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In situ click chemistry generation of candidate ligands for adenosine receptor A2A with a soluble adenosine binding protein template

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Chemistry & Chemical technologies
In situ click chemistry generation of candidate ligands for adenosine receptor A2A with a soluble adenosine binding protein template
click chemistry

Adenosine A2A receptor is a member of GPCR family which is responsible for regulating myocardial blood flow and also plays an important role in the regulation of glutamate and dopamine release, making it a potential therapeutic target for the treatment of conditions such as insomnia, pain, depression, drug addiction and Parkinson's disease.1

In this work, soluble adenosine A2A receptor in 0.01M PBS with detergent was used as in situ template for generation of novel and potent ligands that selectively bind to this protein. The cycloaddition reaction between an anchor alkyne fragment and a library of azides that forms stable 1,2,3-triazoles was used to generate lead compounds. This bioorthogonal reaction is fully compatible with functional groups found in normal physiological environments.2

The process begins with selective binding of molecules containing an alkyne and azide groups to specific areas of the protein binding site. The two fragments that form a high-affinity ligand link together irreversibly within the confines of the template’s binding pocket, thus forming of an energetically favorable complex with the protein. The extent of the triazole formation on A2A template correlated with the affinity of the triazole product for the adenosine ligand binding site. We have modified adenosine via alkyne group and used it as an anchor reacting with over one hundred azides, dividing them into multiple sets. For the detection of triazoles, we used an ion trap mass spectrometry (amaZon SL, Bruker). As a result, eight preliminary hits were found.

In order to find the most affine ligands among these hits we performed one more experiment with A2A receptor binded with ZM241385, a high affinity antagonist ligand selective for the receptor.3 The intensity of found key triazole signal detected via ion trap mass spectrometry decreased at least two fold in comparison with the experiment without ZM241385.


The formation of the molecule was proved by comparison the results of high-resolution TOF ESMS+ spectra for triazole obtained on A2A template and the same molecule synthesized by Cu(II)-catalysed cycloaddition reaction.

[1] Cunha RA, Ferré S, Vaugeois JM, Chen JF. Current Pharmaceutical Design  2008, 14 (15), 1512–24.

[2] Fokin V.V., Shapless K.B., Grimster N.P., Stump B., Fotsing R.F., Weide T., Talley T.T., Yamauchi J.G, Nemecz A., Kim Ch., Ho K.Y. JACS  2012, 134, 6732-6740.

[3] Goren M.A., Morizumi T., Menon  I., Joseph J. S., Dittman J.S., Cherezov V., Stevens R.C., Ernst O.P.,  Menon A.K. Nature Communication s 2014, 5, 5115.