A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Abstract

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.

A practical process for the preparation of [32P]S1P and binding assay for S1P receptor ligands

“…The IC 50 values measured by our protocol are shown in Table 1. The binding affinity for these tested compounds are comparable with previous reports (Kiuchi et al, 2005; Mandala et al, 2002; Osada et al, 2002; Satsu et al, 2013). Figure 2 shows representative fitting curves of the competitive binding assay.…”

Survival benefit of sphingosin‐1‐phosphate and receptors expressions in breast cancer patients

“…Although we did not observe effect of S1P‐S1PR2 binding in the inhibition of colony formation of BCA cells, results of our bioinformatic analyses showed a positive correlation between S1PR2 expression and BCA progression. There is a compound designated CYM‐5520 58 has been shown to more selective for activating S1PR2 and may be suitable for BCA treatment. Although these compounds are currently available only for research, they may become effective regimens for BCA treatment, based on our findings in this study.…”

Inhibitors of sphingosine-1-phosphate metabolism (sphingosine kinases and sphingosine-1-phosphate lyase)

“…CYM5541 S1P3 ML178 S1P4 CYM5038 (ML248) S1P4 (Urbano et al, 2011) XAX162 S1P2 (Satsu et al, 2013) CYM5520 S1P2 (Satsu et al, 2013) Table 3. S1P receptor antagonists (see Figure 4 for structures)…”