Vitaly Ivanovich Kalchenko

Background. Plasma membrane Na+,K+-ATPase plays an important role in maintaining ionic homeostasis and regulating myometrial contractility. Its inhibition may alter uterine smooth muscle responses to uterotonic stimulation. Objective. This study aimed to determine the mechanism by which calix[4]arene C-1130 inhibits Na+,K+-ATPase activity and to evaluate its effects on oxytocin-induced myometrial contractions. Methods. Na+,K+-ATPase activity was studied in suspensions of perforated plasma membranes isolated from myometrial cells. The kinetic parameters of ATP hydrolysis were determined at different ATP and Mg2+ concentrations. The effects of C-1130 on oxytocin-induced isometric and isotonic contractions were exami­ned in myometrial preparations with preserved endometrium. Results. Calix[4]arene C-1130 decreased the maximum rate of Na+,K+-ATPase-mediated ATP hydrolysis without significantly affecting the apparent affinity­ or cooperativity parameters for ATP and Mg2+. At a concentration of 10 μM, C-1130 enhanced oxytocin-induced isometric myometrial contraction and increased the normalized maximal rates of both contraction and relaxa­tion phases. Under isotonic conditions, C-1130 increased the velocity and enhanced the power characteris­tics of muscle contraction. Conclusions. Calix[4]arene C-1130 inhibits myometrial Na+,K+-ATPase­ through a noncompetitive mechanism. The enhancement of oxytocin-induced contractile muscle activi­ty is likely associated with disruption of ionic homeostasis in myocytes caused by inhibition of the plasma membrane Na+,K+-ATPase. Keywords: ATPase kinetic properties, calix[4]arenes, K+-ATPase, myometrium, Na+, plasma membrane, smooth muscle cells

Background. Calixarenes are supramolecular compounds with a unique three-dimensional structure, the biological activity of which is determined by the chemical groups on the upper or lower rim. It was found that thiacalix[4]arene C-1087 selectively (compared to other ATPases) inhibits plasma membrane Ca2+,Mg2+-ATPase activity that playіs a crucial role in maintaining Ca2+ homeostasis in myometrial cell. To expand our understanding of the thiacalix[4]arene C-1087 effects on Ca2+ exchange in myometrial cells, it is necessary to analyze its possible effects on Ca2+ exchange in intracellular pools. Objective. Therefore, the aim of this study was to investigate whether thiacalix[4]arene C-1087 affects Ca ion exchange in myometrial mitochondria. Methods. Mitochondria from myometrium of non-pregnant rats were isolated. Ionized calcium concentration in the mitochondria matrix was determined using the fluorescent probe Fluo-4 AM. Baseline Ca2+ concentration in the absence of exogenous (added) Ca2+ and Ca2+ accumulation in its presence were determined. Ca2+ concentration in the incubation medium was determined using fluorescent probe 1 µM Fluo5F, Pentapotassium Salt. Relative value of mitochondria membrane potential (Δψ) was assayed using a fluorescent probe 100 nM TMRM. The Microsoft Office Excel software package was used for data processing. Results. It was proven that mitochondria incubation in Mg2+– and Mg2+,АТР-medium with 10 μM thiacalix[4]arene C-1087 was accompanied by an increase of both the baseline Са2+ concentration and accumulation of this cation in the matrix and did not affect Са2+ efflux from mitochondria. Mitochondria incubation with 10 μM thiacalix[4]arene C-1087 resulted in reducing of TMRM-loaded mitochondria fluorescence intensity, indicatings the depolarizing effect of this compound. Conclusions. The results obtained indicate the possibility of thiacalix[4]arene C-1087 modulating effect on the Са2+ exchange in mitochondria and polarization of its membranes. Keywords: membrane polarization, mitochondria, thiacalix[4]arene C-1087, Са2+ accumulation and efflux, Са2+ concentration

The kinetics of the stepwise alkaline hydrolysis of three isomeric mononitrofluorescein diacetates, containing the nitro group in the residue of the phthalic acid, were investigated in water and organized aqueous solutions of cationic surfactants and a cationic calix[4]arene. The reaction follows a consecutive two-stage mechanism involving the formation of a spectroscopically distinguishable monoacetate intermediate. UV-visible spectrophotometric monitoring allowed for the separate determination of the pseudo-first-order rate constants for both hydrolytic steps. The catalytic efficiency of the organized systems was found to increase in the order: cetyltrimethylammonium bromide (CTAB) < N,N-dimethyl-N-hydroxyethyl-4-dodecyloxybenzylammonium chloride (C12-Bn-Ch) < 5,11,17,23-Tetra(N,N-dimethyl-N-hydroxyethylammonium)methylene-25,26,27,28-tetradodecyloxy calix[4]arene tetrachloride (C12-C4A-Ch), with acceleration factors reaching nearly three orders of magnitude for the 3'-nitro derivative. The positioning of the nitro substituent markedly influenced the relative rates of the two hydrolysis stages, with the k2’/k1' ratio being governed by the interplay between intrinsic electronic effects and the nature of the organized environment. These results suggest that supramolecular assemblies based on calixarene derivatives provide a highly efficient microenvironment for ester hydrolysis, likely through a synergy between specific host-guest recognition within the macrocyclic cavity and collective electrostatic effects at the aggregate interface.

