Molecular requirements involving the human platelet protease-activated receptor-4 mechanism of activation by peptide analogues of its tethered-ligand.

Thrombin is the most potent agonist of human platelets and its effects are primarily mediated through the protease-activated receptors (PARs)-1 and -4. Although PAR-1 has higher affinity for thrombin than PAR-4, both receptors contribute to thrombin-mediated actions on platelets. Recently, a potent and selective PAR-1 antagonist (vorapaxar) was approved for clinical use in selected patients. In contrast, despite the fact that several PAR-4 antagonists have been developed, few of them have been tested in clinical trials. The aim of the present study was to elucidate the molecular requirements involving the PAR-4 mechanism of activation by peptide analogues of its tethered-ligand. Eight synthetic PAR-4 tethered-ligand peptide analogues were synthesized and studied for their agonistic/antagonistic potency and selectivity toward human washed platelet aggregation, using light transmittance aggregometry. In addition, in silico studies were conducted to describe the receptor-peptide interactions that are developed following PAR-4 exposure to the above analogues. To provide a first structure-activity relationship rationale on the bioactivity profiles recorded for the studied analogues, molecular docking was applied in a homology model of PAR-4, derived using the crystal structure of PAR-1. The following peptide analogues were synthesized: AYPGKF-NH2 (1), GYPGKF-NH2 (2), Ac-AYPGKF-NH2 (3), trans-cinnamoyl-AYPGKF-NH2 (4), YPGKF-NH2 (5), Ac-YPGKF-NH2 (6), trans-cinnamoyl-YPGKF-NH2 (7), and caffeoyl-YPGKF-NH2 (8). Peptide (1) is a selective PAR-4 agonist inducing platelet aggregation with an IC50 value of 26.2 µM. Substitution of Ala-1 with Gly-1 resulted in peptide (2), which significantly reduces the agonistic potency of peptide (1) by 25-fold. Importantly, substitution of Ala-1 with trans-cinnamoyl-1 resulted in peptide (7), which completely abolishes the agonistic activity of peptide (1) and renders it with a potent antagonistic activity toward peptide (1)-induced platelet aggregation. All other peptides tested were inactive. Tyr-2, residue, along with its neighboring environment was a key determinant in the PAR-4 recognition mode. When the neighboring residues to Tyr-2 provided an optimum spatial ability for the ligand to enter into the binding site of the transmembrane receptor, a biological response was propagated. These results were compared with the predicted binding poses of small molecule antagonists of PAR-4, denoted as YD-3, ML-354, and BMS-986120. π-π stacking interaction with Tyr-183 appears to be critical and common for both small molecules antagonists and the peptide trans-cinnamoyl-YPGKF-NH2. Conclusively, the lipophilicity, size, and aromatic nature of the residue preceding Tyr-2 are determining factors on whether a human platelet PAR-4 tethered-ligand peptide analogue will exert an agonistic or antagonistic activity.

Comparative study of interactions of aliskiren and AT1 receptor antagonists with lipid bilayers.

The renin-angiotensin-aldosterone system (RAAS) plays a key role in the regulation of blood pressure. Renin is the rate limiting enzyme of the RAAS and aliskiren is a highly potent and selective inhibitor of the human renin. Renin is known to be active both in the circulating blood stream as well as locally, when bound to the (pro)-renin receptor ((P)RR). In this study we have investigated a possible mechanism of action of aliskiren, in which its accumulation in the plasma membrane is considered as an essential step for effective inhibition. Aliskiren's interactions with model membranes (cholesterol rich and poor) have been investigated by applying different complementary techniques: differential scanning calorimetry (DSC), Raman spectroscopy, magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy and small- and wide-angle X-ray scattering (SAXS and WAXS). In addition, in silico molecular dynamics (MD) calculations were applied for further confirmation of the experimental data. Aliskiren's thermal effects on the pre- and main transition of dipalmitoyl-phosphatidylcholine (DPPC) membranes as well as its topographical position in the bilayer show striking similarities to those of angiotensin II type 1 receptor (AT1R) antagonists. Moreover, at higher cholesterol concentrations aliskiren gets expelled from the membrane just as it has been recently demonstrated for the angiotensin receptor blocker (ARB) losartan. Thus, we propose that both the AT1R and the (P)RR-bound renin active sites can be efficiently blocked by membrane-bound ARBs and aliskiren when cholesterol rich membrane rafts/caveolae are formed in the vicinity of the receptors.

