Katarzyna Cieślik-Boczula

Chemical structure of phenothiazines and their biological activity.

Phenothiazines belong to the oldest, synthetic antipsychotic drugs, which do not have their precursor in the world of natural compounds. Apart from their fundamental neuroleptic action connected with the dopaminergic receptors blockade, phenothiazine derivatives also exert diverse biological activities, which account for their cancer chemopreventive-effect, as: calmodulin- and protein kinase C inhibitory-actions, anti-proliferative effect, inhibition of P-glycoprotein transport function and reversion of multidrug resistance. According to literature data on relations between chemical structure of phenothiazines and their biological effects, the main directions for further chemical modifications have been established. They are provided and discussed in this review paper.

Interactions of Dihydrochloride Fluphenazine with DPPC Liposomes: ATR-IR and 31P NMR Studies

The influence of dihydrochloride fluphenazine (FPh) on the dipalmitoylphosphatidylcholine (DPPC) bilayer structure was investigated using ATR-IR and (31)P NMR methods. The ATR-IR results indicate an increase in conformational disorder in the hydrophobic part compared with pure DPPC liposomes and a decrease in temperature of the chain-melting phase transition in FPh/DPPC liposomes. These effects depended on the concentration of the drug in the DPPC bilayer. The dihydrochloride fluphenazine molecules form H-bonds with the proton-acceptor carbonyl groups of DPPC molecules. At a higher concentration of the drug, the lipid bilayer structure is destroyed, and an isotropic phase is observed using (31)P NMR spectroscopy. The interactions between FPh and the lipid bilayer have a crucial role in MDR (multidrug-resistant) activity of this drug. These results improve one possible strategy of cancer chemoprevention with FPh accompanied by fluidization and destabilization of the model lipid bilayer structure.

The effect of 3-pentadecylphenol on DPPC bilayers ATR-IR and 31P NMR studies

The influence of 3-pentadecylphenol (PDP) on the structure and physicochemical properties of the lipid bilayers of DPPC liposomes was studied using ATR-IR and (31)P NMR methods. On the basis of analysis of the bands assigned to the CH(2) stretching, CH(2) scissoring, C=O stretching, and PO(2)(-) stretching vibrations it was revealed that PDP influences both the hydrophobic and hydrophilic parts of the DPPC liposome bilayer. Analysis of the (31)P NMR line-shape indicated a lamellar to non-lamellar phase transition in PDP-doped DPPC dispersions. It was shown that PDP/DPPC isotropic aggregates have similar nuC=O and nu(as)PO(2)(-) band positions and lower gauche populations in the hydrophobic chain region compared with the DPPC bilayer in the liquid-crystal phase.

Phase separation in phosphatidylcholine membrane caused by the presence of a pyrimidine analogue of fluphenazine with high anti-multidrug-resistance activity.

Phenothiazine compounds are known as effective inhibitors of a multidrug resistance (MDR) of tumor cells to chemotherapeutic agents. This group consists of many important substances used in human medicine such as antipsychotic drugs in the case of fluphenazine (FPh) or chlorpromazine (CPZ). Fluphenazine was on the World Health Organization (WHO) list of Essential Medicines of 2009, and its new pyrimidine analog (FPh-prm) presented in this work has been documented to have a high anti-MDR activity. In order to discover the character of alterations of the lipid bilayer structure caused by the presence of FPh-prm inside the lipid membrane, which is responsible for the essential increase of an anti-MDR activity of FPh-prm, microcalorimetric (differential scanning calorimetry), Laurdan fluorescence, (31)P nuclear magnetic resonance spectroscopy (NMR), and attenuated total reflectance Fourier transfer infrared spectroscopy (FTIR-ATR) were used for dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes mixed with a different concentration of amine analogue. It was stated that the formation of domains with different content of FPh-prm/DPPC can be a reason for the membrane-related mechanism of chemoprevention associated with the inhibition of the outward transport of anticancer drugs by the glycoprotein P (Pgp) in cancer cells by the pyrimidine analog of FPh. To our best knowledge, this report is the first to show the bilayer structure of domains formed by incomplete miscibility of fluphenazine-related compounds and phospholipid molecules. Our results provide a sound basis for the design of future modifications of anti-MDR drugs by providing very effective inhibitors of the pump activity of Pgp.

MCR-ALS as an effective tool for monitoring subsequent phase transitions in pure and doped DPPC liposomes

The MCR-ALS method was applied to increase the structural information derived from infrared spectra of pure and doped dipalmitoylphosphatidylcholine (DPPC) liposomes. Pure DPPC vesicles and mixed ones with long-chain homologues of the phenol substituted by a tert-butyl moiety were investigated using the attenuated total reflectance Fourier-transfer infrared spectroscopy (FTIR-ATR). A combination of pure spectral profiles with pure concentration ones, which were derived from MCR-ALS calculations, enabled us to discuss in detail the structural characteristics of each pure phase state, which occur during a heating of pure as well as mixed lipid systems. Additionally, alterations of relative concentrations of subsequent components, which were represented by different pure phase states, associated with individual phase transitions, were determined in both pure and mixed DPPC systems. As far as we known, it is the first application of the MCR-ALS calculation in analysis of FTIR-ATR spectra of DPPC membranes.

