O-acetylsalicylhydroxamic acid, a novel acetylating inhibitor of prostaglandin H2 synthase: structural and functional characterization of enzyme-inhibitor interactions.

Aspirin is unique among clinically used nonsteroidal antiinflammatory drugs in that it irreversibly inactivates prostaglandin (PG) H2 synthase (PGHS) via acetylation of an active-site serine residue. We report the synthesis and characterization of a novel acetylating agent, O-acetylsalicylhydroxamic acid (AcSHA), which inhibits PGE2 synthesis in vivo and blocks the cyclooxygenase activity of PGHS in vitro. AcSHA requires the presence of the active-site residue Ser-529 to be active against human PGHS-1; the S529A mutant is resistant to inactivation by the inhibitor. Analysis of PGHS inactivation by AcSHA, coupled with the X-ray crystal structure of the complex of ovine PGHS-1 with AcSHA, confirms that the inhibitor elicits its effects via acetylation of Ser-529 in the cyclooxygenase active site. The crystal structure reveals an intact inhibitor molecule bound in the enzyme's cyclooxygenase active-site channel, hydrogen bonding with Arg-119 of the enzyme. The structure-activity profile of AcSHA can be rationalized in terms of the crystal structure of the enzyme-ligand complex. AcSHA may prove useful as a lead compound to facilitate the development of new acetylating inhibitors.

Structural analysis of NSAID binding by prostaglandin H2 synthase: time-dependent and time-independent inhibitors elicit identical enzyme conformations.

Nonsteroidal antiinflammatory drugs (NSAIDs) block prostanoid biosynthesis by inhibiting prostaglandin H(2) synthase (EC 1.14.99.1). NSAIDs are either rapidly reversible competitive inhibitors or slow tight-binding inhibitors of this enzyme. These different modes of inhibition correlate with clinically important differences in isoform selectivity. Hypotheses have been advanced to explain the different inhibition kinetics, but no structural data have been available to test them. We present here crystal structures of prostaglandin H(2) synthase-1 in complex with the inhibitors ibuprofen, methyl flurbiprofen, flurbiprofen, and alclofenac at resolutions ranging from 2.6 to 2.75 A. These structures allow direct comparison of enzyme complexes with reversible competitive inhibitors (ibuprofen and methyl flurbiprofen) and slow tight-binding inhibitors (alclofenac and flurbiprofen). The four inhibitors bind to the same site and adopt similar conformations. In all four complexes, the enzyme structure is essentially unchanged, exhibiting only minimal differences in the inhibitor binding site. These results argue strongly against hypotheses that explain the difference between slow tight-binding and fast reversible competitive inhibition by invoking global conformational differences or different inhibitor binding sites. Instead, they suggest that the different apparent modes of NSAID binding may result from differences in the speed and efficiency with which inhibitors can perturb the hydrogen bonding network around Arg-120 and Tyr-355.

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.

Vancomycin binding to low-affinity ligands: delineating a minimum set of interactions necessary for high-affinity binding.

Bacterial resistance to vancomycin has been attributed to the loss of an intermolecular hydrogen bond between vancomycin and its peptidoglycan target when cell wall biosynthesis proceeds via depsipeptide intermediates rather than the usual polypeptide intermediates. To investigate the relative importance of this hydrogen bond to vancomycin binding, we have determined crystal structures at 1.0 A resolution for the vancomycin complexes with three ligands that mimic peptides and depsipeptides found in vancomycin-sensitive and vancomycin-resistant bacteria: N-acetylglycine, D-lactic acid, and 2-acetoxy-D-propanoic acid. These, in conjunction with structures that have been reported previously, indicate higher-affinity ligands elicit a structural change in the drug not seen with these low-affinity ligands. They also enable us to define a minimal set of drug-ligand interactions necessary to confer higher-affinity binding on a ligand. Most importantly, these structures point to factors in addition to the loss of an intermolecular hydrogen bond that must be invoked to explain the weaker affinity of vancomycin for depsipeptide ligands. These factors are important considerations for the design of vancomycin analogues to overcome vancomycin resistance.

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 /=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.

