Bioconjugate Chem. 2006, 17, 1460−1463
1460
Biologically Active Amphotericin B-Calix[4]arene Conjugates Vale´rie Paquet, Andreas Zumbuehl, and Erick M. Carreira* Laboratorium fu¨r Organische Chemie, ETH Ho¨nggerberg, CH-8093 Zu¨rich, Switzerland. Received July 10, 2006; Revised Manuscript Received September 24, 2006
In order to provide tools for investigations of amphotericin B ion channels, new conjugates bearing a calix[4]arene scaffold covalently linked to four amphotericin B molecules were synthesized. These macromolecules adopt a cone conformation that mimics the structure of a transmembrane pore. The antifungal activity of the conjugates 3 and 4 was superior or similar to that of native amphotericin B, with minimal inhibitory concentration values of 0.10 and 0.25 µM, respectively. Furthermore, the hemotoxicity of the new conjugates was considerably lower (at least 10 times) than the hemotoxicity of monomeric amphotericin B. Finally, the formation of ion channels in the lipid bilayer by the amphotericin B tetramer was monitored by measuring the K+ efflux from various liposomes.
INTRODUCTION Over the past 50 years, amphotericin B (AmB, 1) has remained the drug of choice against systemic fungal infections especially because of its broad spectrum of activity (1, 2). However, although AmB (1) has been the subject of many studies, its mechanism of action has not been clearly elucidated and is still a subject of debate (3). The most widely accepted model refers to the formation of a barrel-stave structure in the membrane that serves as an ion channel causing electrolyte loss that ultimately leads to cell death (Figure 1) (4). AmB (1) molecules are believed to organize in a fashion such that the hydrophilic hydroxylated portion of each molecule faces the inside of the pore, forming a hydrophilic channel, while correspondingly the polyene section of the molecule interacts with the lipid bilayer by being oriented toward the outside of the channel. Even though this model is a classic in membrane biology, both the formation sequence and the precise structure of the channel remain to be confirmed. For instance, several pore sizes have been reported where the number of AmB (1) molecules required to form a channel ranged from 4 to 12, albeit 8 molecules is generally accepted (5). Moreover, the mechanism of pore formation is sometimes described as independent of the sterol located in the membrane (ergosterol in yeast cells and cholesterol in mammalian cells) (6). However, it is more often asserted that channel formation is sterol-dependent, but again, the reported values for the stoichiometry between the sterol and AmB (1) varies considerably in the literature from 0.7 to 3.9 (7). Furthermore, until recently no evidence was available as to whether a single-length or a double-length barrel was formed in the membrane (8). The myriad of mechanistic hypotheses argues for the necessity of designing and synthesizing AmB (1) conjugates with the purpose of providing additional insight. In an attempt to elucidate the nature of the structure of the AmB (1) channel in lipid bilayers, we sought to assemble AmB (1) molecules on a scaffold that would mimic a single-length ion channel structure (9). In such a system, AmB (1) molecules would be preorganized and covalently bound; consequently the number of AmB units forming the pore is clearly defined. Calixarenes were chosen because they are a class of compound that is already known to form synthetic cation channels in lipid * To whom correspondence should be addressed. E-mail: carreira@ org.chem.ethz.ch.
Figure 1. Barrel-stave model wherein AmB molecules form a transmembrane aqueous channel.
Figure 2. Amphotericin B-calix[4]arene conjugate mimicking transmembrane ion channel.
membranes (10). Furthermore, calix[4]arenes are well-defined scaffolds because of their rigidity, particularly with respect to the cone shape, and practical to work with because they are readily synthesized and functionalized (11). Herein, we report the synthesis of a preformed AmB (1) channel bearing four AmB (1) molecules covalently bound to a calix[4]arene core (Figure 2). When compared to monomeric AmB (1), the novel conjugates retain the ability to induce K+ leakage as well as antifungal activity. Investigation of the hemotoxicity also revealed that these macromolecules are 10 times less toxic than native AmB (1).
