Synthesis and application of the first radioligand targeting the allosteric binding pocket of chemokine receptor CXCR3.
Strategies for the identification of allosteric modulators of chemokine receptors largely rely on various cell-based functional assays. Radioligand binding assays are typically not available for allosteric binding sites. We synthesized, purified, and applied the first tritium-labeled allosteric modulator of the human chemokine receptor CXCR3 (RAMX3, [(3) H]N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-2-[4-fluoro-3-(trifluoromethyl)phenyl]-N-[(1-methylpiperidin-4-yl)methyl]acetamide). RAMX3 is chemically derived from 8-azaquinazolinone-type allosteric modulators and binds to the CXCR3 receptor with a K(d)  value of 1.08 nM (specific activity: 80.4 Ci mmol(-1) ). Radioligand displacement assays showed potent negative cooperativity between RAMX3 and chemokine CXCL11, providing a basis for the use of RAMX3 to investigate other potential allosteric modulators. Additionally, the synthesis and characterization of a number of other full and truncated 8-azaquinazoline analogues were used to validate the binding properties of RAMX3. We demonstrate that RAMX3 can be efficiently used to facilitate the discovery and characterization of small molecules as allosteric modulators of the CXCR3 receptor.
Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstr. 19, 91052 Erlangen, Germany.
Viachaslau
V
Bernat
Markus R
MR
Heinrich
Paul
P
Baumeister
Armin
A
Buschauer
Nuska
N
Tschammer
D000081:Acetamides / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D000494:Allosteric Regulation; D001665:Binding Sites; D054371:Chemokine CXCL11 / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011485:Protein Binding; D011744:Pyrimidinones / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D054367:Receptors, CXCR3 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014316:Tritium / Q000737:chemistry
D000081:Acetamides; D054371:Chemokine CXCL11; D008024:Ligands; D011744:Pyrimidinones; C576504:RAMX3; D054367:Receptors, CXCR3; D014316:Tritium