The host-guest complexation of cone-shaped calix[4]arene-hydroxymethylphosphonic acid (CPA) and calix[4]arene-hydroxymethyldimethylphosphine oxide (CPO) with active pharmaceutical ingredients of antiviral drugs Remdesivir, Nevirapine, Vesatolimod, and Bictegravir in the aqueous-organic mobile phase on a Zorbax CN column has been studied using RP HPLC method. By analyzing the dependence of the drug capacity values on the concentration of calixarene in the mobile phase, the stability constants (КА = 3672 - 6884 M-1) of the complexes formed have been determined. Quantum-chemical calculations show that the drugs studied form supramolecular exo-complexes with CPA and CPO molecules. These complexes are stabilized by intermolecular hydrogen bonds of proton donor groups P(O)(OH)2 CPA and proton acceptor groups Me2P=O CPO with the amino group of Remdesivir, the amide group of Nevirapine, the amino group and amide group of Vesatolimod, and the amide group of Bictegravir.

The extraction of radionuclides 137Cs, 90Sr, 90Y, and UO22+ was studied using solid-phase extractants SPE-TBP, SPE-C45 and SPE-CIP67 depending on the acidity of the aqueous solution. SPE-C45 and SPE-CIP67 are porous styrene-divinylbenzene copolymer pellets impregnated with calix[4]arene tetraphosphine oxide C45 (5,11,17,23-tetrakis-diethylphosphinoylmethyl-25,26,27,28-tetrapropoxycalix[4]arene) and CIP67 (5,11,17,23-tetrakis-dipropylphosphinoylmethyl-25,26,27,28-tetrapropoxycalix[4]arene). Commercial solid-phase extractant SPE-TBP based on tributylphosphate was used for comparison. The influence of the radionuclides contact time with extractant on the establishment of extraction equilibrium was analyzed. It was shown that the equilibria in the systems using SPE are established in 3-4 hours. A significant influence of the acidity of the aqueous solution on the extraction efficiency of the radionuclides was found: an increase in acidity leads to an increase in the distribution coefficients. The influence of the nature of cations on the extraction efficiency is shown. The extraction of the radionuclides increases in the following series: UO22+ > 90Y > 90Sr > 137Cs. It was revealed that the extraction of the radionuclides depends on the length of the alkyl radical at the phosphorus atom of the macrocyclic skeleton of calix[4]arene phosphine oxide. Cations are extracted more efficiently with calix[4]arene of shorter alkyl radical length at phosphorus atom. The ability of SPE-calixarenes towards the studied radionuclides exceeds the extraction ability of SPE-TBP and varies in the following order: SPE-TBP< SPE-CIP67< SPE-C45. The conducted studies have shown the possibility of using the studied solid-phase-liquid extraction systems in the organization of radiation control of natural waters. In this case, the method of solid-phase extraction is convenient for radionuclide extraction, as it is simple and allows minimizing waste.

Background. Nа+, K+-ATPase (sodium pump) is an electrogenic Са2+-independent Mg2+, Na+, K+-ATP-dependent transporting system of plasma membrane (PM), which conducts active transfer of univalent ions of Na and K and thus maintains their electrochemical gradients, required for normal functioning of the cell. It was proven that in some widesperad pathologies the activity of the sodium pump is disrupted. Therefore, the search for effectors - selective inhibitors and activators that would be able to specifically affect Na+, K+-ATPase, restoring its activity in pathological conditions, is very promising. The aim of this study was to investigate the action of thiacalix[4]arene С-1193 on the dependence of Nа+, K+-ATPase activity of PM of myometrium cells on the concentration of ATP and Mg ions, as well as on the isotonic contractile activity of the myometrium. Materials and Methods. The experiments were conducted using outbred white non-pregnant rats. The inhibitory action of thiacalix[4]arene C-1193 (25,27-dibutoxythiacalix[4]arene-bis-hydroxymethylphosphonic acid) on the kinetic traits of Na+, K+-ATPase activity was studied in the experiments, conducted using the suspension of perforated plasma membranes of the myometrium cells. The registration of the contractile acti­vity in the preparations of longitudinal smooth muscles of uterine horns with preserved endothelium was done in the isotonic mode. The study of the contractile activity of muscle preparations was carried out using mechanokinetic analysis methods. Results. The study demonstrated that while inhibiting Na+, K+-ATPase, thiacalix[4]arene С-1193 did not change the kinetic parameters (Km, nH) of the dependence of the reaction velocity on the substrate concentration. Calix[4]arene C-1193 had practically no action on the affinity between Na+, K+-ATPase and ATP, which demonstrated the absence of competition between the binding centers for ATP and C-1193. There was no effect on the affinity and cooperative action of Mg ions either. Both cases demonstrated a considerable reduction in the maximal velocity of ATP hydrolysis. It was found that thiacalix[4]arene С-1193 (in the concentration of 10 μM) modulated the isotonic reactions of pluricellular preparations of myometrium, induced via the pathways of electro- and pharmacomechanic coupling. It was also determined that under the effect of С-1193, there was an increase both in the amplitude of contractions and in the mechanokinetic parameters: contractions (ΔLmax, ΔLC and ΔLR) and velocities (VC and VR). The norm-setting for VC and VR regarding the amplitude of contractions at the action of С-1193 resulted in the loss of statistically significant differences between the maximal velocities of the contraction and relaxation phases. Conclusions. The results of our research indicate that thiacalix[4]arene С-1193, has a non-competitive mechanism of inhibiting Nа+, K+-ATPase activity and has no specific action on Са2+-transporting systems of uterine myocytes.