Interactions of the potent synthetic AT1 antagonist analog BV6 with membrane bilayers and mesoporous silicate matrices.

The present work describes the drug:membrane interactions and a drug delivery system of the novel potent AT1 blocker BV6. This designed analog has most of the pharmacological segments of losartan and an additional biphenyltetrazole moiety resulting in increased lipophilicity. We found that BV6:membrane interactions lead to compact bilayers that may in part explain its higher in vitro activity compared to losartan since such environment may facilitate its approach to AT1 receptor. Its high docking score to AT1 receptor stems from more hydrophobic interactions compared to losartan. X-ray powder diffraction (XRPD) and thermogravimetric analysis (TGA) have shown that BV6 has a crystalline form that is not decomposed completely up to 600°C. These properties are desirable for a drug molecule. BV6 can also be incorporated into a mesoporous silicate drug-delivery matrix SBA-15. The properties of the obtained drug-delivery system have been inspected by XRD, (13)C CP/MAS, TGA and nitrogen sorption experiments.

Interactions of the AT1 antagonist valsartan with dipalmitoyl-phosphatidylcholine bilayers.

Valsartan is a marketed drug with high affinity to the type 1 angiotensin (AT1) receptor. It has been reported that AT1 antagonists may reach the receptor site by diffusion through the plasma membrane. For this reason we have applied a combination of differential scanning calorimetry (DSC), Raman spectroscopy and small and wide angle X-ray scattering (SAXS and WAXS) to investigate the interactions of valsartan with the model membrane of dipalmitoyl-phosphatidylcholine (DPPC). Hence, the thermal, dynamic and structural effects in bulk as well as local dynamic properties in the bilayers were studied with different valsartan concentrations ranging from 0 to 20 mol%. The DSC experimental results showed that valsartan causes a lowering and broadening of the phase transition. A splitting of the main transition is observed at high drug concentrations. In addition, valsartan causes an increase in enthalpy change of the main transition, which can be related to the induction of interdigitation of the lipid bilayers in the gel phase. Raman spectroscopy revealed distinct interactions between valsartan with the lipid interface localizing it in the polar head group region and in the upper part of the hydrophobic core. This localization of the drug molecule in the lipid bilayers supports the interdigitation view. SAXS measurements confirm a monotonous bilayer thinning in the fluid phase, associated with a steady increase of the root mean square fluctuation of the bilayers as the valsartan concentration is increased. At high drug concentrations these fluctuations are mainly governed by the electrostatic repulsion of neighboring membranes. Finally, valsartans' complex thermal and structural effects on DPPC bilayers are illustrated and discussed on a molecular level.

Comparative binding effects of aspirin and anti-inflammatory Cu complex in the active site of LOX-1.

(1)H NMR Saturation Transfer Difference (STD) experiments were applied to study the binding of aspirin and of an anti-inflammatory complex of Cu(I), namely [Cu(tpp)(pmt)](2) [pmt = 2-mercaptopyrimidine), synthesized in an attempt to develop novel metallotherapeutic molecules. While aspirin showed only very weak binding, the complex [Cu(tpp)(pmt)](2) clearly favored binding to LOX-1. In silico docking experiments in LOX-1 showed that aspirin does only weakly bind to LOX-1, while the complex binds with high affinity. In addition, docking experiments and molecular dynamics (MD) simulations showed that the complex binds via hydrogen bonding (HB), to an allosteric site of LOX-1, revealing that this enzyme has more than one accessible site for complex metallotherapeutic molecules. When aspirin was added in the solution containing LOX and the complex [Cu(tpp)(pmt)](2), the former was shown to hinder the binding of the Cu complex significantly. This may be interpreted as the copper complex aiding the transfer of aspirin through an acid-base reaction at the LOX enzyme which subsequently blocks its binding.

