Unknown Downstream Outcomes from Receptor-based Bias
While bias most often is detected at the level of the receptor, the
stabilized receptor active state conformation may code for unknown
events further on down into the cytosol. In light of the complexity of
signaling in cells, it is possible that the benefcial effects of a
biased agonist may be lost in the milieu of signals and biochemical
cascades. Dependence on a single readout of bias at the receptor level
may not identify molecules that produce unique effects further down the
signaling cascade. For example, BRET association of receptors with
β-arrestin indicates increased association but does not necessarily
augur receptor internalization and/or signaling (vide infra ).
Recent data with β-arrestin suggest that heterogeneity in β-arrestin
conformations may lead to trafficking of stimulus in various ways after
receptor-β-arrestin interaction (Chen et al, 2023). Specifically, while
BRET analysis of receptor association with beta arrestin may be found,
it may be critical which beta arrestin confromation is involved (i.e.
‘tail’ or ‘core’, (Cahill et al, 2017) to further delineate
internalization vs internalization for the formation of a ‘supercomplex’
of the receptor and β-arrestin in endosomes providing sustained
signaling (Chen et al, 2023). Therefore, a program seeking an internally
signaling agonist for prolonged cellular activity might test compounds
for preferential β-arrestin bias (assuming β-arrestin is the vehicle for
transport to the endoplasmic reticulum) but further experiments might
discern agonist activity toward that desired endpoint. For example, fig
3 shows that agonist 11 is identified as the most biased for β-arrestin.
However, further testing could be done at this stage to differentiate
which β-arrestin conformation is preferred; in this case, it may be that
agonist 11 would not be the preferred compound as it is not biased
toward the predicted β-arrestin conformation that could lead to ER
signaling. In general, testing sub-groups of biased agonists may further
characterize useful activity, especially in cases where an initial
testing of a biased agonist does not provide a more informative outcome.
Follow up studies with other biased molecules could provide a clearer
answer; as shown in the figure, identfication of agonist 11 as an
exemplar biased agonist in this case would not yield the required
signaling as the stimulus bifurcates throughout the cell whereas
agonists 13 to 16 might have provided a better choice.
Bias translation involves the synoptic nature of pharmacologic response
(i.e. the necessary partnership of the activated receptor with complex
signaling patterns) and brings into consideration the cellular milieu of
the active state receptor-cell mixture. In addition to the production of
a variety of ligand-bound receptor active states, comes the subsequent
interaction of these states with another constellation of (for example)
possible β-arrestin conformations. Specifically, the conformational
flexibility of β-arrestin allows GPCR-induced conformational
rearrangement to expose distinct binding surfaces that allow recruitment
of different effectors for specialized signaling complexes (Haider et
al, 2023; Luttrell et al, 2018; Shukla et al, 2014). This can lead to
considerable heterogeneity in cytosolic signaling and factorial
combinations of outcomes are possible. Possible other dissimulations
with respect to expected therapeutic outcomes at this stage include:
- Different signaling partners in different cell lines Agonist
activity on receptors transfected into different cell lines have shown
differences in relative agonist potency. For example, transfections of
calcitonin receptors into two different host cell lines (CHO cells,
COS cells) show large differences in the relative potency for porcine
Cal, human Cal and h CGRP. Specifically, in CHO cells the relative
potency is hCGRP/hCal/pCal of 1/ 10/500 whereas in CHO cells the
potency ratios are 1/2/8 (Christmanson et al, 1994). Total synoptic
agonist response can be revealed through label free assay formats such
as cell impedance; these have been to used to measure the relative
potency of dopamine agonists in two types of cells (U-2 cell, SK-N-MC
cells) where the cell type made a 4-fold difference in the relative
potency of agonists dopamine and A77636 (Peters and Scott, 2009). This
translates into differences in cell bias as function of cell type
- Variant stoichiometry between receptors and signaling
components . The most obvious variable operative here is the relative
stoichiometry of receptors and signaling components in various cells.
