Fluorescent A and A Adenosine Receptor Antagonists As Flow Cytometry Probes

Overview

Journal Purinergic Signal

Publisher Springer

Date 2022 Jun 10

PMID 35687212

Authors

Kiran S Toti

Ryan G Campbell

Hobin Lee

Veronica Salmaso

R Rama Suresh

Zhan-Guo Gao

Kenneth A Jacobson

Affiliations

Soon will be listed here.

Abstract

Adenosine receptor (AR) ligands are being developed for metabolic, cardiovascular, neurological, and inflammatory diseases and cancer. The ease of drug discovery is contingent on the availability of pharmacological tools. Fluorescent antagonist ligands for the human A and AARs were synthesized using two validated pharmacophores, 1,3-dipropyl-8-phenylxanthine and triazolo[1,5-c]quinazolin-5-yl)amine, which were coupled to eight reporter fluorophores: AlexaFluor, JaneliaFluor (JF), cyanine, and near infrared (NIR) dyes. The conjugates were first screened using radioligand binding in HEK293 cells expressing one of the three AR subtypes. The highest affinities at AAR were K 144-316 nM for 10, 12, and 19, and at AAR affinity of K 21.6 nM for 19. Specific binding of JF646 conjugate MRS7774 12 to the HEK293 cell surface AAR was imaged using confocal microscopy. Compound 19 MRS7535, a triazolo[1,5-c]quinazolin-5-yl)amine containing a Sulfo-Cy7 NIR dye, was suitable for AAR characterization in whole cells by flow cytometry (K 11.8 nM), and its bitopic interaction mode with an AAR homology model was predicted. Given its affinity and selectivity (11-fold vs. AAR, ~ 50-fold vs. AAR and AAR) and a good specific-to-nonspecific binding ratio, 19 could be useful for live cell or potentially a diagnostic in vivo NIR imaging tool and/or therapy targeting the AAR.

Citing Articles

Beerkens B, Snijders I, Snoeck J, Liu R, Tool A, Le Devedec S J Med Chem. 2023; 66(16):11399-11413.

PMID: 37531576 PMC: 10461224. DOI: 10.1021/acs.jmedchem.3c00854.

References

Baues M, Klinkhammer B, Ehling J, Gremse F, van Zandvoort M, Reutelingsperger C . A collagen-binding protein enables molecular imaging of kidney fibrosis in vivo. Kidney Int. 2019; 97(3):609-614. PMC: 7115881. DOI: 10.1016/j.kint.2019.08.029. View

Esteoulle L, Daubeuf F, Collot M, Riche S, Durroux T, Brasse D . A near-infrared fluorogenic dimer enables background-free imaging of endogenous GPCRs in living mice. Chem Sci. 2020; 11(26):6824-6829. PMC: 7504901. DOI: 10.1039/d0sc01018a. View

Jacobson K, Ukena D, Kirk K, Daly J . [3H]xanthine amine congener of 1,3-dipropyl-8-phenylxanthine: an antagonist radioligand for adenosine receptors. Proc Natl Acad Sci U S A. 1986; 83(11):4089-93. PMC: 323672. DOI: 10.1073/pnas.83.11.4089. View

Grimm J, Muthusamy A, Liang Y, Brown T, Lemon W, Patel R . A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat Methods. 2017; 14(10):987-994. PMC: 5621985. DOI: 10.1038/nmeth.4403. View

Ciruela F, Fernandez-Duenas V, Jacobson K . Lighting up G protein-coupled purinergic receptors with engineered fluorescent ligands. Neuropharmacology. 2015; 98:58-67. PMC: 4608850. DOI: 10.1016/j.neuropharm.2015.04.001. View

Sacramento J, Martins F, Rodrigues T, Matafome P, Ribeiro M, Olea E . A Adenosine Receptors Mediate Whole-Body Insulin Sensitivity in a Prediabetes Animal Model: Primary Effects on Skeletal Muscle. Front Endocrinol (Lausanne). 2020; 11:262. PMC: 7198774. DOI: 10.3389/fendo.2020.00262. View

Kim Y, de Zwart M, Chang L, Moro S, Von Frijtag Drabbe Kunzel J, Melman N . Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) having high potency at the human A2B and A3 receptor subtypes. J Med Chem. 1998; 41(15):2835-45. PMC: 11687614. DOI: 10.1021/jm980094b. View

