Functional analysis of experimental data in the field of modern researches of neutral macrocyclic antimycotic filipin
Biological, physical and chemical properties of macrocyclic compounds based on the researches of membrane activity. Finding of the certain concentration of filipin for increase of conductivity, definition of selectivity and single ionic filipin channels.
| Рубрика | Химия |
| Вид | статья |
| Язык | английский |
| Дата добавления | 22.02.2021 |
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Institute of Botany, Azerbaijan National Academy of Sciences
FUNCTIONAL ANALYSIS OF EXPERIMENTAL DATA IN THE FIELD OF MODERN RESEARCHES OF NEUTRAL MACROCYCLIC ANTIMYCOTIC FILIPIN
Baghirova Arifa
Summary
ionic filipin chemical macrocyclic
Biological, physical and chemical properties of macrocyclic compounds based on the researches of membrane activity are studied. So all these substances have antifungal properties and apply in the practical medicine that needs their detail research. They have names of polyene antibiotics (PA) because their chemical formula has certain number (n) conjugated double bonds( 4-7 ones). Among them electrically neutral pentaen antimycotic (5 double bonds) PA-filipin takes special place. Previous experiments of filipin fix disrupt of cell membrane in the presence of this antibiotic. Nevertheless it is possible to find the certain concentration of filipin for increase of conductivity, definition of selectivity and single ionic filipin channels and hybrid ionic channels nystatin-filipin and amphotericin- filipin. So it is confirmed filipin-sterol interaction and formation of filipin-sterol complexes for new ionic channels. The spectral analysis of filipin and research of its action was in parallel carried out on cell cultures infected by Influenza virus. The results of experiments confirmed literature information about action of filipin on viruses that may be prerequisite for the subsequent use of preparation as pharmaceutical.
Keywords: macrocyclic compounds, filipin, polyene antibiotics, ionic channels, hybrid ionic channels.
Abbreviation: PA- polyene antibiotics; BLM- bilayer lipid membranes; VSV- vesicular stomatitis virus.
INTRODUCTION
Macrocyclic polyene antibiotics use in clinical therapy for some decades as medicines against pathogenic infections particularly against fungal infections [Zotchev, 2003]. Application of these drugs in medical practice is based on the research of structure-functional dependence of these compounds and study of molecular-biological mechanism of their activity in cell membranes. In parallel with cellular membranes their alternative artificial models were used. In this case bilayer lipid membranes (BLM) were used. Their properties are identical to the properties if native ones. Now there are known more than 200 representatives of PA. From the medical point of view amphotericin B, nystatin, trichomycin and levorin are the most demanded. It is shown that they have high antifungal activity. Antifungal activity is based on the interaction of antibiotic with sterol compounds of plasmatic membranes. Mechanism of interaction is the formation of antibiotic-sterol complexes. This sterol is localized in the membrane. Then formation of ionic channels that are permeable for ions and organic substances with small molecular sizes takes place [Kasumov, 2009]. Among PA filipin has special status. It was extracted by Whitefield and others from soil ac- tinomycets Streptomyces filipensis [Whitefield, 1955]. So it was named filipin and identified as representative of PA group. As pentaene PA for many years it is proposed that this antibiotic disrupts cell membrane and doesn't interact with sterol. Detail study of physical, chemical properties and biological activity of filipin on BLM makes possible to define the concentration of increasing of conductance of membranes and concentration of individual ionic channels. There are data in the literature confirmed about action of filipin on enveloped viruses in particular on Newcastle Disease Virus, Influenza Virus, Vesicular Stomatitis Virus, NiemannPick Disease and Rausher leukemia [Vanier M.T., Latour Ph., 2015]. We research filipin in these aspects.
MATERIALS AND METHODS
Experiments were carried out by Muller methods[Mueller et al, 1963] Membranes were formed on the hole in teflon cup with diameter 0,5 mm and wall thickness about 30 mkm. We used some membrane solutions for preparing: 1) solution of total phospholipids of bull brain in the chloroform/ methanol with initial concentration 12 mg/ml; 2) solution of oxidated cholesterol in heptaene with initial concentration 10/ml. Before experience the initial membrane mixes were evaporated into heptaene solution and formed bimolecular membranes. PA filipin was kindly presented by St-Petersburg Scient.-Res. Institute of Antibiotics and Enzymes. Antibiotic dissolved in dimethyl sulfoxide and added to water-salt solution. We used KCl solution.
