An investigation of the molecular pharmacology of G protein-coupled receptor 35

Mackenzie, Amanda Elaine (2015) An investigation of the molecular pharmacology of G protein-coupled receptor 35. PhD thesis, University of Glasgow.

Abstract

G protein-coupled receptors (GPCRs) are seven-pass integral membrane proteins that act as transducers of extracellular signals across the lipid bilayer. Their location and
involvement in basic and pathological physiological processes has secured their role as key targets for pharmaceutical intervention. GPCRs are targeted by many of the best-selling
drugs on the market and there are a substantial number of GPCRs that are yet to be
characterised; these could offer interest for therapeutic targeting. GPR35 is one such
receptor that, as a result of gene knockout and genome wide association studies, has
attracted interest through its association with cardiovascular and gastrointestinal disease.
Elucidation of the basic physiological function of GPR35 has, however, been difficult due a
paucity of potent and selective ligands in addition to a lack of consensus on the endogenous ligand. Herein, a focussed drug discovery effort was carried out to identify agonists of GPR35. Various in vitro cellular assays were employed in conjunction with N- or C-terminally
manipulated forms of the receptor to investigate GPR35’s signalling profile and to provide an
assay format suitable for the characterisation of newly identified ligands. Although GPR35
associates with both Gαi/o and Gα13 families of small heterotrimeric G proteins, the G
protein-independent β-arrestin-2 recruitment format was found to be the most suited to
drug screening efforts. Small molecule compound screening, carried out in conjunction with
the Medical Research Council Technology, identified compound 1 as the most potent ligand
of human GPR35 reported at that time. However, the lower efficacy and potency of
compound 1 at the rodent species orthologues of GPR35 prevented its use in in vivo studies. A subsequent effort, carried out with Novartis, focused on mast cell stabilisers as putative
agonists of GPR35, revealed lodoxamide and bufrolin as highly potent agonists that activated
human and rat GPR35 with equal potency. This finding offered–for the first time–the
opportunity to employ the same GPR35 ligand between species at a similar concentration, an important factor to
consider when translating rodent in vivo functional studies to those in man. Additionally,
using molecular modelling and site directed mutagenesis studies, these newly identified
compounds were used to aid characterisation of the ligand binding pockets of human and
rat GPR35 to reveal the molecular basis of species selectivity at this receptor. In summary, this research effort presents GPR35 tool compounds that can now be used to dissect the
basic biology of GPR35 and investigate its contribution to disease.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: G protein-coupled receptor, GPCR, orphan, GPR35, drug discovery, pharmacology, zaprinast, lodoxamide, bufrolin, amlexanox, cromolyn, pamoic acid, kynurenic acid, arrestin, BRET, IP-One, DMR, dynamic mass redistribution, mutagenesis, species selectivity.
• Subjects: Q Science > Q Science (General); R Medicine > RM Therapeutics. Pharmacology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Funder's Name: Biotechnology and Biological Sciences Research Council (BBSRC)
• Supervisor's Name: Milligan, Prof. Graeme
• Date of Award: 2015
• Depositing User: Miss AE Mackenzie
• Unique ID: glathesis:2015-6392
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 15 Jul 2016 13:12
• Last Modified: 25 Aug 2016 08:10
• URI: https://theses.gla.ac.uk/id/eprint/6392

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