Bitopic fluorescent antagonists of the A_2A adenosine receptor based on pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine functionalized congeners

Abstract

A pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine antagonist of the A2A adenosine receptor (AR) was functionalized as amine congeners, fluorescent conjugates and a sulfonate, and the A2AAR binding modes were predicted computationally. The optimal n-butyl spacer was incorporated into the following A2AAR-selective (Ki, nM) conjugates: BODIPY630/650 derivative 11 (MRS7396, 24.6) and AlexaFluor488 derivative 12 (MRS7416, 30.3). Flow cytometry of 12 in hA2AAR-expressing HEK-293 cells displayed saturable binding (low nonspecific) and inhibition by known A2AAR antagonists. Water-soluble sulfonate 13 was a highly potent (Ki = 6.2 nM) and selective A2AAR antagonist based on binding and functional assays. Docking and molecular dynamics simulations predicted the regions of interaction of the distal portions of these chain-extended ligands with the A2AAR. The BODIPY630/650 fluorophore of 11 was buried in a hydrophobic interhelical (TM1/TM7) region, while AlexaFluor488 of 12 associated with the hydrophilic extracellular loops. In conclusion, we have identified novel high affinity antagonist probes for A2AAR drug discovery and characterization.

Activation of adenosine A2A or A2B receptors causes hypothermia in mice

“…5A-E). To distinguish a central vs peripheral site of action for A 2A AR antagonism, we tested MRS7352 (Duroux et al, 2017), a polar derivative of SCH442416 that is unlikely to reach brain parenchymal A 2A AR. Although less potent than SCH442416, MRS7352 retains selectivity for A 2A AR (Table 1).…”

“…MRS7352 (4-((4-(3-(5-amino-2-(furan-2-yl)-7 H -pyrazolo[4,3- e ][1,2,4]triazolo[1,5- c ]pyrimidin-7-yl)propyl)phenoxy)methyl)benzenesulfonate triethylammonium salt) was synthesized and purified by HPLC as described (Duroux et al, 2017). The following compounds (vehicle) were purchased from Tocris (Minneapolis, MN): CGS-21680 (15% DMSO/15% KolliphorEL/70% saline for i.p.…”

“… *** BAY60-6583 may be an antagonist at A 1 and A 3 AR and a partial agonist at A 2B AR. Superscripts indicate data source. SEM s are included for experimental results. a (Carlin et al, 2017) b (Alnouri et al, 2015) c (Jarvis et al, 1989) d (Nikodijevic et al, 1990) e Current results f (Gao et al, 2001) g (El-Tayeb et al, 2011) h (Hide et al, 1992) i (Kim et al, 1994) j (Todde et al, 2000) k (Duroux et al, 2017) …”

Adenosine A1-A2A Receptor-Receptor Interaction: Contribution to Guanosine-Mediated Effects

“…Adenosine deaminase (ADA) was purchased from Roche Diagnostics (GmbH, Mannheim, Germany) and zardaverine from Calbiochem (San Diego, CA, USA). MRS7396, which is a selective fluorescent antagonist at the A 2A R derived from SCH442416, was previously described [19].…”

“…We used a fluorescent A 2A R antagonist (MRS7396) that is able to engage in a BRET process upon interacting with a cell surface A 2A R tagged with the NanoLuciferase (NL) at its N-terminus (i.e., A 2A R NL ) ( Figure 2A). MRS7396 is a BODIPY630/650 derivative of SCH442416 [19], which upon A 2A R binding can act as an acceptor chromophore for NanoLuciferase emission (490 nm) in a BRET process. Thus, we challenged stable A 2A R NL -expressing cells with increasing concentrations of MRS7396, in the presence/absence of non-labelled SCH442416.…”