EXPERIMENTAL PROCEDURES Susceptibility Assays. The minimal inhibitory concentration (MIC) values were determined for the Saccharomyces cereVisiae wild type (BY4741, a derivative of S288C) (12). The assays were inspired by the standard protocol approved by the National Committee of Clinical Laboratory Standards (NCCLS) but using
10.1021/bc060205i CCC: $33.50 © 2006 American Chemical Society Published on Web 10/20/2006
Bioconjugate Chem., Vol. 17, No. 6, 2006 1461
Amphotericin B-Calix[4]arene Conjugates Scheme 1
YEPD liquid medium instead of the RPMI-1640 medium (13). The strains were cultivated overnight in 5 mL of YEPD liquid medium at 30 °C with constant shaking. The saturated cultures were diluted to an ODB600B of 0.1 (3 × 107 cells/mL). For the 24-well plate, each well was prepared by adding 1% (12 µL) of DMSO solution of the tested compound with 1% (12 µL) yeast cells solution and completed with YEPD (1.176 mL). The plates were sealed with Parafilm and then incubated for 18 h at 30 °C. The optical density was read at 600 nm using 1.5 mL cuvettes. The MIC value was defined as the drug concentration needed to inhibit growth at less than 5% compared to a drugfree culture. Hemolytic Assays (14). Human blood, anticoagulated with citrate or EDTA, was centrifuged (2000g) at 4 °C for 10 min. The pellets were washed three times with PBS buffer (pH 7.2 with 2 g/L glucose) and then diluted to a concentration of 4% (4 × 108 cells/mL). All experiments were done in triplicate, and the total volume for the hemolysis tubes was 1.4 mL. The solutions were prepared by adding 1% (14 µL) of DMSO solution of the tested compound with 736 µL of PBS buffer and completed with 750 µL of 4% erythrocytes. After 1 h of incubation at 37 °C, the samples were centrifuged (1500g) at 4 °C for 5 min and the absorbance of the supernatant was measured at 560 nm. The concentration that led to 50% hemolysis (EH50) was interpolated graphically. The value for the 100% hemolysis was obtained by the treatment with 100 µM AmB. K+ Efflux Assays. The K+ efflux measurements were made following a procedure previously described (15). The appropriate liposome suspensions were prepared using POPC, cholesterol, and ergosterol. Then the liposomes were sized by extrusion through two 400, 200, and finally 100 nm pore size membranes (10 times for each pore size). The resultant “100 nm” unilamellar liposomes were dialyzed against 600 mL of 150 mM NaCl, 5 mM HEPES (pH 7.4) buffer. Afterward, the suspension was diluted with 150 mM NaCl, 5 mM HEPES (pH 7.4) buffer to 1 mM overall lipid concentration (phospholipids + sterols). For each efflux measurement 10 mL of this liposome suspension was placed in a small beaker. Potentiometric measurements were performed every second with a 16-channel electrode monitor in magnetically stirred solutions at ambient temperature. The reference electrode was a Metrohm double-junction Ag/AgCl reference electrode with 3 M KCl as the reference electrolyte and 1 M LiOAc as the bridge electrolyte. After recording the amphotericin-induced potassium efflux, liposomes were lysed
Table 1. MIC and EH50 Values for AmB (1) and Conjugates 3 and 4 compd
MIC BY4741 (µM)a
EH50 (µM)b
1 (AmB) 3 4
0.30 0.10 0.25
4.0 50 40
a Assayed with S. cereVisiae (BY4741) following the NCCLS protocol. See Supporting Information for details. b Determined according to the procedure in ref 14. EH50: concentration causing 50% hemoglobin release in human erythrocytes. MIC: minimal inhibitory concentration.
by adding sodium cholate (172 mg). The resulting reading (taken after 1 h) was used to quantify the 100% K+ release.
RESULTS AND DISCUSSION Our group has previously reported the synthesis of a readily accessible piperazine linker as a synthetic anchor for the conjugation of various entities to AmB (1), such as fluorescein (15). On the basis of this approach, we envisioned the use of the AmB derivative 2 in a series of amide coupling reactions with a calix[4]arene precursor bearing several acyl chloride functionalities (Scheme 1). This synthetic route conveniently allows the use of natural and unprotected AmB (1) because the four coupling reactions are chemoselective. The yields reported correspond to pure single isomers of the calixarene AmB conjugates as shown. The conical shape of the calix[4]arene scaffold is preserved throughout the synthesis because it was fixed during a prior alkylation step (16). The conical conformation in the final products was also confirmed by the symmetry found in the aromatic region of the 1H NMR spectra of the conjugates 3 and 4. After the isolation of conjugates 3 and 4, we could then examine if such a preorganized macromolecular arrangement of AmB (1) molecules would display biological activity. First, to evaluate the antifungal activities of 3 and 4, the minimal inhibitory concentrations (MIC, the concentration needed to inhibit growth at less than 5% compared to a drug-free culture) against Saccharomyces cereVisiae were determined (Table 1). Interestingly, there was no diminution in the antifungal activity observed for the conjugates in comparison to native AmB (1). In fact, both MIC values for 3 and 4 (0.10 and 0.25 µM, respectively) were slightly better than for monomeric AmB (1) (MIC ) 0.30 µM). These results are consistent with the ion channel mechanism because the AmB conjugate that mimics pore structure retains good antifungal activity. However, they do not rule out completely the possibility of other mechanisms
1462 Bioconjugate Chem., Vol. 17, No. 6, 2006
Paquet et al.
