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In the present study, we developed a high-throughput and sensitive assay for interactions of amphotericin B (AmB) with two model lipid membranes, which mimicked mammal cell membrane and fungal membrane using surface plasmon resonance (SPR). The binding kinetics of AmB to the membrane could be analyzed by multiple sensorgrams obtained at different AmB concentrations, indicating that the binding properties could be clarified for an approximately 7-fold concentration range. AmB showed an approximately 18-fold higher affinity for ergosterol-containing membrane than for cholesterol-containing membrane. We also optimized the procedure for the reproducible immobilization of liposome containing the sterols and the estimation of binding kinetics of AmB to the lipid membranes, and the sensitivity of AmB to membrane interaction was 20-fold higher, compared with the reported method. The throughput of the established method for the binding kinetics characterization was calculated to be 10 compounds a day. The results demonstrate that the established SPR method could be a valuable tool for predicting selective binding to sterol-containing membranes.Key words amphotericin B; surface plasmon resonance; lipid membrane Amphotericin B (AmB), an antifungal polyene macrolide antibiotic produced by Streptomyces nodosus, is known as the gold-standard drug for the treatment of many systemic fungal infections or "deep mycoses" that could be life-threatening for specific populations, such as immunocompromised, transplant and cancer patients.1) The route of administration for antifungal therapies with AmB is intravenous (i.v.) infusion. This treatment requires hospitalization and has numerous side effects, such as nephrotoxic potential.2-4) Therefore, the development of new antifungal antibiotics is desired for more simplified treatment and reduction of side effects.AmB has a highly hydrophobic, water-insoluble molecule with amphiphilic properties, and the drug product formulation of AmB is quite challenging. 5) Although there are various ways to formulate new drug products based on the AmB skeleton, no formulation of AmB is currently available for systemic treatment of infection after oral administration. It is generally accepted that an ion-permeable channel formed across the bilayer membrane is responsible for the biological activity of AmB.6,7) The antibiotic activity of AmB is believed to be primarily due to a higher affinity for ergosterol, a primary fungal sterol, than for cholesterol in mammalian membranes.8-11) Thus, in order to develop a new drug based on the AmB skeleton with better antibiotic efficacy and fewer side effects, such as nephrotoxicity, diverse drug candidates need to be examined for their membrane affinity.The affinity of AmB for lipid membranes is one of the critical factors influencing selectivity and potency. Surface plasmon resonance (SPR)-based biosensors have been already applied to study the membrane-binding properties of drugs with the aim of gaining insight into their mechanism of action. 12,13) We and ou...

Xenopus laevis is an excellent animal for analyzing early vertebrate development. Various effects of glycosaminoglycans (GAGs) on growth factor-related cellular events during embryogenesis have been demonstrated in Xenopus. To elucidate the relationship between alterations in fine structure and changes in the specificity of growth factor binding during Xenopus development, heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) chains were isolated at four different embryonic stages and their structure and growth factor-binding capacities were compared. The total amounts of both HS and CS/DS chains decreased from the pre-midblastula transition to the gastrula stage, but increased exponentially during the following developmental stages. The length of HS chains was not significantly affected by development, whereas that of CS/DS chains increased with development. The disaccharide composition of GAGs in embryos also changed during development. The degree of sulfation of the HS chains gradually decreased with development. The predominant sulfation position in the CS/DS chains shifted from C4 to C6 of GalNAc during embryogenesis. Growth factor-binding experiments using a BIAcore system demonstrated that GAGs bound growth factors including fibroblast growth factors-1 and -2, midkine, and pleiotrophin, with comparable affinities. These affinities significantly varied during development, although the correlation between the structural alterations of GAGs and the change in the ability to bind growth factors remains to be clarified. The expression of saccharide sequences, which specifically interact with a growth factor, might be regulated during development.