Losartan's affinity to fluid bilayers modulates lipid-cholesterol interactions.

Losartan is an angiotensin II receptor antagonist mainly used for the regulation of high blood pressure. Since it was anticipated that losartan reaches the receptor site via membrane diffusion, the impact of losartan on model membranes has been investigated by small angle X-ray scattering. For this purpose 2-20 mol% losartan was incorporated into dimyristoyl-phosphatidylcholine (DMPC) and palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayers and into their binary mixtures with cholesterol in the concentration range of 0 to 40 mol%. Effects of losartan on single component bilayers are alike. Partitioning of losartan into the membranes confers a negative charge to the lipid bilayers that causes the formation of unilamellar vesicles and a reduction of the bilayer thickness by 3-4%. Analysis of the structural data resulted in an estimate for the partial area of losartan, A(Los) ≈ 40 Å(2). In the presence of cholesterol, differences between the effects of losartan on POPC and DMPC are striking. Membrane condensation by cholesterol is retarded by losartan in POPC. This contrasts with DMPC, where an increase of the cholesterol content shifts the partitioning equilibrium of losartan towards the aqueous phase, such that losartan gets depleted from the bilayers from 20 mol% cholesterol onwards. This indicates (i) a chain-saturation dependent competition of losartan with lipid-cholesterol interactions, and (ii) the insolubility of losartan in the liquid ordered phase of PCs. Consequently, losartan's action is more likely to take place in fluid plasma membrane patches rather than in domains rich in cholesterol and saturated lipid species such as in membrane rafts.

Interactions at the bilayer interface and receptor site induced by the novel synthetic pyrrolidinone analog MMK3.

This work presents a thorough investigation of the interaction of the novel synthetic pyrrolidinone analog MMK3 with the model membrane system of dipalmitoylphosphatidylcholine (DPPC) and the receptor active site. MMK3 has been designed to exert antihypertensive activity by functioning as an antagonist of the angiotensin II receptor of subtype 1 (AT(1)). Its low energy conformers were characterized by 2D rotating-frame Overhauser effect spectroscopy (ROESY) in combination with molecular dynamics (MD) simulations. Docking study of MMK3 shows that it fits to the AT(1) receptor as SARTANs, however, its biological activity appears to be lower. Thus, differential scanning calorimetry (DSC), Raman spectroscopy and small angle X-ray scattering (SAXS) experiments on the interaction of MMK3 with DPPC bilayers were carried out and results demonstrate that the drug is well incorporated into the membrane leaflets and furthermore causes partial bilayer interdigitation, although less effective than SARTANs. Thus, it appears that the nature of the bilayer matrix and the stereoelectronic active site requirements of the receptor are responsible for the low bioactivity of MMK3.

Conformational analysis of the ΜΒΡ83-99 (Phe91) and ΜΒΡ83-99 (Tyr91) peptide analogues and study of their interactions with the HLA-DR2 and human TCR receptors by using molecular dynamics.

The two new synthetic analogues of the MBP(83-99) epitope substituted at Lys(91) (primary TCR contact) with Phe [MBP(83-99) (Phe(91))] or Tyr [MBP(83-99) (Tyr(91))], have been structurally elucidated using 1D and 2D high resolution NMR studies. The conformational analysis of the two altered peptide ligands (APLs) has been performed and showed that they adopt a linear and extended conformation which is in agreement with the structural requirements of the peptides that interact with the HLA-DR2 and TCR receptors. In addition, Molecular Dynamics (MD) simulations of the two analogues in complex with HLA-DR2 (DRA, DRB1*1501) and TCR were performed. Similarities and differences of the binding motif of the two analogues were observed which provide a possible explanation of their biological activity. Their differences in the binding mode in comparison with the MBP(83-99) epitope may also explain their antagonistic versus agonistic activity. The obtained results clearly indicate that substitutions in crucial amino acids (TCR contacts) in combination with the specific conformational characteristics of the MBP(83-99) immunodominant epitope lead to an alteration of their biological activity. These results make the rational drug design intriguing since the biological activity is very sensitive to the substitution and conformation of the mutated MBP epitopes.

