{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/374181"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/374181","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Exploring the molecular signatures of the ligand-receptor-Gi/o protein interactions of adenosine A1 and A3 receptors","abstract":"Human adenosine A1 receptor (hA1R) and adenosine A3 receptors (hA3R) are two closely related class A Gi/o-coupled receptors. Both of them represented valuable therapeutic targets and various types of ligands have been developed for regulating their activities. hA1R has important implications in pain regulation, epilepsy, neurodegenerative diseases and the cardiorespiratory system, while hA3R is a crucial player in neuropathic pain, immune diseases and tumor progression. The first part of this thesis was to investigate the receptor-ligand interactions between the hA1R/hA3R and their potential antagonists. Based on previous hit compounds K18 and A17, new heterocyclic carbonyloxycarboximidamides-based derivatives and pyrazolo[3,4-c] pyridine analogues were synthesised and characterised. Through the profiling of their binding affinity, binding kinetics and subtype selectivity, several high-affinity candidates with about 10-fold improvement in affinity from their precursors were identified as hA3R-selective (e.g., 39), hA1R-selective (e.g., R1) or dual hA1R/hA3R antagonists (e.g., R8). Also, the binding pocket of 39 at hA3R was explored through extensive mutagenesis studies and the key molecular signatures accounting for the improved affinity of 39 were decoded. These new lead compounds represented excellent hAR antagonist candidates for investigating the structural selectivity filers in the orthosteric binding pockets of hA1R and hA3R as well as for future drug development for treating diseases such as heart failure, asthma and cancer. Following the exploration of the interactions between antagonists and hA1R/hA3R, the actions of agonists in activating the hA1R/hA3R were measured at the level of the activation of individual G proteins. Being primarily coupled to the inhibitory G proteins, hA1R/hA3R showed little activation at the non-inhibitory G proteins (Gss, GsL, Gq, G11, G15, G12 and G13). It has been discovered in this part of the thesis that hA1R/hA3R showed intrinsic receptor biases within the inhibitory G protein family, where hA1R activates all the Gi/o proteins while hA3R preferentially activates Gi1, Gi2 and Gi3 over Goa, Gob and Gz in the presence of agonists. Also, hA1R showed much higher constitutive activity than hA3R in activating all six inhibitory G proteins. To investigate the structural mechanisms underlying this differential preference in activating the Gi/o proteins between hA1R and hA3R, the roles of 32 non-conserved positions in hA1R/hA3R which could interact with the G proteins were investigated. These positions were reciprocally switched between hA1R and hA3R in mutagenesis and the resulted mutants were tested in their Gi/o protein activation level. Several key positions were found contributing to the complicated networks for regulating the levels of Gi/o protein activation by hA1R and hA3R. For example, it was shown that the alanine4.42, glutamine5.68, isoleucine6.33 and glutamine8.48 in hA1R were crucial in maintaining the high constitutive activity in activating Gi/o proteins through different structural mechanisms. For hA3R, asparagine5.72, glutamic acid6.22 and threonine6.33 prevented the wild type hA3R from activating the GoA and GoB while the valine34.51 and phenylalanine6.31 formed part of the barriers for hA3R to activate Gz. This throughout mutagenesis study has provided a solid foundation for understanding the structural dynamics and activation mechanisms in the hA1R/hA3R-Gi/o protein interaction network. In summary, this thesis has included the identification and characterisation of the key molecular signatures involved in the dynamical interactions between orthosteric ligands with hA1R/hA3R as well as between the hA1R/hA3R and the inhibitory G proteins. The understandings of these interactions have important implications in the future design and development of high-affinity selective AR ligands with desired Gi/o protein biases targeting the therapeutically beneficial downstream pathways specifically.","abstract_html":"Human adenosine A1 receptor (hA1R) and adenosine A3 receptors (hA3R) are two closely related class A Gi/o-coupled receptors. Both of them represented valuable therapeutic targets and various types of