Nesheva, Desislava
(2022)
Examining the mechanism of action of small-molecule negative allosteric modulators of the human chemokine receptor CXCR2.
PhD thesis, University of Nottingham.
Abstract
The CXC chemokine receptor 2 (CXCR2) is a G protein-coupled receptor (GPCR) with key functions in neutrophil trafficking and activation both during normal homeostasis, and in acute and chronic inflammation. In addition, CXCR2 signalling promotes tumour survival through the mediation of cell proliferation and metastasis, angiogenesis and immune suppression. Despite the therapeutic potential of inhibiting CXCR2 for the treatment of inflammatory conditions and cancer, there is currently not an approved treatment at the receptor. This is largely due to the challenging task of balancing the successful treatment of inflammation or cancer suppression whilst maintaining the homeostatic function of the immune system intact when blocking CXCR2 (Cheng et al., 2019a).
There are a range of structurally distinct negative allosteric modulators (NAMs) of CXCR2 compounds that bind to the receptor at an intracellular pocket overlapping with the site of G protein coupling. Two compounds – navarixin and AZD5069 remain in clinical trials as combination therapies for the treatment of cancer. These compounds, in particular, have been reported to have slow dissociation kinetics at CXCR2.
This thesis generated new approaches to explore the in vitro pharmacology of candidate CXCR2 NAMs, in particular to understand their mechanism of action in more depth. A number of key questions were identified to address – first, the ability of NAMs to regulate CXCR2 signalling through different effector proteins (e.g. arrestins as well as G proteins); second, the extent to which different NAMs can regulate CXCR2 conformation and modulate chemokine binding, as well as blocking effector coupling; and third, the extent to which NAM binding kinetics at the intracellular site, as well as the allosteric nature of the mechanism, influenced the functional profile of their antagonism over time.
First, we co-expressed the human CXCR2 receptor tagged C- terminally with the LgBiT fragment, and β-arrestin2 and mini Gαo effectors with the SmBit fragment of the Nanoluciferase enzyme to generate a luciferase complementation assay (NanoBiT) for CXCR2-effector interactions in stably transfected HEK293 cells. These assays provided live-cell real time readouts of the agonist chemokine CXCL8 activation, and the effects over time of NAM inhibition. For the range of NAM pharmacophores explored, these approaches demonstrated their equivalent inhibition of both mini G protein and arrestin receptor interactions. We also identified differences among the NAMs in their ability to supress the basal receptor activation and in the surmountability of their effects. Using mathematical modelling approaches and comparison of close homologues (enantiomers) of navarixin, NAMs functional effects were attributed to their binding kinetics properties showing that slow koff NAMs insurmountably supress receptor-effector interactions, due to the insufficient time of binding equilibrium to be established. In contrast, fast koff NAMs promoted rightward shifts in the CXCL8 concentration-response curves likely due to negative binding cooperativity between the NAM and the orthosteric agonist.
Next, a commercially available AF647 labelled CXCL8 peptide was used to establish a non-radiolabelled CXCR2 binding assay format via both imaging and TR-FRET methodologies, applicable in whole-cells and in membrane preparations. NAMs fully inhibited tracer binding at CXCR2 in high sodium- conditions suggesting stabilisation of the inactive receptor conformation and apparently mutually exclusive binding of the NAM and chemokine, despite the difference in their topography of binding sites. Under conditions in which receptor transition to an active conformation would be better promoted (low sodium), an allosteric effect of NAM inhibition was demonstrated, and an influence on labelled chemokine dissociation kinetics measured in the real time homogeneous TR-FRET assay.
In developing novel receptor-effector interaction and fluorescent ligand approaches applicable to real time studies of binding and signalling, these results provide new information on the action of intracellular NAMs at the CXCR2 receptor. Key findings include the ability of NAMs to prevent CXCR2 coupling with multiple effectors, and a role for NAMs in allosteric modulation of chemokine affinity through conformational selection (supported by recent structural studies) – as well as steric blockade of effector interaction.
Finally, our data reveal the importance of slow binding kinetics, as well as non-competitive interactions in generating insurmountable inhibition – a feature of CXCR2 antagonism which may be beneficial under inflammatory conditions involving a cytokine storm. This increased understanding may aid future in vitro optimisation of CXCR2 NAM compounds, to titrate the desire for blockade that is therapeutically effective while managing the risk of side effects.
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