Efomycine M, a new specific inhibitor of selectin, impairs leukocyte adhesion and alleviates cutaneous inflammation

To identify small-molecule compounds that interfered with leukocyte adhesion, we screened a library of 20,000 natural compounds. We found such activity in fermentation material from Streptomyces BS1261, in which we identified by fast atom bombardment (FAB) mass spectrometry a group of macrolides that we termed efomycines. In adhesion assays evaluating the binding of thrombin-activated human platelets to neutrophils, the efomycines (at a mixed total concentration of 10^-7−10^-4 M in the fermentation supernatant) significantly reduced binding in a concentration-dependent fashion. At an efomycine concentration of 2 10^-5 M, neutrophil−platelet adhesion was inhibited by 86% (P < 0.01 compared to controls) (Fig. 1a). This was not due to nonspecific toxic events or to interference with platelet activation, as similar results were obtained when neutrophils were fixed with paraformaldehyde and when efomycines were added to the platelets either before or after thrombin activation (data not shown). As adhesion between activated platelets and neutrophils is mediated by P-selectin¹¹, these results indicated that efomycines might interfere with P-selectin-dependent adhesion.

Figure 1. Efomycines inhibit selectin-mediated functions in vitro. a, Neutrophil/platelet adhesion assays in normal culture medium (first column), in the presence of EDTA to chelate divalent cations (second column) or of Streptomyces BS1261 fermentation material containing the indicated total concentrations of efomycines A, B, E and G (columns 3−8). Experiment is representative of six experiments showing similar results. b, Binding of carcinoembryonic antigen (CEA, bearing the sialyl LewisX moiety) to purified srE-selectin assessed in the presence of efomycine M at different concentrations (), a function-blocking antibody against E-selectin () or a mixture of 10-6 M efomycine M and antibody against E-selectin (). Bound CEA was determined by ELISA. c, Adhesion of human neutrophilic granulocytes metabolically labeled with [35S]methionine to srP-selectin was assessed in the presence of efomycine M at different concentrations as indicated (), a function-blocking antibody against P-selectin () or a mixture of 10-6 M efomycine M and the antibody against P-selectin (). Bound radioactivity was determined by scintillation counting after SDS lysis of bound neutrophils. (*, P < 0.05; ** P < 0.01) Full Figure and legend (37K)

In another series of experiments, we coated microtiter plate wells with srE-selectin and assessed the binding of carcinoembryonic antigen (CEA)—which bears the sialyl Lewis^X moiety, a natural selectin ligand—by enzyme-linked immunosorbent assay (ELISA). Efomycines significantly inhibited the E-selectin-dependent CEA binding, by 69% ( 3.6, P < 0.01; data not shown). Thus, efomycines interfered with both P- and E-selectin-mediated adhesive interactions.

To assess the ultrastructural conformation of efomycines required for inhibition of leukocyte adhesion, we separated natural efomycines from Streptomyces BS1261 fermentation material by high-performance liquid chromatography (HPLC) and analyzed them by thin-layer chromatography (TLC), reverse-phase HPLC, ¹H-NMR spectroscopy and FAB mass spectrometry. The compounds isolated were termed efomycines A (M_{r =} 1,038), B (M_{r =} 1,052), E (M_{r =} 1,024) and G (M_{r =} 1,010). In addition, we synthesized a fifth compound, efomycine M, by base-catalyzed -elimination of the deoxyfucose side chains (M_{r =} 728; Fig. 2).

Figure 2. Structures of efomycine G (natural species isolated from the fermentation material of Streptomyces BS1261) and efomycine M (synthesized by defucosylation), typical members of the efomycine family. Full Figure and legend (16K)

To investigate this effect further, we coated microtiter plates with srE-selectin and assessed the binding of CEA to the plates in cell-free adhesion experiments. In two experiments, efomycine M (10^-6 M) significantly inhibited CEA binding to E-selectin by 73.4% 3.6 and 64.8% 2.9, respectively (P < 0.01 in both cases as compared to medium controls). A function-blocking monoclonal antibody against E-selectin had similar effects (P < 0.01), and simultaneous incubation with efomycine M and antibody against anti-E-selectin did not increase the inhibitory effect (Fig. 1b).

