Quantifying biased agonism of triazoloquinazolines: dual target A_2AR/PDE10A ligands

I. Winfield^1,2, L. Kalash¹, S. Carvalho², D. Safitri², A. Bender¹, G. Ladds². ¹Center for Molecular Sciences Informatics, University of Cambridge, Cambridge, United Kingdom, ²Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

Introduction: Compounds targeting both A_2AR and PDE10A show particularly promising prospects for therapeutic intervention for Huntingdon disease (1, 2). We utilised an in silico docking strategy to identify A_2AR agonists from a database of known PDE10A inhibitors (3), subsequently characterising identified target compounds in vitro.

Methods: Identified compounds were initially screened for receptor activation in yeast cells expressing the A₁R, A_2AR or A_2BR (4). Active compounds were then validated in CHO-K1 cells, stably expressing the A_2AR (CHO-K1-A_2AR), for their ability to modulate cAMP production and activation of ERK1/2. We further quantified the extent of biased agonism between both pathways, as well as testing compounds in ST14a cells, a model for medium spiny neurones.

Results: Through screening test compounds in yeast we ascertain the ability of triazoloquinazolines to act as agonist of the Adenosine receptors (ARs). Through further validation in CHO-K1 cells expressing either the A_2AR, or A₃R, we identify 3 compounds to display selectivity for the A_2AR and one to be a pan-AR agonist in terms of mediating cAMP production (Table 1). In addition we observe the ability of 3 compounds to mediate activation of ERK1/2 in an A_2AR-dependent manner, in CHO-K1-A_2AR cells (Table 1). Through quantifying the extent of biased agonism between these two pathways we discover that all agonists tested display bias towards activation of ERK1/2 (Figure 1) Further, we also identify the ability of our agonists to mediate A_2AR dependent signalling in ST14a cells, a model for medium spiny neurones.

Conclusions: We have identified a series of triazoloquinazolines, a class of compound traditionally associated with being AR antagonists (5), to act as A_2AR agonists. Further we have highlighted the ability of these compounds to mediate cAMP production and activate ERK1/2, showing them to be biased towards the activation of ERK1/2, a pathway that may promote proliferation (6) or cytoprotection (7).

References:

[1] Latini and Pedata (2001). J. Neurochem 79:463-84;

[2] Giampà et al (2009). Neurobiol. Dis. 34:450-56;

[3] Kehler et al (2011). Bioorg. Med. Chem. Lett. 21:3738-42;

[4] Knight et al (2016). J. Med. Chem. 59:947-64;

[5] Ongini et al (1999). Naunyn. Schmiedebergs. Arch. Pharmacol. 359:7-10;

[6] Jacques-Silva et al (2004). Oncology. 67:450-59;

[7] Baltos et al (2016). Biochem. Pharmacol. 99:101-12.