle creatine kinase and myosin heavy chain) myogenic markers were reduced by transiently expressed Gα(z)QL. Membrane attachment of Gα(z) was apparently required for the suppressive effects because a fatty acylation-deficient Gα(z) mutant could not inhibit myogenin expression. Introduction of siRNA against Gα(z) enhanced myogenin-driven luciferase activity and increased myosin heavy chain expression. Immunostaining of C2C12 cells over-expressing Gα(z) showed delayed nuclear expression of myogenin and severe myotube deformation. Gα(z) expression was accompanied by reduced levels of Rock2, RhoA and RhoGAP, enhanced expression of Rnd3, and a reduction of serum-responsive factor-driven reporter activity. These results support a novel role of Gα(z) in restraining myogenic differentiation through the disruption of Rho signaling.

Galpha16 activates Ras by forming a complex with tetratricopeptide repeat 1 (TPR1) and Son of Sevenless (SOS).

Many G protein-coupled receptors (GPCRs) are known to modulate cell growth and differentiation by stimulating the extracellular signal-regulated protein kinases (ERKs). In growth factor signaling, ERKs are typically stimulated through an elaborate network of modules consisting of adaptors, protein kinases, and the small GTPase Ras. The mechanism by which G protein signals tap into the ERK signaling pathway has thus far remain elusive. Members of the Gq family of G proteins, in particular Galpha16, have been shown to associate with tetratricopeptide repeat 1 (TPR1), an adaptor protein which preferentially binds to Ras. Here, we examined if TPR1 is indeed the missing link between Galpha16 signaling and Ras activation. Expression of Galpha16QL, a constitutively active mutant of Galpha16, in HEK 293 cells led to the formation of GTP-bound Ras and the subsequent phosphorylation of ERK. Likewise, stimulation of endogenou G16-coupled CCR1 chemokine receptors produced the same responses in human erythroleukemia cells. siRNA-mediated knockdown of TPR1 or expression of a dominant negative mutant of TPR1 effectively abolished the ability of Galpha16QL to induce Ras activation in HEK 293 cells. In contrast, these manipulations had no inhibitory effect on Galpha16QL induced activation of phospholipase Cbeta. Galpha16QL-induced phosphorylations of downstream targets including ERK, signal transducer and activator of transcription 3, and IkappaB kinase were significantly suppressed upon expression of the dominant negative mutant of TPR1. Furthermore, SOS2, a Ras guanine nucleotide exchange factor, was found to form a complex with TPR1 and Galpha16QL. Expression of SOS2 enhanced Galpha16QL-induced Ras activation and its subsequent signaling. Collectively, our results suggest that Galpha16 regulates multiple signaling pathways by activating Ras through its association with TPR1, but TPR1 is not required for Galpha16 to stimulate phospholipase Cbeta.

In vitro intestinal absorption and first-pass intestinal and hepatic metabolism of cycloastragenol, a potent small molecule telomerase activator.

Cycloastragenol (CAG) is the aglycone derivative of astragaloside IV which has recently been demonstrated to activate telomerase and represents a potential drug candidate for the treatment of degenerative diseases. In the present study, intestinal absorption and metabolism of CAG were examined using the Caco-2 model and liver microsomes, respectively. The results showed that CAG rapidly passes through the Caco-2 cell monolayer by passive diffusion. Four different glucuronide conjugates and two oxidized CAG metabolites were found in the apical and basolateral sides of Caco-2 monolayer, suggesting that first-pass intestinal metabolism of CAG might occur upon passage through the intestinal epithelium. CAG underwent extensive metabolism in rat and human liver microsomes with only 17.4% and 8.2%, respectively, of the starting amount of CAG remaining after 30 min of incubation. Monohydroxylation of the parent and oxidization of the hydroxylated CAG were found in the liver samples. The present study indicates that CAG is efficiently absorbed through intestinal epithelium. However, extensive first-pass hepatic metabolism would limit the oral bioavailability of this compound.

In search of novel and therapeutically significant melatoninergic ligands.

Melatonin, the pineal gland hormone, is widely distributed in mammalian tissues and exerts its action via two melatonin receptor sub-types, MT(1) and MT(2). Melatonin is known to play functional roles in regulating circadian rhythms and seasonal reproduction. In recent years, growing evidence has also linked melatonin to a variety of other body systems and disease states, thus highlighting its significance as a therapeutic agent. However, due to its properties, melatonin is ineffective in clinical use, thus prompting the development of melatoninergic ligands that mimic the actions of melatonin but in a manner that is more potent and specific for melatonin receptors. An additional focus has been to develop ligands that exhibit receptor subtype selectivity. While there are over seventy patents on melatoninergic ligands, success in developing therapeutically effective melatoninergic ligands have been varied. However, the recent approval of Ramelteon for treatment of sleep disorders and the evaluation of other compounds in clinical trials have highlighted their clinical importance. In this review an overview of recently developed novel melatoninergic ligands is provided including recently filed patents and compounds undergoing clinical evaluation.

Co-expressions of different opioid receptor types differentially modulate their signaling via G(16).

Combinations of two different types of opioid receptors - delta-, kappa-, mu-opioid receptors (DOR, KOR, and MOR) and opioid receptor-like receptor 1 (ORL(1)) - were co-expressed with the alpha subunit of G(16) in COS-7 cells, and the ability of various selective agonists to induce activation of phospholipase Cbeta was examined. Nociceptin/orphanin FQ-induced response was enhanced when ORL(1) was co-expressed with MOR or KOR but not DOR. The kappa-agonist U50,488H induced a modest inositol phosphate formation when KOR was expressed alone or with MOR, but the response was attenuated when co-expressing with either DOR or ORL(1). It is suggested that the co-expressions of two different opioid receptor types indeed modify their downstream signaling events.