We also assessed the effect of efomycine M on P-selectin-mediated adhesion using srP-selectin and radiolabeled human neutrophils. In two experiments, efomycine M inhibited neutrophil binding to srP-selectin in a dose-dependent manner. At 10^-5 efomycine M, binding was inhibited by 51.3% ( 2.1) and 47.9% ( 1.9), respectively for each experiment (P < 0.05). An antibody against P-selectin inhibited binding by 70.0% ( 4.5) (Fig. 1c). Thus, efomycine M interfered with adhesive interactions mediated by endothelial selectins, but this effect was more pronounced with E-selectin than with P-selectin.

Given that efomycine M specifically inhibited selectin-mediated leukocyte adhesion, we investigated leukocyte rolling in vivo by intravital microscopy of mouse ear microvessels. In untreated mice injected into the aortic arch with intravitally labeled human lymphocytes^{12, 13}, T cells had a mean rolling fraction of 13.6% 3.7 (n = 9 vessels/3 mice). Intravascular injection of efomycine M (5 mg/kg) resulted in immediate, significant reduction of rolling lymphocytes by 72.8% (rolling fraction 3.7%, 1.1, P < 0.03). Function-blocking monoclonal antibodies against E- and P-selectin did not further inhibit the proportion of rolling cells (rolling fraction 2.9%, 1.5), consistent with the hypothesis that selectin-mediated functions were inhibited by efomycine M (Fig. 3). When mice (n = 3) were first injected with the selectin-directed antibodies followed by efomycine M, the antibody injection reduced lymphocyte rolling by 69.7% (rolling fraction 12.2% 2.5 in untreated mice as compared to 3.7% 2.1 in mice injected with antibodies; n = 10 vessels/3 mice, P < 0.03). Additional injection of efomycine M did not enhance this inhibitory effect (rolling fraction 4.3% 3.5) (Fig. 3). Differences in lymphocyte rolling were not due to changes of blood flow, as determined by velocity of non-interacting cells (V_free; data not shown). Thus, selectin-mediated functions seemed to be inhibited by efomycine M in vivo.

Figure 3. Efomycine M inhibits selectin-mediated leukocyte rolling in vivo. Rolling human T lymphocytes were assessed by intravital microscopy in mouse ear microvessels. Top, rolling T cells were assessed in untreated mice (n = 3) after intravascular single-shot injection of efomycine M (5 mg/kg) and additional injection of function-blocking antibodies against E-selectin and P-selectin as indicated. Bottom, rolling was assessed in untreated mice (n = 3), and then the same treatments given in reverse order (injection of antibodies against E-selectin and P-selectin followed by efomycine M). Neither efomycine M nor function-blocking antibodies to endothelial selectins enhanced the inhibitory effect of the other. **, P<0.03. Full Figure and legend (34K)

As indicated by the microscopic morphology of HDMEC, the efomycine mixture (Streptomyces BS1261 fermentation material) and the purified efomycines A, B, E and G were highly toxic at concentrations of 10^-7 M and above. Because efomycine M at concentrations up to 10^-5 M did not alter the endothelial cell morphology, we assessed the impact of efomycines G and M upon HDMEC viability, apoptosis and proliferation. Although efomycine M did not induce significant necrosis at concentrations as high as 3 10^-5 M for up to 72 h, efomycine G induced marked cytotoxicity within 24 h at concentrations as low as 5 10^-8 M (Fig. 4a). Efomycine G also significantly inhibited HDMEC proliferation at concentrations of more than or equal to 10^-7 M, although efomycine M did so only at concentrations above 10^—5 M (Fig. 4b). Finally, efomycine M moderately induced apoptosis of HDMEC at concentrations of 3 10^-5 M. The apoptotic effects of efomycine G could not be evaluated because of its direct cytotoxicity (data not shown). Thus, the toxicity to cultured HDMEC of efomycine M was lower by a factor of approximately 100 than that of its analog efomycine G.