Hydrogels containing water soluble conjugates of silver(I) ions with amino acids, metabolites or natural products for non infectious contact lenses.

The poor handling and hygiene practices of contact lenses are the key reasons for their frequent contamination, and are responsible for developing ocular complications, such as microbial keratitis (MK). Thus there is a strong demand for the development of biomaterials of which contact lenses are made, combined with antimicrobial agents. For this purpose, the known water soluble silver(I) covalent polymers of glycine (GlyH), urea (U) and the salicylic acid (SalH2) of formulae [Ag3(Gly)2NO3]n (AGGLY), [Ag(U)NO3]n (AGU), and dimeric [Ag(salH)]2 (AGSAL) were used. Water solutions of AGGLY, AGU and AGSAL were dispersed in polymeric hydrogels using hydroxyethyl-methacrylate (HEMA) to form the biomaterials pHEMA@AGGLY-2, pHEMA@AGU-2, and pHEMA@AGSAL-2. The biomaterials were characterized by X-ray fluorescence (XRF) spectroscopy, thermogravimetric differential thermal analysis (TG-DTA), differential scanning calorimetry (DTG/DSC), attenuated total reflection spectroscopy (FT-IR-ATR) and single crystal diffraction analysis. The antibacterial activity of AGGLY, AGU, AGSAL, pHEMA@AGGLY-2, pHEMA@AGU-2 and pHEMA@AGSAL-2 was evaluated against the Gram negative species Pseudomonas aeruginosa (P. aeruginosa) and Gram positive ones Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus), which mainly colonize in contact lenses. The in vitro toxicity of the biomaterials and their ingredients was evaluated against normal human corneal epithelial cells (HCECs) whereas the in vitro genotoxicity was evaluated by the micronucleus (MN) assay in HCECs. The Artemia salina and Allium cepa models were applied for the evaluation of in vivo toxicity and genotoxicity of the materials. Following our studies, the new biomaterials pHEMA@AGGLY-2, pHEMA@AGU-2, and pHEMA@AGSAL-2 are suggested as efficient candidates for the development of antimicrobial contact lenses.

Organotin derivatives of cholic acid induce apoptosis into breast cancer cells and interfere with mitochondrion; Synthesis, characterization and biological evaluation.

Organotin(IV) derivatives of cholic acid (CAH) with the formulae R3Sn(CA) (R = Ph- (1), n-Bu- (2)) and R2Sn(CA)2 (R = Ph- (3), n-Bu- (4) and Me- (5)) were synthesized. The compounds were characterized in solid state by melting point, FT-IR, 119Sn Mössbauer, X-ray fluorescence (XRF) spectroscopy and in solution by 1H NMR, UV-Vis spectral data and by Electrospray Ionisation Mass spectrometry (ESI-MS), High Resolution Mass spectrometry (HRMS), and atomic absorption analysis. The in vitro bioactivity of 1-5 against human breast adenocarcinoma cancer cells MCF-7 (positive to hormone receptors) and MDA-MB-231 (negative to hormone receptors) reveal that triorganotin derivatives 1-2 exhibit significantly stronger activity than the corresponding diorganotin ones. Compound 5 is inactive against both cell lines at the concentrations tested. Triorganotins 1-2 inhibit selectively MCF-7 than MDA-MB-231 cells, suggesting hormone mimetic behavior of them. Organotins 1-4 inhibit both cancerous cell lines, stronger than cisplatin which rise up to 55-fold against MCF-7 and 170-fold against MDA-MB-231. The in vitro toxicity of 1-4 was evaluated on normal human fetal lung fibroblast cells (MRC-5), while their genotoxicity in vitro by micronucleus assay (MN). Moreover, the in vivo toxicity of 1-4 was tested by Artemia salina assay and their in vivo genotoxicity with Allium cepa test. The mechanism of action of 1-4 against MCF-7 was clarified in vitro by the means of cell morphology studies, cell cycle arrest, Acridine Orange/Ethidium Bromide (AO/EB) Staining, mitochondrial membrane permeabilization test and by their binding affinity toward the calf thymus (CT) DNA.

pHEMA@AGMNA-1: A novel material for the development of antibacterial contact lens.

