Calixarene methylene bisphosphoniC aCids as promising effeCtors of bioChemiCal proCesses

this interdisciplinary study, performed with participation of research workers of Palladin Institute of Biochemistry and Institute of organic chemist ry of NaS of ukraine, is devoted to analysis of biochemical effects of some calixarene methylene bisphosphonic acids (cyclic phenol oligomers) on two well-known biological phenomenons – mg2+-dependent atP hydrolysis (myosin subfragment-1 of myometrium smooth muscle was used as an example) and fibrin polymerization. calix[4]arene С-97 (calix[4]arene methylene bisphosphonic acids) is a macrocyclic substance, which contains intramolecular highly ordered lipophilic cavity formed by four aromatic rings, one of which is functionalized at the upper rim with methylene bisphosphonic group. at concentration of 100 μm, this substance was shown to effectively inhibit atPase activity of pig myometrium myosin subfragment-1 (inhibition coefficient І0.5 = 83 ± 7 μm). at the same time, this calix[4]arene causes significant (vs. control) increase of myosin subfragment-1 hydrodynamic diameter, which may indicate formation of an intermolecular complex between calixarene and myosin head. computer simulation methods (docking and molecular dynamics with addition of grid technologies) enabled to elucidate the grounds of intermolecular interactions between calix[4]arene С-97 and myometrium myosin subfragment-1, that involve hydrophobic, electrostatic and π-π-stacking interactions, some of which are close to the atPase active centre. In view of the ability of calixarenes to penetrate into the cell and their low toxicity, the results obtained may be used as a basis for further development of a new generation of supramolecular effectors (starting from the above mentioned substances, in particular calix[4]arene С-97) for regulation of smooth muscle contractile activity at the level of atP dependent actin-myosin interaction. calix[4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization. the most potent inhibitor proved to be calix[4]arene tetrakis-methylene-bis-phosphonic acid (c-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52·10-6 m (Ic50 ). at this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1. For the case of desaB fibrin polymerization, the Ic50 was 1.26·10 -6 m at a molar ratio of c-192 to fibrin monomer of 4 : 1. Dipropoxycalix[4]-arene bis-methylene-bis-phosphonic acid (c-98) inhibited fibrin desaB polymerization with an Ic50 = 1.31·10 -4 m. We hypothesized that c-192 blocks fibrin formation by combining with polymerization site ‘a’ (aa17–19), which ordinarily initiates protofibril formation in a ‘knob-hole’ manner. this suggestion was confirmed by an hPLc assay, which showed a host–guest inclusion complex of c-192 with the synthetic peptide gly-Pro-arg-Pro, an analogue of site ‘a’. Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident. calixarene c-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13·10-5 and 1.10·10-5 m, respectively. these experiments demonstrate that c-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.


this interdisciplinary study, performed with participation of research workers of Palladin Institute of Biochemistry and Institute of organic chemist ry of NaS of ukraine, is devoted to analysis of biochemical effects of some calixarene methylene bisphosphonic acids (cyclic phenol oligomers) on two well-known biological phenomenons -mg
-dependent atP hydrolysis (myosin subfragment-1 of myometrium smooth muscle was used as an example) and fibrin polymerization.
calix [4]arene С-97 (calix [4]arene methylene bisphosphonic acids) is a macrocyclic substance, which contains intramolecular highly ordered lipophilic cavity formed by four aromatic rings, one of which is functionalized at the upper rim with methylene bisphosphonic group.at concentration of 100 μm, this substance was shown to effectively inhibit atPase activity of pig myometrium myosin subfragment-1 (inhibition coefficient І 0.5 = 83 ± 7 μm).at the same time, this calix [4]arene causes significant (vs.control) increase of myosin subfragment-1 hydrodynamic diameter, which may indicate formation of an intermolecular complex between calixarene and myosin head.computer simulation methods (docking and molecular dynamics with addition of grid technologies) enabled to elucidate the grounds of intermolecular interactions between calix [4]arene С-97 and myometrium myosin subfragment-1, that involve hydrophobic, electrostatic and π-π-stacking interactions, some of which are close to the atPase active centre.In view of the ability of calixarenes to penetrate into the cell and their low toxicity, the results obtained may be used as a basis for further development of a new generation of supramolecular effectors (starting from the above mentioned substances, in particular calix [4]arene С-97) for regulation of smooth muscle contractile activity at the level of atP dependent actin-myosin interaction.
