Most GPCRs are pleiotropically coupled to multiple signaling pathways in cells and the availability of functional assays to separately measure agonist-induced activation of these pathways has revealed that agonists do not activate all pathways in a uniform manner, i.e., some activate selected pathways at the expense of others and thus are ‘biased’ toward some signals in the cell. The way in which cells mix these signals for an overall cellular response also reveals that efficacy has ‘quality’ as well as quantity for different agonists. This has led to a revolution in GPCR candidate selection since an important further selection criterion for agonism, in the form of bias determination, now offers new ways to advance molecules.
There Are Four Distinct Reasons Why Bias Measurements Are Important
- Bias can make better drugs: The identification of biased ligands can lead to molecules that (a) emphasize beneficial signaling pathways, (b) de-emphasize harmful pathways and (3) de-emphasize harmful pathways and preclude the natural signaling system from activating those same harmful pathways. In addition, bias can expand pursuit of targets otherwise thought excluded from consideration due to activation of harmful signaling (i.e. dysphoria for k-opioid agonists).
- Bias identifies molecules for progression from high throughput screens: Counter-screens of hits found in high throughput screens in biased assays can identify hits that are truly different on a molecular level thereby optimizing the subsequent identification of useful therapeutic phenotypes in animal models and more complex therapeutic assays.
- Bias reduces efficacy fingerprints to measurable scales that may then be optimized through medicinal chemistry: It is now known that efficacy has quality as well as quantity which arises from the mixture of elemental signaling pathways in the cytosol. Radar plots or clustering has been used to represent these efficacy types but bias measurements further allow the reduction of these complex phenotypes to graded activation of signaling pathways. Therefore, an identified favorable efficacy in therapeutic cells could be deconstructed to a combination of these scales to allow medicinal chemists to optimize the pattern and amplify the favorable phenotype.
- Bias can more effectively quantify selectivity: Selective agonism on more than one signaling pathway can identify possibly false levels of selectivity or at least offer a rational scheme for determining selectivity based on all known signaling for a given molecule.
In general, and at little extra cost, the measurement of signaling bias increases value in already known lead compounds and can assist in the prediction of the activity of these compounds in other, including therapeutic, assays.
DiscoverX provides the largest portfolio of GPCR assays and a unique service that utilizes state of the art tools developed by Professor Terry Kenakin at the University of North Carolina School of Medicine for characterization of ligand bias. With the appropriate β-arrestin, internalization, and second messenger assays to quantify selective response and statistical tools to scale these effects, harnessing bias to produce selective ligands is now made simple.
Emphasis of Beneficial Signaling Pathways
Parathyroid hormone (PTH) alternately builds and degrades bone physiologically; it has been shown that PTH does not build bone in β-arrestin knockout mice (Ferrari et al, 2005; Gesty-palmer et al, 2006; 2009) suggesting that PTH agonists biased toward β-arrestin signaling would be optimally beneficial in the treatment of osteoporosis.
De-Emphasis of Harmful Signaling Pathways
Opioid agonists such as morphine provide useful analgesia but debilitating respirator depression. It has been shown in β-arrestin knockout mice that morphine produces less respiratory depression thereby suggesting that an opioid agonist biased toward G protein signaling and away from β-arrestin may be a superior analgesic (Raehal et al, 2005; DeWire et al, 2013; Charfi et al, 2015).
De-Emphasis of Harmful Pathways with Inhibition of Natural Activation of Those Same Harmful Pathways
The increase of angiotensin signaling in congestive heart failure, resulting in elevation of arterial pressure, is a normal response to decreased organ perfusion; this leads to short term improvement in organ perfusion. However, long term elevation in arterial pressure is deleterious to a failing myocardium, therefore decreased angiotensin responsiveness, in the form of angiotensin receptor blockade with drugs such as losartan, is indicated therapy. However, angiotensin also provides beneficial effects through β-arrestin signals and these are lost during standard losartan therapy. New angiotensin antagonists such as TRV120027 block the deleterious effects of angiotensin but also provide beneficial β-arrestin signals through biased signaling leading to an improved profile for congestive heart failure therapy (Violin et al, 2006; 2010).
Making k-Agonism a Viable Therapeutic Option
k-Opioid agonist activity has been implicated in the modulation of reward, mood, cognition, and perception making these molecules of possible benefit as antidepressants and anxiolytics in affective disorders, drug addiction, and psychotic disorders. However, k-opioid agonists also produce serious dysphoria thereby precluding their application to these therapies. Biased k-opioid agonists may circumvent these limitations by reducing dysphoric effects and emphasizing beneficial effects (White et al,2014).
Ligands with Different Bias in Signaling Are Different on a Molecular Level
Biased signaling is the result of the stabilization of unique ensembles of receptor states by agonists (Kenakin and Morgan, 1989; Kenakin, 1995; 2002). Therefore, if two agonists are found to produce signaling with different bias it is likely they are producing different receptor active states which will behave very differently in vivo. Under these circumstances, these two agonists would also likely produce different signaling patterns in complex assays such as in vivo animal models.
Radar Plots Display Unique Patterns of Efficacy
Multiple agonist activities can be displayed on multiple ordinate axes in the form of radar plots to show two dimensional arrays of ‘webs of efficacy’ to distinguish agonists, i.e., see such webs for β-adrenoceptor agonists (Evans et al, 2010 ) and k-opioid receptors (Zhou et al, 2013). Interestingly, each agonist tested reveals a different web, thereby indicating the uniqueness of agonist efficacy quality profiles; these may relate to the unique therapeutic phenotypic responses to these agonists.
Clustering Displays Agonist Efficacy Phenotypes
Through testing agonists in multiple functional assays, measures of efficacy for each signaling pathway (in the form of transducer coefficients, (Kenakin et al, 2012)) can be used in clustering programs to group agonists in terms of their efficacy (Kenakin, 2015). Such clusters may enable simple manipulation of these phenotypes through medicinal chemistry.
Therapeutically Relevant Receptor Selectivity
β2-Adrenoceptor agonists for agonist therapy require high activity at β2-adrenoceptors and correspondingly low activity at cardiac β1-adrenoceptors. The β2-adrenoceptor bronchodilator clenbuterol has a 500-fold selectivity of β2– over β1– activating capability for cyclic AMP, indicating a favorable profile (Casella et al, 2011). However, this selectivity reduces to 5.7-fold when the receptor-mediated β-arrestin effects for these same receptors are measured. The measurement of selectivity for different signaling pathways may be critical to the assessment of true selectivity for new agonists for therapy.
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