The synthesis and characterization of analogs of the antimicrobial compound squalamine: 6 beta-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.

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 Schiff's 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.

Dissociation constants for dihydrofolic acid and dihydrobiopterin and implications for mechanistic models for dihydrofolate reductase.

The dissociation constants (pKa) for the pteridine ring system of dihydrofolate (H2folate) have been redetermined, and those for dihydrobiopterin (H2biopterin) have been determined. Determination of the pKa for N5 of H2folate is complicated by the low solubility and instability of H2folate at pH 2-4, and other complicating factors. The initial rate of absorbance change due to degradation is a maximum at pH 2.5, and the products depend on the oxygen concentration: under aerobic conditions, (p-aminobenzoyl)glutamic acid and 7,8-dihydropterin-6-carboxaldehyde are major products. H2Biopterin is much more soluble and more stable at low pH. For protonation of N5, the pKa is 2.56 +/- 0.01 for H2biopterin and 2.59 +/- 0.03 for H2folic acid. Spectrophotometric determination of the pKa for the N3-O4 amide group of H2folate is subject to serious errors when a wavelength between 220 and 235 nm is used. These errors arise from the pH-dependent absorbance of mercaptoethanol often present in the preparation. The amide group has a pKa of 10.41 +/- 0.04 in H2biopterin and 10.85 +/- 0.04 in H2folate. The redetermined value for the pKa of N5 of H2folate has implications for mechanistic models for dihydrofolate reductase, and revised kinetic constants have been calculated for one model.

13C and 15N nuclear magnetic resonance evidence of the ionization state of substrates bound to bovine dihydrofolate reductase.

The state of protonation of substrates bound to mammalian dihydrofolate reductase (DHFR) has significance for the mechanism of catalysis. To investigate this, dihydrofolate and dihydropteroyl-pentaglutamate have been synthesized with 15N enrichment at N-2. 15N NMR studies have been performed on the binary complexes formed by bovine DHFR with these compounds and with [5-15N]dihydrobiopterin. The results indicate that there is no protonation at N-5 in the binary complexes, and this was confirmed by 13C NMR studies with folate and dihydrofolate synthesized with 13C enrichment at C-6. The chemical shift displacements produced by complex formation are in the same direction as those which result from deprotonation of the N-3/C-4-O "amide" group and are consistent with at least partial loss of the proton from N-3. This would be possible if, as crystallographic data indicate, there is interaction of N-3 and the 2-amino group of the bound ligands with the carboxylate of the active site glutamate residue (Glu30).

Effects of potassium on lipid-protein interactions in light sarcoplasmic reticulum.

This study shows the effect of K+ on phospholipid-protein interactions in light sarcoplasmic reticulum (LSR) as measured by 31P NMR. In the presence of 110 mM K+, a substantial effect of the membrane protein on the behavior of the phospholipids was detected. Subtracting the spectrum of the LSR lipid extract from the spectrum of the intact LSR membrane produced a difference spectrum of much greater breadth than the normal phospholipid bilayer powder pattern. This powder pattern is indicative of a phospholipid domain considerably more motionally restricted than the phospholipids in a normal phospholipid bilayer. The apparent axially symmetric powder pattern is consistent with axial diffusion. In a reconstituted membrane containing the calcium pump protein at a lipid/protein ratio much less than in the light sarcoplasmic reticulum, the broad component was more prominent. The relative resonance intensity of the broad component appeared to be proportional to the lipid/protein ratio of the membrane. In 10 mM K+, no broad powder pattern is observed in the corresponding difference spectrum. Thus, in the absence of potassium, the membrane protein has much less influence on the phospholipid of the membrane, as measured by 31P NMR. In addition to the effects of K+ on the membrane structure of the sarcoplasmic reticulum, K+ modulated the function of the calcium pump. The rate of calcium-dependent ATP hydrolysis increased in light sarcoplasmic reticulum when [K+] increased from 10 to 110 mM. The rate of calcium transport was also stimulated by an increase in K+.

In vivo nuclear magnetic resonance studies of hepatic methoxyflurane metabolism. I. Verification and quantitation of methoxydifluoroacetate.

