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Piracetam, Nootropil (2-oxo-1-pyrrolidine acetamide), is a drug promoting erythrocyte deformability. To establish the mode of action of this compound, we have investigated its influence on the organization of model phospholipid membranes. 31P NMR data show that the drug induces a structural modification in liposomes made of phosphatidylcholine and phosphatidylethanolamine. Our conformational analysis results have allowed the interpretation of the effect of piracetam on these model membranes: the specific interaction between the drug molecules and the phosphate headgroups induces a new organization of the lipids favouring formation of mobile drug-phospholipid complexes that exhibit an isotropic-type signal in the 31P NMR spectra.

Aminoglycoside antibiotics accumulate in lysosomes of kidney and cultured cells and cause an impairment of phospholipid catabolism which is considered to be an early and significant step in the development of their toxicity. Using liposomes, wer previously demonstrated that the activity of lysosomal phospholipases A1 and A2 towards phosphatidylcholine was markedly enhanced by the inclusion of phosphatidylinositol in the bilayer, and that gentamicin impaired this activity by binding to phosphatidylinositol. Since gentamicin-induced inhibition was inversely related to the amount of phosphatidylinositol included in the liposomes, we proposed that gentamicin impairs activity of phospholipases by decreasing the quantity of available negative charges carried by the bilayer surface (Mingeot-Leclercq et al., Biochem Pharmacol 37: 591-599, 1988). We now extend these observations to phosphatidylserine and phosphatidic acid, and compare the inhibition caused by gentamicin, amikacin and streptomycin towards lysosomal phospholipases on the hydrolysis of phosphatidylcholine in the presence of each of these acidic phospholipids. Inclusion of phosphatidic acid in liposomes, and, to a lesser extent, phosphatidylserine, caused a larger increase in phospholipases activity than phosphatidylinositol. In parallel, the three aminoglycosides tested were found less inhibitory towards phospholipases activity measured on phosphatidic acid-or phosphatidylserine-containing liposomes than was previously observed with phosphatidylinositol, even though equilibrium dialysis experiments failed to demonstrate significant difference in binding parameters of the drug towards each of these liposomes populations. Yet, as for phosphatidylinositol-containing liposomes, the inhibition was inversely related to the amount of phosphatidic acid or phosphatidylserine included in the bilayer and the inhibitory potency of the three drugs was consistently gentamicin greater than amikacin greater than streptomycin with the three types of negatively-charged liposomes used. We conclude that impairment of lysosomal phospholipases activity towards phosphatidylcholine included in negatively-charged membranes by aminoglycoside antibiotics is dependent upon drug binding to the bilayer, but that it is modulated by the nature of the acidic phospholipid that binds the drug as well as by that of the drug itself. A companion paper (Mingeot-Leclercq et al., Biochem Pharmacol 40: 499-506, 1990) will examine by computer-aided conformational analysis the parameters (drug-phospholipid energy of interaction, position of the drug in a monolayer and its accessibility to the aqueous phase) which may be important for these effects.