ligands have been developed for regulating their activities. hA1R has important implications in pain regulation, epilepsy, neurodegenerative diseases and the cardiorespiratory system, while hA3R is a crucial player in neuropathic pain, immune diseases and tumor progression. The first part of this thesis was to investigate the receptor-ligand interactions between the hA1R/hA3R and their potential antagonists. Based on previous hit compounds K18 and A17, new heterocyclic carbonyloxycarboximidamides-based derivatives and pyrazolo[3,4-c] pyridine analogues were synthesised and characterised. Through the profiling of their binding affinity, binding kinetics and subtype selectivity, several high-affinity candidates with about 10-fold improvement in affinity from their precursors were identified as hA3R-selective (e.g., 39), hA1R-selective (e.g., R1) or dual hA1R/hA3R antagonists (e.g., R8). Also, the binding pocket of 39 at hA3R was explored through extensive mutagenesis studies and the key molecular signatures accounting for the improved affinity of 39 were decoded. These new lead compounds represented excellent hAR antagonist candidates for investigating the structural selectivity filers in the orthosteric binding pockets of hA1R and hA3R as well as for future drug development for treating diseases such as heart failure, asthma and cancer. Following the exploration of the interactions between antagonists and hA1R/hA3R, the actions of agonists in activating the hA1R/hA3R were measured at the level of the activation of individual G proteins. Being primarily coupled to the inhibitory G proteins, hA1R/hA3R showed little activation at the non-inhibitory G proteins (Gss, GsL, Gq, G11, G15, G12 and G13). It has been discovered in this part of the thesis that hA1R/hA3R showed intrinsic receptor biases within the inhibitory G protein family, where hA1R activates all the Gi/o proteins while hA3R preferentially activates Gi1, Gi2 and Gi3 over Goa, Gob and Gz in the presence of agonists. Also, hA1R showed much higher constitutive activity than hA3R in activating all six inhibitory G proteins. To investigate the structural mechanisms underlying this differential preference in activating the Gi/o proteins between hA1R and hA3R, the roles of 32 non-conserved positions in hA1R/hA3R which could interact with the G proteins were investigated. These positions were reciprocally switched between hA1R and hA3R in mutagenesis and the resulted mutants were tested in their Gi/o protein activation level. Several key positions were found contributing to the complicated networks for regulating the levels of Gi/o protein activation by hA1R and hA3R. For example, it was shown that the alanine4.42, glutamine5.68, isoleucine6.33 and glutamine8.48 in hA1R were crucial in maintaining the high constitutive activity in activating Gi/o proteins through different structural mechanisms. For hA3R, asparagine5.72, glutamic acid6.22 and threonine6.33 prevented the wild type hA3R from activating the GoA and GoB while the valine34.51 and phenylalanine6.31 formed part of the barriers for hA3R to activate Gz. This throughout mutagenesis study has provided a solid foundation for understanding the structural dynamics and activation mechanisms in the hA1R/hA3R-Gi/o protein interaction network. In summary, this thesis has included the identification and characterisation of the key molecular signatures involved in the dynamical interactions between orthosteric ligands with hA1R/hA3R as well as between the hA1R/hA3R and the inhibitory G proteins. The understandings of these interactions have important implications in the future design and development of high-affinity selective AR ligands with desired Gi/o protein biases targeting the therapeutically beneficial downstream pathways specifically.","abstract_has_math":false,"creators":["Huang, Xianglin"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Ladds, Graham"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-07-18","date_published":"2024-07-18","updated_at":"2026-07-22T22:24:28Z","subjects":["adenosine receptor","GPCR"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f86c8969-6634-4a93-8977-4536df83b7d6/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0009000329477638"],"render_values":[{"text":"0009-0003-2947-7638","href":"https://orcid.org/0009-0003-2947-7638","code":true}]}]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.112348","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Ladds, Graham"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["Cambridge