Terminology summary
Reference agonist(s) for Emax: When no single reference ligand
with full efficacy across all compared pathways is available, different
reference ligands can be used to define Emax in each pathway. The
reference ligand and all tested ligands should have their efficacy
compared (e.g. % or value 0-1) to the reference agonist(s) for Emax.
Special recommendation for biased inverse agonism in
compared
pathways
Recommendation 14: When a ligand acts as an inverse agonist in
two pathways compared for bias, a bias factor can in theory be
calculated in the same way as for agonists. However, the reference
ligand would need to be another inverse agonist, i.e., in this case it
would be warranted to not use the endogenous ligand, as it typically is
an agonist (agouti is a rare example of an endogenous inverse agonist).
Reason: Inverse agonists inhibiting the non-ligand-dependent
constitutive activity of a receptor may, just as well as biased agonists
do, act differentially on pathways by stabilizing distinct receptor
conformations. This is only evident for receptors with constitutive
activity in the absence of an agonist. The minimum condition needed to
quantify bias would be concentration-response curves in two pathways and
this condition can be met for an inverse agonist ligand. For receptors
with a high constitutive activity, biased inverse agonism could be
valuable to understand fundamental signaling and to exploit this
therapeutically.
Further reading: Specific discussion of the actual methods to
quantify bias for inverse agonists are beyond the scope of this paper,
as they involve differences in agonist-mediated and constitutively
mediated efficacy (Ehlert, Suga & Griffin, 2011). Specifically, it is
known that constitutively active receptors themselves possess an
efficacy that can be different from agonist-mediated efficacy; this is
manifest in the phenomenon of protean agonism whereby a low efficacy
partial agonist demonstrates positive agonism in quiescent systems and
inverse agonism in constitutively active systems (Chidiac, Nouet &
Bouvier, 1996; Kenakin, 1997). This is due to the fact that the
agonist-mediated active state is of lower efficacy than the
constitutively active state. Such phenomena must be considered to
ascribe an efficacy to an inverse agonist.
Special recommendation for bias-inducing allosteric
modulators
Recommendation 15: For bias-inducing allosteric modulators, it
is essential to specify which combination of allosteric modulator and
orthosteric ligand was used to determine bias of the latter (Slosky,
Caron & Barak, 2021).
Reason: For bias-inducing allosteric modulators, which do not
convey agonism on their own, the functional outcome can vary for
different orthosteric ligands, in line with the probe dependency of
bias. As no concentration-response curves can be measured for such an
allosteric ligand on its own, bias cannot be attributed to it
individually.
Comparing ligand bias across studies and systems using rank
orders
Recommendation 16: We recommend using ligand rank orders of
bias factors (rather than quantitative bias values) for comparisons of
ligand bias across studies using different experimental systems. When
comparing more than two pathways, the preferentially activated pathway
must be identical and the second pathway should be the next-most
efficacious, which could differ for two ranked ligands or studies.
Reason: Bias values obtained from different experimental
systems are typically not comparable on a quantitative level. For
example, a bias value above 2.0 in one system may be below 2.0 for the
same pathways when studied in another system differing by e.g., cell
line, measured molecules or process (Figure 3). Achieving a more
consistent assessment of which ligand is the most biased towards a given
pathway is important to identify functionally selective probes that can
be used to dissect a distinct effect. This provides information about
which pathways should be targeted or avoided in the design of drugs with
higher efficacy and fewer side effects.
Disclaimer: The relative ligand bias rank orders may also
differ across systems (Figure 3B). However, they are expected to differ
less than detailed quantitative values.
Special recommendation for agonism versus antagonism in
compared pathways (‘non-quantitative
bias’)
Recommendation 17: When agonism and no agonism (neutral
antagonism or inverse agonism), respectively, are observed in two
pathways compared, it is not possibly to quantify bias using the above
models. This is because calculation of a quantitated bias factor
requires two concentration-response curves with the same modality
(agonism or inverse agonism). However, the bias can be described as a
non-quantitative term, “non-quantitative bias”.
Alternatively, it can be approximated by measuring an affinity to limit
bias or describe it in a “bias is larger than” relationship (Kenakin,
2015; Stahl, Ehlert & Bohn, 2019; Stahl, Zhou, Ehlert & Bohn, 2015).
Specifically, the affinity (determined from functional antagonism) is
used to determine receptor occupancy and a very low level of efficacy is
assumed to generate a simulated curve (i.e. maximal response of 5%)
which is then used to calculate bias. This yields the lowest possible
bias (it could be greater than this if the efficacy is lower than the
assumed one giving 5% maximal response).
Reason: In this case, there is no need to quantify bias to
claim bias. Thus, a non-quantitative statement would be good enough to
specify the case.
Disclaimer: Although a very low ligand efficacy cannot be
detected in one system, it may be detected in another functional system
with higher sensitivity. Hence, the statement should be understood as a
practical qualification, in the sense that the efficacy of the ligand is
close to zero within the detection limits of the given system. I.e.,
some partial agonists may appear to be neutral antagonists.