Signalling bias at GPR84
With the discovery of GPR84 agonist MCFAs, lipid mimetics, and other
natural products, functional assays began to uncover signalling bias.
This began with the exploration of structure-activity relationships
(SAR) around DIM derivatives with regard to the canonical G-protein and
β-arrestin pathways. Most of the compounds were biased towards the
G-protein pathway, exemplified by PSB-16671 (Fig 2), while only a few
compounds were found to have limited bias towards the β-arrestin pathway
(Pillaiyar, Köse et al. , 2017). These derivatives are also known
to act allosterically (Mahmud, Jenkins et al. , 2017), adding
further texture to the downstream signalling of GPR84. Further SAR
around 6-OAU lipid mimetics highlighted the variability of cAMP versus
β-arrestin signalling, ranging from unbiased compounds such as PSB-1584,
to compounds such as PSB-16434 (Fig 2) which had a 79-fold pathway
selectivity towards cAMP (Pillaiyar, Köse et al. , 2018).
Additionally, a cyclopropane-containing MCFA isolated from the marine
bacterium Labrenzia sp. 011 has been shown to recruit β-arrestin
without affecting cAMP production in GPR84 stable cell lines,
demonstrating bias towards β-arrestin from natural product MCFAs (Amiri
Moghaddam, Dávila-Céspedes et al. , 2018). Biased signalling
resulting from natural agonists has been shown to underlie
context-specific signalling in other inflammation associated GPCRs,
which are exemplified by the 20 chemokine receptors and their distinct
responses to the 50 described chemokines (Eiger, Boldizsar et
al. , 2021).
A ligand-based virtual screen and subsequent hit optimisation led to the
discovery of DL-175, which is a potent and selective G-protein biased
agonist with no detectable recruitment of β-arrestin (Lucy, Purviset al. , 2019). As an in vitro tool this compound has found
use in dissecting G-protein versus β-arrestin pathway effects in
macrophages and neutrophils (Fredriksson, Holdfeldt et al. , 2022;
Lucy, Purvis et al. , 2019; Mårtensson, Sundqvist et al. ,
2021). In its initial biological characterisation, it was found that
DL-175 confers distinct functional responses in macrophages when
compared to the balanced agonist 6-OAU (Lucy, Purvis et al. ,
2019).
The currently described range of GPR84 agonists point to a marked system
bias of this receptor in favour of Gi-mediated pathways
over the β-arrestin pathway (Fig 3). We and others have observed trends
that favour the G-protein pathway within the chemical series of the
orthosteric agonists 6-OAU (Pillaiyar, Köse et al. , 2018),
DL-175, and some MCFAs (Lucy, Purvis et al. , 2019; Mikkelsen,
Arora et al. , 2022; Peters, Rabe et al. , 2020). Indeed, it
has been suggested that this system bias is a physiological property of
GPR84 in its proposed function in innate immunity (Peters, Rabe et
al. , 2020).
In contrast, compounds with bias towards the β-arrestin pathway are more
commonly seen within the DIM class of allosteric agonists, although
these are also micromolar potency and low affinity compounds (Köse,
Pillaiyar et al. , 2020; Pillaiyar, Köse et al. , 2017). The
absence of a physiological agonist with which to set as a reference
ligand when determining bias factors remains a challenge to medicinal
chemistry projects, though the evidence to date indicates that GPR84 is
inherently poorly coupled to the β-arrestin pathway. With few
exceptions, GPR84 agonists with sub-micromolar cAMP potencies are also
biased towards cAMP (Fig 3). Furthermore, activity in the β-arrestin
pathway is also highly variable, as analogues within the major ligand
classes of MCFAs, 6-OAU derivatives, and DIM derivatives have been shown
to have greater correlation between binding and cAMP potency than
between binding and β-arrestin potency (Köse, Pillaiyar et al. ,
2020). Nonetheless, multiple distinct methods have shown that GPR84 can
indeed couple to β-arrestins and it remains an important pathway to
investigate. This system bias is another mechanism by which cellular
contexts dictate the resulting signals and physiological responses and
have been observed in other inflammatory GPCR pairings such as CXCR4 and
the arrestin-coupled ACKR3, CCR2 and the arrestin-coupled D6R, and the
C5a1 receptor and the arrestin-coupled C5a2 receptor (Pandey, Kumariet al. , 2021; Yen, Schafer et al. , 2022).