Trust China Scholarship Council"]},{"key":"dc:creator","label":"Author","values":["Huang, Xianglin"]},{"key":"dc:creator.authoridentifier","label":"Author Identifier","values":["0009000329477638"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-07-18"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/374181"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["adenosine receptor","GPCR"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f86c8969-6634-4a93-8977-4536df83b7d6/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.112348"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/61a1996e-4bbb-4eb8-823e-1487fbb4f2b6/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Human adenosine A1 receptor (hA1R) and adenosine A3 receptors (hA3R) are two closely related class A Gi/o-coupled receptors. Both of them represented valuable therapeutic targets and various types of ligands have been developed for regulating their activities. hA1R has important implications in pain regulation, epilepsy, neurodegenerative diseases and the cardiorespiratory system, while hA3R is a crucial player in neuropathic pain, immune diseases and tumor progression. The first part of this thesis was to investigate the receptor-ligand interactions between the hA1R/hA3R and their potential antagonists. Based on previous hit compounds K18 and A17, new heterocyclic carbonyloxycarboximidamides-based derivatives and pyrazolo[3,4-c] pyridine analogues were synthesised and characterised. Through the profiling of their binding affinity, binding kinetics and subtype selectivity, several high-affinity candidates with about 10-fold improvement in affinity from their precursors were identified as hA3R-selective (e.g., 39), hA1R-selective (e.g., R1) or dual hA1R/hA3R antagonists (e.g., R8). Also, the binding pocket of 39 at hA3R was explored through extensive mutagenesis studies and the key molecular signatures accounting for the improved affinity of 39 were decoded. These new lead compounds represented excellent hAR antagonist candidates for investigating the structural selectivity filers in the orthosteric binding pockets of hA1R and hA3R as well as for future drug development for treating diseases such as heart failure, asthma and cancer. Following the exploration of the interactions between antagonists and hA1R/hA3R, the actions of agonists in activating the hA1R/hA3R were measured at the level of the activation of individual G proteins. Being primarily coupled to the inhibitory G proteins, hA1R/hA3R showed little activation at the non-inhibitory G proteins (Gss, GsL, Gq, G11, G15, G12 and G13). It has been discovered in this part of the thesis that hA1R/hA3R showed intrinsic receptor biases within the inhibitory G protein family, where hA1R activates all the Gi/o proteins while hA3R preferentially activates Gi1, Gi2 and Gi3 over Goa, Gob and Gz in the presence of agonists. Also, hA1R showed much higher constitutive activity than hA3R in activating all six inhibitory G proteins. To investigate the structural mechanisms underlying this differential preference in activating the Gi/o proteins between hA1R and hA3R, the roles of 32 non-conserved positions in hA1R/hA3R which could interact with the G proteins were investigated. These positions were reciprocally switched between hA1R and hA3R in mutagenesis and the resulted mutants were tested in their Gi/o protein activation level. Several key positions were found contributing to the complicated networks for regulating the levels of Gi/o protein activation by hA1R and hA3R. For example, it was shown that the alanine4.42, glutamine5.68, isoleucine6.33 and glutamine8.48 in hA1R were crucial in maintaining the high constitutive activity in activating Gi/o proteins through different structural mechanisms. For hA3R, asparagine5.72, glutamic acid6.22 and threonine6.33 prevented the wild type hA3R from activating the GoA and GoB while the valine34.51 and phenylalanine6.31 formed part of the barriers for hA3R to activate Gz. This throughout mutagenesis study has provided a solid foundation for understanding the structural dynamics and activation mechanisms in the hA1R/hA3R-Gi/o protein interaction network. In summary, this thesis has included the identification and characterisation of the key molecular signatures involved in the dynamical interactions between orthosteric ligands with hA1R/hA3R as well as between the hA1R/hA3R and the inhibitory G proteins. The understandings of these interactions have important implications in the future design and development of high-affinity selective AR ligands with desired Gi/o protein biases targeting the therapeutically beneficial downstream pathways specifically."