published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

Class B GPCRs are emerging target in drug research. Currently these receptors serve as drug targets for several drug discovery companies and more than 50 percent of the drugs in the market targets GPCRs. Secretin receptor is found to be expressed in various tissues. Secretin regulates many bodily functions from energy to water homeostasis through both central and peripheral system. Though it holds a history of 100 years, the major drawback is its structural insights. In evidence of its integrated role in physiology as a potential target, the lookout for a novel agonist and / or antagonist for secretin receptor is initiated. As this target is in the primary state of drug research, it is also necessary to develop the appropriate screening platforms. Due to the lack of experimental structure of secretin receptor-ligand, a 3D virtual homology model is developed using multiple template approach. Besides virtual docking, a non-radioactive FRET competitive binding assay is also developed and substantiated to enable the receptor-ligand interaction studies. Both peptide and non-peptide analogs were screened for virtual docking, in vitro binding and functional response. For the peptide analogs, the modifications were made either in the N or C terminal portion of the peptide based on the previous findings that C-terminal portion is involved in receptor binding followed by allosteric modifications and N-terminal portion is involved in activation. These peptide analogs exhibited binding affinity in the virtual model. Paradoxically it did not exhibit in vitro binding as predicted. Along with this, the agonistic and antagonistic functional responses of these peptide analogs were also found to be negative. SPECS natural product library of 500 non-peptide analogs were screened virtually against secretin receptor and 32 hits were identified. Of these hits glycyrrhizin’s functions were comparable to secretin was screened for receptor binding and functional response. These in vitro assays did not exert anything positive; however an IP-GTT on WT, 〖SCT〗^(-/- )and 〖SCTR〗^(-/-) mice with acute treatment of glycyrrhizin at 10 mg/kg and chronic treatment of 5 mg/kg exhibited an interesting profile with negligible effect on 〖SCT〗^(-/- )mice whereas in WT and 〖SCTR〗^(-/-) mice it displayed a better profile with improved glucose tolerance. The chronic study serum analysis on day 28 exhibited substantial reduction in blood glucose while significant increase in serum secretin and insulin levels. As glycyrrhizin promotes secretin secretion, its acute effect on blood pressure in WT mice was also analyzed at 10 mg/kg; remarkably exhibited a significant drop in blood pressure. In summary modifications in the peptide analogs lead to instability in the receptor-ligand binding complex in the in vitro system leading to loss of binding efficiency. In case of non-peptides, though glycyrrhizin could not exhibit in vitro response, its supplementary mechanism through secretin pathway of increased secretin release is confirmed using the WT, 〖SCT〗^(-/- )and 〖SCTR〗^(-/-) mice. The hypotensive effect with an acute treatment in WT is also revealed. Discovery of this new mechanism of an old drug could broaden the research for a new class of drug, “secretin sensitizers / promoters”. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Osmoregulation is critical to life and is tightly regulated by integrated physiological and behavioral responses to maintain the osmolality of body fluid. In particular, this involves recovery from dehydration both at the intracellular and extracellular levels. To achieve appropriate body fluid balance, three major hormones namely secretin (SCT), angiotensin II (ANGII) and vasopressin (VP) are responsible. Of note, SCT and ANGII share overlapping physiological roles including similar expression pattern within the brain, dipsogenic actions and activation of VP expression and/or release in mice. However, it remains unclear how their receptor pathways may cross-interact to aid osmoregulation. In recent years, G protein-coupled receptor (GPCR) oligomerization has been implicated to play roles in regulating processes such as expression, pharmacological diversity, signal transduction and internalization. Though not as extensively studied, class B GPCRs are also gaining merit in their oligomerization abilities, within which the wealth of available information is focused on SCT receptor (SCTR) homomers and heteromers. Moreover, there is also evidence indicating the ability for ANGII receptors to oligomerize. On the basis of this information, this project predominantly aims to explore the molecular association between SCTR and ANGII receptors via in vitro experiments and provide insights into its physiological relevance. In this study, bioluminescence resonance energy transfer (BRET) assays revealed SCTR and ANGII type 1a receptor (AT1aR) to form hetero-complexes. This oligomerization event was found by BRET competition to be contributed predominantly by transmembrane (TM) domain regions 2 and 4 in SCTR, and TM1 and TM4 in AT1aR. Within which, combinational use of mutant TM peptides and SCTR chimeras revealed the importance of lipid-exposed residues, particularly Leu204 and Ser205 in SCTR TM2 as key contact points for formation of the SCTR/AT1aR complex. Morphologically, the heteromers were visualized by confocal FRET imaging at the cell surface and found have a role in modulating AT1aR trafficking. It was also found that the SCTR/AT1aR complex affected Gαs signaling specifically, reducing maximal response values by 24.3 ± 2.8 % compared to CHO-K1 cells transfected with only SCTR. While, this negative effect could be abolished by co-application of SCT and ANGII peptides, use of constitutively active AT1aR mutants or disruption of the hetero-complex using SCTR mutants. Taken together, the SCTR/AT1aR complex was proposed to impose conformational restraints on the SCTR that could be overcome upon activation of the AT1aR. Physiologically, hyperosmolality isovolemic induced drinking could be attenuated by central administration of TM peptides and the protein kinase A pathway blocker H-89, indicating receptor oligomerization to have a role in neural osmoregulation via a Gαs dependent pathway. This study presents novel findings regarding the receptor oligomerization of SCTR and AT1aR, which may be the molecular basis to the overlapping roles of SCT and ANGII in water homeostasis. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

 As a neuropeptide, both secretin and secretin receptor are expressed in the central nervous system (CNS). It has been revealed that the activities of secretin on hypothalamic cells of rodents are important for osmoregulation and food intake. In the present study, embryonic mouse hypothalamic cell line N42 was used to study the promoter activity of mouse secretin receptor (mSR). By 5′ deletion analysis, a promoter element was identified within ?282 to ?443, relative to the ATG codon, and it contains a GC-box (-297 to -286), a ras responsive element (RRE) (-289 to -276) and an E-box (-416 to -411). Electrophoretic mobility shift assay (EMSA) and supershift analyses showed that Sp1 interacted with the GC-box, another zinc finger As a neuropeptide, both secretin and secretin receptor are expressed in the central nervous system (CNS). It has been revealed that the activities of secretin on hypothalamic cells of rodents are important for osmoregulation and food intake. In the present study, embryonic mouse hypothalamic cell line N42 was used to study the promoter activity of mouse secretin receptor (mSR). By 5′ deletion analysis, a promoter element was identified within ?282 to ?443, relative to the ATG codon, and it contains a GC-box (-297 to -286), a ras responsive element (RRE) (-289 to -276) and an E-box (-416 to -411). Electrophoretic mobility shift assay (EMSA) and supershift analyses showed that Sp1 interacted with the GC-box, another zinc finger / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy