Differential scanning calorimetry investigation of the interaction of cationic amphiphilic model compounds and muscarinic allosteric agents with phospholipid bilayers

Abstract

The essential biomembrane components, phospholipids, interact with various drug substances among others. The physicochemical properties of the phospholipids are not only a prerequisite for their formation of bilayers; these properties also enhance interactions with amphiphilic substances. Ligand binding to muscarinic acetylcholine receptors can be modulated by various substances. These allosteric modulators are classified as typical and atypical on the basis of radioligand binding experiments. <br /> We aimed at using phospholipid bilayers to study whether selected typical and atypical modulators differ in their ability to interact with hydrophobic/lipophilic interphases. We used the method of differential scanning calorimetry (DSC) and measured drug effects on the phase transition temperature, Tt, of modulator-containing liposome suspensions compared with the suspension of pure phospholipids. The typical modulators used were Wduo3 (1,1'-(1,3-propandiyl)-bis[4,4'-phthalimidomethoxyl-iminomethyl-pyridin-ium]-dibromide), W84 (hexane-1,6-bis(dimethyl-3´-phthalimidopropyl-ammonium) di-bromide) and its derivative naphmethonium (a phthalimidopropyl residue in W84 is replaced by the more voluminous naphthylimido-ß-dimethylpropyl residue). The atypical modulators were TD5 (a silicon-containing derivative of W84 with a quaternary ammonium nitrogen replaced by silicon) and Duo3 (1,1'-(1,3-propandiyl)-bis[4,4'-(2,6-dichlorbenzoxyl)-iminomethyl-pyridinium]-dibromide). The phospholipids used were dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidic acid (DPPA). The buffer, a 14mM TES/14mM histidine buffer adjusted with HCl to pH 6 [TES = N-tris (hydroxymethyl)-2-aminoethane-sulfonic acid] ensured that DPPA was ionic while DPPC was neutral under the experimental conditions. That way, the role played by the difference in headgroups could be examined. The average Tt values of pure phospholipid liposome suspensions were 41.5 ± 0.21°C (0 ± SEM, n = 30) and 63.4 ± 0.15°C (0 ± SEM, n = 35) for DPPC and DPPA, respectively. Measurements were first carried out using a set of systematically varied phenylpropylamines to establish structure-activity relationships. The findings suggested that the drug-induced Tt depends to a great extent on the depth of penetration into the phospholipid bilayer and also on the structure of the penetrating lipophilic moiety.<br /> On the basis of these findings the results of experiments with the allosteric modulators were interpreted. The typical modulator W84 induced a reduction of Tt in DPPA liposomes to the Tt value of pure DPPC. The substance thus eliminated only the head-group specificity of DPPA and does not seem to penetrate into the bilayer. This was confirmed by the absence of an effect by W84 on the Tt when measured with DPPC liposomes (ΔTt = 0,0 ± 0.06°C [0 ± SEM, n = 3]). The substance naphmethonium acted similar to W84, though the presence of the larger lipophilic substituent caused a reduction in Tt of DPPA to a value slightly lower than that of pure DPPC (Tt = 38.5 ± 0.11°C [0 ± SEM, n = 5]). The substance had a marginal effect on the Tt of DPPC liposomes (ΔTt = 0,2 ± 0.10°C [0 ± SEM, n = 2 values at molar ratio of 1.0]). The effect of the silicon-containing atypical modulator TD5 on DPPA liposomes was a reduction that was molar ratio-dependent, contrary to the uniform molar ratio-independent values obtained from W84 and naphmethonium experiments with DPPA. At the substance to DPPA molar ratio of 1.0, TD5 produced a signal with a peak seemingly resulting from the superimposition of two peaks with a Tt value lower than that of pure DPPC (Tt = 33.8 ± 0.20°C [0 ± SEM, n = 3 values at molar ratio of 1.0 from 3 experiments]). This was the greatest reduction measured among the allosteric modulators. A reduction in Tt of DPPC to the value obtained from the DPPA experiment was also achieved (Tt = 33.9 ± 0.10°C [0 ± SEM, n = 3]). TD5 was the only substance that reduced the Tt of both DPPA and DPPC liposomes to approximately the same level at a molar ratio of 1.0. The effect of the atypical bispyridinium-type modulator Duo3 on the Tt of DPPA was complex; an initial reduction was followed beyond a molar ratio of 0.5 by an increase in Tt above that of pure DPPA. Besides, this was the only compound that did not reduce the Tt of DPPA to a value as low as that of pure DPPC. But the substance did reduce the value of DPPC (Tt = 37.1 ± 0.42°C [0 ± SEM, n = 3]). While W84 had no effect on the Tt of DPPC and the bispyridinium-type modulator Wduo3, which is a typically acting allosteric agent only had a marginal effect (Tt = 40.1 ± 0.18°C [0 ± SEM, n = 2]), Duo3 did induce a clearly significant reduction in the Tt of DPPC (Tt = 37.1 ± 0.42°C [0 ± SEM, n = 3]). Taken together, the finding reveal that typical and atypical modulators differ in their ability to interact with hydrophilic/lipophilic phospholipid interphases. It is tempting to suggest that the molecular mode of interaction with hydrophilic/lipophilic interphases of the muscarinic receptor protein is likewise different between the compounds and that this difference may be involved in atypical versus typical allosteric actions.