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["87eda9de84448d1f82354d60eee3eb5f","c55a1ce5963825aacc72182f0d4861d8"]},{"key":"dc:title","label":"Title","values":["Exploring the molecular signatures of the ligand-receptor-Gi/o protein interactions of adenosine A1 and A3 receptors"]}]}],"canonical_facts":{"dc:contributor.advisor":["Ladds, Graham"],"dc:contributor.sponsor":["Cambridge Trust China Scholarship Council"],"dc:creator":["Huang, Xianglin"],"dc:creator.authoridentifier":["0009000329477638"],"dc:date.issued":["2024-07-18"],"dc:description.abstract":["Human adenosine A1 receptor (hA1R) and adenosine A3 receptors (hA3R) are two closely related class A Gi/o-coupled receptors. 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Also, the binding pocket of 39 at hA3R was explored through extensive mutagenesis studies and the key molecular signatures accounting for the improved affinity of 39 were decoded. These new lead compounds represented excellent hAR antagonist candidates for investigating the structural selectivity filers in the orthosteric binding pockets of hA1R and hA3R as well as for future drug development for treating diseases such as heart failure, asthma and cancer. Following the exploration of the interactions between antagonists and hA1R/hA3R, the actions of agonists in activating the hA1R/hA3R were measured at the level of the activation of individual G proteins. Being primarily coupled to the inhibitory G proteins, hA1R/hA3R showed little activation at the non-inhibitory G proteins (Gss, GsL, Gq, G11, G15, G12 and G13). It has been discovered in this part of the thesis that hA1R/hA3R showed intrinsic receptor biases within the inhibitory G protein family, where hA1R activates all the Gi/o proteins while hA3R preferentially activates Gi1, Gi2 and Gi3 over Goa, Gob and Gz in the presence of agonists. Also, hA1R showed much higher constitutive activity than hA3R in activating all six inhibitory G proteins. To investigate the structural mechanisms underlying this differential preference in activating the Gi/o proteins between hA1R and hA3R, the roles of 32 non-conserved positions in hA1R/hA3R which could interact with the G proteins were investigated. These positions were reciprocally switched between hA1R and hA3R in mutagenesis and the resulted mutants were tested in their Gi/o protein activation level. Several key positions were found contributing to the complicated networks for regulating the levels of Gi/o protein activation by hA1R and hA3R. For example, it was shown that the alanine4.42, glutamine5.68, isoleucine6.33 and glutamine8.48 in hA1R were crucial in maintaining the high constitutive activity in activating Gi/o proteins through different structural mechanisms. For hA3R, asparagine5.72, glutamic acid6.22 and threonine6.33 prevented the wild type hA3R from activating the GoA and GoB while the valine34.51 and phenylalanine6.31 formed part of the barriers for hA3R to activate Gz. This throughout mutagenesis study has provided a solid foundation for understanding the structural dynamics and activation mechanisms in the hA1R/hA3R-Gi/o protein interaction network. In summary, this thesis has included the identification and characterisation of the key molecular signatures involved in the dynamical interactions between orthosteric ligands with hA1R/hA3R as well as between the hA1R/hA3R and the inhibitory G proteins. The understandings of these interactions have important implications in the future design and development of high-affinity selective AR ligands with desired Gi/o protein biases targeting the therapeutically beneficial downstream pathways specifically."],"dc:format.checksum.md5":["87eda9de84448d1f82354d60eee3eb5f","c55a1ce5963825aacc72182f0d4861d8"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.112348"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/61a1996e-4bbb-4eb8-823e-1487fbb4f2b6/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/374181"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/f86c8969-6634-4a93-8977-4536df83b7d6/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:subject":["adenosine receptor","GPCR"],"dc:title":["Exploring the molecular signatures of the ligand-receptor-Gi/o protein interactions of adenosine A1 and A3 receptors"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:28Z"}