The relative stoichiometry of receptors and signaling proteins is a
well established variable in functional pharmacology and this brings
into play the role of the host cell in bias measurement and detection.
Thus, a paucity of an important signaling partner (i.e. G protein)
could negate a bias seen in a system where this is not the case i.e.
Eason et al, 1992. This is particularly relevant to low efficacy
agonists where response could disappear with low signaling coupler in
a given cell; this may be a factor in the bias seen with the biased
opioid receptor TRV130 (Singleton et al, 2021). An even more
surprising effect can be seen for truly biased agonists in receptor
systems without limitations in coupling proteins. For example, the
relative potency of the full calcitonin agonists eel and porcine
calcitonin for human calcitonin receptors transfected into in wild
type HEK 293 cells is
EC50(eel Cal)/
EC50 (pCal) = 0.4; Co-transfection of Gαs protein to
enrich the natural Gαs content produces a complete
reversal of the relative potency of eel and porcine calcitonin. In
this enriched cell line, the relative potency is reversed to
EC50 (eel Cal)/ EC50 (pCal) = 8 (a
32-fold difference) (Watson et al, 2000). Reduction of key signaling
components in a cell clearly can limit low efficacy agonists from
utilizing pathways but a recently interesting variation on the theme
of receptor-signaling protein relative stoichiometry suggests that
actual increases in cellular receptor can affect the observed bias (Li
et al, 2023). Differences in receptor expression levels in cells also
can introduce a temporal dissociation for response as in the case of
GPR84 where a delayed and suppressed activation of Akt was found in
low expressing cell lines (Luscombe et al, 2023).
- Variant Stoichiometry of GRKs and β-Arrestin: There are
striking cases of ligand-directed signaling to the β-arrestin system
through targeting GRKs. For example, the chemokine receptor CCR7 has
two natural agonists (CCL19, CCL21) and while CCL19 leads to receptor
phosphorylation and β-arrestin recruitment through GRK2 and GRK6,
CCL21 activates only GRK6. This differential activity on GRKs leads to
different cytosolic consequential responses for these two chemokines
(Zidar et al, 2009). Other studies indicate with receptor structural
studies that the interaction of the neurotensin 1 receptor with GRK2
guides the receptor-β-arrestin interaction for signaling (Duan et al,
2023). GRK may not be the only player as β-arrestin/GRK complexes also
may require the presence of ubiquitin to direct receptor selectivity
(Liu et al, 2023). In general, data with GRK knockout cells reveal the
importance of GRKs as major players in the cytosolic signaling of
GPCRs (Drube et al, 2022) therefore variation in cellular GRK levels
can modify GPCR signaling from initial in vitro bias
determinations in other cell lines.
- Varying temporal differentiation of agonist signals in cellsIn vitro assays are snapshots in time with no real regard to
the timescale of real life physiology but rather are optimized for
accuracy of measurement. The two main in vitro assays used to
detect bias (second messenger G protein vs β-arrestin) have very
different timescales for steady-state and maximal effect making
comparisons possibly dependent on when the measurements are made. For
example, a study of dopamine D1 receptor agonist bias on cyclic AMP
and β-arrestin shows a discernible temporal difference thus
introducing a possible dissimulation in the assessment of signaling
bias (Klein Herenbrink et al, 2016). These temporal differences extend
to the cell where G protein activation of ERK is rapid and transient
and β-arrestin activation of ERK are more sustained involving
translocation to the nucleus (Liu et al, 2023). Temporal dissociations
extend beyond short acute timespan responses to the production of
transcription effects leading to protein expression. In general, the
time bias measurements are taken yield somewhat arbitrary indices of
differential signaling that may have unpredictable effects in real
time physiology. These dissimulations in time emanate from the
cellular translation of receptor activation and not necessarily from
the timescale of ligand-receptor interaction. In fact it has been
shown that the bias of opioid agonists are independent of the rate of
interaction of the molecules with the receptor (Pedersen et al, 2020).
