G-protein coupled receptor kinases (GRKs) are key regulators of GPCR signalling, and the search continues for potent and selective GRK inhibitors, for example, for the treatment of congestive heart failure. Compound101 (cmpd101), a GRK inhibitor, was developed by the Takeda Pharmaceuticals Company, and it exhibits some selectivity for the GRK2/3 subfamily. In this thesis, a number of novel cmpd101 analogues were investigated to determine their abilities to inhibit agonist-induced arrestin-3 recruitment to the µ opioid receptor (MOPr) and δ opioid receptor (DOPr) (as a proxy of GRK-induced phosphorylation of the receptor). In addition, the selectivity of cmpd101 and novel analogues towards different GRK isoforms was investigated following cellular overexpression of the GRK isoforms. To further explore the general selectivity of these novel analogues, their effects on agonist-induced Gi activation and MOPr internalisation were also explored. In DAMGO-stimulated cells, novel compounds BU14016, BU16005 and BU16007 showed significantly greater inhibition of arrestin-3 recruitment to MOPr than compound101, whilst compounds BU14013, BU14014 showed significantly less inhibition. In contrast, in cells treated with the lower efficacy MOPr agonist morphine, compound101 did not inhibit arrestin-3 recruitment, whilst only compounds BU16005 and BU14016 had any inhibitory capacity against morphine. Thus, the inhibitory effect of the novel analogues on GRK phosphorylation and following arrestin-3 recruitment to MOPr is apparently agonist-dependent. Interestingly, the novel analogues were not effective at reducing SNC80- or DADLE-induced arrestin-3 recruitment to DOPr. Thus, the effects of the novel analogues may also be GPCR subtype-dependent. Moreover, GRK isoform-overexpression studies with subsequent arrestin-3 recruitment measurements, suggested that the novel analogues show little or no selectivity between the isoforms, although this reflects the key role of GRK2 in promoting initial phosphorylation of MOPr. Overall, this thesis provides new evidence regarding the selectivity of cmpd101 and novel analogues for inhibition of GRK/arrestin function in HEK293 cells.
|Date of Award||21 Jan 2021|
- The University of Bristol
|Supervisor||Eamonn P Kelly (Supervisor) & Graeme Henderson (Supervisor)|
- Mu opioid receptor, Delta opioid receptor, Arrestins, GRKs, GRK2
- GRK inhibitors, compound101