1. Development of a NanoBRET-Based Sensitive Screening Method for CXCR4 Ligands
Takuya Kobayakawa, Hirokazu Tamamura, Maxwell M Sakyiamah, Wataru Nomura Bioconjug Chem . 2019 May 15;30(5):1442-1450. doi: 10.1021/acs.bioconjchem.9b00182.
A critical part of the development of CXCR4 modulators is to have a simple and sensitive assay system to complement the search by screening and evaluating the binding affinity. Herein, a NanoBRET assay system was developed, and its feasibility as a high-throughput screening tool for potent CXCR4 ligands was ascertained. TAMRA-Ac-TZ14011, a fluorescent-labeled CXCR4 antagonist, was adopted as a fluorescent acceptor of bioluminescent energy from N-terminally fused NanoLuc-CXCR4 stably expressed in CHO cells. The ratio of fluorescence at 620 nm to the luminescence at 460 nm represents the interaction between test compounds and CXCR4. We have demonstrated in the present study that the NanoBRET assay system is applicable for the evaluation of CXCR4 ligands using the combination of TAMRA-Ac-TZ14011 as an acceptor and NanoLuc tagged to CXCR4 as a bioluminescent donor expressed in living cells. IC50values of known CXCR4 ligands were determined and found to be compatible with the values obtained by other existing and sensitive methods, such as SDF-1:3.2 nM, Ac-TZ14011:15.3 nM, and FC131:4.5 nM, which confirmed the feasibility of our system ( Z' values ≥0.5). The introduction of an IL-6 secretory signaling peptide (secNluc-CXCR4) further enhanced the expression and trafficking of the tagged receptor, which, in turn, increased the dynamic range of the NanoBRET assay system. Thus, we have successfully developed a NanoBRET system in living cells, which is simple, homogeneous, and useful in multiwell plate screening of potent CXCR4 ligands.
2. Molecular modeling study of cyclic pentapeptide CXCR4 antagonists: new insight into CXCR4-FC131 interactions
Toshio Furuya, Yasushi Yoshikawa, Nobutaka Fujii, Kazuya Kobayashi, Shinya Oishi Bioorg Med Chem Lett . 2012 Mar 15;22(6):2146-50. doi: 10.1016/j.bmcl.2012.01.134.
CXCR4 is a G-protein coupled receptor that is associated with many diseases such as breast cancer metastasis, HIV infection, leukemic disease and rheumatoid arthritis, and is thus considered an attractive drug target. Previously, we identified a cyclic pentapeptide, FC131, that is a potent antagonist for CXCR4. In this study, we constructed a three dimensional model of the CXCR4-FC131 complex. To investigate the backbone flexibility of FC131, we performed molecular dynamics simulations of FC131 based on the NMR structure of FC131, and obtained snapshot structures from the trajectories which were used to model the docking pose of FC131 into CXCR4. Our final model of the CXCR4-FC131 complex is partially different from the X-ray crystal structure of CXCR4-CVX15 and suggests water-mediated interactions. Nevertheless, this docking pose is consistent with the experimental data. We believe our model will aid in the discovery and development of small-molecule antagonists for CXCR4.
3. Therapeutic potential of the chemokine receptor CXCR4 antagonists as multifunctional agents
Satoshi Ueda, Hirokazu Tamamura, Nobutaka Fujii, Kenichi Hiramatsu, Hiroyuki Masuno, Takanobu Araki, Nami Ohashi, Hiroshi Tsutsumi, Tomohiro Tanaka, Shinya Oishi Biopolymers . 2007;88(2):279-89. doi: 10.1002/bip.20653.
The chemokine receptor CXCR4 possesses multiple critical functions in normal and pathologic physiology. CXCR4 is a G-protein-coupled receptor that transduces signals of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-1). The interaction between CXCL12 and CXCR4 plays an important role in the migration of progenitors during embryologic development of the cardiovascular, hemopoietic, central nervous systems, and so on. This interaction is also known to be involved in several intractable disease processes, including HIV infection, cancer cell metastasis, leukemia cell progression, rheumatoid arthritis (RA), and pulmonary fibrosis. It is conjectured that this interaction may be a critical therapeutic target in all of these diseases, and several CXCR4 antagonists have been proposed as potential drugs. Fourteen-mer peptides, T140 and its analogues, were previously developed in our laboratory as specific CXCR4 antagonists that were identified as HIV-entry inhibitors, anti-cancer-metastatic agents, anti-chronic lymphocytic/acute lymphoblastic leukemia agents, and anti-RA agents. Cyclic pentapeptides, such as FC131 [cyclo(D-Tyr-Arg-Arg-L-3-(2-naphthyl)alanine-Gly)], were also previously found as CXCR4 antagonist leads based on pharmacophores of T140. This review article describes the elucidation of multiple functions of CXCR4 antagonists and the development of a number of low-molecular weight CXCR4 antagonists involving FC131 analogues and other compounds with different scaffolds including linear-type structures.