calix [4]arenes bearing two or four methylenebisphosphonic acid groups at the macrocyclic upper rim have been studied with respect to their effects on fibrin polymerization.the most potent inhibitor proved to be calix [4]arene tetrakis-methylene-bis-phosphonic acid (c-192), in which case the maximum rate of fibrin polymerization in the fibrinogen + thrombin reaction decreased by 50% at concentrations of 0.52 •10 -6 m (Ic 50 ).at this concentration, the molar ratio of the compound to fibrinogen was 1.7 : 1.For the case of desaB fibrin polymerization, the Ic 50 was 1.26 •10 -6 m at a molar ratio of c-192 to fibrin monomer of 4 : 1. Dipropoxycalix [4]-arene bis-methylene-bis-phosphonic acid (c-98) inhibited fibrin desaB polymerization with an Ic 50 = 1.31 •10 -4 m.We hypothesized that c-192 blocks fibrin formation by combining with polymerization site 'a' (aa17- 19), which ordinarily initiates protofibril formation in a 'knob-hole' manner.this suggestion was confirmed by an hPLc assay, which showed a host-guest inclusion complex of c-192 with the synthetic peptide gly-Pro-arg-Pro, an analogue of site 'a'.Further confirmation that the inhibitor was acting at the initial step of the reaction was obtained by electron microscopy, with no evidence of protofibril formation being evident.calixarene c-192 also doubled both the prothrombin time and the activated partial thromboplastin time in normal human blood plasma at concentrations of 7.13•10 -5 and 1.10•10 -5 m, respectively.these experiments demonstrate that c-192 is a specific inhibitor of fibrin polymerization and blood coagulation and can be used for the design of a new class of antithrombotic agents.K e y w o r d s: methylene bisphosphonic acid, calixarenes, atPase activity, docking, fibrin polymerization, fibronogen, fibrin, inhibition.
O ne of most important methodological ap proaches, applied in molecular and cellu lar technologies in biochemistry, molecular biology and biophysics, that is focused on the study of structural and functional properties of biomole cules, supramolecular complexes and subcellular and membrane structures, is based on the use of natural and artificial effectors e (activators and inhibitors) for modifying activities of enzymatic, transport, regulator and signaling proteins.There fore, studying physicochemical and biochemical basis and mechanisms involving interactions of BiOChemisTry anD BiOTeChnOlOgy fOr mODern meDiCine small effector molecules with biomacromolecules and biomembranes has become currently one of the basic problems of so called "contact biology".it would be no exaggeration to say that "contact" events play a crucial role in realization of practi cally all fundamental biological phenomena that are studied by transdisciplinary sciences of chemi cobiological direction.
it is obvious that an artificial effector e pre tending to form a "molecular platform" able to effectively regulate the functional activity of an in tracellular protein must satisfy, as minimum, the following conditions: 1) to be nontoxic and non immunogenic for the organism; 2) to penetrate rather well through the plasmatic cellular mem brane; 3) to function as a reversible mechanism; 4) to show a specific (selective) effect exactly on a specific protein; 5) to be characterized by a rela tively high affinity towards a target protein (effec tor constant K е < 10 5 m).
in the context of the above mentioned, much attention of researches is currently devoted to phe nol cyclic oligomers, calixarenes, as perspective ar tificial effectors for different biochemical processes.
Calixarenes [1] are vase shaped macrocyclic compounds produced synthetically by precise cyc locondensation of psubstituted phenols and for maldehyde, which possess intramolecular lipophilic cavities formed by aromatic rings of the macrocy clic skeleton (fig.1).Calixarenes may be easily functionalized at the upper or lower rims of the macrocyclic skele ton.
Due to their ability to form a variety of in teractions -multisite hydrogen bonding, specific stacking, and generalized electrostatic interac tions -calixarene derivatives can be applied as valid recognition motifs when developing synthetic supramolecular systems.They have the unique ability to recognize and bind to the hostguest supramolecular complexes, cations, anions and neutral molecules of appropriate size and architec ture [2].These properties open wide perspectives for practical application of calixarenes in different branches of chemistry, physics and biology [3,4].