In a companion paper (Mingeot-Leclercq et al. Biochem Pharmacol 40: 489-497, 1990), we showed that the inhibitory potency of gentamicin on the activity of lysosomal phospholipases, measured towards phosphatidylcholine included in negatively-charged liposomes, is markedly influenced by the nature of the acidic phospholipid used (phosphatidylinositol, phosphatidylserine, phosphatidic acid), whereas the binding of the drug to the three types of liposomes is similar. This result challenged previous conclusions pointing to a key role exerted by drug binding to phospholipid membranes and presumably charge neutralization, for phospholipases inhibition (Carlier et al. Antimicrob Agents Chemother, 23: 440-449, 1983; Mingeot-Leclercq et al., Biochem Pharmacol 37:591-599, 1988). Conformational analysis of mixed monolayers of gentamicin and each of the three acid phospholipids shows that gentamicin systematically adopts an orientation largely parallel to the hydrophobic-hydrophilic interface, but that (i) the energies of interaction are largely different (phosphatidylinositol greater than phosphatidylserine greater than phosphatidic acid), and (ii) the apparent accessibility of the bound drug to water varies in an inverse relation with the energies of interaction. Amikacin, a semisynthetic derivative of kanamycin A with a lower inhibitory potential towards phospholipases than gentamicin in the three types of liposomes used, also showed similar differences in energies of interaction and accessibility to water, but constantly exhibited an orientation perpendicular to the hydrophobic-hydrophilic interface. We conclude that impairment of lysosomal phospholipase activities towards phosphatidylcholine included in negatively-charged membranes by aminoglycoside antibiotics is indeed dependent upon drug binding to the bilayer, but is also modulated by (i) the nature of the acidic phospholipid, which influences the energy of interaction and the accessibility of the drug with respect to the hydrophilic phase, and (ii) the orientation of the drug, which it itself related to its chemical structure. Inasmuch as phospholipases inhibition is related to aminoglycoside nephrotoxicity, these findings may help in better defining the molecular determinants and mechanisms responsible for this adverse effect.

Conformational analysis of gallopamil was performed in order to gain insight into the molecular determinant of its calcium-antagonistic property. Whereas the neutral form of gallopamil was characterized by a single, largely predominant configuration, the protonated form of the drugs yielded several conformers, some of which were characterized by a readily accessible ionized site. The capacity of gallopamil to inhibit ionophore-mediated calcium translocation in a two-phase bulk system was inversely related to the pH of the aqueous phase. These findings indicate that the capacity of gallopamil to interfere with the transport of cations is critically dependent on the availability of a protonated configuration of the drug.

Previous studies [Laurent et al., Biochem. Pharmac. 31, 3861 (1982)] have demonstrated that aminoglycoside antibiotics bind to negatively charged phospholipid bilayers and inhibit the activity of lysosomal phospholipases. This inhibition also occurs in vivo in animal and man. It is considered to be an early and significant step in the development of aminoglycoside-induced nephrotoxicity. The binding of 6 aminoglycosides in current clinical use (dibekacin, gentamicin, tobramycin, kanamycin A, amikacin and streptomycin) to phosphatidylinositol has been studied by gel filtration technique and by conformational analysis. Variation of the phosphatidylinositol content from 0 to 27% of total phospholipids causes a cooperative increase in aminoglycoside binding. At fixed phosphatidylinositol concentration, the binding of the different aminoglycosides is related to the number of aminogroups carried by the drug (viz., gentamicin greater than kanamycin A greater than streptomycin) and is largely, but not entirely dependent upon electrostatic interactions. Conformational analysis of the interaction of aminoglycosides with phosphatidylinositol monolayers was made by a step-wise computation approach. We first have taken into account the Vander Waals, torsional and electrostatic energies and we have calculated the hydrophobic and hydrophilic centers of each molecule. Assembly was then computed by successive association of one molecule of drug and up to 4 molecules of phosphatidylinositol. The calculated interaction energies varied from -8.5 kcal/mol (gentamicin) to -4.9 kcal/mol (amikacin) and -3.9 kcal/mol (streptomycin).(ABSTRACT TRUNCATED AT 250 WORDS)

The prostaglandins PGB2, PGE2 and PGF2 alpha were found to translocate calcium in a modified Pressman cell. At pH 7.40, PGB2 was more potent than PGE2 and than PGF2 alpha. When incorporated at a 1% molar ratio in liposomes made of cholesterol and different diacyl phosphatidyl choline, prostaglandins are able to mediate a slow calcium exchange diffusion. A significant prostaglandin-mediated calcium release that depends on the lipid matrix rigidity is observable at 37 degrees but not at 22 degrees. Conformational analysis of the complex formed by two molecules of prostaglandins and one calcium atom, either at a simulated membrane-water interface or in a simulated bulk lipid phase reveals rigid complexes with great distances between hydrophilic and hydrophobic gravity centres that predict low ionophoretic properties.