The complexation of tetrahydroxycalix[4]arene-methyldimethylphosphine oxide (CMPO), tetrahydroxythiacalix[4]arene-methyldimethylphosphine oxide (TCMPO), and tetrapropoxycalix[4]arene-methyldiethylphosphine oxide (CEPO) with active pharmaceutical ingredients of antiviral drugs Remdesivir, Nevirapine, Vesatolimod, Bictegravir, Emtricitabine, and Tenofovir in the water medium was studied using the RP HPLC method. By analyzing the dependence of the drug capacity values on the concentration of calixarenes in the chromatographic mobile phase, the stability constants (KA = 1100 - 12000 M-1) of the complexes formed were determined. Quantum-chemical calculations show that the antiviral drugs form supramolecular exo-complexes with the calixarene-phosphine oxide molecules. These complexes can be stabilized by intermolecular hydrogen bonds between the proton acceptor P=O groups and the proton donor groups of antiviral drugs.

Background. According to existing knowledge, thiacalix[4]arene С-1087 is highly capable of inhibiting Са²⁺-pump (Са²⁺, Mg²⁺-АТРase) of the plasma membrane; at the same time it inhibits the functioning of Са²⁺-pump of the sarcoplasmic reticulum of uterine smooth muscles to some degree. The aim of this research was to study the effects of С-1087 on the concentration of Ca²⁺ ions and contractile activity of the rat myometrium cells using an inhibitor of Са²⁺-pump of the sarcoplasmic reticulum – thapsigargin. Materials and Methods. The experiments were conducted using outbred white non-pregnant rats. The contractile activity in the preparations of longitudinal SM of uterine horns with preserved endothelium was registered in the isometric mode. To determine the changes in [Ca²⁺]_i level, myocytes were treated with probes Hoechst 33342 (to test the nucleus of the cell) and fluo-4 AM (to test the change in Са²⁺-concentration in the cell). Results. The tenzometric studies with the subsequent mechanokinetic analysis demonstrated that under the action of thapsigargin (0.5 µM), thiacalix[4]arene C-1087 (10 µM) caused considerable changes in the kinetics of the spontaneous contractile activity processes in the myometrium of rats, including the decrease in the maximal contraction velocity and the increase in the maximal relaxation velocity. By means of confocal microscopy with Ca²⁺-sensitive fluorescent probe fluo-4, it was demonstrated that the application of thiacalix[4]arene С-1087 to immobilized myocytes of the uterus against the background of thapsigargin caused a transient spike of Са²⁺-signal with the subsequent turn of the intracellular concentration of Ca ions to the stable increased level. The effects of С-1087 under the action of thapsigargin regarding the relaxation phase in the spontaneous myometrium contractions were removed after the preliminary blocking of nitric oxide synthases L-NAME (100 µM). Under the action of L-NAME, thiacalix[4]arene С-1087 (10 µM) caused complete inhibition of the relaxation process in the contraction of myometrium preparations, induced by high-potassium solution (80 mM). Conclusions. The primary reason for changes in the contractile activity and Ca²⁺-signal in uterine myocytes under the effect of thiacalix[4]arene С-1087 is its ability to inhibit Са²⁺-pump of the plasma membrane; further С-1087-induced changes in the smooth muscle tissues may be caused by the increased level of Са²⁺ concentration in myocytes. The obtained results demonstrate thiacalix[4]arene С-1087 is a promising compound for the elaboration of pharmacological preparations for modulating the contractile activity in smooth muscles, including myometrium.