Lipid Trolling to Optimize A₃ Adenosine Receptor-Positive Allosteric Modulators (PAMs)

Area of Science:

• Medicinal Chemistry
• Pharmacology
• Molecular Modeling

Background:

• A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs) enhance agonist efficacy.
• Previous A3AR PAMs showed limited allosteric effects due to concurrent orthosteric antagonism.
• A putative lipid-exposed allosteric binding site on the cytosolic side was proposed.

Purpose of the Study:

• To design and synthesize novel A3AR PAMs with improved allosteric activity.
• To investigate the role of a lipid-exposed allosteric binding site in modulating A3AR function.
• To explore structure-activity relationships for optimizing A3AR PAM efficacy.

Main Methods:

• Mutagenesis and homology modeling to characterize the allosteric binding site.
• Chemical synthesis and functionalization of the imidazo[4,5-c]quinolin-4-amine scaffold.
• Functional assays ([35S]GTPγS binding) and molecular dynamics simulations.

Main Results:

• Functionalization with linear alkyl-alkynyl chains bearing terminal cationic groups enhanced A3AR PAM activity.
• Compound 26 (MRS8247) and its homologues increased the Emax and potency of the A3AR agonist Cl-IB-MECA.
• Molecular dynamics simulations supported a 'lipid trolling' mechanism involving electrostatic anchoring to phospholipids.

Conclusions:

• Rational design targeting an extrahelical, lipidic binding site significantly improved A3AR PAM activity.
• The novel A3AR PAMs demonstrate potential for therapeutic applications.
• The findings provide new insights into allosteric modulation mechanisms at G protein-coupled receptors.