New techniques are being used to explore this variant in bias, i.e.
genetically-encoded fluorescent biosensors have been employed to
illuminate spatiotemporal biased signaling (Kayser et al, 2023).
- Location bias: Agonists that target receptors to β-arrestin
leading to internalization can show bias with respect to the location
(and function) of the internalized species (Eiger et al, 2022;Wang et
al, 2023). For example, the chemokines RANTES and AOP-RANTES both
internalize CCR5 receptor (for prevention of HIV-1 infection) but
whereas RANTES internalizes the receptor which then rapidly re-emerges
through recycling, AOP-RANTES internalizes the receptor to shunt it to
lysosomal destruction (Mack et al, 1998). Thus the actual receptor
conformation stabilized by the agonist may determine the fate of
β-arrestin bound receptors. Location bias also has been noted for
GLP-1 agonists where in studies all GLP-1 agonists activate nuclear
ERK1/2 activity but the agonists liraglutide and oxyntomodulin (biased
towards pERK1/2 relative to cAMP when compared to GLP-1 and
exendin-4), show spatiotemporal control by also stimulating pERK1/2
activity in the cytosol (Fletcher et al, 2018).
- Variation of the magnitude of bias significant with respect to
the overall cell response While ‘bias’ can be detected in in
vitro systems, there is no guide as to the significance of that bias
to whole body physiology. Bias indices can range from fairly modest
(2-3 fold) to values >10-20 raising the question, what
level of bias is physiologically significant? While this probably will
be system dependent, a measure of how powerful apparently small bias
values can be is demonstrated by diazepam, an anxiolytic with known
prominent therapeutic activity. Specifically, diazepam produces a mild
two-fold sensitization of GABA response but this translates to an 80%
increase in GABA response and a well-known significant physiological
effect (Skerrit and MacDonald, 1984). This suggests that bias values
of 2 or greater might have a significantly affect on agonist
phenotypic activity. Calculation of the bias of the opioid analgesic
TRV130 for cyclic AMP over β-arrestin using
ΔΔLog(max/EC50) values (Kenakin, 2017) yields a value
of 3.39 (Singleton et al, 2021), ostensibly a relatively low value but
of possible significance in light of data with diazepam.
It should also be noted that
the low efficacy of TRV130 for β-arrestin signaling (TRV130 has 33%
of the efficacy of morphine for G protein and only 15% of the
efficacy of morphine on β-arrestin) may significantly contribute to
the beneficial profile of this molecule.
Considering a relatively ‘balanced’ agonist that generally does not
distinguish signaling proteins versus a biased agonist that does, the
question arises does the degree of bias influence variability in terms
of translation (i.e. variation in potency with differences in cell
type and/or tissue sensitivity)? A theoretical model of a single
receptor interacting with two coupling proteins (Kenakin, 2003) (Fig
4A) indicates that the potency ratio of two balanced agonists (or two
agonists of identical bias) will not deviate in two cells lines of
varying G protein make-up (CellA = [G1]/[G2] = 1 ; CellB =
[G1]/[G2] = 10; Fig 4B). In contrast, for two agonists of
different bias , the difference in G protein composition can produce
radical differences in relative potency (Fig 4C). It can be seen that
the relative potencies of non biased agonists remains the same whereas
the relative potencies of the agonists of different bias actually
reverses with the change in G protein composition. These simulations
suggest that the degree of bias may contribute to the variability of
translation of agonist effect in different cell types.
