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Determination of the binding mode for the cyclopentapeptide CXCR 4 antagonist FC 131 using a dual approach of ligand modifications and receptor mutagenesis
Author(s) -
Thiele S,
Mungalpara J,
Steen A,
Rosenkilde M M,
Våbenø J
Publication year - 2014
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/bph.12842
Subject(s) - cxcr4 antagonist , mutagenesis , docking (animal) , receptor , cxcr4 , chemokine receptor , chemistry , mutant , ligand (biochemistry) , stereochemistry , antagonist , chemokine , biochemistry , biology , microbiology and biotechnology , medicine , nursing , gene
Background and Purpose The cyclopentapeptide FC 131 (cyclo(‐ L ‐ A rg 1 ‐ L ‐ A rg 2 ‐L‐2‐ N al 3 ‐ G ly 4 ‐ D ‐ T yr 5 ‐)) is an antagonist at the CXC chemokine receptor CXCR 4, which plays a role in human immunodeficiency virus infection, cancer and stem cell recruitment. Binding modes for FC 131 in CXCR 4 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. Experimental Approach Heterologous 125 I ‐12 G 5‐competition binding and functional assays (inhibition of CXCL 12‐mediated activation) of FC 131 and three analogues were performed on wild‐type CXCR 4 and 25 receptor mutants. Computational modelling was used to rationalize the experimental data. Key Results The A rg 2 and 2‐ N al 3 side chains of FC 131 interact with residues in TM ‐3 ( H is 113 , A sp 171 ) and TM ‐5 (hydrophobic pocket) respectively. A rg 1 forms charge‐charge interactions with A sp 187 in ECL ‐2, while D ‐ T yr 5 points to the extracellular side of CXCR 4. Furthermore, the backbone of FC 131 interacts with the chemokine receptor‐conserved G lu 288 via two water molecules. Intriguingly, T yr 116 and G lu 288 form a H ‐bond in CXCR 4 crystal structures and mutation of either residue to Ala abolishes CXCR 4 activity. Conclusions and Implications Ligand modification, receptor mutagenesis and computational modelling approaches were used to identify the binding mode of FC 131 in CXCR 4, which was in agreement with binding modes suggested from previous SAR studies. Furthermore, insights into the mechanism for CXCR 4 activation by CXCL 12 were gained. The combined findings will facilitate future design of novel CXCR 4 antagonists.