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Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology

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Abstract

Rapid phosphoester hydrolysis of endogenous purine and pyrimidine nucleotides has challenged the characterization of the role of P2 receptors in physiology and pathology. Nucleotide phosphoester stabilization has been pursued on a number of medicinal chemistry fronts. We investigated the in vitro and in vivo stability and pharmacokinetics of prototypical nucleotide P2Y1 receptor (P2Y1R) agonists and antagonists. These included the riboside nucleotide agonist 2-methylthio-ADP and antagonist MRS2179, as well as agonist MRS2365 and antagonist MRS2500 containing constrained (N)-methanocarba rings, which were previously reported to form nucleotides that are more slowly hydrolyzed at the α-phosphoester compared with the ribosides. In vitro incubations in mouse and human plasma and blood demonstrated the rapid hydrolysis of these compounds to nucleoside metabolites. This metabolism was inhibited by EDTA to chelate divalent cations required by ectonucleotidases for nucleotide hydrolysis. This rapid hydrolysis was confirmed in vivo in mouse pharmacokinetic studies that demonstrate that MRS2365 is a prodrug of the nucleoside metabolite AST-004 (MRS4322). Furthermore, we demonstrate that the nucleoside metabolites of MRS2365 and 2-methylthio-ADP are adenosine receptor (AR) agonists, notably at A3 and A1ARs. In vivo efficacy of MRS2365 in murine models of traumatic brain injury and stroke can be attributed to AR activation by its nucleoside metabolite AST-004, rather than P2Y1R activation. This research suggests the importance of reevaluation of previous in vitro and in vivo research of P2YRs and P2XRs as there is a potential that the pharmacology attributed to nucleotide agonists is due to AR activation by active nucleoside metabolites.

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Abbreviations

AST-004:

(1R,2R,3S,4R,5S)-4-(6-amino-2-(methylthio)-9H-purin-9-yl)-1-(hydroxymethyl)bicyclo[3.1.0]hexane-2,3-diol

CCPA:

2-Chloro-N6-cyclopentyladenosine

CGS21680:

2-(4-(2-carboxyethyl)phenethylamino)-5′-N-ethylcarboxamidoadenosine

CHO:

Chinese hamster ovary 

2-MeS-ADP:

2-methylthio-adenosine 5′-diphosphate

HEK:

Human embryonic kidney

I-AB-MECA:

[125I]N6-(4-amino-3-iodobenzyl)adenosine-5′-N-methyluronamide

LLOQ:

Lower limit of quantitation

NECA:

Adenosine-5′-N-ethyluronamide

R-PIA:

[3H]N6-R-phenylisopropyladenosine

PSB-603:

8-[4-[4-(4-chlorophenzyl)piperazide-1-sulfonyl)phenyl]]-1-propylxanthine

ULOQ:

Upper limit of quantitation.

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Acknowledgments

We thank Jeanne Rumsey, Christin Vielmuth and Angelika Fischer for skillful technical assistance.

Funding

Research reported in this publication was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under Award Number R41NS093756, the NIDDK Intramural Research Program (ZIADK31117) and the Deutsche Forschungsgemeinschaft (DFG).

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Authors and Affiliations

  1. Astrocyte Pharmaceuticals Inc., Suite 1800, 245 First Street, Cambridge, MA, 02142, USA

    Theodore E. Liston, William S. Korinek & Dane A. Sethre-Hofstad

  2. PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, Bonn, Germany

    Sonja Hinz & Christa E. Müller

  3. University of Texas Health at San Antonio, San Antonio, TX, USA

    Deborah M. Holstein & James D. Lechleiter

  4. Inotiv, Maryland Heights, MO, USA

    Jay Wendling, Roger J. Melton & Mary Campbell

  5. National Institute of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda, MD, USA

    R. Rama Suresh, Zhan-Guo Gao, Dilip K. Tosh & Kenneth A. Jacobson

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  1. Theodore E. Liston
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Significance statement

Extensive chemical efforts have attempted to stabilize nucleotide phosphoester groups to prevent hydrolysis and allow investigation of the in vitro and in vivo effects of P2Y and P2X receptor ligands. Our research demonstrates that prototypical P2Y1 receptor agonists containing an (N)-methanocarba ring system to impede α-phosphoester hydrolysis are rapidly metabolized to nucleoside metabolites with affinity for adenosine receptors, and antagonists are similarly unstable. This research suggests that some P2YR pharmacology observed to date may actually be due in part to adenosine receptor activation.

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Liston, T.E., Hinz, S., Müller, C.E. et al. Nucleotide P2Y1 receptor agonists are in vitro and in vivo prodrugs of A1/A3 adenosine receptor agonists: implications for roles of P2Y1 and A1/A3 receptors in physiology and pathology. Purinergic Signalling 16, 543–559 (2020). https://doi.org/10.1007/s11302-020-09732-z

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