Big Data Challenges Targeting Proteins in GPCR Signaling Pathways; Combining PTML-ChEMBL Models and [35S]GTPγS Binding Assays

Forty-two extracts of folk medicinal plant organs from Pakistan were tested in competition binding assays for their interaction with the specific ligand recognition sites on the human α2-adrenoceptor subtypes α2A, α2B and α2C. Strong binding of the extracts (40 mg/ml) from Acacia nilotica (L.) Delile leaves (88-98% displacement of radiolabel) and Peganum harmala seeds (89-96% displacement) on three subtypes prompted us to extract these plant materials with 40% and 80% methanol, ethanol, and acetone. The extraction results indicated an absence of α2-adrenoceptor binding activity in the stalk of A. nilotica and A. tortils, whereas the leaves of both plants contained activity. The extracts of A. nilotica leaves showed a slight, but consistent, preference for the α2C-adrenoceptor, whereas the leaves of A. tortils were slightly more active on the α2B subtype. The extract of P. harmala stalks was less active than that of its seeds. The binding activities of A. nilotica leaves and P. harmala seeds were mainly concentrated in the water and 30% methanol fractions and further sub-fractions. In a functional activity assay, the active fractions inhibited epinephrine-stimulated 35S-GTPγS binding, thus indicating a predominantly antagonistic nature of the compounds with α2-adrenoceptor affinity in these fractions. Among the known major alkaloids of P. harmala (demissidine, harmaline, harmine, 6-methoxyharmalan, and norharmane), only 6-methoxyharmalan showed moderate affinity (dissociation constant (Ki) of 530 ± 40 nᴍ for α2A subtype). This study is a first systematic attempt towards the discovery of potential drug candidates from these plant materials for treating α2-adrenoceptor related diseases

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Potential Utility of Biased GPCR Signaling for Treatment of Psychiatric Disorders

International Journal of Molecular Sciences ◽

10.3390/ijms20133207 ◽

2019 ◽

Vol 20 (13) ◽

pp. 3207 ◽

Cited By ~ 6

Author(s):

Hidetoshi Komatsu ◽

Mamoru Fukuchi ◽

Yugo Habata

Keyword(s):

Psychiatric Disorders ◽

Signaling Pathways ◽

Brain Function ◽

Marked Reduction ◽

G Protein Coupled Receptors ◽

Detailed Understanding ◽

Gpcr Signaling ◽

Downstream Signaling ◽

Multiple Signaling ◽

G Protein Coupled

Tremendous advances have been made recently in the identification of genes and signaling pathways associated with the risks for psychiatric disorders such as schizophrenia and bipolar disorder. However, there has been a marked reduction in the pipeline for the development of new psychiatric drugs worldwide, mainly due to the complex causes that underlie these disorders. G-protein coupled receptors (GPCRs) are the most common targets of antipsychotics such as quetiapine and aripiprazole, and play pivotal roles in controlling brain function by regulating multiple downstream signaling pathways. Progress in our understanding of GPCR signaling has opened new possibilities for selective drug development. A key finding has been provided by the concept of biased ligands, which modulate some, but not all, of a given receptor’s downstream signaling pathways. Application of this concept raises the possibility that the biased ligands can provide therapeutically desirable outcomes with fewer side effects. Instead, this application will require a detailed understanding of the mode of action of antipsychotics that drive distinct pharmacologies. We review our current understanding of the mechanistic bases for multiple signaling modes by antipsychotics and the potential of the biased modulators to treat mental disorders.

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A Crucial Role for Gαq/11, But Not Gαi/o or Gαs, in Gonadotropin-Releasing Hormone Receptor-Mediated Cell Growth Inhibition

Molecular Endocrinology ◽

10.1210/me.2008-0122 ◽

2008 ◽

Vol 22 (11) ◽

pp. 2520-2530 ◽

Cited By ~ 18

Author(s):

Colin D. White ◽

Marla Coetsee ◽

Kevin Morgan ◽

Colleen A. Flanagan ◽

Robert P. Millar ◽

...

Keyword(s):

Cell Growth ◽

Crucial Role ◽

Direct Evidence ◽

Gnrh Receptor ◽

Binding Assays ◽

Erk Activation ◽

Cellular Effects ◽

Mapk Cascades ◽

Gtpγs Binding ◽

Gonadotropin Releasing Hormone Receptor

Abstract GnRH acts on its cognate receptor in pituitary gonadotropes to regulate the biosynthesis and secretion of gonadotropins. It may also have direct extrapituitary actions, including inhibition of cell growth in reproductive malignancies, in which GnRH activation of the MAPK cascades is thought to play a pivotal role. In extrapituitary tissues, GnRH receptor signaling has been postulated to involve coupling of the receptor to different G proteins. We examined the ability of the GnRH receptor to couple directly to Gαq/11, Gαi/o, and Gαs, their roles in the activation of the MAPK cascades, and the subsequent cellular effects. We show that in Gαq/11-negative cells stably expressing the GnRH receptor, GnRH did not induce activation of ERK, jun-N-terminal kinase, or P38 MAPK. In contrast to Gαi or chimeric Gαqi5, transfection of Gαq cDNA enabled GnRH to induce phosphorylation of ERK, jun-N-terminal kinase, and P38. Furthermore, no GnRH-mediated cAMP response or inhibition of isoproterenol-induced cAMP accumulation was observed. In another cellular background, [35S]GTPγS binding assays confirmed that the GnRH receptor was unable to directly couple to Gαi but could directly interact with Gαq/11. Interestingly, GnRH stimulated a marked reduction in cell growth only in cells expressing Gαq, and this inhibition could be significantly rescued by blocking ERK activation. We therefore provide direct evidence, in multiple cellular backgrounds, that coupling of the GnRH receptor to Gαq/11, but not to Gαi/o or Gαs, and consequent activation of ERK plays a crucial role in GnRH-mediated cell death.

