title: Pharmacological characterization of [3H]VUF11211, a novel radiolabeled small-molecule inverse agonist for the chemokine receptor CXCR3.
abstract: Chemokine receptor CXCR3 has attracted much attention, as it is thought to be associated with a wide range of immune-related diseases. As such, several small molecules with different chemical structures targeting CXCR3 have been discovered. Despite limited clinical success so far, these compounds serve as interesting tools for investigating receptor activation and antagonism. Accumulating evidence suggests that many of these compounds are allosteric modulators for CXCR3. One feature of allosteric ligands is that the magnitude of the mediated allosteric effect is dependent on the orthosteric probe that is used. Consequently, there is a risk for incorrect assessment of affinity for allosteric modulators with orthosteric radioligands, which has so far been the most applied approach for chemokine receptors. Therefore, we aimed to use a small-molecule allosteric ligand from the piperazinyl-piperidine class, also known as VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide]. VUF11211 acts as an inverse agonist at a constitutively active mutant of CXCR3. Radiolabeling of VUF11211 gave [(3)H]VUF11211, which in radioligand binding studies shows high affinity for CXCR3 (Kd = 0.65 nM) and reasonably fast association (kon= 0.03 minute(-1)nM(-1)) and dissociation kinetics (koff = 0.02 minute(-1)). The application of the [(3)H]VUF11211 to assess CXCR3 pharmacology was validated with diverse classes of CXCR3 compounds, including both antagonists and agonists, as well as VUF11211 analogs. Interestingly, VUF11211 seems to bind to a different population of CXCR3 conformations compared with the CXCR3 agonists CXC chemokine ligand 11 (CXCL11), VUF11418 [1-((1R,5S)-6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-N-((2'-iodobiphenyl-4-yl)methyl)-N,N-dimethylmethanaminium Iodide], and VUF10661 [N-(6-amino-1-(2,2-diphenylethylamino)-1-oxohexan-2-yl)-2-(4-oxo-4-phenylbutanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide]. These findings, taken together, indicate that this allosteric inverse agonist radioligand for CXCR3 may facilitate the discovery, characterization, and optimization of allosteric modulators for the chemokine receptor CXCR3.
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Danny J
authors.initials: DJ
authors.lastname: Scholten
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Maikel
authors.initials: M
authors.lastname: Wijtmans
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: van Senten
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Custers
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Ailas
authors.initials: A
authors.lastname: Stunnenberg
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Iwan J P
authors.initials: IJ
authors.lastname: de Esch
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Martine J
authors.initials: MJ
authors.lastname: Smit
authors.affiliation: Amsterdam Institute for Molecules Medicines and Systems, Division of Medicinal Chemistry, Faculty of Science, VU University Amsterdam, Amsterdam, The Netherlands r.leurs@vu.nl.
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
mesh_terms: D000494:Allosteric Regulation; D054314:Drug Inverse Agonism; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology*; D011869:Radioligand Assay; D019275:Radiopharmaceuticals / Q000494:pharmacology*; D054367:Receptors, CXCR3 / Q000235:genetics / Q000378:metabolism*; D014316:Tritium
chemical_list: C586675:5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide; C515187:CXCR3 protein, human; D010879:Piperazines; D019275:Radiopharmaceuticals; D054367:Receptors, CXCR3; D014316:Tritium; D009536:Niacinamide