The Metal Organic Framework (MOF) of formula {[Ag6(µ3-HMNA)4(µ3-MNA)2]2-·[(Et3NH)+]2·(DMSO)2·(H2O)} (AGMNA), a known efficient antimicrobial compound which contains the anti-metabolite, 2-thio-nicotinic acid (H2MNA), was incorporated in polymer hydrogels using, hydroxyethyl-methacrylate (HEMA). The material pHEMA@AGMNA-1 was characterized by X-ray fluorescence (XRF) spectroscopy, X-ray powder diffraction analysis (XRPD), Scanning Electron Microscopy (SEM), Energy-dispersive X-ray spectroscopy (EDX), Thermogravimetric Differential Thermal Analysis (TG-DTA), Differential Scanning Calorimetry (DTG/DSC), attenuated total reflection spectroscopy (FT-IR-ATR) and Ultrasonic Imaging. The antimicrobial capacity of pHEMA@AGMNA-1 was evaluated against the Gram negative bacterial strain Pseudomonas aeruginosa and the Gram positive ones of the genus of Staphylococcus epidermidis and Staphylococcus aureus, which are the etiology of the microbial keratitis. The % bacterial viability of P. aeruginosa, S. epidermidis and S. aureus upon their incubation with pHEMA@AGMNA-1 discs is significantly low (0.4 ± 0.1%, 1.5 ± 0.4% and 7.7 ± 0.5% respectively). The inhibition zones (IZ) caused by pHEMA@AGMNA-1 discs against P. aeruginosa, S. epidermidis and S. aureus are 14.0 ± 1.1, 11.3 ± 1.3 and 11.8 ± 1.8 mm respectively. Furthermore, pHEMA@AGMNA-1 exhibits low toxicity. Thus, pHEMA@AGMNA-1 might be an efficient candidate for the development of antimicrobial active contact lenses.

An Efficient Disinfectant, Composite Material {SLS@[Zn3(CitH)2]} as Ingredient for Development of Sterilized and Non Infectious Contact Lens.

The [Zn3(CitH)2] (1) (CitH4= citric acid), was dispersed in sodium lauryl sulphate (SLS) to form the micelle of SLS@[Zn3(CitH)2] (2). This material 2 was incorporated in hydrogel made by hydroxyethyl-methacrylate (HEMA), an ingredient of contact lenses, toward the formation of pHEMA@(SLS@[Zn3(CitH)2]) (3). Samples of 1 and 2 were characterized by UV-Vis, 1H-NMR, FT-IR, FT-Raman, single crystal X-ray crystallography, X-ray fluorescence analysis, atomic absorption and TG/DTA/DSC. The antibacterial activity of 1-3 as well as of SLS against Gram-positive (Staphylococcus epidermidis (St. epidermidis) and Staphylococcus aureus (St. aureus)) and Gram-negative (Pseudomonas aeruginosa (PAO1), and Escherichia coli (E. coli)) bacteria was evaluated by the means of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and inhibitory zone (IZ). 2 showed 10 to 20-fold higher activity than 1 against the bacteria tested. Moreover the 3 decreases the abundance of Gram-positive microbes up to 30% (St. aureus) and up to 20% (PAO1) the Gram-negative ones. The noteworthy antimicrobial activity of the obtained composite 3 suggests an effective antimicrobial additive for infection-free contact lenses.

