title: Identification of overlapping but differential binding sites for the high-affinity CXCR3 antagonists NBI-74330 and VUF11211.
abstract: CXC chemokine receptor CXCR3 and/or its main three ligands CXCL9, CXCL10, and CXCL11 are highly upregulated in a variety of diseases. As such, considerable efforts have been made to develop small-molecule receptor CXCR3 antagonists, yielding distinct chemical classes of antagonists blocking binding and/or function of CXCR3 chemokines. Although it is suggested that these compounds bind in an allosteric fashion, thus far no evidence has been provided regarding the molecular details of their interaction with CXCR3. Using site-directed mutagenesis complemented with in silico homology modeling, we report the binding modes of two high-affinity CXCR3 antagonists of distinct chemotypes: VUF11211 [(S)-5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide] (piperazinyl-piperidine) with a rigid elongated structure containing two basic groups and NBI-74330 [(R)-N-(1-(3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl)ethyl)-2-(4-fluoro-3-(trifluoromethyl)phenyl)-N-(pyridin-3-ylmethyl)acetamide] (8-azaquinazolinone) without any basic group. Here we show that NBI-74330 is anchored in the transmembrane minor pocket lined by helices 2 (W2.60, D2.63), 3 (F3.32), and 7 (S7.39, Y7.43), whereas VUF11211 extends from the minor pocket into the major pocket of the transmembrane domains, located between residues in helices 1 (Y1.39), 2 (W2.60), 3 (F3.32), 4 (D4.60), 6 (Y6.51), and 7 (S7.39, Y7.43). Mutation of these residues did not affect CXCL11 binding significantly, confirming the allosteric nature of the interaction of these small molecules with CXCR3. Moreover, the model derived from our in silico-guided studies fits well with the already published structure-activity relationship data on these ligands. Altogether, in this study, we show overlapping, yet different binding sites for two high-affinity CXCR3 antagonists, which offer new opportunities for the structure-based design of allosteric modulators for CXCR3.
authors.affiliation: Division of Medicinal Chemistry, Faculty of Science, Amsterdam Institute for Molecules, Medicines, and Systems, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Danny J
authors.initials: DJ
authors.lastname: Scholten
authors.forename: Luc
authors.initials: L
authors.lastname: Roumen
authors.forename: Maikel
authors.initials: M
authors.lastname: Wijtmans
authors.forename: Marlies C A
authors.initials: MC
authors.lastname: Verkade-Vreeker
authors.forename: Hans
authors.initials: H
authors.lastname: Custers
authors.forename: Michael
authors.initials: M
authors.lastname: Lai
authors.forename: Daniela
authors.initials: D
authors.lastname: de Hooge
authors.forename: Meritxell
authors.initials: M
authors.lastname: Canals
authors.forename: Iwan J P
authors.initials: IJ
authors.lastname: de Esch
authors.forename: Martine J
authors.initials: MJ
authors.lastname: Smit
authors.forename: Chris
authors.initials: C
authors.lastname: de Graaf
authors.forename: Rob
authors.initials: R
authors.lastname: Leurs
mesh_terms: D000081:Acetamides / Q000737:chemistry* / Q000494:pharmacology; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D001665:Binding Sites; D003198:Computer Simulation; D057809:HEK293 Cells; D006801:Humans; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009536:Niacinamide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D010879:Piperazines / Q000737:chemistry* / Q000494:pharmacology; D011743:Pyrimidines / Q000737:chemistry* / Q000494:pharmacology; D011869:Radioligand Assay; D054367:Receptors, CXCR3 / Q000037:antagonists & inhibitors* / Q000235:genetics
chemical_list: C586675:5-chloro-6-(4-(1-(4-chlorobenzyl)piperidin-4-yl)-3-ethylpiperazin-1-yl)-N-ethylnicotinamide; D000081:Acetamides; C515187:CXCR3 protein, human; C501976:NBI-74330; D010879:Piperazines; D011743:Pyrimidines; D054367:Receptors, CXCR3; D009536:Niacinamide