Bias Translation: Possible Reasons for Failure
In terms of translation of biased agonism found in human cells in
vitro , there are two considerations. The first relates to drug
development in that a given pharmacologic profile must be linked to a
potential therapeutic indication and often that is done through testing
in animal models. Therefore, this involves the transfer of bias from a
human receptor to an animal orthologue receptor. For orthosteric ligands
this usually is not a serious impediment as residues required for
natural ligand binding and structural integrity of receptors are highly
conserved whereas residues critical for allosteric signaling are poorly
conserved ( Leandera et al, 2020). In general, orthosteric natural
agonist recognition sites for families of receptors with a common
agonist (such as the 5 member family of acetylcholine receptors) is
difficult due to the similarity of the conserved acetylcholine binding
sites (Gentry et al, 2013; Myslivecek, 2022). However, since bias
involves protein allostery, there are data to suggest that bias
transduction could be more sensitive to receptor structure and a.a.
homology than standard orthosteric agonism even through nuances of
agonists binding differently at the same binding site (metabotropic
glutamate receptor 5 (Hellyer et al, 2020). In fact allosteric effects
have been shown to be particularly sensitive to minor differences in
a.a. homology possibly because the residues involved in allosteric
function go beyond those involved in agonist recognition. Therefore,
differences in residues distant from the natural agonist binding site
can be critical to the effect of a ligand produced at the receptor
binding site and single amino acid mutations have been shown to produce
serious effects on allosteric molecular function. For example, the
muscarinic receptor positive allosteric modulator BQCA produces
>10 fold potentiation of acetylcholine effects in native
receptors but a single amino acid mutation (Y3816.51A)
in the receptor completely negates the effects of BQCA (Abdul-Ridha et
al, 2014). Similar differences in biased signaling are seen with the
dopamine D1 receptor agonist bias for agonists at dopamine D2 receptors.
While in the Wild Type Dopamine D1 Receptor DPAT D1 is biased toward
ERK, a single a.a. mutation reverses DPAT bias toward cAMP ( Tschammer
et al, 2011). Differences in a.a. sequence, as seen with human and
animal receptor orthologues, can show large differences in allosteric
modulators as in the case of allosteric potentiating modulators of the
glutamate receptor Type 1; these show large differences in activity
between human and rat receptors (Cho et al, 2014). Since bias is
allosteric modulation, this may pose special problems testing
translation of in vitro human signaling bias to animal
therapeutic models to assess significance.
The second issue with bias translation does not inolve species
differences but rather, translation across different human systems.
Progress in new technologies has brought vast improvements in screening
hit rates and the development of new molecules but in spite of these
advances, the success rate of actual drug candidates that are useful
therapeutically is still surprisingly low. One estimate suggests that
50% of all new drug candidates fail because of lack of efficacy
(Arrowsmith, 2011) where here efficacy is defined as the candidate
performed as required in human therapeutic settings. Aside from
commercial and safety issues, this rate of failure indicates a serious
shortcoming in the drug discovery process in that it represents the fact
that, after rigorous state of the art application of pharmacology and
discovery science, seemingly optimal candidate molecules still do not do
what they were supposed to do in humans. Literature analyses suggest
that the difficulties may be related to failure to verify compound
exposure and to demonstrate physical target engagement in the relevant
therapeutic tissue (Morgan et al, 2012; 2018; Bunnage et al, 2013; Cook
et al, 2014) but with biased ligands, there may be other issues. Obvious
reasons for miscalculation are failure to recognize what efficacy is
needed to treat the disease, and/or a wrong choice of biological or
chemical target. However, another factor that may be under-estimated is
a failure to adequately characterize the true efficacies of the
candidate molecule; this may especially be true in the case of biased
molecules. From this standpoint the question could be asked, is it
enough to characterize receptor-mediated bias for a candidate molecule
without further classifying possible texture in biased signaling in the
cell?
There are a number of possible dissimulations between initial bias
estimates at the receptor level and complex in vivo signaling
profiles; it is worth considering these. An in vitroidentification of biased signaling furnishes a premise that the ligand
in question will produce differential signaling compared to the natural
agonist. Historically, initial ideas on how bias could improve therapy
(see Kenakin, 2019) were gained from simplistic comparison of agonism in
two quantifiable assay systems, cyclic AMP and β-arrestin. Presently
there are several more sophisticated analyses to predict favorable
prospective biased signaling than previously considered when only G
protein and β-arrestin signaling were the options (vide infra ).