During the last 10 years, calixarene deriva tives, especially water soluble and amphiphilic ones, have been the subject of growing interest in the field of biology.highly diverse biomedical applications of these molecules now include an tibacterial, anticancer, antiviral, antithrombotic, membranotropic activities, selective enzyme block ing and mimicking, as well as protein complexa tion [5].Calixarenes, substituted at the upper or/ and lower macrocyclic rim with biologically ac tive functional groups, are considered as promising compounds for treating many diseases [6].
We were interested in modifying the calix arene platform by bioinspired methylene bispho sphonates.Being structural analogs of natural pyrophosphate, these compounds demonstrate versatile bioactivities [7,8].The bioactivities of bisphosphonates were investigated in detail by s.Komisarenko in the Palladin institute of Bio chemistry, nas of Ukraine.There were revealed and carefully examined antitumor and immu nomodulating activities of methylene bisphospho nic acid (mBPha) (fig.2) [9,10].
Phosphonates and bisphosphonates are struc tural analogs of inorganic pyrophosphate (PP i ) and phosphate, accordingly; they were chosen Fig. 1.Synthesis and structure of calix [4]arene s. v. KOmisarenKO, s.O. KOsTerin, e. v. lUgOvsKOy, v. i. KalChenKO for the study a priori in consideration of the fact that PP i takes part as a product or substrate in a large number of most important enzymatic reac tions.it also may be found in many metabolites.The presence of P-C bonds in phosphonates and Р-С-Р bonds in bisphosphonates, which usual ly cannot be hydrolyzed by enzymes, and strong complex forming abilities of phosphonates suggest that these substances may be used for perturbation of cell metabolic pathways; although, according to some literature data, bisphosphonates do not influ ence the activity of a large quantity of enzymes, in particular pyrophosphatase.Unlike this opinion, s.Komisarenko et al. showed that bisphosphonates were effective inhibitors of inorganic pyrophos phatase and some other enzymes in reactions in volving inorganic pyrophosphate [11][12][13][14][15][16].There was also shown antitumor effect of mBPha -the property, which needed further analysis and pre clinical investigations.in particular, there were investigated the mechanism of bisphosphonate ef fect and dependence of enzyme activities on the structure of bisphosphonates.mBPha was shown to possess immunomodulatory activity.Thus, when introduced to animals, mBPha caused inhibition of both biosynthesis of antibodies to Тdependent antigens and cell immune response reactions.it also inhibited biosynthesis of igm, ige and, es pecially, iggclass antibodies but did not influ ence the composition of lymphocyte subpopula tion.Other examined bisphosphonates (oxy and aminoderivatives of mBPha) were strong complex forming substances but did not possess immu nomodulatory effect.mBPha and its analogsoxyethylidene bisphosphonic acid, aminomBPha and phosphonoacetic acid -showed no toxicity and did not inhibit lymphocyte proliferation in culture when stimulated by mitogens.mBPha did not es sentially influence synthesis of specific interleukins by lymphocytes.it could not go through placental barrier and showed no embryo toxic effect.Use of 14 СmBPha made it possible to reveal mBPha tropism towards lymphoid cells and determine ki netic and thermodynamic parameters of mBPha transport into cells.The data obtained suggest that mBPha could penetrate into lymphocytes with a transporter through the concentration gradient by means of the facilitated diffusion mechanism.such transport may be competitively inhibi ted by РР і and oxyethylidene bisphosphonic acid (but no Р і ) and does not depend on the intensity of aTP syn thesis in the cell.There were calculated and deter mined types of structural complexes that contain bisphosphonates inside cells and analyzed com plexes with biologically important metals , which may interact with cell enzymatic systems [17].substances, which differed in a number of phosphoryl groups, type of bonds (Р-С, Р-О or Р-n) or charge and molecular size, were used to study the mechanism by which bisphospho nates and phosphonates may influence the activ ity of a number of key enzymes responsible for transformation of РР і or molecules including РР і .s. Komisarenko et al. came to the conclusion that tropism for lymphocytes is the basis of immu nomodulatory effects of phosphonates (first of all, mBPha).Transport and accumulation of mBPha in lymphocytes results in inhibition of inorganic pyrophpsphatase activity and increase of local РР і concentration; later, different ligand complexes of bisphosphonates with bivalent metal ions and with РР і are formed and some enzymes change their activity, in particular, Dnadependent rnapoly merase ii, enzymes of purine metabolism and so on [18].
On the basis of bisphosphonates, several pro totypes of medicinal substances were developed [19][20][21].Thus, a polyurethane composition was synthesized that might serve as an immobilized immunomodulator with local antiinflammatory and immunosuppressive effects.it was shown that disodium salt of methylene bisphosphonic acid had antitumor properties, which resulted in develop ment of a new antitumor preparation meBifOn that was tested clinically and is currently produced in Kiev by the Public company farmaK vaT.With the use of immunoglobulins (antibodies) and phosphoorganic complexons (aminobisphos phonates), there were synthesized immunovector molecules with antibody activities that conserved complex forming properties.such structures were proposed, in particular, for radioimmunolocali sation of antigens.it should be noted that the abovementioned investigations under the direc tion of s.Komisarenko were reali zed together with the following research workers of the Pal ladin institute of Biochemistry, nas of Ukraine: n. m. gula, g. g. gaivoronska, m. g.Juravsky, n.P. Karlova, І. М. Kolesnikova, О. P. Penezina, g.М. fomovska and others.Bisphosphonate and phosphonate preparations were synthesized in the institute of Organic Chemistry, nas of Ukraine by a. m.Borisevich with the participation of pro fessors P. s. Pelkis and m.O. losinsky and in the engelgardt institute of molecular Biology, as of the Ussr by n.B. Tarusova with the participation of P. m. Khomutov, the corresponding member of as of the Ussr.synthesis of the polyurethane composition was realized together with a group of scien tists form the institute of highmolecular Compounds Chemistry, nas of Ukraine under the guidance of D.sc.g.O. Pkhakadze.

BiOChemisTry anD BiOTeChnOlOgy fOr mODern meDiCine
This article describes the results of joint pro ject of the Palladin institute of Biochemistry, nas of Ukraine and the institute of Organic Chemis try, nas of Ukraine focused on the synthesis, eluci dation of biological activity and biopharmaceuti cal application of the calixarenes С97 and C192 functionalized with one or four biophoric methy lene bisphosphonic acid groups in the upper rim (fig.2).

І. synthesis and molecular structure of calixarene methylene bisphosphonic acids
The main method for synthesis of metylene bisphosphonic acids consists in using arbuzov reac tion of geminal dihalogenoalkanes with trial kyl phosphites followed by hydrolysis of the esters formed [1].however, at synthesis of the calixarene methylene bisphosphonic acids, this method is lim ited by availability of appropriate dihalogenoalkyl calixarenes.
Calixarene methylene bisphosphonic acids C-97 [23] is synthesized from monoformyl calixa rene 1 (scheme 1).On the first stage, diethylphos phite sodium salt is added to the C=O bond of monoformyl calixa rene 1, according to the clas sical scheme of the abramov reaction, with for mation of αhydroxyphosphonate 2.Then, in the presence of sodium, hydroxyphosphonate 2 diethyl phosphite cleaves water molecule to form a phos phorylated quinonmethyde as intermediate (omit ted in the scheme).The quinonmethyde further reacts with diethyl phosphite to form calixa rene methylene bisphosphonate 3. acid C-97 is obtained by consecutive treatment of the ester 3 with tri methylbromosilane and methanol.
Calixarene tetrakismethylenebisphosphonic acid C-192 is synthesized by the reaction of tetra formylcalixarene 4 with sodium salt of diisopropyl phosphite with formation of tetrakisbisphospho nate 5, which after subsequent dealkylation due to treatment with trimetylbromosilane and methanol gives C-192 (scheme 2) [2].
according to the 1 h nmr spectra and mo lecular modeling data, the acids C-97 and C-192 adopt the cone conformations.The both confor mations are stabilized by intramolecular hydrogen bonds Oh••••O at the lower rim of macrocycle.The conformation of C192 is additionally stabilized by the intramolecular hydrogen bonds between dis tal dihydroxyphosphoryl groups of the upper rim (fig.3).The upper rim bonding makes the cone C-192 more regular compared with C-97 one.
