title: Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis.
abstract: OBJECTIVE
  The cyclopentapeptide FC131 (cyclo(-L-Arg(1) -L-Arg(2) -L-2-Nal(3) -Gly(4) -D-Tyr(5) -)) is an antagonist at the CXC chemokine receptor CXCR4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC131 in CXCR4 have previously been suggested based on molecular docking guided by structure-activity relationship (SAR) data; however, none of these have been verified by in vitro experiments.
  
  
  METHODS
  Heterologous (125) I-12G5-competition binding and functional assays (inhibition of CXCL12-mediated activation) of FC131 and three analogues were performed on wild-type CXCR4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data.
  
  
  RESULTS
  The Arg(2) and 2-Nal(3) side chains of FC131 interact with residues in TM-3 (His(113) , Asp(171) ) and TM-5 (hydrophobic pocket) respectively. Arg(1) forms charge-charge interactions with Asp(187) in ECL-2, while D-Tyr(5) points to the extracellular side of CXCR4. Furthermore, the backbone of FC131 interacts with the chemokine receptor-conserved Glu(288) via two water molecules. Intriguingly, Tyr(116) and Glu(288) form a H-bond in CXCR4 crystal structures and mutation of either residue to Ala abolishes CXCR4 activity.
  
  
  CONCLUSIONS
  Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC131 in CXCR4, which was in agreement with binding modes suggested from previous SAR studies. Furthermore, insights into the mechanism for CXCR4 activation by CXCL12 were gained. The combined findings will facilitate future design of novel CXCR4 antagonists.
authors.affiliation: Laboratory for Molecular Pharmacology, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, The Panum Institute, University of Copenhagen, Copenhagen, Denmark.
authors.forename: S
authors.initials: S
authors.lastname: Thiele
authors.forename: J
authors.initials: J
authors.lastname: Mungalpara
authors.forename: A
authors.initials: A
authors.lastname: Steen
authors.forename: M M
authors.initials: MM
authors.lastname: Rosenkilde
authors.forename: J
authors.initials: J
authors.lastname: Våbenø
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D019556:COS Cells; D054377:Chemokine CXCL12 / Q000494:pharmacology; D002522:Chlorocebus aethiops; D006801:Humans; D008024:Ligands; D062105:Molecular Docking Simulation; D016296:Mutagenesis; D010456:Peptides, Cyclic / Q000494:pharmacology*; D011485:Protein Binding; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*
chemical_list: C527276:CXCR4 protein, human; D054377:Chemokine CXCL12; D008024:Ligands; D010456:Peptides, Cyclic; D019718:Receptors, CXCR4; C496951:cyclo(tyrosyl-arginyl-arginyl-3-(2-naphthyl)alanyl-glycyl)