The elimination and metabolism of the fluorinated inhalation anesthetic methoxyflurane (2,2-dichloro-1,1-difluoroethyl methyl ether) in rats has been monitored using in vivo 19F nuclear magnetic resonance at 8.45 T. The elimination of methoxyflurane from rat liver as measured using a surface coil is a first order process when measured beginning 2-3 hr after the end of methoxyflurane anesthesia over a period of 12 hr. The rate constant for hepatic methoxyflurane elimination is dependent upon the duration of anesthesia, varying from 0.24 hr-1 for 15 min of anesthesia to 0.07 hr-1 for 1 hr of anesthesia. Methoxyflurane was shown to be metabolized in the liver to methoxydifluoroacetate using the surface coil method. No resonance for hepatic fluoride ion could be observed in vivo. Pure sodium methoxydifluoroacetate was synthesized in order to confirm the identity of the resonances in liver and urine. 19F NMR spectra of urine collected from anesthetized rats contain resonances for two methoxyflurane metabolites, methoxydifluoroacetate and inorganic fluoride. Studies with liver homogenates imply that fluoride is quickly cleared from the liver and eliminated from the body through the urine, explaining the inability to observe hepatic fluoride using a surface coil. The 19F NMR resonance for inorganic fluoride in urine was found to be broadened by interaction with metal ions, since the broadening could be eliminated by treatment with chelating resin.

In vivo nuclear magnetic resonance studies of hepatic methoxyflurane metabolism. II. A reevaluation of hepatic metabolic pathways.

Methoxyflurane (2,2-dichloro-1,1-difluoro-ethyl methyl ether) is believed to be metabolized via two convergent metabolic pathways. The relative flux through these two metabolic pathways has been investigated using a combination of in vivo surface coil NMR techniques and in vitro analyses of urinary metabolites. Analysis of the measured concentrations of inorganic fluoride, oxalate, and methoxydifluoroacetate in the urine of methoxyflurane-treated rats for 4 days after anesthesia indicates that the anesthetic is metabolized primarily via dechlorination to yield methoxydifluoroacetate. The methoxydifluoroacetate is largely excreted without further metabolism, although a small percentage of this metabolite is broken down to yield fluoride and oxalate, as determined by urine analysis of rats dosed with synthetic methoxydifluoroacetate. At early times after methoxyflurane exposure, the relative concentrations of methoxyflurane metabolites indicate that a significant fraction of the metabolic flux occurs via a different pathway, presumably demethylation, to yield dichloroacetate as an intermediate. Direct analysis of dichloroacetate in the urine using water-suppressed proton NMR indicates that the level of this metabolite is below the detection threshold of the method. Measurements made on the urine of rats dosed directly with dichloroacetate indicate that this compound is quickly metabolized, and dichloroacetate levels in urine are again found to be below the detection threshold. These results demonstrate the quantitative importance of the dechlorination pathway in the metabolism of methoxyflurane in rats.

Effects of TRIS and HEPES on function of rabbit muscle light sarcoplasmic reticulum.

ATP hydrolysis activity and calcium transport activity were determined on light sarcoplasmic reticulum from rabbit skeletal muscle. The effects of two buffers, TRIS and HEPES, were compared. Titration of TRIS into sarcoplasmic reticulum preparations in HEPES provided evidence for TRIS inhibition of ATPase activity and TRIS stimulation of calcium transport activity.

Cross polarization P-31 nuclear magnetic resonance of phospholipids.

