Barry S. Selinsky

The synthesis and characterization of analogs of the antimicrobial compound squalamine: 6β-hydroxy-3-aminosterols synthesized from hyodeoxycholic acid

Analogs of the aminosterol antimicrobial agent squalamine have been synthesized beginning from hyodeoxycholic acid. After carboxylic acid esterification and oxidation of both alcohol functions to ketones, the A/B ring junction was converted from cis to trans by acid-catalyzed isomerization. Different polyamines were added to the 3-keto group by reductive amination, yielding both the 3 alpha and 3 beta addition products. The synthetic products exhibited potent, broad-spectrum antimicrobial activity similar to that of the parent compound. Changing the identity of the polyamine or the stereochemistry of addition has little effect upon antimicrobial activity but appears to change the selectivity of the agents. The analogs are synthesized with high yield from inexpensive starting materials and are promising alternatives to squalamine as potential antibiotics.

The synthesis of spermine analogs of the shark aminosterol squalamine

Aminosterols isolated from the dogfish shark Squalus acanthias are promising therapeutic agents in the treatment of infection and cancer. One of these, MSI-1436, has been shown to possess antimicrobial activity slightly better than squalamine. In this study, a series of analogs of MSI-1436 have been synthesized from stigmasterol. The 7 alpha-hydroxy substituent of MSI-1436 was either omitted or the stereochemistry modified to the 7 beta position. Also, analogs of MSI-1436 with 24-sulfate, 24-amino, and 24-hydroxy substituents were synthesized in order to assess the importance of the side chain functional group on antimicrobial activity. All of the analogs possess significant antimicrobial activity, suggesting that substitution at C7 and C24 of the aminosterols plays a minor role in their antimicrobial potency.

The aminosterol antibiotic squalamine permeabilizes large unilamellar phospholipid vesicles

The ability of the shark antimicrobial aminosterol squalamine to induce the leakage of polar fluorescent dyes from large unilamellar phospholipid vesicles (LUVs) has been measured. Micromolar squalamine causes leakage of carboxyfluorescein (CF) from vesicles prepared from the anionic phospholipids phosphatidylglycerol (PG), phosphatidylserine (PS), and cardiolipin. Binding analyses based on the leakage data show that squalamine has its highest affinity to phosphatidylglycerol membranes, followed by phosphatidylserine and cardiolipin membranes. Squalamine will also induce the leakage of CF from phosphatidylcholine (PC) LUVs at low phospholipid concentrations. At high phospholipid concentrations, the leakage of CF from PC LUVs deviates from a simple dose-response relationship, and it appears that some of the squalamine can no longer cause leakage. Fluorescent dye leakage generated by squalamine is graded, suggesting the formation of a discrete membrane pore rather than a generalized disruption of vesicular membranes. By using fluorescently labeled dextrans of different molecular weight, material with molecular weight </=4000 g/mol is released from vesicles by squalamine, but material with molecular weight >/=10,000 is retained. Negative stain electron microscopy of squalamine-treated LUVs shows that squalamine decreases the average vesicular size in a concentration-dependent manner. Squalamine decreases the size of vesicles containing anionic phospholipid at a lower squalamine/lipid molar ratio than pure PC LUVs. In a centrifugation assay, squalamine solubilizes phospholipid, but only at significantly higher squalamine/phospholipid ratios than required for either dye leakage or vesicle size reduction. Squalamine solubilizes PC at lower squalamine/phospholipid ratios than PG. We suggest that squalamine complexes with phospholipid to form a discrete structure within the bilayers of LUVs, resulting in the transient leakage of small encapsulated molecules. At higher squalamine/phospholipid ratios, these structures release from the bilayers and aggregate to form either new vesicles or squalamine/phospholipid mixed micelles.

2.0 Å structure of prostaglandin H2 synthase-1 reconstituted with a manganese porphyrin cofactor

Prostaglandin H2 synthase (EC 1.14.99.1) is a clinically important drug target that catalyzes two key steps in the biosynthesis of the eicosanoid hormones. The enzyme contains spatially distinct cyclooxygenase and peroxidase active sites, both of which require a heme cofactor. Substitution of ferric heme by MnIII protoporphyrin IX greatly diminishes the peroxidase activity, but has little effect on the cyclooxygenase activity. Here, the 2.0 A resolution crystal structure of the MnIII form of ovine prostaglandin H2 synthase-1 is described (R = 21.8%, Rfree = 23.7%). Substitution of MnIII for FeIII causes no structural perturbations in the protein. However, the out-of-plane displacement of the manganese ion with respect to the porphyrin is greater than that of the iron by approximately 0.2 A. This perturbation may help to explain the altered catalytic properties of the manganese enzyme.