Figure 4. Low in vitro toxicity and low in vivo plasma clearance of efomycine M. a, HDMEC were incubated with vehicle, efomycine G or efomycine M for 72 h. Direct cytotoxicity of efomycines on HDMECs was assessed fluorometrically. Values represent percentage of cell viability in control vehicle-treated cultures ( s.d.). Experiment is representative of three independent experiments showing similar results. b, Proliferation of HDMECs was assessed fluorometrically in triplicate cultures incubated with efomycine G or efomycine M as indicated. Values shown represent percentage fluorescence as compared to untreated cultures ( s.d.). c, Female scid/scid mice (n = 3) were injected intraperitoneally with efomycine M (5 mg/kg), and plasma concentrations of the compound were determined at various time points. Values indicate mean plasma concentrations s.d. , efo M 24 h; , efo M 48 h; , efo M, 72 h; , efo G 24 h; , efo G 48 h; , efo G 72 h. Full Figure and legend (38K)

Because of its selective inhibition of selectin-dependent leukocyte adhesion and low toxicity, we assessed efomycine M as a candidate for in vivo applications. In initial pharmacokinetic studies, plasma concentrations of efomycine M increased rapidly, reaching a maximum of 3 10^-6 M (2.16 mg/l; s.d., 0.033) at 30 min after a single i. p. injection of scid/scid mice (n = 3) with 5 mg/kg (Fig. 4c). The maximum bioavailability calculated as the area under the curve (AUC) was 3,510 g h/l. Normalized with respect to body weight, the AUC_norm was 0.702 kg h/l. The subsequent decrease of plasma concentrations indicated an elimination half-life of more than 2 h, with a calculated plasma clearance of 1.4 l/h/kg and a blood clearance of 1.5 l/h/kg. Thus, efomycine M had a moderate to low clearance, was well absorbed from the peritoneal cavity and reached plasma concentrations of more than 100 g/l (10^-6 M) 4 h after a single injection of 5 mg/kg. Because these concentrations were within the ranges that had been effective in vitro in inhibiting leukocyte−endothelial cell adhesion, we assessed efomycine M in disease models in vivo.

We first used a T cell−mediated mouse model of hyperproliferative inflammatory skin disorders such as psoriasis¹⁰. In this system, the transfer of major histocompatibility (MHC)−matched but minor histocompatibility−mismatched CD4⁺CD45RB^Hi donor T lymphocytes induced psoriasiform skin inflammation in scid/scid mice, in addition to the intestinal inflammation that follows the transfer of syngeneic donor cells^{20, 21}. As expected¹⁰, in untreated recipient mice, the psoriasiform skin lesions worsened over time, whereas treatment with cyclosporin A (starting 7 weeks after T cell transfer) resulted in significant alleviation of the hyperproliferative inflammatory skin alterations. Similar, though somewhat weaker, therapeutic effects were seen when the recipient mice were treated by intraperitoneal (i.p.) injection of antibodies (200 g) against E-selectin or CD11b²². Notably, daily injection of 5 mg/kg i.p. efomycine M also resulted in rapid, pronounced improvement of the psoriasiform skin alterations (Fig. 5b). In fact, the improvement in clinical score and the reduction of ear thickness by efomycine M were apparent as early as 2−3 days after the initiation of treatment and were as pronounced as those seen with cyclosporin A. As assessed by histopathological analysis and immunohistochemistry, the clinical improvement was associated with a significant reduction in epidermal thickness and a marked reduction in infiltrating T lymphocytes, neutrophils and other leukocytes (Fig. 5a). In addition, epidermal expression of intercellular adhesion molecule-1 (ICAM-1), MHC class II and integrin ₆ were markedly reduced in recipient mice treated with efomycine M (data not shown). The concomitant intestinal inflammation also was alleviated by efomycine M, as indicated by a marked reduction of the inflammatory infiltrate within the intestinal lamina propria (n = 3 mice/group; data not shown).

