TY - JOUR

T1 - Pharmacological characterization of [3H]VUF11211, a novel radiolabeled small-molecule inverse agonist for the chemokine receptor CXCR3

AU - Scholten, Danny J

AU - Wijtmans, Maikel

AU - van Senten, Jeffrey R

AU - Custers, Hans

AU - Stunnenberg, Ailas

AU - de Esch, Iwan J P

AU - Smit, Martine J

AU - Leurs, Rob

N1 - Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

PY - 2015/4

Y1 - 2015/4

N2 - 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.

AB - 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.

KW - Allosteric Regulation

KW - Drug Inverse Agonism

KW - HEK293 Cells

KW - Humans

KW - Mutation

KW - Niacinamide/analogs & derivatives

KW - Piperazines/pharmacology

KW - Radioligand Assay

KW - Radiopharmaceuticals/pharmacology

KW - Receptors, CXCR3/genetics

KW - Tritium

U2 - 10.1124/mol.114.095265

DO - 10.1124/mol.114.095265

M3 - Journal article

C2 - 25576486

VL - 87

SP - 639

EP - 648

JO - Molecular Pharmacology

JF - Molecular Pharmacology

SN - 0026-895X

IS - 4

ER -