RESULTS AND DISCUSSION
Chemical structure of antibiotic is presented on the fig. 1 There are 5 double bonds and some carboxyl and methyl groups in the chemical structure of filipin.
Fig. 1 Chemical structure of filipin (C35H580u)
Mechanism of action of filipin is based on De Kruyff's sterol hypothesis [De Kruyff, Demel, 1974]. According to this hypothesis PA interact with the sterol of cytoplasmic cell membrane and form conducting ionic channels that are permeable for ions and nonelectrolytes. It is shown that filipin totally corresponds this hypothesis. Experimental data show that this antibiotic forms complexes with cholesterol in cell membranes. Stoichiometry of antibiotic-cholesterol interaction and formation of complexes shows that one molecule of cholesterol connects with one molecule of antibiotic. By this concept cholesterol interacts with system of double bonds of filipin molecule in lipid bilayer. By the data of atomic-force microscope filipin forms clusters in the phospholipid membranes [Ostroumova et al.,
Fig. 2 Kinetics of conductivity ofphospholipid membranes in the presence of filipin (concentration 1x10 6M)
2012a;Ostroumova, et al., 2012b]. Localization of fili- pin-cholesterol complexes proposes the existence of two types of models: first type is parallel, second one is perpendicular location of filipin in relation to the membrane surface. In first type of orientation OH-groups of filipin may locate in parallel of bilayer surface. In second type filipin-cholesterol complexes locate perpendicular in relation to the bilayer one [De Kruyff, De- mel,1974].In this case hydrophilic side of molecule of antibiotic is in the hydrophobic part of membrane and interacts with hydrophilic side of second complex and forms regular row of filipin-cholesterol complexes. Absence of charged groups in the filipin molecule makes possible of translocation of all complexes in the mem- brane[De Kruyff,Demel,1974]. It was proposed recently the disruption of membrane associate with this process. However detail research of this antibiotic shows that membranes treated by filipin are rather stable about 2 hours at the membrane potential 200 mV[Samedova, Kasumov, 2009].Filipin in the concentration 2x10-6M was added by both sides of membrane increase the conductivity in many orders. There is monotonous kinetics of increase in conductivity of membranes with reaching stationary level without inactivation.
There is typical kinetics of the increase of membrane conductivity at the presence of filipin on the fig.2. Increase of conductivity depends of type of ions in the solution and doesn't depend of the value of membrane potential. Increase of membrane current is followed by consecutive spasmodic changes (fig.3). Addition of filipin on one side of membrane and nystatin and amphotericin B on another one leads to increase of conductivity [fig.4].
Fig. 3 Single ionic filipin channels in phospholipid membranes (concentration2x108M)
Membrane structure: phospholipid-cholesterol= 2:1. Experimental conditions: 2MKCl, t0= 220C, pH=7,0..Membranepotential = +200mV[Samedova,Kasumov 2009].
Single ionic channels with conductivity 15-20 pS were fixed in cholesterol-containing membranes in KCl water solution (concentration 1x 10-8M) and presented on fig.3. This value approximately by 3-4 times exceeds conductivity of amphotericin B channels. It is shown that filipin and amphotericin B channels have two states: conducting and nonconducting. During the life-time of the channel in membrane there are shorttime transitions from conducting state to noncoducting one.
Membrane structure: phospholipid:cholesterol= 20:1,. Experimental conditions: 2MKCl, t0= 220C, pH=7,0..Membrane potential = +200mV[Samedova, Kasumov, 2009].
Research of filipin shows that if filipin is added from one side of membrane and ampbotericin B or nystatin in the same concentration (1x10-6M) from another side there is increase of integral conductivity in both cases. We try to fix the work of combined ionic channels (fig.4). Conductivity of combined channels of filipin and amphpotericin B is 25-30 pS. It is nearly in 1,5 times higher “pure” filipin channels and in 5 times higher “pure” amphotericin B ones.