Figure 5. Efomycine M effectively alleviates hyperproliferative inflammatory skin lesions in a T cell−mediated mouse model of psoriasis. a, scid/scid recipient mice were reconstituted with CD4+CD45RBHi T lymphocytes. Skin sections of non-reconstituted scid/scid mice (top) and of recipients treated with vehicle (middle) and with efomycine M (bottom), harvested after 3 weeks of treatment, were stained with hematoxylin and eosin (H&E) or for leukocyte antigens as indicated. Scale bar, 20 m. b, Hyperproliferative inflammatory skin lesions were induced by reconstitution of scid/scid mice with CD4+CD45RBHi T cells. Ear thicknesses were monitored as the clinical parameter of the resulting psoriasiform skin disorder. Groups of reconstituted mice were treated by daily i.p. injections (for b and c): , uninjected;, vehicle; , cyclosporine A; efomycine M; , anti-CD62E; , anti-CD11b. Arrow, beginning of treatment; error bars, s.d. c, scid/scid mice were reconstituted with CD4+CD45RBHi T lymphocytes and treated by i.p. injection immediately after reconstitution (arrow, beginning of treatment), repeated on 3 consecutive days. The ear thicknesses as clinical parameter of the resulting psoriasiform skin disorder were monitored. d, Alleviation of psoriatic tissue alterations in transplanted human skin by efomycine M. Lesional psoriatic skin from a patient with chronic plaque psoriasis transplanted onto the back of a scid/scid mouse was treated with vehicle, dexamethasone or efomycine M as indicated. Tissues were harvested after 4 weeks of treatment, embedded in paraffin and stained with H&E. The tissue samples shown are from the same patient and are representative of psoriatic skin obtained from three different patients showing similar results. The insert in the left panel highlights the inflammatory infiltrate and leukocyte exocytosis in the papillar region. Scale bar, 20 m. Full Figure and legend (221K)

In the second model used to assess the therapeutic efficacy of efomycine M, scid/scid mice (n = 3 mice/group) transplanted with lesional psoriatic skin from three donors^{9, 15} were treated for four weeks with subcutaneously (s.c.) injected vehicle or efomycine M (5 mg/kg) or with orally administered dexamethasone (0.2 mg/kg). Vehicle-treated grafts retained all characteristics of lesional psoriatic skin, with a uniform pronounced acanthosis, elongated rete ridges and prominent parakeratosis. An inflammatory infiltrate persisted within the dermis that was most pronounced within the papillary region (insert in Fig. 5d), and epidermal microabscesses (a hallmark of psoriasis) were readily detectable. In contrast, dexamethasone treatment resulted in profound reduction of these features. Epidermal thickness decreased markedly (from 363 m 148 m to 175 m 51 m; P < 0.01) (Fig. 5d). In grafts treated with efomycine M, the epidermal thickness also was markedly reduced (181 m 50 m; P < 0.01) (Fig. 5d). In addition, immunohistochemical analyses confirmed a substantial reduction of infiltrating T lymphocytes and neutrophils (data not shown). Thus, efomycine M had a marked therapeutic effect in the treatment of human psoriatic skin lesions, similar to that seen with dexamethasone.

Figure 6. NMR-based molecular modeling shows similar three-dimensional structures of efomycine M and sialyl LewisX. a, Spatial conformations of efomycine M and sialyl Lewisx were obtained by 1H-NMR analysis and molecular modeling procedures (see Methods). When hydroxyl groups reported to be pivotal structures for adhesive functions of sialyl LewisX were defined as anchor structures, marked similarities became apparent between the two molecules. b, Schematic demonstration of the function-bearing groups spanning the macrolide ring system and the acyclic side chain of efomycine M. Full Figure and legend (70K)