values previously observed, all compounds rapidly induce complete K+ efflux from vesicles containing ergosterol (red line). The channel formation process seems unaffected by the nature of the substituent distally substituted on the calixarene because both conjugates 3 and 4 retain the ability to induce K+ leakage in the same fashion as a free monomeric AmB (1) molecule. However, derivatives 3 and 4 caused significantly reduced efflux with cholesterol-containing vesicles (blue line), indicating improved selectivity over AmB (1). This superior differentiation between ergosterol and cholesterol containing lipid bilayers is consistent with the observation that compounds 3 and 4 are less hemotoxic than AmB (1), since the nonselective binding of AmB (1) in membranes incorporating ergosterol (fungal) and cholesterol (mammalian) has been considered the main cause of toxicity. In conclusion, the first preorganized AmB single-length ion channels based on a calix[4]arene scaffold were synthesized. Conjugates 3 and 4 not only are as active as native AmB (1) against S. cereVisiae but also retain the ability to induce K+ leakage from vesicles, suggesting an efficient channel formation in the membrane. The fact that these molecules are active is unusual, as previous assumptions have called for 8-12 AmB molecules per channel, while the compounds reported herein consist of only 4 AmB units. Moreover, the minimal hemotoxicity observed for these tetramers is noteworthy. Finally, molecular architectures such as AmB-calix[4]arene discussed herein can serve as an interesting starting point for transmembrane pore models.
ACKNOWLEDGMENT Figure 3. K+ efflux from vesicles (LUVET100) prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (pink line), POPC with 13% ergosterol (red line), and POPC with 30% cholesterol (blue line) caused by AmB (1), 3, and 4, added in DMSO to a suspension of vesicles to give a final concentration of 1.0 µM (a-c).
such as internalization, membrane disruption, and peroxidation. However, in contrast to the prevailing models, which call for 8-12 AmB staves, it would seem that a large number of AmB (1) molecules are not required to form an active ion channel because an arrangement containing only four AmB (1) units is sufficient to preserve activity in the calixarene that forms the basis of our study. The toxicity of the AmB (1) derivatives toward erythrocytes was next examined (Table 1), for which an EH50 value (the concentration of an agent that causes 50% hemoglobin release) can be measured using an established assay (14). AmB derivative 3 (EH50 ) 50 µM) was 10 times less toxic than AmB (1) (EH50 ) 4.0 µM), while 4 displayed even less toxicity (EH50 ) 40 µM). Previous observations have shown that AmB aggregates were more toxic for mammalian cells than the corresponding monomer. However, our results suggest that the spatial arrangement of the complex might be an important factor for controlling the toxicity, with head-to-tail complexes showing higher toxicity than head-to-head complexes such as seen in this communication (17). Differential interactions between ergosterol found in fungal membranes and cholesterol in mammalian cells and the concurrent membrane preorganization are often considered the basis for the selectivity of AmB (1) as an antifungal agent. A convenient assay for studying the channel formation process involves the measurement of induced K+ efflux in liposomes (18). We then studied the ability of derivatives 3 and 4 to lead to K+ efflux from large unilamellar vesicles (LUVs) prepared from POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) admixed with sterols, mimicking conditions found in natural biomembranes (Figure 3) (19). In accordance with the MIC
We acknowledge Oliver Scheidegger from LOC Mass Spectrometry Service for mass measurements. This work was supported by internal ETH Grant TH 0-20201-04 and by a postdoctoral fellowship from NSERC (Canada). Supporting Information Available: Experimental procedures and compound characterization data for all compounds. This material is available free of charge via the Internet at http:// pubs.acs.org.