Fig. 4 Combined ionic channels formed at the same concentrations of filipin and amphotericin B (2x10- 6M) added to different sides of BLM [Samedova,Kasumov 2009]
Selective permeability of filipin channels is mainly cationic.There is potential difference of + 18± 2 mV on membrane at the 10-fold gradient in KCl solution. Reversion of membrane potential is not shownat the presence of filipin. Orientation of filipin on model membranes was confirmed by circular dichroism method. Filipin is located perpendicularly to membrane surface in the cholesterol amount on membrane is about 33% and antibiotic is distributed evenly in a lipid matrix. Formation of filipin-sterol complexes is proved on different biological objects. For example, cells of Mycoplasma gallisepticum, need sterols for growth. These cells aren't damage on the medium without sterols for 5 days however the growth is suppressed in the presence of sterols. These cells grow on the mediums without sterols but cells merge quickly last ones in the case of addition of sterol into the medium. Their growth is suppressed by filipin in the presence of cholesterol and vice versa. Cells Acholeplasma laidlawiii on the cholesterol-containing medium also are sensitive to filipin [De Kruyff, Demel, 1974]. The cholesterol content in cells of lens of humans [Van Marle, 2000], in nuclear membranes of Erlich ascites carcinoma cells, in membranes of photoreceptor cells [Cooper, 1984], in plasmatic membranes of cells of some animals epidermis, in the Golgi tubes etc. is determined with the help of filipin as marker by cytochemical method [Lampen, 1966]. Filipin-sterol niches are visible in the form “niches” of size 25-30 nm in plasmatic membranes of follicles of mice, in membranes of cells of endoplas- matic reticulum of pancreas [Lampen, 1966; Van Marle, 2000].
Filipin may be used also for detection of sterols in membranes of plant cells. In this case unlike of amphotericin B filipin also as well as nystatin does not suppress of activity of the photosystem I in the chloroplasts [Cooper, 1984]. Sensitivity of subcellular organellas to filipin depends of sterol content in membranes [Kasumov, 2009].
From cytological point of view the action of filipin on succinate-cytochrom S-reductase (this enzyme contains the residues of succinic acid) in rats mitochondria and in fungi Neuorpspora. In this case the presence of sterols is necessary for binding of antibiotic with cell membranes. It is known that PA act on membrane enzymes. Filipin also acts on this process. So the inhibition of ATP-ase activity at the concentration of filipin more than 0,2mM for 1 mg of protein [Solo- vyeva,1977]. Inhibition of membrane enzymes activity is caused by disruption of cholesterol-phospholipid bonds or disruption of association between enzymes and membrane lipids[Solovyeva,1977].It has shown that filipin activates K+ - ATP-ase [Gassner, Komnick, 1983]. Process of activation of follows by the increase of membrane permeability without of disruption of structure of vesicular membranes. Increase of filipin concentration in cholesterol-containing lipid membranes (ratio filipin / cholesterol= 10) leads to increase of amount of filipin-cholesterol complexes and active lateral dislocation of complexes that as a result leads to the equal redistribution of these ones in membrane. There is inhibition of Na+- K+ - ATP-ase at the ratio of filipin / cholesterol >2 in dependence of temperature. Possibly the formation of filipin / sterol complexes in intracellular pools takes place in sterol-containing cells [Gassner, Komnick,1983].
Some properties of filipin began to reveal later. So, for example, effect of this drug on vector-borne infections (vector-borne spongy encephalopathy) became clear. This disease is caused by prion proteins [Marella, 2003]. In this case normal prion protein (PrP-sen) associates with membranes and transforms to pathological isomerous resistant form (PrP-res) that leads to vector- borne spongy encephalopathy. It has shown that filipin interacts with sterol of neuroblastoma cells. Formation of filipin complex inhibits process of endocytosis of normal prtion protein PrP-sen and reduces its amount in cell membranes. Normal protein PrP-sen transforms to pathological form PrP-res where the formation of resistant protein is suppressed by filipin in case of infection of neuroblastoma cells by vector-borne spongy encephalopathy. Thus filipin may be potential inhibitor of formation of pathological protein in prion infections [Marella, 2000]
It was known about influence of PA on reproductive activity of some viruses and as a result reduce the titres of infectivity. Among these antibiotics most efficient is amphotericin B. This one acts on reproductive properties of some viruses including hepatitis, influenza, herpes, HIV, vesicular stomatitis and enterovirus [Clayette, 2002]. Nevertheless there are fixed the action of filipin on reproduction properties and reduction of infectivity of some ones. It is possible to analyse some results of filipin action based on experimental data. For examplethere is shown the action of filipin on activity on enveloped viruses such VSV, influenza, Newcatle disease virus and virus of leukemia by Rausher. It has shown that filipin interacts with membrane-binding cholesterol. There is inclusion of filipin into virions. There is not release of lipids or proteins from virions after treatment by this antibiotic. Morphological changes were induced by filipin in influenza viruses and Rausher leukemia and differed from virions of VSV. Infectivity of virions of VSV decreased in 500 times but influenza virions were resistant to treatment by filipin. Thus there were fixed filipin-cholesterol interaction [Majuk, 1977].
