The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors
Beta-adrenoceptors belong to the superfamily of G-protein coupled receptors (GPCR) and remain an important target for drug discovery. The complexity of GPCR pharmacology in terms of its signaling profile has led to a desire to further the study of receptor-ligand interaction and obtain more detaile...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2017
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| Online Access: | https://eprints.nottingham.ac.uk/40085/ |
| _version_ | 1848795981848510464 |
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| author | Santu, L.T. |
| author_facet | Santu, L.T. |
| author_sort | Santu, L.T. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Beta-adrenoceptors belong to the superfamily of G-protein coupled receptors (GPCR) and remain an important target for drug discovery. The complexity of GPCR pharmacology in terms of its signaling profile has led to a desire to further the study of receptor-ligand interaction and obtain more detailed information regarding ligand affinity and efficacy. Development of selective fluorescent ligands targeted at human β1 and β2–adrenoceptors may facilitate drug discovery programs in terms of understanding receptor pharmacology and receptor localisation in both recombinant and primary cells from healthy and diseased tissue. Fluorescent ligands are usually designed and synthesized by tethering the ligand to a fluorophore via a linker to form a conjugate. This thesis reports the synthesis of a series of novel dipeptide-linked congeners which, when coupled to commercially available fluorophore active esters (BODIPY-X-630/650 or BODIPY–FL), afford a series of seventeen red- and green-emitting dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors.
Pharmacological characterization of the dipeptide-linked fluorescent ligands was achieved using the NanoBRET assay, a novel proximity-based assay. The most promising synthesised compounds propranolol-Gly-Ala FL and propranolol-Gly-Ser-FL (both nanomolar range KD), showed a respective 87-fold and 26-fold selectivity for the β2–adrenoceptor versus the β1–adrenoceptor [pKD = 8.59 ± 0.11 and 7.74±0.03 (β2); 6.65±0.09 and 6.32±0.20 (β1)]. Additionally, these compounds were used in a NanoBRET displacement binding experiment as tracer ligands, with known unlabelled compounds (such as CGP20712a, cimaterol, propranolol (hydrochloride) and ICI 118551) and newly-synthesised acetylated ligands at the Nluc β2 AR in order to determine their KD in this system. The displacement binding data agreed with literature values obtained by whole-cell binding assay in both CHO-K1 and Nluc HEK cells. Furthermore, [3H]-CGP 12177 whole-cell binding experiments were conducted in Nluc HEK 293 and CHO-K1 β1 and β2-AR and the results show good correlation with the NanoBRET saturation data and data obtained from another assay, the CRE-SPAP reporter gene assay. In cells expressing β2-AR, confocal microscopy studies revealed specific membrane labelling with selected ligands which was inhibited by propranolol and ICI 118551.
These novel ligands have potential as tools for exploring the pharmacology of β–adrenoceptors in native systems where more than one receptor subtype is present in terms of imaging and in providing a replacement for radioligands in binding studies. |
| first_indexed | 2025-11-14T19:40:44Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-40085 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T19:40:44Z |
| publishDate | 2017 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-400852025-02-28T13:39:53Z https://eprints.nottingham.ac.uk/40085/ The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors Santu, L.T. Beta-adrenoceptors belong to the superfamily of G-protein coupled receptors (GPCR) and remain an important target for drug discovery. The complexity of GPCR pharmacology in terms of its signaling profile has led to a desire to further the study of receptor-ligand interaction and obtain more detailed information regarding ligand affinity and efficacy. Development of selective fluorescent ligands targeted at human β1 and β2–adrenoceptors may facilitate drug discovery programs in terms of understanding receptor pharmacology and receptor localisation in both recombinant and primary cells from healthy and diseased tissue. Fluorescent ligands are usually designed and synthesized by tethering the ligand to a fluorophore via a linker to form a conjugate. This thesis reports the synthesis of a series of novel dipeptide-linked congeners which, when coupled to commercially available fluorophore active esters (BODIPY-X-630/650 or BODIPY–FL), afford a series of seventeen red- and green-emitting dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors. Pharmacological characterization of the dipeptide-linked fluorescent ligands was achieved using the NanoBRET assay, a novel proximity-based assay. The most promising synthesised compounds propranolol-Gly-Ala FL and propranolol-Gly-Ser-FL (both nanomolar range KD), showed a respective 87-fold and 26-fold selectivity for the β2–adrenoceptor versus the β1–adrenoceptor [pKD = 8.59 ± 0.11 and 7.74±0.03 (β2); 6.65±0.09 and 6.32±0.20 (β1)]. Additionally, these compounds were used in a NanoBRET displacement binding experiment as tracer ligands, with known unlabelled compounds (such as CGP20712a, cimaterol, propranolol (hydrochloride) and ICI 118551) and newly-synthesised acetylated ligands at the Nluc β2 AR in order to determine their KD in this system. The displacement binding data agreed with literature values obtained by whole-cell binding assay in both CHO-K1 and Nluc HEK cells. Furthermore, [3H]-CGP 12177 whole-cell binding experiments were conducted in Nluc HEK 293 and CHO-K1 β1 and β2-AR and the results show good correlation with the NanoBRET saturation data and data obtained from another assay, the CRE-SPAP reporter gene assay. In cells expressing β2-AR, confocal microscopy studies revealed specific membrane labelling with selected ligands which was inhibited by propranolol and ICI 118551. These novel ligands have potential as tools for exploring the pharmacology of β–adrenoceptors in native systems where more than one receptor subtype is present in terms of imaging and in providing a replacement for radioligands in binding studies. 2017-07-17 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en arr https://eprints.nottingham.ac.uk/40085/1/Lea%20Santu.pdf Santu, L.T. (2017) The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors. PhD thesis, University of Nottingham. Fluorescent ligands Beta adrenoceptors binding asays |
| spellingShingle | Fluorescent ligands Beta adrenoceptors binding asays Santu, L.T. The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors |
| title | The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors |
| title_full | The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors |
| title_fullStr | The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors |
| title_full_unstemmed | The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors |
| title_short | The design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors |
| title_sort | design, synthesis and characterisation of subtype selective dipeptide-linked fluorescent ligands for human β1 and β2–adrenoceptors |
| topic | Fluorescent ligands Beta adrenoceptors binding asays |
| url | https://eprints.nottingham.ac.uk/40085/ |