DESCRIPTION: Squalamine is a novel aminosterol isolated from Squalus acanthias which exhibits broad spectrum antimicrobial activity. The mechanism of how squalamine kills microbial cells is yet to be determined. Squalamine preferentially lyses vesicles comprised of anionic lipids over vesicles comprised of zwitterionic lipids. However, additional studies are necessary to understand the mechanism of membrane lysis by squalamine, and to explain some interesting observations recorded in the preliminary work. The general aim of this proposal is to understand the mechanism of the antimicrobial activity of squalamine. The specific aims of this proposal are: 1. Squalamine has been demonstrated to cause the leakage of fluorescent dyes from phospholipid vesicles. The membrane defect generated by squalamine will be characterized by determining the mode of dye leakage (either all-or-none or graded). Also, the size of the defect will be estimated by comparing the ability of squalamine to induce the leakage of dyes with different molecular sizes from lipid vesicles. 2. Squalamine will be synthetically tritiated, and the radiolabeled squalamine used to measure sterol binding to phospholipid vesicles using equilibrium dialysis followed by scintillation counting. 3. Preliminary studies of squalamine-induced dye leakage from phosphatidylcholine vesicles can be interpreted as the result of membrane fusion. The fusogenic potential of squalamine will be directly measured in different phospholipid systems. Vesicle fusion will be measured either by monitoring the mixing of vesicle contents, using two populations of vesicles loaded with a fluorophore and quenching agent, respectively, or by a lipid mixing assay using fluorescence resonance energy transfer. 4. Analogs of squalamine have been synthesized which exhibit antimicrobial activity. The synthesis of spectroscopic probes of squalamine will be undertaken. Specifically, squalamine probes will be synthesized containing 15N (in the polyamine chain), 19F (within the sterol ring), or 13C (in the alkyl ring substituent), which can be used as NMR probes of squalamine orientation in oriented systems.