A putative bioactive conformation for the altered peptide ligand of myelin basic protein and inhibitor of experimental autoimmune encephalomyelitis [Arg91, Ala96] MBP87-99.

[Arg(91), Ala(96)] MBP(87-99) is an altered peptide ligand (APL) of myelin basic protein (MBP), shown to actively inhibit experimental autoimmune encephalomyelitis (EAE), which is studied as a model of multiple sclerosis (MS). The APL has been rationally designed by substituting two of the critical residues for recognition by the T-cell receptor. A conformational analysis of the APL has been sought using a combination of 2D NOESY nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) calculations, in order to comprehend the stereoelectronic requirements for antagonistic activity, and to propose a putative bioactive conformation based on spatial proximities of the native peptide in the crystal structure. The proposed structure presents backbone similarity with the native peptide especially at the N-terminus, which is important for major histocompatibility complex (MHC) binding. Primary (Val(87), Phe(90)) and secondary (Asn(92), Ile(93), Thr(95)) MHC anchors occupy the same region in space, whereas T-cell receptor (TCR) contacts (His(88), Phe(89)) have different orientation between the two structures. A possible explanation, thus, of the antagonistic activity of the APL is that it binds to MHC, preventing the binding of myelin epitopes, but it fails to activate the TCR and hence to trigger the immunologic response. NMR experiments coupled with theoretical calculations are found to be in agreement with X-ray crystallography data and open an avenue for the design and synthesis of novel peptide restricted analogues as well as peptide mimetics that rises as an ultimate goal.

The role of the anticancer drug vinorelbine in lipid bilayers using differential scanning calorimetry and molecular modeling.

Differential scanning calorimetry (DSC) has been employed to investigate the thermal changes caused by the anticancer alkaloid drug vinorelbine in dipalmytoylphosphatidylcholine (DPPC) bilayers. The total enthalpy change was increased by the presence of the drug molecule, indicating a partial interdigitation of the lipid alkyl chains. The presence of cholesterol in DPPC bilayers including vinorelbine induced an obstruction of the interdigitation, since cholesterol interrupts the upraise of enthalpy change. Vinorelbine's interdigitation ability and stabilizing properties with the active site of the receptor have been compared with those of similar in structure amphipathic and bulky alkaloid vinblastine. The obtained results may in part explain their similar mechanism of action but different bioactivity.

Effects of cannabinoids in membrane bilayers containing cholesterol.

The thermotropic and dynamic properties of the biologically active Delta(8)-tetrahydrocannabinol (Delta(8)-THC) and its inactive congener O-methyl-Delta(8)-tetrahydrocannabinol (Me-Delta(8)-THC) in DPPC/cholesterol (CHOL) bilayers have been studied using a combination of DSC and solid-state NMR spectroscopy. The obtained results showed differential effects of the two cannabinoids under study. These are summarized as follows: (a) the presence of the active compound fluidizes more significantly the DPPC/CHOL bilayers than the inactive analog as it is revealed by DSC and NMR spectroscopy results; (b) cholesterol seems to play a significant role in the way cannabinoids act in membrane bilayers; (c) the observed additional peaks in (13)C/MAS-NMR spectra which were cannabinoid specific offer an evidence of their different dynamic properties in membranes. In particular, the aromatic part of the inactive cannabinoid appears more mobile than that of the active one. This finding is in agreement with previously obtained X-ray data which locate the inactive cannabinoid in the hydrophobic core of the bilayer while the active one in the polar region; and (d) the observed downfield shift of C-1 carbon in the preparation containing the active cannabinoid is a strong evidence that Delta(8)-THC resides nearby the polar region where also cholesterol is well known to locate itself. Such downfield shift is absent when Me-Delta(8)-THC is resided in the membrane bilayer. These differential effects of the two cannabinoids propose that the phospholipid/cholesterol core of the membrane may play an important role in the mode of cannabinoid action by regulating their thermotropic and dynamic properties.

Differential membrane fluidization by active and inactive cannabinoid analogues.