Interaction of Piperidin Derivative of Mannich Base with DPPC Liposomes

The long chain Mannich bases, especially with the piperidine and morpholine groups, display very promising antimicrobial activity. In order to extend our knowledge on their impact on biological systems, we examined the interactions of the 5-pentadecyl-2-((piperidin-1-yl)methyl)phenol (PPDP) with model lipid membrane by means of differential scanning calorimetry (DSC) and fluorescence measurements. The small unilamellar vesicles of dipalmitoylophosphatidylcholine (DPPC) with different piperidine Mannich base concentration were investigated as a function of the increase of temperature. The phase separation accompanied by the rise of the transition enthalpy of both subcomponents, the increase of the function of the GP values of Laurdan versus the wavelength of excitation in the gel phase of PPDP/DPPC systems, and no remarkable differences in the fluorescence anisotropy of PPDP molecules in lipid environment for different mixtures of PPDP/DPPC was observed. Additionally, it was shown that PPDP itself interdigitated in solid state.

Anesthetic-dependent changes in the chain-melting phase transition of DPPG liposomes studied using near-infrared spectroscopy supported by PCA

The effect of inhalation anesthetics (enflurane, isoflurane, sevoflurane or halothane) on the lipid chain-melting phase transition of negatively charged phospholipid membranes was studied using near-infrared (NIR) spectroscopy supported by Principal Component Analysis (PCA). NIR spectra of anesthetics-mixed dipalmitoylphosphatidylglycerol (DPPG) membranes were recorded in a range of the first overtone of the symmetric and antisymmetric stretching vibrations of CH2 groups of lipid aliphatic chains as a function of increasing temperature. Anesthetic-dependent changes in the trans to gauche conformers ratio of CH2 groups in the hydrocarbon lipid chains were characterized in detail and compared with the zwitterionic lipid membranes, which were built of dipalmitoylphosphatidylcholine (DPPC) molecules.

Alpha-helix to beta-sheet transition in long-chain poly-l-lysine: Formation of alpha-helical fibrils by poly-l-lysine

The temperature-induced α-helix to β-sheet transition in long-chain poly-l-lysine (PLL), accompanied by the gauche-to-trans isomerization of CH2 groups in the hydrocarbon side chains of Lys amino acid residues, and formation of β-sheet as well as α-helix fibrillar aggregates of PLL have been studied using Fourier-transform infrared (FT-IR) and vibrational circular dichroism (VCD) spectroscopy, and transmission electron microscopy (TEM). In a low-temperature alkaline water solution or in a methanol-rich water mixture, the secondary structure of PLL is represented by α-helical conformations with unordered and gauche-rich hydrocarbon side chains. Under these conditions, PLL molecules aggregate into α-helical fibrils. PLLs dominated by extended antiparallel β-sheet structures with highly ordered trans-rich hydrocarbon side chains are formed in a high-temperature range at alkaline pD and aggregate into fibrillar, protofibrillar, and spherical forms. Presented data support the idea that fibrillar aggregation is a varied phenomenon possible in repetitive structural elements with not only a β-sheet-rich conformation, but also an α-helical-rich conformation.

New fluphenazine analogues as inhibitors of P-glycoprotein in human lymphocyte cultures

Aim of the study To evaluate the inhibitory effect of 17 new analogues of FPh on the Pgp transport function, by estimation of the rhodamine 123 (Rod-123) accumulation inside cultured lymphocytes. Material and methods Lymphocyte were cultured in the presence of a lectin (PHA; 2%, v/v), incubated with benzo[α]pyrene (B[α]P; 7.5 µM, 48 h) to induce genotoxic damage and to increase Pgp expression in the cells. Lymphocytes cultured without the tested compounds were considered as controls. Results It was established that 10 analogues of FPh, among 17 tested, significantly increased Rod-123 accumulation in lymphocytes at the concentration of 10 µM. As compared to the control cultures the Pgp transport function was the most strongly inhibited by 1a, 1b, 1d, 3f, 3h and 3i analogues (approximately by 25%). Conclusions FPh analogues 1a, 1b, 1d, 3f, 3h and 3i should be further studied as promising candidates for adjuvant cancer chemotherapeutics.

The PPII-to-α-helix transition of poly-l-lysine in methanol/water solvent mixtures accompanied by fibrillar self-aggregation: An influence of fluphenazine molecules

Fourier-transform infrared, vibrational circular dichroism spectroscopy and transmission electron microscopy are used to follow the structural changes of pure and fluphenazine (FPh)-mixed poly-l-lysine (PLL) triggered by variations of the methanol to water ratio in solvent mixtures. FPh molecules are used as an effective psychotic drug but with a strong Parkinsons-related side effect. To answer the question whether FPh molecules can modify the fibril development, the PLL polypeptide was used as a model of α-helix- and PPII-rich fibrils. It was stated that the presence of FPh molecules did not inhibit the creation of both types of PLL fibrils with clustering features. The methanol-poor aqueous solutions promote the formation of extended polyproline II (PPII) helices; however, the methanol-rich aqueous solutions induce the development of α-helices of both pure and FPh-mixed PLL. Unpredicted and interesting features of PLL fibrillogenesis are evidenced by the formation of uncommon fibrillar aggregates, which are developed in methanol/water solvents from PLL molecules rich in either α-helix or PPII structures. Possibility of PLL molecules to form β-sheet-, α-helix- and PPII-rich fibrils demonstrating that fibrillogenesis is a common phenomenon, and fibrillar aggregates can be based on all of the basic protein secondary structures.