In Vitro Pharmacological Profile of KW-6356, a Novel Adenosine A<sub>2A</sub>Receptor Antagonist/Inverse Agonist

Ohno, Yutaro; Suzuki, Mina; Asada, Hidetsugu; Kanda, Tomoyuki; Saki, Mayumi; Miyagi, Hikaru; Yasunaga, Mai; Suno, Chiyo; Iwata, So; Saito, Junichi; Uchida, Shunya

doi:10.1124/molpharm.122.000633

Cited by 11 publications

(7 citation statements)

References 59 publications

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“…Importantly, the A 2A -StaR2 construct that has been used for the vast majority of A 2A AR co-crystal structures 16 contains two mutations inside the ligand binding pocket (T88 3.36 A and S277 7.42 A) preventing the discovery of this hydrogen bond. In fact, out of the 24 different A 2A AR antagonists for which co-crystal structures have been solved to date (see Supplementary Table 1) only five have been determined with A 2A AR constructs harboring the native T88 3.36 (ZM241384 21 , "cmpd-1" 36 , PSB-2113 16 , PSB-2115 16 , and most recently KW-6356 37 ). Nevertheless, the structure of a modified A 2A -StaR2-bRIL construct (that has the S277 7.42 A mutation reverted to the wt residue) in complex with Etrumadenant revealed nearly identical binding poses of Etrumadenant, despite the T88 3.36 A mutation.…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction

et al. 2023

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The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations.

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Section: Discussionmentioning

confidence: 99%

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction

et al. 2023

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“…In addition, the side chain of E169 ECL2 is rotated toward the ligand and interacts with the imidazole moiety of LUF5833 (Figure a). In the A 2A AR structure in complex with LUF5834, H264 ECL3 forms an ionic lock to E169 ECL2 instead (Figures a and a), which represents a frequent feature in A 2A AR crystal structures when crystals grew at acidic or neutral pH values. , The p K a value of the H264 ECL3 side chain imidazole was calculated to 6.90 using the propKa , online tool. Thus, it is almost fully protonated at the pH value used for crystallization (pH 5.2) and is roughly 25% protonated at a physiological pH value of 7.4.…”

Section: Resultsmentioning

confidence: 99%

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A_2A Receptor

Claff,

Mahardhika,

Vaaßen

et al. 2024

ACS Pharmacol. Transl. Sci.

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The adenosine A 2A receptor (A 2A AR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A 2A AR agonist 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-p-hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A 2A AR construct A 2A -PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p-hydroxy group of LUF5834 with T88 3.36 was observed, an amino acid that was mutated to alanine in the most frequently used A 2A AR crystallization constructs thus preventing the discovery of its interactions in most of the previous A 2A AR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N-ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S91 3.39 K mutation, although with an affinity lower than that at the native receptor. This could explain the compound's partial agonistic activity: while full A 2A AR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs.

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“…47 Zebrafish possess two A 2A R genes named adora2a.1 and adora2a.2, which share 62 and 74% amino acid identity to mammalian A 2A R, respectively. 48 The key residues involved in the binding of istradefylline on human A 2A R 49 are conserved in zebrafish orthologs. Since blockade of A 2A R receptors has been previously reported to increase zebrafish locomotion, 50 we evaluated the influence of light on the effect of istradefylline on the free-swimming behavior of nai ̈ve 5 day old zebrafish larvae.…”

Section: Istradefylline Exhibits Rapidmentioning

confidence: 99%

Optical Control of Adenosine A_2A Receptor Using Istradefylline Photosensitivity

Dumazer,

Gómez-Santacana,

Malhaire

et al. 2024

ACS Chem. Neurosci.

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In recent years, there has been growing interest in the potential therapeutic use of inhibitors of adenosine A 2A receptors (A 2A R) for the treatment of neurodegenerative diseases and cancer. Nevertheless, the widespread expression of A 2A R throughout the body emphasizes the importance of temporally and spatially selective ligands. Photopharmacology is an emerging strategy that utilizes photosensitive ligands to attain high spatiotemporal precision and regulate the function of biomolecules using light. In this study, we combined photochemistry and cellular and in vivo photopharmacology to investigate the light sensitivity of the FDA-approved antagonist istradefylline and its potential use as an A 2A R photopharmacological tool. Our findings reveal that istradefylline exhibits rapid trans-to-cis isomerization under near-UV light, and prolonged exposure results in the formation of photocycloaddition products. We demonstrate that exposure to UV light triggers a time-dependent decrease in the antagonistic activity of istradefylline in A 2A R-expressing cells and enables real-time optical control of A 2A R signaling in living cells and zebrafish. Together, these data demonstrate that istradefylline is a photoinactivatable A 2A R antagonist and that this property can be utilized to perform photopharmacological experiments in living cells and animals.

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In Vitro Pharmacological Profile of KW-6356, a Novel Adenosine A_2AReceptor Antagonist/Inverse Agonist

Cited by 11 publications

References 59 publications

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A_2A Receptor

Optical Control of Adenosine A_2A Receptor Using Istradefylline Photosensitivity

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In Vitro Pharmacological Profile of KW-6356, a Novel Adenosine A2AReceptor Antagonist/Inverse Agonist

Cited by 11 publications

References 59 publications

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction

Crystal structure of adenosine A2A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A2A Receptor

Optical Control of Adenosine A2A Receptor Using Istradefylline Photosensitivity

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Product

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In Vitro Pharmacological Profile of KW-6356, a Novel Adenosine A_2AReceptor Antagonist/Inverse Agonist

Structural Insights into Partial Activation of the Prototypic G Protein-Coupled Adenosine A_2A Receptor

Optical Control of Adenosine A_2A Receptor Using Istradefylline Photosensitivity