Below, as examples of biological activity of calixarenes, we describe our own transdiscipli nary investigations, which illustrate the use of calixa rene methylene bisphosphonic acids (calix [4] arenes С97 and С192) as inhibitors of two sepa rate biochemical processes: the reaction of mg 2+ dependent aTP hydrolysis, catalyzed by uterine smooth muscle myosin sudfragment1, and the process of fibrin polymerization and formation of fibrin network of thrombus.

ІІ. structure-functional basis of intermolecular interactions of calixarene methylene bisphosphonic acid C-97 with myometrium myosin subfragment-1
it is of importance to understand molecular and membrane mechanisms that control the con tractile function of smooth muscle, in particular myometrium, in normal state and at pathological states with different disorders; therefore, inves tigation of these questions is rather actual.The main function of uterus in the female organism is baby carrying and delive ry.Disturbance of uterine smooth muscle motility in females often leads to various pathologies -weak labour of childbirth, premature childbirth, miscarriage etc. in this con nection, there is need for development of effective methods of correcting disturbed contractile activi ty of myometrium and, particularly, for investigations aimed at developing and examining new pharma cological highly efficient substances capable of normalizing uterine motility.
it should be noted that at present research workers of the Department of muscle Biochemis try (Palladin institute of Biochemistry, nas 1 2

C-97
Scheme 1 of Ukraine) and the Department of Phospho rane Chemistry (institute of Organic Chemistry, nas of Ukraine) have accumulated a number of interesting experimental results concerning the influence of calixarenes on transport and enzy matic activi ty of membraneassociated energy dependent cationtransporting systems of plas matic membrane, sarcoplasmic reticulum and mitochondria of uterine myocytes.it was shown that some calix [4]arenes may serve as rather se lective and highaffinity inhibitors of mg 2+ , АТР dependent sodium pump (calix [4]arenes С97, С99 and С107; І 0.5 < 100 nМ) and calcium pump (calix [4]arene С90; І 0.5 = 2030 μМ) in the plasma membrane with Ca 2+ accumulation in mitochondria, which is impossible in the presence of protonofore СССР (calix [4]arene С91) (here codes are given for calixarene compounds).such membraneassociated energydependent transport systems are directly involved in the control of in tracellular cation homeostasis (first of all, Ca 2+ homeostasis).nevertheless, in case of muscles, in particular smooth ones, of great importance for maintaining processes of electro and pharmaco mechanical coupling is functioning of both cation BiOChemisTry anD BiOTeChnOlOgy fOr mODern meDiCine transporting aTPdependent systems and aTP hydrolase systems of contractile proteins.Different pathological states of the female reproductive sys tem (including the ones caused by hormonal dis balance) may lead to disfunction of the uterine contractile complex, which is revealed in abnormal superprecipitation (in vitro) and changes of acto myosin АТРase activi ty. at some pathologies (e.g., hypertrophy) with disordered contractile function of smooth muscles, including myometrium, some changes may take place in the expression of myosin isoforms and kinetics of actinmyosin interaction.

5
Scheme 2 accordingly, screening of reversible affine effectors of actinmyo sin interaction (inhibitors, activators) is important for developing new pharmacological preparations capable of normalizing uterine con tractile function at pathological state of myome trium.

C-192
myosin АТРase transforms chemical energy, stored in АТР macroergic bonds, into mechanical work that is accompanied by directed movement; that is why this enzyme is also called a molecular motor [25].myosin subfragment1 (head, s1) is n terminal part of myosin heavy chain and consists of two domains: nterminal globular motor (cata lytic) domain, which contains an active center of aTPase, and a site of actin binding, and regulatory domain or leverarm that is responsible for move ment of myosin relative to actin.
