Bitopic A₃ Adenosine Receptor Molecular Probes: Positive Allosteric Modulation and Noncanonical Activation.

Area of Science:

• Medicinal Chemistry
• Pharmacology
• Molecular Biology

Background:

• 1H-Imidazo[4,5-c]quinolin-4-amines function as lipid-facing, positive allosteric modulators (PAMs) of the Gi-coupled A3 adenosine receptor (A3AR).
• These bitopic PAMs utilize elongated amino-alkyl chains to anchor to anionic phospholipids in the inner leaflet of cell membranes.

Purpose of the Study:

• To rationally expand the structure-activity relationship (SAR) of bitopic A3AR PAMs.
• To investigate the impact of terminal functionalities, reporter groups, and N1-alkylation on A3AR modulation.
• To develop novel molecular probes for studying A3AR.

Main Methods:

• Synthesis and chemical modification of 1H-Imidazo[4,5-c]quinolin-4-amine derivatives.
• Functional assays including [35S]GTPγS binding to assess potency and efficacy.
• Molecular docking and molecular dynamics simulations to predict binding modes and interactions.

Main Results:

• Varied terminal groups and N1-alkylation significantly enhanced human A3AR agonist (Cl-IB-MECA) potency and efficacy, revealing ago-PAM activity.
• Compound 38 (MRS8435) showed high agonist efficacy (~300% Emax) without ago-PAM activity.
• Specific N1-benzyl derivatives (35, 42) demonstrated high ago-PAM efficacy (~77% Cl-IB-MECA Emax), while substitutions (4-methyl 36, 4-iodo 46) increased Cl-IB-MECA potency significantly.

Conclusions:

• The study successfully expanded the SAR of bitopic A3AR PAMs through rational design.
• Incorporation of diverse functionalities, including reporter groups, yielded potent modulators and potential molecular probes.
• Molecular simulations confirmed stable interactions with phospholipids and the allosteric binding site.