As β-arrestins are part of the canonical desensitisation and
internalisation pathways of GPCRs, an inherent system bias away from
this pathway has broad implications for receptor regulation and drug
design. The lack of efficacy of DL-175 in the GPR84-β-arrestin pathway
has been demonstrated using tagged receptor and arrestin systems by
chemiluminescent enzyme fragment complementation (Lucy, Purvis et
al. , 2019; Mårtensson, Sundqvist et al. , 2021) and BRET
(Marsango, Ward et al. , 2022), as well as by measuring arrestin
translocation to the membrane in enhanced bystander BRET assays
(Fredriksson, Holdfeldt et al. , 2022). It is now also known that
two key threonine residues in intracellular loop 3 are phosphorylated by
GRK2/3 following agonist stimulation with ZQ-16, but not DL-175
(Marsango, Ward et al. , 2022). Phosphorylation of these residues,
Thr263 and Thr264, allow for
subsequent interactions with β-arrestin and β-arrestin-2 (Marsango, Wardet al. , 2022). This is consistent with the idea that bias is
orchestrated by GRK proteins in response to certain agonist-induced
receptor conformations, which then influence β-arrestin interactions and
signalling, rather than directly favouring or disfavouring interactions
with β-arrestin itself (Choi, Staus et al. , 2018; Zidar, Violinet al. , 2009).
Functional results in primary cells using the GRK2/3 inhibitor cmpd101
support the physiological involvement of GRK2/3 in GPR84
desensitisation. The label-free impedance response of BMDMs stimulated
with DL-175 is more prolonged than the response to 6-OAU, but
interestingly pre-treatment with cmpd101 results in a 6-OAU response
that mirrors DL-175 alone (Lucy, Purvis et al. , 2019). In
neutrophils, the ROS response induced by ZQ-16 was also prolonged by
pre-treatment with cmpd101, whereas the response of DL-175 was not
affected (Fredriksson, Holdfeldt et al. , 2022). Further work is
needed to determine the involvement of GRK2/3 on the kinetics of these
responses and direct readouts such as live-cell cAMP or β-arrestin
recruitment would be especially valuable. However, both the impedance
response of BMDMs and ROS production in neutrophils when stimulated with
DL-175 were terminated in relatively short timeframes, suggesting an
alternative mechanism of desensitisation. By comparison to FPR2, a
direct coupling of GPR84 to the actin cytoskeleton has been hypothesised
(Fredriksson, Holdfeldt et al. , 2022). The importance of
desensitisation in drug design can be highlighted by the use of
S1P1-desensitising agonists for the treatment of
multiple sclerosis. Agonists causing persistent S1P1signalling are sought for their endothelial protective properties
(Grailhe, Boutarfa-Madec et al. , 2020). Likewise, in infection
research, agonist-induced internalisation of the HIV coreceptor CCR5 is
an effective strategy for viral entry inhibition, and a further
desirable effect is to achieve this with minimal receptor activation and
the concomitant inflammatory response (Kazmierski, Bifulco et
al. , 2003).
GPR84 has been shown to activate transducer proteins including
Gi/o, G12/13, G15,
GRK2/3, and β-arrestin1/2 (Gaidarov, Anthony et al. , 2018;
Marsango, Ward et al. , 2022; Peters, Rabe et al. , 2022; J.
Wang, Wu et al. , 2006) which couple to effector molecules
including β-catenin, DOK3, NLRP3, and phospholipase C (Dietrich, Yanget al. , 2014; Gao, Qu et al. , 2020; Peters, Rabe et
al. , 2022; Zhang, Chen et al. , 2022), small GTPases such as
ras/rho and dynamin (Peters, Rabe et al. , 2022), kinases PI3K,
Akt, ERK1/2, JNK, and p38 (Gao, Qu et al. , 2020; Meng, Zhanget al. , 2017; Park, Yoon et al. , 2018; Recio, Lucyet al. , 2018), and the transcription factors NF-κB and STAT3
(Recio, Lucy et al. , 2018; Yin, Cheng et al. , 2020).
Further investigation into the effector proteins downstream of GPR84 may
be important when assessing physiological efficacy.
For example, positive allosteric modulators at the
Gq-coupled M1 muscarinic acetylcholine
receptor with similar selectivity profiles and similar effects on the
binding and calcium responses of acetylcholine were still found to
differentially potentiate, i.e. bias, receptor coupling to
phospholipases C and D (Marlo, Niswender et al. , 2009). Further
evaluation revealed that PLD activity is necessary for
M1-dependent long-term depression in the prefrontal
cortex, an effect that is implicated in targeting M1 for the treatment
of cognitive deficits in schizophrenia and Alzheimer’s disease (Moran,
Xiang et al. , 2019).
In the case of GPR84, the best evidence to the importance of bias is the
suggestion that the Gi-based agonist DL-175 promotes
equivalent levels of phagocytosis but not chemotaxis as 6-OAU (Lucy,
Purvis et al. , 2019). However, given the differences in assay
time points, cell types, and concentration-response profiles between
these assays and those for cAMP production and β-arrestin recruitment,
it remains a possibility that other effectors are involved in the
distinct responses between phagocytosis and chemotaxis. For example, it
has recently been shown that GPR84 couples to Gα15 which
results in phospholipase C activity, ERK phosphorylation, and calcium
signalling (Peters, Rabe et al. , 2022). Given that GPCRs can not
only promiscuously couple to a number of G-proteins, but can also
‘switch’ in coupling preferences over a signalling time course (Cawston,
Redmond et al. , 2013), the Gαi and
Gα15 pathways may be important pharmacological
descriptors to monitor in future experiments.