The effects of the two cannabinomimetic drugs (-)-2-(6a,7,10,10a-tetrahydro-6,6,9-trimethyl-1-hydroxy-6H-dibenzo[b,d]pyranyl-2-(hexyl)-1,3-dithiolane (AMG-3) and its pharmacologically less active 1-methoxy analogue (AMG-18) on the thermotropic and structural properties of dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) liposomes have been studied by X-ray diffraction and differential scanning calorimetry (DSC). DSC data revealed that the incorporation of the drugs affect differently the thermotropic properties of DPPC. The presence of the more active drug distinctly broadened and attenuated both the pretransition and main phase transition of DPPC bilayers, while the inactive analogue had only minor effects. Small and wide angle X-ray diffraction data showed that the two cannabinoids have different effects on the lipid phase structures and on the hydrocarbon chain packing. The pharmacologically active analogue, AMG-3, was found to efficiently fluidize domains of the lipids in the L(beta)' gel phase, and to perturb the regular multibilayer lattice. In the liquid crystalline L(alpha) phase, AMG-3 was also found to cause irregularities in packing, suggesting that the drug induces local curvature. At the same concentration, the inactive AMG-18 had only minor structural effects on the lipids. At about 10-fold or higher concentrations, AMG-18 was found to produce similar but still less pronounced effects in comparison to those observed by AMG-3. The dose-dependent, different thermotropic and structural effects by the two cannabinoid analogues suggest that these may be related to their biological activity.

Topography of alphaxalone and delta 16-alphaxalone in membrane bilayers containing cholesterol.

We have used small-angle X-ray diffraction and differential scanning calorimetry (DSC) to study the topographies of alphaxalone and its biologically inactive analog delta 16-alphaxalone in dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol model membranes. Diffraction patterns were obtained and analyzed for preparations of bilayers without and with the steroids. Temperature dependence of the total period repeat distance (d-spacing) allowed us to identify equivalent temperatures at which the preparations had similar d-spacing and were in the same mesomorphic state. The combination of X-ray and DSC data showed that the anesthetic steroid alphaxalone broadens the membrane phase transition and increases the ratio of gauche: trans conformers in the membranes in contrast to the inactive steroid delta 16-alphaxalone which affects the membranes only marginally. In model DMPC membranes alphaxalone and delta 16-alphaxalone are located near the bilayer interface. This location is maintained by alphaxalone when cholesterol is incorporated in the bilayer as evidenced by the X-ray measurements. However, when delta 16-alphaxalone is incorporated in cholesterol containing bilayers, a decrease in the electron density profile of the preparation is observed. This can be explained by invoking the formation of a delta 16-alphaxalone-cholesterol complex. The delta 16-alphaxalone complex shows no periodicity and is therefore, not detected in the X-ray diffraction experiment. Presumably, this complex forms aggregates either on the surface or inside the bilayer. This explanation corroborates DSC results which show that delta 16-alphaxalone sharpens the phase transition of DMPC/cholesterol preparations, an indication that some cholesterol is excluded from the bilayer preparation after the addition of the biologically inactive steroid.

Small angle X-ray diffraction and differential scanning calorimetric studies on O-methyl-(-)-delta 8-tetrahydrocannabinol and its 5' iodinated derivative in membrane bilayers.

We have previously studied and compared the location of (-)-delta 8-tetrahydrocannabinol (delta 8-THC) with that of O-methyl-delta 8-THC (Me delta 8-THC) in the membrane using partially hydrated dimyristoylphosphatidylcholine (DMPC) bilayers ((Mavromoustakos et al. (1990) Biophys. Acta 1024, 336-344; Yang et al. (1993) Life Sci. 53, 117-122). delta 8-THC was found to be located near the membrane interface with its phenolic hydroxyl group anchored near the carbonyl groups of DMPC while the more lipophilic Me-delta 8-THC is located deeper in the membrane bilayer. Parallel experiments using Me-delta 8-THC and its 5'-iodo analog (5'-I-Me-delta 8-THC) allowed us to determine the topography of these two molecules in the bilayer. Our results from small angle X-ray diffraction and differential scanning calorimetry (DSC) combined with previous data on the orientation of Me-delta 8-THC in model membranes, led us to the conclusion that these molecules intercalate between contiguous acyl chains in the lipophilic moiety of the membrane bilayer. The terminal iodo group in 5'-I-Me-delta 8-THC was found to reside in a region extending approx. +/- 5 A from the center of the bilayers. The location of Me-delta 8-THC in the membranes as well as its orientation may explain its inability to effectively perturb the bilayer lipid chains.