May L, Bridge L, Stoddart L, Briddon S, Hill S . Allosteric interactions across native adenosine-A3 receptor homodimers: quantification using single-cell ligand-binding kinetics. FASEB J. 2011; 25(10):3465-76. PMC: 3177574. DOI: 10.1096/fj.11-186296. View

Kose M, Gollos S, Karcz T, Fiene A, Heisig F, Behrenswerth A . Fluorescent-Labeled Selective Adenosine A Receptor Antagonist Enables Competition Binding Assay by Flow Cytometry. J Med Chem. 2018; 61(10):4301-4316. DOI: 10.1021/acs.jmedchem.7b01627. View

10.

Comeo E, Kindon N, Soave M, Stoddart L, Kilpatrick L, Scammells P . Subtype-Selective Fluorescent Ligands as Pharmacological Research Tools for the Human Adenosine A Receptor. J Med Chem. 2019; 63(5):2656-2672. DOI: 10.1021/acs.jmedchem.9b01856. View

11.

Halgren T, Murphy R, Friesner R, Beard H, Frye L, Pollard W . Glide: a new approach for rapid, accurate docking and scoring. 2. Enrichment factors in database screening. J Med Chem. 2004; 47(7):1750-9. DOI: 10.1021/jm030644s. View

12.

Boison D, Rho J . Epigenetics and epilepsy prevention: The therapeutic potential of adenosine and metabolic therapies. Neuropharmacology. 2019; 167:107741. PMC: 7220211. DOI: 10.1016/j.neuropharm.2019.107741. View

13.

Sastry G, Adzhigirey M, Day T, Annabhimoju R, Sherman W . Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments. J Comput Aided Mol Des. 2013; 27(3):221-34. DOI: 10.1007/s10822-013-9644-8. View

14.

Vernall A, Stoddart L, Briddon S, Ng H, Laughton C, Doughty S . Conversion of a non-selective adenosine receptor antagonist into A3-selective high affinity fluorescent probes using peptide-based linkers. Org Biomol Chem. 2013; 11(34):5673-82. DOI: 10.1039/c3ob41221k. View

15.

Duroux R, Ciancetta A, Mannes P, Yu J, Boyapati S, Gizewski E . Bitopic fluorescent antagonists of the A adenosine receptor based on pyrazolo[4,3-][1,2,4]triazolo[1,5-]pyrimidin-5-amine functionalized congeners. Medchemcomm. 2017; 8(8):1659-1667. PMC: 5729930. DOI: 10.1039/C7MD00247E. View

16.

Tosh D, Salmaso V, Rao H, Bitant A, Fisher C, Lieberman D . Truncated (N)-Methanocarba Nucleosides as Partial Agonists at Mouse and Human A Adenosine Receptors: Affinity Enhancement by -(2-Phenylethyl) Substitution. J Med Chem. 2020; 63(8):4334-4348. PMC: 7443318. DOI: 10.1021/acs.jmedchem.0c00235. View

17.

Claff T, Klapschinski T, Tiruttani Subhramanyam U, Vaassen V, Schlegel J, Vielmuth C . Single Stabilizing Point Mutation Enables High-Resolution Co-Crystal Structures of the Adenosine A Receptor with Preladenant Conjugates. Angew Chem Int Ed Engl. 2022; 61(22):e202115545. PMC: 9310709. DOI: 10.1002/anie.202115545. View

18.

Stoddart L, Kindon N, Otun O, Harwood C, Patera F, Veprintsev D . Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells. Commun Biol. 2020; 3(1):722. PMC: 7695831. DOI: 10.1038/s42003-020-01451-w. View

19.

Jacobson K, Tosh D, Jain S, Gao Z . Historical and Current Adenosine Receptor Agonists in Preclinical and Clinical Development. Front Cell Neurosci. 2019; 13:124. PMC: 6447611. DOI: 10.3389/fncel.2019.00124. View

20.

Friesner R, Murphy R, Repasky M, Frye L, Greenwood J, Halgren T . Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes. J Med Chem. 2006; 49(21):6177-96. DOI: 10.1021/jm051256o. View