There were extracted 4 amphotericin B-resistant mutants and 2 filipin-resistant mutants from infusorians (Paramecium caudatum) [Katsumi, Haruoki, 2001].Mutant cells of Chinese hamster free of gene of Nieman-Pick disease- gene NPCI (genetic disease caused by disturbance of lipid metabolismand accumulation of lipids in liver, spleen, marrow and brain) are characterized probably by low content of total absence of cholesterol in plasmatic membranes [Katsumi, Haruoki, 2001].Filipin may be used as test at the Nie- man-Pick disease [VanierM.T.,Latour Ph.,2015]. This disease takes place in the result of mutational changes. Major factor of these pathogenesis is low exit of cholesterol from late endosomes / lysosomes. Concentration of cholesterol may be defined by fluorescent microscopy after staining by filipin test. Well carried out test is the main one for the Nieman-Pick disease diagnosis for patients with the use of chicken fibroblasts culture. It is also effective and functional way to the study of this disease and pathogenicity of new mutations [Vanier M.T., LatourPh.,2015].
Genome of actinomyces Streptomyces filipinensis is the subject of some changes [Payero T.D., Vicente C.M. et al.,2015], So there is produced filipin III which has more biological activity that initial antibiotic and another filipin components. All transformed components of filipin at the mininmal concentration inhibit the action of strains Candida utilis n Saccharomyces cerevisiae. Among this components hydroxofilipin-I and filipin II reveal essential antifungal activity. These results show that there are two alternative directions for formation of filipin III that open new opportunities for creation of biologically active filipin and its derivatives with perfect properties [Payero T.D., Vicente C.M. et al.,2015].
It was fixed the formation of filipin, nystatin, le- vorin and etruskomycin complexes with sterol compound of cell membrane in the infection of cell cultures by influenza virus (strain WSN; cell cultures: FL - human amnion cells ; CF- chicken fibroblasts) It has shown the process of formation of PA-sterol complexes by spectral analysis [Samedova, Kasumov, 2009].
CONCLUSION
The research of macrocyclic polyene antimycotic filipin shows that identically with others PA this preparation is membrane-active compound. It is fixed the increase of conductivity of phospholipid membranes in the presence of this antibiotic in water-salt solution. Analysis of experimental data shows that filipin forms complexes with sterol compounds in plasmatic membranes of different type of cells. Thus mechanism of action of this substance is based on the interaction with sterols in cytoplasmic membranes of cells and BLM. This process leads to the formation of molecular size ion channels conducting for ions and organic substances. This antibiotic meets to a “sterol“ hypothesis. Filipin channel has equal number of filipin and cholesterol molecules. There is monotonous kinetics of conductivity in membranes with reaching stationary level without inactivation for filipin. Researches of lipid membranes show that conductivity of single ion channels is 15-20 pS and combined (filipin-amphoteri- cin B) ion channels is 25-30 pS. Selective permeability of filipin channels is mainly cationic and its value is +18 mV. Cytological researches show that this antibiotic activates latent K+- ATP-se.
Analysis of experimental data showed inhibiting action of filipin on reproductive activity of some viruses. Titre of infectivity for some enveloped viruses decreases in many orders under the action of filipin.
Filipin may be potential inhibitor for formation of pathological protein in chronically infectedcells at the vector-borne prion infections.
Considering functional features of this macrocy- clic antibiotic and analyzing experimental data in the perspective filipin may be used as medicine at various viral, fungal and prion infections
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