Squalamine is not a proton ionophore

Squalamine, an aminosterol antibiotic isolated from the dogfish shark, creates relatively large defects in phospholipid bilayers, allowing the unrestricted translocation of small molecules across these compromised membranes (B.S. Selinsky, Z. Zhou, K.G. Fotjik, S. R. Jones, N.R. Dollahon, A.E. Shinnar, Biochim. Biophys. Acta 1370 (1998) 218-234). However, an aminosterol structurally similar to squalamine was found to act as a proton ionophore in anionic phospholipid vesicles. In contrast with squalamine, gross membrane disruption was not observed with this synthetic analog (G. Deng, T. Dewa, S.L. Regen, J. Am. Chem. Soc. 118 (1996) 8975-8976). In this report, the ionophoric activity of squalamine was tested in anionic and zwitterionic phospholipid vesicles. No ionophoric activity was observed for squalamine in vesicles comprised of phosphatidylglycerol (PG), phosphatidylcholine (PC), or a mixture of the two lipids. Experiments using radiolabeled squalamine indicated that all of the squalamine added to PG vesicles remained with the vesicles, while approximately one-half of the squalamine added to PC vesicles was incorporated. We have synthesized the aminosterol analog of squalamine possessing ionophoric activity, and its ionophoric activity in PG vesicles was confirmed. The synthetic compound possessed no measurable lytic activity when added to preformed phospholipid vesicles. As both compounds possess significant antimicrobial activity, these results suggest that either multiple mechanisms for the antimicrobial activity of aminosterols exist, depending upon the aminosterol structure, or possibly an unrelated common mechanism for antimicrobial activity remains to be discovered.

19F nuclear magnetic resonance analysis of trifluoroethanol metabolites in the urine of the Sprague-Dawley rat

2,2,2-Trifluoroethanol (TFE) is a common industrial solvent and a known metabolite of the inhalation anesthetics fluroxene (2,2,2-trifluoroethyl vinyl ether) and halothane (2-bromo-2-chloro-1,1,1-trifluoroethane). The water-soluble metabolites of TFE were identified in the urine of Sprague-Dawley rats using 19F NMR spectroscopy. In rats dosed with 0.21 g TFE/kg body weight, approximately one-half of the administered TFE was excreted as the trifluoroethyl glucuronide. The remaining TFE was oxidized, primarily to trifluoroacetaldehyde hydrate, with a small percentage of the aldehyde oxidized further to trifluoroacetate. One additional fluorinated compound was found; after investigation, this was identified as a Schiffs base compound resulting from the addition of trifluoroacetaldehyde to urea. The time-dependent excretion of TFE metabolites was measured as a function of ethanol induction of hepatic enzymes. This study demonstrates the utility of 19F NMR for the analysis of drug metabolism in laboratory animals. In addition, the resistance of trifluoroacetaldehyde hydrate to further oxidation, coupled with its reactivity with common cellular amines, indicates the potential toxicity of this metabolite to mammalian tissues.

Bacterial and algal orthologs of prostaglandin H2 synthase: novel insights into the evolution of an integral membrane protein

Prostaglandin H₂synthase (PGHS; EC 1.14.99.1), a bi-functional heme enzyme that contains cyclooxygenase and peroxidase activities, plays a central role in the inflammatory response, pain, and blood clotting in higher eukaryotes. In this review, we discuss the progenitors of the mammalian enzyme by using modern bioinformatics and homology modeling to draw comparisons between this well-studied system and its orthologs from algae and bacterial sources. A clade of bacterial and algal orthologs is described that have salient structural features distinct from eukaryotic counterparts, including the lack of a dimerization and EGF-like domains, the absence of gene duplicates, and minimal membrane-binding domains. The functional implications of shared and variant features are discussed.

Structure–activity relationships in aminosterol antibiotics: The effect of stereochemistry at the 7-OH group

Squalamine and three aminosterol analogs have been shown to inhibit bacterial cell growth and induce lysis of large unilamellar phospholipid vesicles. The analogs differ in the identity of the polyamine attached at C3 of the sterol, and the stereochemistry of a hydroxyl substituent at C7. Analogs with a tetraammonium spermine polyamine are somewhat more active than analogs with a shorter trisammonium spermidine polyamine, and analogs with an axial (α) hydroxyl substituent at C7 are more active than analogs with the corresponding equatorial (β) hydroxyl group. There is some variability noted; the 7β-OH spermine analog is the most active compound against Escherichia coli, but the least effective against Pseudomonas aeruginosa. Lytic activity correlates well with antimicrobial activity of the compounds, but the lytic activity varies with the phospholipid composition of the vesicles.