Faculty Collaboration Database

Extrahelical Binding Site for a 1H-Imidazo[4,5-c]quinolin-4-amine A₃ Adenosine Receptor Positive Allosteric Modulator on Helix 8 and Distal Portions of Transmembrane Domains 1 and 7. Mol Pharmacol 2024 Feb 15;105(3):213-223

Date

01/06/2024

Pubmed ID

38182432

Pubmed Central ID

PMC10877738

DOI

10.1124/molpharm.123.000784

Scopus ID

2-s2.0-85185222449 (requires institutional sign-in at Scopus site)   1 Citation

Abstract

This study describes the localization and computational prediction of a binding site for the A₃ adenosine receptor (A₃AR) positive allosteric modulator 2-cyclohexyl-1H-imidazo[4,5-c]quinolin-4-(3,4-dichlorophenyl)amine (LUF6000). The work reveals an extrahelical lipid-facing binding pocket disparate from the orthosteric binding site that encompasses transmembrane domain (TMD) 1, TMD7, and Helix (H) 8, which was predicted by molecular modeling and validated by mutagenesis. According to the model, the nearly planar 1H-imidazo[4,5-c]quinolinamine ring system lies parallel to the transmembrane segments, inserted into an aromatic cage formed by π-π stacking interactions with the side chains of Y284^7.55 in TMD7 and Y293^8.54 in H8 and by π-NH bonding between Y284^7.55 and the exocyclic amine. The 2-cyclohexyl group is positioned "upward" within a small hydrophobic subpocket created by residues in TMDs 1 and 7, while the 3,4-dichlorophenyl group extends toward the lipid interface. An H-bond between the N-1 amine of the heterocycle and the carbonyl of G29^1.49 further stabilizes the interaction. Molecular dynamics simulations predicted two metastable intermediates, one resembling a pose determined by molecular docking and a second involving transient interactions with Y293^8.54; in simulations, each of these intermediates converges into the final bound state. Structure-activity-relationships for replacement of either of the identified exocyclic or endocyclic amines with heteroatoms lacking H-bond donating ability were consistent with the hypothetical pose. Thus, we characterized an allosteric pocket for 1H-imidazo[4,5-c]quinolin-4-amines that is consistent with data generated by orthogonal methods, which will aid in the rational design of improved A₃AR positive allosteric modulators. SIGNIFICANCE STATEMENT: Orthosteric A₃AR agonists have advanced in clinical trials for inflammatory conditions, liver diseases, and cancer. Thus, the clinical appeal of selective receptor activation could extend to allosteric enhancers, which would induce site- and time-specific activation in the affected tissue. By identifying the allosteric site for known positive allosteric modulators, structure-based drug discovery modalities can be enabled to enhance the pharmacological properties of the 1H-imidazo[4,5-c]quinolin-4-amine class of A₃AR positive allosteric modulators.

Author List

Fisher CL, Pavan M, Salmaso V, Keyes RF, Wan TC, Pradhan B, Gao ZG, Smith BC, Jacobson KA, Auchampach JA

Authors

John A. Auchampach PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
Robert Keyes PhD Research Scientist II in the Biochemistry department at Medical College of Wisconsin
Brian C. Smith PhD Associate Professor in the Biochemistry department at Medical College of Wisconsin
Tina C. Wan PhD Research Scientist II in the Pediatrics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Allosteric Regulation
Allosteric Site
Amines
Binding Sites
Lipids
Molecular Docking Simulation
Molecular Dynamics Simulation
Receptors, Purinergic P1