- Different levels of assessment of bias within the
stimulus-response cascade in the cytosol: When considering bias it is
also relevant to think about where in the cytosolic signaling cascade
the bias may make a difference. Standard in vitro bias assays
generally assess differences at the receptor level but as signals
bifurcate throughout the cell, the emphasis on discrete pathways may
vary. For example, Fig 5 shows the effect of seven dopamine agonists
measured from the point of view of six response pathways. When bias is
assessed for each pathway through
ΔΔLog(max/EC50)
values, it is interesting to note that the magnitudes of the bias
indices vary with pathway indicating that as the signal propagates
from the cell, it is differentially modified in an agonist dependent
manner (Klein Herenbrink et al, 2016). These types of effects reveal
the texture of bias as a function of the number of vantage points used
to make the measurements. For example, G protein selective PTH analogs
build bone through cAMP and β-arrestin selective analogs would not be
predicted to be as efficacious since the β−arrestin activity
terminates G protein signals. However, paradoxically, β-arrestin
selective analogs also build bone mass in vivo largely through
regulation of cell-cycle, survival and migration/cytoskeletal dynamics
(Luttrell et al, 2018). Arrestin-focused response signatures can
further be explored through arrestin-dependent transcriptome
signatures to predicted outcomes of biased agonism (Maudsley et al,
2016).
Improving Bias Translation:
There are numerous theoretical and practical hurdles to the accurate
translation of in vitro bias to complex in vivo systems
raising the question, how can these be minimized to optimally design
biased agonist programs for success? The first step is to identify bias
in a molecule and this can be done through cross-screening in two assays
and comparing the results with a bias plot. Considering the complexity
of allosteric differences with different receptor conformations, it
probably is not too important which two pathways are chosen; G protein
signaling and β-arrestin historically have been the standards. The main
function of this first step is the identification of a candidate biased
molecules which may produce a useful agonist phenotypes in vivoby stabilizing unique receptor conformations. However, out of an array
of biased candidates, their ‘robustness’ in terms of resilience of bias
to varying cellular conditions could be tested:
- Identify ‘Efficacy-based’ over ‘Affinity-based’ bias : Bias
based on differences in efficacy (Rajagopal et al, 2011) are more
resilient to changes in cell sensitivity than those based on affinity.Experiment : Test bias for immutability in cells of varying
sensitivity (i.e. varying levels of receptor expression).
- Measure the intrinsic efficacy of the candidates in single
pathways : Low efficacy in a negative pathway can be useful to
strengthen bias under a range of in vivo conditions and a
measure of agonist efficacy can be obtained through manipulation of
levels of receptor expression (Jiang et al, 2022). Experiment :
Measure the relative efficacy of the candidate to the natural agonist
with the operational model.
- Measure variation of bias in different cellular backgrounds:Bias could be measured in a range of host cell lines to gauge
variation in biased signaling. For example, GRKs have been shown to
affect signaling bias and cells have variable GRK levels and variable
levels of GRKs can be used as a variable to assess the impact of GRK
levels on agonist bias (Matthees et al, 2021). Experiment:Assess bias in cells with varying levels of GRKs and/or varying cell
types.
- Measure the temporal dependence of bias estimates: Some
agonists yield time dependent estimates of bias which could make them
unstable predictors of in vivo bias; for instance, while
dopamine cAMP/β-arrestin bias is stable when measurements are made
over 90 min, aripiprazole changes by a factor of 10 (Klein Herenbrink
et al, 2016). Experiment: Measure bias at two separated
timepoints.
- Apply more textured estimates of signaling heterogeneity:While simple assays such as cyclic AMP and β-arrestin BREThave been used to good measure in this field, the availability of
first line assays to further differentiate active state signaling can
offer advantages. Thus while cyclic AMP may augur effects of the
agonist-activated receptor on Gαs signaling, assays that differentiate
all G protein signaling (such as TRUPATH, (Olsen et al, 2020) may
offer rapid first-line separation of agonist profiles.Experiment: Utilize more textured G protein assays (Soave et
al, 2020) as the first differentiator of bias.
- Measure agonist receptor off-rates: Slow dissociation of
molecules from receptors can cause pharmacodynamic-pharmacokinetic
dissociation and favorable in vivo target coverage. This is a
property separate from potency and/or bias but is essential in the
characterization of a future in vivo candidate.Experiment: Measure agonist off-rates.