LITERATURE CITED (1) (a) Donovick, R., Gold, W., Pagano, J. F., Stout, H. A. (1955) Amphotericins A and B, antifungal antibiotics produced by a streptomycete. I. In vitro studies. Antibiot. Annu. 3, 579-586. (b) Hartsel, S. C., Bolard, J. (1996) Amphotericin B: new life for an old drug. Trends Pharmacol. Sci. 12, 445-449. (c) Gallis, H. A., Drew, R. H., Pickard, W. W. (1990) Amphotericin B: 30 years of clinical experience. ReV. Infect. Dis. 12, 308-329. (d) Lemke, A., Kiderlen, A. F., Kayser, O. (2005) Amphotericin B. Appl. Microbiol. Biotechnol. 68, 151-162. (e) Brajtburg, J., Bolard, J. (1996) Carrier effects on biological activity of amphotericin B. Clin. Microbiol. ReV. 9, 512-531. (2) Wu, W., Wieckowski, S., Pastorin, G., Benincasa, M., Klumpp, C., Briand, J.-P., Gennaro, R., Prato, M., Bianco, A. (2005) Targeted delivery of amphotericin B to cells by using functionalized carbon nanotubes. Angew. Chem., Int. Ed. 44, 6358-6362. (3) (a) Brajtburg, J., Powderly, W. G., Kobayashi, G. S., Medoff, G. (1990) Amphotericin B: current understanding of mechanisms of action. Antimicrob. Agents Chemother. 34, 183-188. (b) Fujii, G., Chang, J.-E., Coley, T., Steere, B. (1997) The formation of amphotericin B ion channel in lipid bilayers. Biochemistry 36, 49594968. (c) Resat, H., Baginski, M. (2002) Ion passage pathways and thermodynamics of the amphotericin B membrane channel. Eur. Biophys. J. 31, 294-305. (d) Gruszecki, W. I., Gagos, M., Herec, M., Kernen, P. (2003) Organization of antibiotic amphotericin B in model lipid membranes. A mini review. Cell. Mol. Biol. Lett. 8, 161-170. (e) Odds, F. C., Brown, A. J. P., Gow, N. A. R. (2003) Antifungal agents: mechanisms of action. Trends Microbiol. 11, 272-279.
Bioconjugate Chem., Vol. 17, No. 6, 2006 1463
Amphotericin B-Calix[4]arene Conjugates (4) De Kruijff, B., Demel, R. A. (1974) Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cell and lecithin liposomes. III. Molecular structure of the polyene antibioticcholesterol complexes. Biochim. Biophys. Acta 339, 57-70. (5) (a) Andreoli, T. E. (1974) The structure and function of amphotericin B cholesterol pores in lipid bilayer membranes. Ann. N.Y. Acad. Sci. 235, 448-468. (b) Moreno-Bello, M., Bonilla-Marin, M., Gonzalez-Beltran, C. (1988) Distribution of pore sizes in black lipid membranes treated with nystatin. Biochim. Biophys. Acta. 944, 97100. (c) Bonilla-Marin, M., Moreno-Bello, M., Ortega-Blake, I. (1991) A microscopic electrostatic model for the amphotericin B channel. Biochim. Biophys. Acta 1061, 65-77. (6) Hartsel, S. C., Benz, S. K., Peterson, R. P., Whyte, B. S. (1991) Potassium-selective amphotericin B channels are predominant in vesicles regardless of sidedness. Biochemistry 30, 77-82. (7) (a) De Kruijff, B., Gerritsen, W. J., Oerlemans, A., van Dijck, P. W., Demel, R. A., van Deenen, L. L. (1974) Polyene antibioticsterol interactions in membranes of Acholesplasma laidlawii cells and lecithin liposomes. II. Temperature dependence of the polyene antibiotic-sterol complex formation. Biochim. Biophys. Acta 339, 44-56. (b) Hartsel, S. C., Hatch, C., Ayenew, W. (1993) How does amphotericin B work? Studies on model membrane systems. J. Liposome Res. 3, 377-408. (c) Milhaud, J., Ponsinet, V., Takashi, M., Michels, B. (2002) Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol. Biochim. Biophys. Acta 1558, 95108. (8) Matsuoka, S., Ikeuchi, H., Matsumori, N., Murata, M. (2005) Dominant formation of a single-length channel by amphotericin B in dimyristoylphosphatidylcholine membrane evidence by 13C-31P rotation echo double resonance. Biochemistry 44, 