To determine whether efomycine M is efficacious in the treatment of inflammatory disorders, we have focused on psoriasis, a common T cell−mediated autoimmune disorder¹⁴. These proof-of-principle studies used two complementary mouse models that have been used in screening anti-psoriatic drugs^{24, 25, 26}. In these models, efomycine M was as efficacious as cyclosporin A or antibodies against E-selectin or _M2 integrin in the transfer model, or dexamethasone in the xenotransplantation model. Although psoriasis as well as the skin disorder in the CD4⁺CD45RB^Hi transfer model are mediated primarily by T cells^{10, 27}, other leukocytes, such as neutrophils, are also involved^{2, 14, 17, 18}. As selectin-mediated rolling initiates the tissue localization of both T cells and neutrophils, efomycine M presumably interfered with extravasation of both (or more) cell types, thus having a twofold effect on key pathophysiological events. Notably, efomycine M administered immediately after reconstitution of scid/scid mice prevented disease development, consistent with its preventing the initial migration of pathogenic T cells. It seems less likely that efomycine M modulated other pathogenic events during the course of this skin disorder, such as local T cell activation or leukocyte retention. In any case, in both models, various parameters of psoriasiform cutaneous inflammation were greatly alleviated by efomycine M treatment.

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Fermentations of Streptomyces BS1261 were separated by reversed-phase HPLC, and the compounds detected by UV adsorption (250 nm) were analyzed by TLC (RP18 plate, 9:1 acetonitrile/water) and FAB mass spectrometry. The peak areas indicated a ratio of efomycines G/E/A/B of 14:68:16:2. Efomycines were further characterized by TLC, analytical reverse-phase chromatography (RPC) on an RP18-HPLC column, ¹H-NMR spectroscopy and FAB mass spectrometry. Efomycine M was synthesized by base-catalyzed deglycosylation leading to -elimination of the deoxyfucose chains as described⁴³.

For assessment of endothelial cell−neutrophil adhesion, TNF-stimulated HUVECs and HDMECs⁴⁴ were incubated with efomycines at concentrations from 10^-6 to 2 10^-5 M for 20 min. Neutrophils intravitally labeled with BCECF according to the manufacturer's instructions (Sigma, Deisenhofen, Germany) were added (2 10⁵ cells/well) and allowed to adhere for 20 min. Adherent cells were lysed by 0.5% CTAB (cetyl-trimethyl-ammonium bromide), and fluorescence was measured at 485 nm in an ELISA reader.

For assessment of adhesion of CEA to srE-selectin, CEA (0.3 g/ml) was added in the presence or absence of efomycine M (10^-7−10^-5 M) or an antibody against E-selectin (68-5H11, PharMingen, Heidelberg, Germany; 2 g/well) onto srE-selectin (gift of M. Adamczewski) coated at 10 g/ml onto microtiter plates. Bound CEA was detected by ELISA.

For assessment of neutrophil adhesion to srP-selectin, human neutrophils (2 10⁵/well) intravitally labeled with [³⁵S]methionine (Amersham, Freiberg, Germany; 0.2 mCi per 10⁷ cells) were added for 1 h to microtiter plates coated with 50 l of srP-selectin (Serotec/Biozol, Eching, Germany; 10 g/ml). Adhesion was blocked by efomycine M (10^-7−10^-5 M) or the monoclonal antibody of the function-blocking anti-P-selectin monoclonal antibody AK-4 (PharMingen; 2 g/well). Bound cells were lysed using 2% SDS, and radioactivity was quantified by scintillation counting.

Female scid/scid mice (n = 3/group) received a single intraperitoneal injection of 5 mg/kg efomycine M. Ten blood samples were drawn between 5 min and 24 h after compound administration. Urine was collected for 24 h. The plasma concentration of efomycine M was determined after liquid−liquid extraction; the quantification limit was 5 g/l.

Reconstitution of scid/scid mice with CD4⁺CD45RB^Hi T lymphocytes was done as described¹⁰. Recipients were treated 6−7 weeks after reconstitution by daily i.p. injections for 2 weeks (5 mg/kg efomycine M, 30 mg/kg cyclosporin A, 200 g of antibody against E-selectin or _M integrin). In another experiment, recipients were treated immediately after reconstitution and on 3 consecutive days. Mice were monitored for 7 weeks, and histopathological and immunohistochemical analyses were done as described¹⁰.

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