Abstract
Allosteric antagonism by bitopic ligands, as reported for many receptors, is a distinct modulatory mechanism. Although several bitopic A2A adenosine receptor (A2AAR) ligand classes were reported as pharmacological tools, their receptor binding and functional antagonism patterns, i.e., allosteric or competitive, were not well characterized. Therefore, here we systematically characterized A2AAR binding and functional antagonism of two distinct antagonist chemical classes. i.e., fluorescent conjugates of xanthine amine congener (XAC) and SCH442416. Bitopic ligands were potent, weak, competitive or allosteric, based on the combination of pharmacophore, linker and fluorophore. Among antagonists tested, XAC, XAC245, XAC488, SCH442416, MRS7352 showed Ki binding values consistent with KB values from functional antagonism. Interestingly, MRS7396, XAC-X-BY630 (XAC630) and 5-(N,N-hexamethylene)amiloride (HMA) were 9–100 times weaker in displacing fluorescent MRS7416 binding than radioligand binding. XAC245, XAC630, MRS7396, MRS7416 and MRS7322 behaved as allosteric A2AAR antagonists, whereas XAC488 and MRS7395 antagonized competitively. Schild analysis showed antagonism slopes of 0.42 and 0.47 for MRS7396 and XAC630, respectively. Allosteric antagonists HMA and MRS7396 were more potent in displacing [3H]ZM241385 binding than MRS7416 binding. Sodium site D52N mutation increased and decreased affinity of HMA and MRS7396, respectively, suggesting possible preference for different A2AAR conformations. The allosteric binding properties of some bitopic ligands were rationalized and analyzed using the Hall two-state allosteric model. Thus, fluorophore tethering to an orthosteric ligand is not neutral pharmacologically and may confer unexpected properties to the conjugate.
Highlights
The A2A adenosine receptor (A2A AR), a major target of caffeine, is an attractive drug target for many conditions
To examine and analyze mechanistically the patterns of functional antagonism by various antagonists of different chemical entities, cells expressing relatively low A2A AR levels with minimal signaling amplification were used, as recommended [26], since receptor overexpression is related to the constitutive activity, and signaling amplification has been linked to changing the patterns of antagonism [28]
In order to examine the pattern of antagonism by the putative bitopic ligands, we first examined the effects two known monotopic A2A AR antagonists, xanthine amine congener (XAC) and ZM241385, in antagonizing agonist
Summary
The A2A adenosine receptor (A2A AR), a major target of caffeine, is an attractive drug target for many conditions. Both A2A AR agonists and antagonists have been approved for clinical use [1,2,3]. Despite the tremendous therapeutic opportunities associated with important A2A AR physiological functions, most A2A AR ligand clinical trials have failed [1,2], which is due at least in part to the lack of understanding of mechanisms related to receptor binding and activation, which is relevant to both therapeutic and side effects. Further understanding of its A2A AR function and regulation, especially its allosteric modulation could provide an important basis for development of agonists, antagonists and allosteric modulators of the A2A AR with better therapeutic effects and fewer side effects
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
