Rational Design of Sulfonated A₃ Adenosine Receptor-Selective Nucleosides as Pharmacological Tools To Study Chronic Neuropathic Pain

Abstract

(N)-Methanocarba (bicyclo[3.1.0]hexane)-adenosine derivatives were probed for sites of charged sulfonate substitution, which precludes diffusion across biological membranes, e.g. blood brain barrier. Molecular modeling predicted that sulfonate groups on C2-phenylethynyl substituents would provide high affinity at both mouse (m) and human (h) A3 adenosine receptors (ARs), while a N6-p-sulfo-phenylethyl substituent would determine higher hA3AR vs. mA3AR affinity. These modeling predictions, based on steric fitting of the binding cavity and crucial interactions with key residues, were confirmed by binding/efficacy studies of synthesized sulfonates. N6-3-Chlorobenzyl-2-(3-sulfophenylethynyl) derivative 7 (MRS5841) bound selectively to h/m A3ARs (Ki hA3AR 1.9 nM) as agonist, while corresponding p-sulfo isomer 6 (MRS5701) displayed mixed A1/A3AR agonism. Both nucleosides administered i.p. reduced mouse chronic neuropathic pain that was ascribed to either A3 or A1/A3ARs using A3AR genetic deletion. Thus, rational design methods based on A3AR homology models successfully predicted sites for sulfonate incorporation, for delineating adenosine’s CNS vs. peripheral actions.

Rigidified A3 Adenosine Receptor Agonists: 1-Deazaadenine Modification Maintains High in Vivo Efficacy

“…The C2 group of 20 was accommodated in an exofacial interface region generated by the outward movement of TM2 in the hybrid A 3 AR model, as previously proposed for derivatives bearing rigid and extended C2 substituents. 12,13 Thus, the major conserved recognition points for A 3 AR agonists were preserved in the 1-deaza analogues, and as expected, the N1 of adenosine is not required for binding. Several different N 6 substitutions can be tolerated in this series, slightly modulating the affinity depending on their accommodation in a region a Effect shown for ipsilateral hind paw; there is no effect on the contralateral side.…”

“…11 The observation that nanomolar A 3 AR affinity could be maintained in the 1-deaza series was explored structurally through molecular modeling using a hybrid homology model 5,6 of the receptor (methodological details have been previously reported). 12 Figure 2 shows the docking pose of compound 20 at the hA 3 AR model.…”

Peripheral Adenosine A3 Receptor Activation Causes Regulated Hypothermia in Mice That Is Dependent on Central Histamine H1 Receptors

“…Hypothermia was caused by intraperitoneal dosing of MRS5841, an intrinsically impermeant compound that does not penetrate the CNS (Paoletta et al, 2013). In contraposition, although mast cells are found in the brain and account for a considerable fraction of brain histamine content (Tabarean, 2015), dosing MRS5841 centrally did not produce hypothermia.…”

“…MRS5698, 2-(3,4-difluorophenylethynyl)-N 6 -(3-chlorobenzyl)-(N)-methanocarbaadenosine-59-methyluronamide , was dissolved in 4:1 (v/v) PEG300/warm Solutol HS15, then an equal volume of 10% 2-hydroxypropyl-b-cyclodextrin in water was added during vortexing. MRS5841, N 6 -3-chlorobenzyl-2-(3-sulfophenylethynyl) (N)-methanocarbaadenosine-59-methyluronamide, was dissolved in saline (Paoletta et al, 2013). MRS5980, (1S,2R,3S,4R,5S)-4-(2-((5-chlorothiophen-2-yl) ethynyl)-6-(methylamino)-9H-purin-9-yl)-2,3-dihydroxy-N-methylbicyclo [3.1.0]hexane-1-carboxamide (Tosh et al, 2014), was dissolved in dimethyl sulfoxide and adjusted to 30% dimethyl sulfoxide/0.5% methylcellulose/water.…”

“…The effects of intraperitoneal versus intracerebroventricular dosing were compared using MRS5841, a sulfonated A 3 AR agonist with selectivity and potency similar to MRS5698 but improved aqueous solubility and restricted central nervous system (CNS) penetration (Paoletta et al, 2013). MRS5841 was not detectably permeable across an artificial membrane, although it is probably a transporter substrate (Caco-2 efflux ratio 5 13) (Table 1).…”