P-31 single-pulse and cross-polarization (CP) nuclear magnetic resonance spectra were obtained of aqueous dispersions of pure phospholipids. Dimyristoyl phosphatidylcholine, dipalmitoylphosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, egg phosphatidylcholine, bovine brain sphingomyelin, and transphosphatidylated (from egg phosphatidylcholine) phosphatidylethanolamine were studied. The spectra from all the phospholipids, taken in the usual single-pulse mode, showed the pseudo-axially symmetric powder pattern typical of phospholipids in a hydrated lamellar form. P-31 CP spectra of all the phosphatidylcholines and phosphatidylethanolamine revealed a decrease in intensity in the vicinity of the isotropic chemical shift as long as the lipid was above the gel-to-liquid crystalline phase transition temperature. This intensity pattern has been observed previously for C-13 CP spectra of molecules rotating rapidly about a single well-defined axis (e.g., solid benzene) (Pines, A., M.G. Gibby, and J.S. Waugh, 1973, J. Chem. Phys., 59:569-590). Pure lipid dispersions below their gel-to-liquid crystalline phase transition temperature, including dipalmitoylphosphatidylcholine and sphingomyelin, do not exhibit a local minimum in the CP spectrum at the position of the isotropic chemical shift. Thus, below the phase transition temperature, there is not the same rapid rotation of the headgroup about a well-defined axis. A dramatic change in the rate of headgroup rotation is shown to take place at the pretransition of dipalmitoylphosphatidylcholine. P-31 CP spectra were also obtained for bovine rod outer segment disk membranes, rabbit muscle sarcoplasmic reticulum membranes, a total lipid extract of the latter, and a recombined membrane containing human erythrocyte glycophorin. The CP spectra were similar to the single-pulse spectra, indicating a substantial difference in behavior from pure phospholipid dispersions. This is interpreted in terms of a slower headgroup rotation.

Phospholipid exchange between restricted and nonrestricted domains in sarcoplasmic reticulum vesicles.

Phosphorus nuclear magnetic resonance spectra of rabbit muscle light sarcoplasmic reticulum membranes consist of two overlapping resonances, one much broader than the other. The broad resonance arises from phospholipids motionally restricted, probably by association with the Ca2+-ATPase, while the narrow resonance arises from phospholipid only slightly perturbed by the presence of the protein. (Selinsky, B.S. and Yeagle, P.L. (1984) Biochemistry 23, 2281-2288). The rate of exchange between the two phospholipid domains represented by the resonances was determined by measuring the transfer of magnetization from the broad resonance to the narrow resonance. The rate of exchange of phospholipids from the restricted domain to the nonrestricted domain was determined to be 1 s-1.

Perturbations of phospholipid head groups by membrane proteins in biological membranes and recombinants.

P-31 nuclear magnetic resonance (NMR) spin-lattice relaxation times (T1) have been used to probe the behavior of phospholipid head groups in the presence of membrane proteins. Measurements have been made on rabbit muscle sarcoplasmic reticulum and recombinants of the Ca2+ Mg2+ ATPase, rod outer segment disk membranes and recombinants of rhodopsin, and human erythrocyte ghosts and recombinants of human erythrocyte glycophorin. Recombined membranes with lipid/protein ratios greater than or equal to that found in biological membranes showed T1 behavior similar to the biological membranes and pure phosphatidylcholine. However, recombined membranes with a low lipid/protein ratio exhibited a T1 that was dramatically shorter than any of the other systems. Analysis of the relaxation mechanism and the factors contributing to it implicate a phospholipid head group conformation change at high protein content. It is suggested that this is due to trapping of phospholipid between proteins and is not the same phenomenon as motional restriction at the lipid-protein interface at higher lipid contents.

Two populations of phospholipids exist in sarcoplasmic reticulum and in recombined membranes containing Ca-ATPase.

Phosphorus nuclear magnetic resonance spectra of sarcoplasmic reticulum membranes from rabbit muscle and of recombined membranes containing the calcium-dependent adenosinetriphosphatase (Ca-ATPase) of sarcoplasmic reticulum reveal two distinguishable, overlapping resonances. One resonance resembles a normal phospholipid bilayer resonance, and the other is much broader. The broader component is not seen in protein-free phospholipid vesicles. In recombined membranes of the Ca-ATPase, the intensity found in the broad component was proportional to the concentration of protein in the vesicles. The two-component spectra are interpreted to arise from at least two different domains of phospholipids, one of which is motionally restricted by the Ca-ATPase. Phospholipids exchange between these two domains at a rate less than 10(3) s-1. A model for protein-lipid interactions in membranes containing the Ca-ATPase is proposed in which some of the phospholipid head groups of the membrane interact directly with the protein.