Abstract
BackgroundWhen stimulated by small molecular agonists, the A3 adenosine receptor (AR) mediates cardioprotective effects without inducing detrimental hemodynamic side effects. We have examined pharmacologically the protective properties of a multivalent dendrimeric conjugate of a nucleoside as a selective multivalent agonist for the mouse A3AR.ResultsA PAMAM dendrimer fully substituted by click chemistry on its peripheral groups with 64 moieties of a nucleoside agonist was shown to be potent and selective in binding to the mouse A3AR and effective in cardioprotection in an isolated mouse heart model of ischemia/reperfusion (I/R) injury. This conjugate MRS5246 and a structurally related model compound MRS5233 displayed binding Ki values of 0.04 and 3.94 nM, respectively, and were potent in in vitro functional assays to inhibit cAMP production. A methanocarba (bicyclo[3.1.0]hexane) ring system in place of ribose maintained a North conformation that is preferred at the A3AR. These analogues also contained a triazole linker along with 5'-N-methyl-carboxamido and 2-alkynyl substitution, previously shown to be associated with species-independent A3AR selectivity. Both MRS5233 and MRS5246 (1 and 10 nM) were effective at increasing functional recovery of isolated mouse hearts after 20 min ischemia followed by 45 min reperfusion. A statistically significant greater improvement in the left ventricular developed pressure (LVDP) by MRS5246 compared to MRS5233 occurred when the hearts were observed throughout reperfusion. Unliganded PAMAM dendrimer alone did not have any effect on functional recovery of isolated perfused mouse hearts. 10 nM MRS5246 did not improve functional recovery after I/R in hearts from A3AR gene "knock-out" (A3KO) mice compared to control, indicating the effects of MRS5246 were A3AR-specific.ConclusionsCovalent conjugation to a versatile drug carrier enhanced the functional potency and selectivity at the mouse A3AR and maintained the cardioprotective properties. Thus, this large molecular weight conjugate is not prevented from extravasation through the coronary microvasculature.
Highlights
When stimulated by small molecular agonists, the A3 adenosine receptor (AR) mediates cardioprotective effects without inducing detrimental hemodynamic side effects
Conjugation of an A3AR-specific agonist to a PAMAM dendrimer greatly improved its in vitro affinity and potency compared to the monomer itself
Despite the greatly increased size and bulkiness of the G protein-coupled receptor (GPCR)-Ligand Dendrimer (GLiDe) conjugate, it retained its potent cardioprotective effects in mouse isolated perfused hearts, and it was more effective than its smaller monomeric counterpart
Summary
When stimulated by small molecular agonists, the A3 adenosine receptor (AR) mediates cardioprotective effects without inducing detrimental hemodynamic side effects. Polyamidoamine (PAMAM) dendrimers can serve as biocompatible polymeric nanocarriers of bioactive molecules of interest. They are versatile in that their size, shape and placement of functional groups can be customized for various applications [1,2,3,4,5]. An agonist of the A3AR conjugated to a PAMAM dendrimer was characterized pharmacologically and tested for protective effects in an isolated mouse heart model of ischemia/reperfusion (I/R) injury. A1 and A3ARs present in the heart are well known to mediate the cardioprotective effects of adenosine [19,20,21,22]. Unlike the A1AR, when the A3AR is activated by selective agonists, such as Cl-IB-MECA 1 (Figure 1), it does not induce detrimental hemodynamic side effects, such as negative chronotropy, inotropy and dromotropy [20,23,24,25]
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