Aminoglycoside antibiotics such as gentamicin, which are fully hydrophilic, and cationic amphiphilic drugs such as bis(beta-diethylaminoethylether)hexestrol (DEH), are both known to inhibit lysosomal phospholipases and induce phospholipidosis. This enzymatic inhibition is probably related to the neutralization of the surface negative charges on which the lysosomal phospholipases A1 and A2 are dependent to express fully their activities (Mingeot-Leclerq et al., Biochem Pharmacol 37: 591-599, 1988). Using negatively charged liposomes, we show by 31P NMR spectroscopy that both gentamicin and DEH cause a significant restriction in the phosphate head mobility and, in sonicated vesicles, the appearance of larger bilayer structures. Both DEH and gentamicin increased the apparent size of sonicated negatively charged liposomes (but not of neutral liposomes) as measured by quasi-elastic light scattering spectroscopy. Examination of replicas from freeze-etched samples, however, revealed that gentamicin caused aggregation of liposomes, whereas DEH induced their fusion and the formation of intramembranous roundly shaped structures. Only DEH caused a significant decrease of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene, a fluorescent lipid-soluble probe. In addition, DEH, but not gentamicin, interfered with the bilayer to hexagonal phase transition occurring in dioleoyl- and dielaidoylphosphatidylethanolamine liposomes upon warming, and caused the appearance of an isotropic signal suggestive of the formation of inverted micelles. In computer-aided conformational analysis of the molecules at a simulated air-water interface, gentamicin was shown to display a largely-open crescent shape. When surrounded by phosphatidylinositol molecules, it remained as such at the interface which it locally mis-shaped, establishing close contact with the negatively charged phospho groups. In contrast, DEH could be oriented perpendicularly to the interface, with its two cationic groups associated with the phospho groups, and its phenyl- and diethylethandiyl moieties deeply inserted between and interacting with the aliphatic chains. Thus, although both agents cause lysosomal phospholipases inhibition, the differences in their interactions with negatively-charged bilayers is likely to result in a different organization of the phospholipids accumulated in vivo, which could lead to different toxicities.

We report here about the relationship between the destabilization of the lipid organization induced by praziquantel and derivatives and their mode of insertion into the lipid matrix. Measurements of lipid transition temperature and efflux of 6-carboxyfluorescein encapsulated in liposomes establish the lipid destabilizing capacity of praziquantel as compared to praziquantel derivatives. IR spectroscopy (attenuated total reflection technique) applied to oriented lipid bilayers indicates that praziquantel or derivatives do not modify significantly the lipid structure. In order to give a molecular description of the position of the drug into the lipid bilayer, we applied a conformational analysis procedure making it possible to calculate the structure of amphiphilic molecules assembled in aggregates and the mode of insertion of amphiphilic drugs into a lipid layer. The praziquantel lipid destabilizing capacity is explained in terms of the high praziquantel-lipid interaction and the large area occupied per drug molecule in the lipid layer.

Thromboxane A2, prostaglandin H2, a series of chemically stable cyclic endoperoxide analogues (U 46619, U 44069, ONO 11113, 9, 11, diazo PGH2 and SQ 26655) and different isomers of SQ 26655 were analysed for their spatial configuration by conformational analysis in a simulated membrane-water interface environment with a "structure tree" procedure already described for prostaglandins, leukotrienes and lipoxins. The conformers derived from the structure tree and with a high probability of existence are presented. A new method allows one to visualize the surface charge density of the calculated molecules. The spatial configuration and the surface charge density of each molecule are compared to their known order of competition binding to the putative TXA2/PGH2 receptor of platelets. The conformational and charge density analysis merely shows that the different stereochemistry of these molecules lead to spatial conformation, that mimics (agonists), or that are far from (antagonists) the TXA2/PGH2 conformation.