704-710. (9) For synthetic studies in which the polyene subunit was replaced with a biaryl bis, see the following. Rogers, B., Selsted, M. E., Rychnovsky, S. D. (1997) Synthesis and biological evaluation of non-polyene analogs of amphotericin B. Bioorg. Med. Chem. Lett. 7, 3177-3182. (10) (a) De Mendoza, J., Cuevas, F., Prados, P., Meadows, E. S., Gokel, G. W. (1998) A synthetic cation-transporting calix[4]arene derivative active in phospholipid bilayers. Angew. Chem., Int. Ed. 37, 15341537. (b) Yoshino, N., Satake, A., Kobuke, Y. (2001) An artificial ion channel formed by a macrocyclic resorcin[4]arene with amphiphilic cholic acid ether groups. Angew. Chem., Int. Ed. 40, 457459. (c) Maulucci, N., De Riccardis, F., Botta, C. B., Casapullo, A., Cressina, E., Fregonese, M., Tecilla, P., Izzo, I. (2005) Calix[4]arene-cholic acid conjugates: a new class of efficient synthetic ionophores. Chem. Commun. 10, 1354-1356. (11) Gutsche, C. D. (1998) Calixarenes revisited. In Monographs in Supramoleular Chemistry (Stoddart, J. F., Ed.) Royal Society of Chemistry, Cambridge, U.K.
(12) Brachmann, C. B.; Davies, A.; Cost, G. J.; Caputo, E.; Li, J.; Hieter, P.; Boeke, J. D. (1998) Designer deletion strains derived from Saccharomyces cereVisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115-132. (13) (1995) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast, M27-A2, ApproVed Standard, 2nd ed. Vol. 22, Number 15, National Committee of Clinical Laboratory Standards: Wayne, PA. (14) Kinsky, S. C., Avruch, J., Permutt, M., Rogers, H. B. (1962) The lytic effect of polyene antifungal antibiotics on mammalian erythrocytes. Biochem. Biophys. Res. Commun. 9, 503-507. (15) Zumbuehl, A., Jeannerat, D., Martin, S. E., Sohrmann, M., Stano, P., Vigassy, T., Clark, D. D., Hussey, S. L., Peter, M., Peterson, B. R., Pretsch, E., Walde, P., Carreira, E. M. (2004) An amphotericin B-fluorescein conjugate as a powerful probe for biochemical studies of the membrane. Angew. Chem., Int. Ed. 43, 5181-5185. (16) (a) Gutsche, C. S., Levine, J. A. (1982) Calix[4]arenes. 6. Synthesis of a functionalizable calix[4]arene in a conformationally rigid cone conformation. J. Am. Chem. Soc. 104, 2652-2653. (b) Casnati, A., Ting, Y., Berti, D., Fabbi, M., Pochini, A., Ungaro, R., Sciotto, D., Lombardo, G. G. (1993) Synthesis of water soluble molecular receptors from calix[4]arenes fixed in the cone conformation. Tetrahedron 49, 9815-9822. (17) (a) Hartsel, S. C., Bauer, E., Kwong, E. H., Wasan, K. M. (2001) The effect of serum albumin on amphotericin B aggregate structure and activity. Pharm. Res. 9, 1305-1309. (b) Szlinder-Richert, J., Mazerski, J., Cybulska, B., Grzybowska, J., Borowski, E. (2001) MFAME, N-methyl-N-D-fructosyl amphotericin B methyl ester, a new amphotericin B derivative of low toxicity: relationship between self-association and effects on red blood cells. Biochim. Biophys. Acta 1528, 15-24. (c) Janknegt, R., de Marie, S., BakkerWoudenberg, I. A., Crommelin, D. J. (1992) Liposomal and lipid formulations of amphotericin B. Clin. Pharmacol. 4, 279-291. (d) Bolard, J., Legrand, P., Heitz, F., Cybulska, B. (1991) One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium. Biochemistry 23, 5707-5715. (18) Zumbuehl, A., Stano, P., Heer, D., Walde, P., Carreira, E. M. (2004) Amphotericin B as a potential probe of the physical state of vesicle membranes. Org. Lett. 6, 3683-3686. (19) Paquet, M.-J., Fournier, I., Barwicz, J., Tancre`de, P., Auger, M. (2002) The effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers as viewed by 2H NMR. Chem. Phys. Lipids. 119, 1-11. BC060205I