Effects of the anesthetic steroid alphaxalone and its inactive delta 16-analog on the thermotropic properties of membrane bilayers. A model for membrane perturbation.

We have studied in detail the effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on the thermotropic properties of model membranes using differential scanning calorimetry (DSC). The results obtained showed that, for model membranes from hydrated dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), and egg sphingomyelin, the biologically active analog significantly broadened the phase transition, in contrast to the inactive one which produced only marginal effects. Also, alphaxalone abolished the pretransition in these preparations whereas its delta 16-analog only broadened it. However, in DPPE bilayers almost no differences were observed in the effects produced by the two analogs. These results suggest that the ability of the two steroids to perturb membranes is lipid dependent. Comparisons between the effects of the two steroids on lipid/cholesterol model membranes revealed that delta 16-alphaxalone excluded cholesterol from lipid/cholesterol/delta 16-alphaxalone ternary systems whereas alphaxalone enhanced the effects of cholesterol and reduced the cooperativity in the binary phospholipid/cholesterol system. In an attempt to determine whether the different thermotropic effects of the two steroids on model membranes were due to (a) differences in their ability to perturb the bilayers; (b) different extents of incorporation into the bilayer, solid state 2H-NMR was applied using specifically deuterated steroids. The 2H-NMR data showed that alphaxalone incorporated fully into the membrane bilayer up to a molar concentration of 20%, while its inactive analog did only up to a concentration of 1%. To compare the abilities of the two steroids to perturb membrane preparations when both analogs were present in equal amounts in the membrane, the effects of very low steroid concentrations on DPPC bilayers were studied using DSC. The experiment showed that alphaxalone perturbed the membrane bilayers more effectively than its inactive analog. These results strongly suggest that the small structural differences between the two steroids are responsible for the observed differences in their abilities to perturb membranes, possibly because of differences in the packing of these two molecules within the bilayers.

The use of high-resolution solid-state NMR spectroscopy and differential scanning calorimetry to study interactions of anaesthetic steroids with membrane.

We have used a combination of high-resolution solid-state 13C-NMR and DSC (differential scanning calorimetry) to study the distinctively different thermotropic and dynamic properties of the anaesthetic steroid alphaxalone and its inactive congener delta16-alphaxalone in dipalmitoylphosphatidylcholine (DPPC) model membranes. In the solid-state 13C-NMR, the techniques included cross polarization (CP) and/or magic angle spinning (MAS). The observed data revealed the following important results. (a) DSC as a bulk method showed that the active steroid lowers the main phase transition temperature and broadens the pretransition more significantly than the inactive congener. The 13C-CP/MAS experiments allowed us to detect the pretransition temperature in the alphaxalone-containing preparation, which was not discernible in DSC. (b) The chemical shift values varied with temperature, indicating different degrees of trans-gauche isomerization in the lipid acyl chains when the bilayer is in the liquid crystalline phase. (c) Only specific additional peaks appeared in the 13C-CP/MAS spectra when each of the steroids was present in the preparation. delta16-alphaxalone gives rise to more additional peaks than alphaxalone, indicating a different mobility of the corresponding molecular moiety in the phospholipid bilayer environment. (d) The relative intensities of these peaks also confirmed that alphaxalone is fully incorporated in the bilayer, whereas delta16-alphaxalone is only partially so. These results suggest that the differential effects of these two analogues in the membrane may, at least in part, explain the reason for their different biological activities.