Determination of the binding mode for the cyclopentapeptide CXCR4 antagonist FC131 using a dual approach of ligand modifications and receptor mutagenesis

Abstract

BACKGROUND AND PURPOSEThe) 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. EXPERIMENTAL APPROACHHeterologous 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. KEY RESULTS CONCLUSIONS AND IMPLICATIONSLigand 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. Abbreviations2-Nal, 3-(2-naphthyl)alanine; 7TM, 7 transmembrane helix; Aib, 2-aminoisobutyric acid; Cit, citrulline; ECL, extracellular loop; HIV, human immunodeficiency virus; IP, inositol phosphate; SAR, structure-activity relationship; WT, wild type BJP British Journal of Pharmacology

Progress Toward Rationally Designed Small-Molecule Peptide and Peptidomimetic CXCR4 Antagonists

“…Importantly, none of these studies were accompanied by in vitro experiments that verified the proposed binding mode. However, a recent binding mode study of FC131 based on extensive site--directed mutagenesis [104] supports the previously suggested binding models. The collective picture that has emerged from these models is generally consistent with SAR (see section Figure 6A) [105].…”

Probing the Molecular Interactions between CXC Chemokine Receptor 4 (CXCR4) and an Arginine-Based Tripeptidomimetic Antagonist (KRH-1636)

“…23,33,58 However, the R188A mutation did not have any effect on the antagonistic potency of 1 or the two analogs (Table 2); interestingly, the R188A-mutant was also reported to have no effect on the potency of FC131. 35 A possible explanation is that both 1 and FC131 are doubly positively charged, which results in electrostatic repulsion by Arg 188 .…”

“…35 and 1 inhibit binding of the same set of monoclonal antibodies, directed against the extracellular loops of CXCR4, it has been suggested that they have similar binding sites and molecular mode of action. 28,65 As Asp 171 , Asp 262 , and indirectly Glu 288 are also involved in binding of 1 (Figure 6), the present study confirms that these two compounds bind to the same receptor region.…”

“…34 The CXCR4:2 complex provided the first detailed insight into binding of a peptide antagonist to CXCR4, and based on this structure we have recently reported an experimentally verified binding mode for the cyclopentapeptide FC131 ( Figure 1B). 35 Prior to the publication of the X-ray structures of CXCR4, extensive receptor mapping studies were performed for the AMD-compound series, …”

Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5)

“…Unless otherwise stated, 100% accounts for the maximal chemokine-induced response observed at the given mutant, or WT when chemokines were inactive at the mutant, and 0% is the signaling of the given receptor (mutant or WT) in the absence of any ligand. These overall readouts have previously been used effectively for CCR5, CXCR4, and other chemokine receptors (21,22,59,60).…”