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Comprehensive identification of signaling pathways for idiopathic pulmonary arterial hypertension

AJP Cell Physiology ◽

10.1152/ajpcell.00382.2019 ◽

2020 ◽

Vol 318 (5) ◽

pp. C913-C930

Author(s):

Bingxun Liu ◽

Liping Zhu ◽

Ping Yuan ◽

Glenn Marsboom ◽

Zhigang Hong ◽

...

Keyword(s):

Pulmonary Arterial Hypertension ◽

Arterial Hypertension ◽

Intracellular Calcium ◽

Signaling Pathways ◽

Signaling Pathway ◽

Cell Function ◽

Idiopathic Pulmonary Arterial Hypertension ◽

Channel Blockers ◽

Gpcr Signaling ◽

Pulmonary Arterial

Whole exome sequencing (WES) was used in the research of familial pulmonary arterial hypertension (FPAH). CAV1 and KCNK3 were found as two novel candidate genes of FPAH. However, few pathogenic genes were identified in idiopathic pulmonary arterial hypertension (IPAH). We conducted WES in 20 unrelated IPAH patients who did not carry the known PAH-pathogenic variants among BMPR2, CAV1, KCNK3, SMAD9, ALK1, and ENG. We found a total of 4,950 variants in 3,534 genes, including 4,444 single-nucleotide polymorphisms and 506 insertions/deletions (InDels). Through the comprehensive and multilevel analysis, we disclosed several novel signaling cascades significantly connected to IPAH, including variants related to cadherin signaling pathway, dilated cardiomyopathy, glucose metabolism, immune response, mucin-type O-glycosylation, phospholipase C (PLC)-activating G protein-coupled receptor (GPCR) signaling pathway, vascular contraction and generation, and voltage-dependent Ca2+ channels. We also conducted validation studies in five mutant genes related to PLC-activating GPCR signaling pathway potentially involved in intracellular calcium regulation through Sanger sequencing for mutation accuracy, qRT-PCR for mRNA stability, immunofluorescence for subcellular localization, Western blotting for protein level, Fura-2 imaging for intracellular calcium, and proliferation analysis for cell function. The validation experiments showed that those variants in CCR5 and C3AR1 significantly increased the rise of intracellular calcium and the variant in CCR5 profoundly enhanced proliferative capacity of human pulmonary artery smooth muscle cells. Thus, our study suggests that multiple genetically affected signaling pathways take effect together to cause the formation of IPAH and the development of right heart failure and may further provide new therapy targets or putative clues for the present treatments such as limited therapeutic effectiveness of Ca2+ channel blockers.

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Comparative Analysis of Functional Assays for Characterization of Agonist Ligands at G Protein-Coupled Receptors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057103257555 ◽

2003 ◽

Vol 8 (5) ◽

pp. 500-510 ◽

Cited By ~ 22

Author(s):

Anke Niedernberg ◽

Sorin Tunaru ◽

Andree Blaukat ◽

Bruce Harris ◽

Evi Kostenis

Keyword(s):

G Protein ◽

Partial Agonist ◽

A Priori ◽

G Protein Coupled Receptors ◽

Binding Assays ◽

Functional Assays ◽

Gtpγs Binding ◽

Sphingosine 1 Phosphate ◽

Intracellular Ca ◽

G Protein Coupled

A variety of functional assays are available for agonist or antagonist screening of G protein-coupled receptors (GPCRs), but it is a priori not predictable which assay is the most suitable to identify agonists or antagonists of GPCRs with therapeutic value in humans. More specifically, it is not known how a given set of GPCR agonists compares in different functional assays with respect to potency and efficacy and whether the level of the signaling cascade that is analyzed has any impact on the detection of agonistic responses. To address this question, the authors used the recently cloned human S1P5 receptor as a model and compared a set of 3 lipid ligands (sphingosine 1-phosphate [S1P], dihydro sphingosine 1-phosphate [dhS1P], and sphingosine) in 5 different functional assays: GTPγS binding, inhibition of adenylyl cyclase activity, mobilization of intracellular Ca2+ via the FLIPR and aequorin technology, and MAP kinase (ERK1/2) activation. S1P induced agonistic responses in all except the ERK1/2 assays with EC50 values varying by a factor of 10. Whereas dhS1P was identified as a partial agonist in the GTPγS assay, it behaved as a full agonist in all other settings. Sphingosine displayed partial agonistic activity exclusively in GTPγS binding assays. The findings suggest that assays in a given cellular background may vary significantly with respect to suitability for agonist finding and that ligands producing a response may not readily be detectable in all agonist assays. ( Journal of Biomolecular Screening 2003:500-510)

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