Bernd Buchmann

Efomycine M: an inhibitor of selectins?

To the editor: In a recent issue of Nature Medicine, Schön et al. characterized efomycine M (Efo-M) isolated from Streptomyces BS1261 as a specific inhibitor of selectins1. The authors showed that Efo-M at low micromolar concentrations is able to block Eand P-selectin interactions with sialyl Lewisx (sLex)-bearing ligands, for example, carcinoembryonic antigen (CEA). Moreover, Efo-M inhibited selectin-mediated leukocyte rolling in vivo and substantially improved the clinical symptoms of two in vivo inflammatory skin models using transplanted human skin biopsies. Thus, Efo-M was reported to function as a panselectin antagonist with promising therapeutic potential. Following the hypothesis by Schön et al., we performed extended in vitro and in vivo investigations with Efo-M. Although in principle we could confirm the anti-inflammatory profile of Efo-M, our findings suggest a mode of action that is separate from pan-selectin inhibition. As shown in Figure 1a, highly purified Efo-M (Supplementary Fig. 1 online) does not compete with the binding of 3H-labeled, polyvalent sLex (3H–p-sLex) to E-, Land P-selectins, even at concentrations of 100 μM in scintillation proximity assays (SPAs)2. In contrast, an excess of unlabeled p-sLex as well as fucoidan (Supplementary Fig. 2 online) clearly inhibited the binding of 3H–p-sLex, whereas monovalent sLex competed with 3H–p-Lex only in binding to E-selectin (Supplementary Fig. 3 online), as described previously3. As the experimental setup of SPAs described in Figure 1a allows the quantification of binding only to the glycan binding site of selectins, we repeated the experiment with 3H-labeled Efo-M (Fig. 1b), which should allow the detection of Efo-M binding to a glycan-independent binding site. However, no specific (for example, Ca2+-dependent and selectin-specific) binding of 3H-labeled Efo-M to Land P-selectin could be detected. Moreover, measuring direct interactions of Efo-M to selectins in ELISA (Fig. 1c) as well as in highly sensitive Biacore experiments revealed only a weak binding of Efo-M to L-selectin at higher concentrations, and no specific binding of Efo-M to Eand P-selectin in this type of assay was detectable (Fig. 1d). In their original study and a subsequent publication4, Schön et al. showed a model-based alignment of Efo-M and sLex structures, suggesting a comparable orientation of hydroxyl groups in Efo-M and sLex, which are crucial for the adhesive function of selectin ligands. Our analysis using crystallized Efo-M and a liquidstate conformation analysis of Efo-M by nuclear magnetic resonance (NMR) spectroscopy provided no evidence that sLex and Efo-M share a three-point pharmacophore when binding to the selectin surface (Supplementary Fig. 4 online). These results as well as the lack of binding of Efo-M to selectins under different experimental conditions suggest an alternative explanation for the findings of Schön et al. Although we observed anti-inflammatory activity in our in vivo models as well (Fig. 1e), Efo-M clearly exhibited selectinindependent effects on immune cells at low micromolar concentrations. Ex vivo–stimulated spleen cells from dinitrofluorbenzene-challenged and Efo-M–treated mice produced less tumor necrosis factor (TNF)-α (Fig. 1f). Moreover, in the presence of Efo-M (10 μM), activated mouse spleen cells (data not shown) and human peripheral blood mononuclear cells (PBMCs) showed a 50% reduction in cell viability (Fig. 1g), and lipopolysaccharide (LPS) induced secretion of TNF-α (Supplementary Fig. 5 online). At concentrations of >10 μM, Efo-M induced apoptosis in more than 30% of purified human B and T cells (Fig. 1h) and inhibited the maturation of dendritic cells (Supplementary Fig. 6 online). Efo-M was cytotoxic in PBMC cultures at low micromolar concentrations (50% inhibitory concentration (IC50), 4 μM), indicating again the substantial inhibition of cell viability caused by Efo-M, whereas we observed induction of apoptosis in human endothelial cells with higher concentrations of Efo-M (IC50, 27 μM; Supplementary Fig. 7 online) and no apoptosis in human neutrophils (data not shown). Together, our findings suggest that a selectinantagonizing function is probably not responsible for the observed in vivo results by Schön et al. and raise questions regarding how Efo-M inhibits inflammatory processes in the skin.