Paradoxical downregulation of CXC chemokine receptor 4 induced by polyphemusin II-derived antagonists.

CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor implicated in cell entry of T-cell line-tropic HIV-1 strains. CXCR4 and its ligand stromal cell derived factor-1 (SDF-1)/CXCL12 play pivotal parts in many physiological processes and pathogenetic conditions (e.g., immune cell-homing and cancer metastasis). We previously developed the potent CXCR4 antagonist T140 from structure-activity relationship studies of the antimicrobial peptide polyphemusin II. T140 and its derivatives have been exploited in biological and biomedical studies for the SDF-1/CXCR4 axis. We investigated receptor localization upon ligand stimulation using fluorescent SDF-1 and T140 derivatives as well as a specific labeling technique for cellular-membrane CXCR4. Fluorescent T140 derivatives induced translocation of CXCR4 into the perinuclear region as observed by treatment with fluorescent SDF-1. T140 derivative-mediated internalization of CXCR4 was also monitored by the coiled-coil tag-probe system. These findings demonstrated that the CXCR4 antagonistic activity and anti-HIV activity of T140 derivatives were derived (at least in part) from antagonist-mediated receptor internalization.

Fluorescent imaging of high-grade bladder cancer using a specific antagonist for chemokine receptor CXCR4.

We previously reported that the expression of CXC chemokine receptor-4 (CXCR4) was upregulated in invasive bladder cancers and that the small peptide T140 was a highly sensitive antagonist for CXCR4. In this study, we identified that CXCR4 expression was induced in high-grade superficial bladder tumors, including carcinoma in situ and invasive bladder tumors. To visualize the bladder cancer cells using urinary sediments from the patients and chemically induced mouse bladder cancer model, a novel fluorescent CXCR4 antagonist TY14003 was developed, that is a T140 derivative. TY14003 could label bladder cancer cell lines expressing CXCR4, whereas negative-control fluorescent peptides did not label them. When labeling urinary sediments from patients with invasive bladder cancer, positive-stained cells were identified in all patients with bladder cancer and positive urine cytology but not in controls. Although white blood cells in urine were also labeled with TY14003, they could be easily discriminated from urothelial cells by their shape and size. Finally, intravesical instillation of TY14003 into mouse bladder, using N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced bladder cancer model, demonstrated that fluorescent signals were detected in the focal areas of bladder of all mice examined at 12 weeks of BBN drinking by confocal microscopy and fluorescent endoscopy. On the contrary, all the normal bladders were found to be negative for TY14003 staining. In conclusion, these results indicate that TY14003 is a promising diagnostic tool to visualize small or flat high-grade superficial bladder cancer.

Synthesis and biological evaluation of selective CXCR4 antagonists containing alkene dipeptide isosteres.

A set of cyclic peptide analogues of a selective CXCR4 antagonist FC131 [cyclo(-d-Tyr-Arg-Arg-Nal-Gly-)] were synthesized and bioevaluated. Using (E)-alkene and (Z)-fluoroalkene dipeptide isosteres for Arg-Arg and Arg-Nal substructures, indispensable or the partial contribution of the two peptide bonds to the CXCR4 antagonism and anti-HIV activity was demonstrated. FC131 and the analogues were shown to selectively inhibit SDF-1 binding to CXCR4, whereas no inhibition of binding of SDF-1 to CXCR7 was observed.

Optimized method of G-protein-coupled receptor homology modeling: its application to the discovery of novel CXCR7 ligands.

Homology modeling of G-protein-coupled seven-transmembrane receptors (GPCRs) remains a challenge despite the increasing number of released GPCR crystal structures. This challenge can be attributed to the low sequence identity and structural diversity of the ligand-binding pocket of GPCRs. We have developed an optimized GPCR structure modeling method based on multiple GPCR crystal structures. This method was designed to be applicable to distantly related receptors of known structural templates. CXC chemokine receptor (CXCR7) is a potential drug target for cancer chemotherapy. Homology modeling, docking, and virtual screening for CXCR7 were carried out using our method. The predicted docking poses of the known antagonists were different from the crystal structure of human CXCR4 with the small-molecule antagonist IT1t. Furthermore, 21 novel CXCR7 ligands with IC50 values of 1.29-11.4 µM with various scaffolds were identified by structure-based virtual screening.

Development and application of fluorescent SDF-1 derivatives.

BACKGROUND: SDF-1/CXCR4 signaling plays key roles in directed cell migration under physiological and pathological conditions. To develop agonist-based CXCR4 probes for detection of CXCR4 expression on cell lines and metastatic tumors, SAR analyses of fluorescent SDF-1 derivatives were carried out. RESULTS: Several SDF-1 derivatives with a single fluorescent label were designed and synthesized. Modification of the SDF-1 C-terminus with AlexaFluor(®) 488 or tetramethylrhodamine provided potent CXCR4 probes. Using a potent probe, a novel binding inhibition assay was established for biological evaluation of potential CXCR4 ligands. CONCLUSION: SDF-1 derivatives with C-terminal modification exhibit equipotent binding with CXCR4 and an alternative SDF-1 receptor CXCR7 to unlabeled SDF-1. The SDF-1 derivatives are applicable to flow cytometry to detect the receptor expression and identify binding compounds for CXCR4.

Structure-activity relationship study of a CXC chemokine receptor type 4 antagonist, FC131, using a series of alkene dipeptide isosteres.

A structure-activity relationship study on a highly potent CXC chemokine receptor type 4 (CXCR4) antagonist, FC131 [cyclo(-d-Tyr(1)-Arg(2)-Arg(3)-Nal(4)-Gly(5)-)], was carried out using a series of alkene isosteres of the d-Tyr(1)-l/d-Arg(2) dipeptide to investigate the binding mode of FC131 and its derivatives with CXCR4. The structure-activity relationships of isostere-containing FC131 analogues were similar to those of the parent FC131 and its derivatives, suggesting that a trans-conformer of the d-Tyr(1)-Arg(2) peptide bond is the dominant contributor to the bioactive conformations of FC131. Although NMR analysis demonstrated that the two conformations of the peptidomimetic containing the d-Tyr(1)-d-Arg(2) isostere are possible, binding-mode prediction indicated that the orientations of the alkene motif within d-Tyr(1)-MeArg(2) peptidomimetics depend on the chirality of Arg(2) and the ß-methyl group of the isostere unit, which makes the dominant contribution for binding to the receptor. The most potent FC122 [cyclo(-d-Tyr(1)-d-MeArg(2)-Arg(3)-Nal(4)-Gly(5)-)] bound with CXCR4 by a binding mode different from that of FC131.