АТРbinding site contains the sequence gly glUserglyalaglylysThr similar to sequences in active centers of other АТРases [27].АТРase active site is formed in part by the P loop, switch 1 and switch 2 polypeptides that are linked to the 7stranded βsheet; conformations of these structures are sensitive to the position and coordination of γphosphate.АТРase center has been identified as a pocket, from which a gap is stretched to the actin binding site.When АТР binds to the myosin s1, the pocket becomes closed, while the gap expands and disturbs the binding of s1 with actin; as a result, myosin subfragment1 dissociates from actin.When АТР is hydrolyzed by myosin АТРase to aDP and P i , actin can again as sociate with s1, which facilitates releasing of АТР hydroly sis products from the active center.When aTP is hydrolyzed, the presence of associated actin is important for releasing of inorganic phosphate from the nucleotidebinding pocket, since in its absence the salt bridge between arg 238 (switch 1) and glu 459 (switch 2) inhibits the phosphate release [14,28].switch 2 αhelix transmits linear force (arising at aTP hydrolysis) from the active site to the converter domain; the last converts linear force into a torque that rotates the leverarm and enables the shift of myosin relative to actin.
according to available literature, subfrag ment1 is a selfsufficient functional part of myo sin molecule; it preserves the properties of native myosin even in isolated state -that is, it displays aTPase activity and abili ty to interact with actin [29,30].
earlier we showed the inhibiting effect of calix [4]arene С97 on aTPase activity of myo metrium actomyosin (inhibition coefficient І 0.5 = 84 ± 2 μМ) [31]. in this connection, the question arises concerning the mechanisms of ca lix [4]arene С97 effect on aTPhydrolase activity of (exactly) myosin subfragment1.subfragment1 is a convenient model for studying calixarene ef fects due to its high solubility in aqueous solu tions with low ionic strength (unlike myosin) and because its specific aTPase activity is close to the myosin activity.
Below we provide some data obtained in the study of calix [4]arene С97 effect on aTPase ac tivity of myometrium smooth muscle myosin sub fragment1 and consider possible mechanisms of complex formation between this calix [4]arene and myosin subfragment1 (with the use of computer simulation techniques -doking analysis and mo lecular dynamics).
at first, we will give some information con cerning methodological approaches used in the study.
Obtaining actomyosin and myometrium myosin subfragment-1.These studies were performed by r.D. labintseva and О. А. Bevza (Palladin in stitute of Biochemistry, nas of Ukraine).smooth muscle actomyosin was purified from muscle tissue of pig uterus according to Barany [32] and We ber [33] with some modifications.myometrium smooth muscle myosin subfragment1 was obtained through actomyosin splitting by αchymotrypsin according to the method of Weeds and Taylor [34] with some modifications.separation and purifica tion of subfragment1 was performed by ion ex change chromatography using column packed with Deaesepharose Cl6B and chromatographic system Biorad (Usa).Protein concentration was determined by Bradford method [35]; also, concen trational dependence of light absorption at 280 nm was used with the curve standardized by serum albumin.Purity of smooth muscle myosin subfrag ment1 was controlled by electrophoresis in poly acrylamide gel with sodium dodecyl sulphate ac cording to laemmli electrophoretic technique [36]. in accord with electrophoretic data, myosin head had Мr ≈ 100 kDa, which corresponded to litera ture results [37].subfragment1 was identified as a fraction having a rather high aTPase activi ty -68 ± 9 μmol Р і /min per 1mg of protein (М ± m, n = 8).
Study of calix [4]arene С-97 effect on ATPase activity of myometrium myosin subfragment-1.Kinetic measurements.These experiments were car ried out by r.D. labintseva, О. А. Bevza and s.O. Kosterin (Palladin institute of Biochemistry, nas of Ukraine).To study calixarene С97 effect on aTPase activity of isolated myosin subfrag ment1, aliquots of calixarene solution in 50 mm trisНСl buffer (рН 7.2) with the initial concentra tion 10 mm were added to the standard incubation medium (see above).final calixarene concentra tion in the sample was 10-100 μm.The value of aTP hydrolase activity, determined at the absence of calixarene in the incubation medium, was taken for 100% ("zero point").at studying concentration dependence of calix [4]arene С97 effect on the en zymatic activity of myosin subfragment1 aTPase, the values of the apparent inhibition coefficient І 0.5 and hill coefficient n h were calculated with the use of linearized hill graphs [39] according to the empirical equation where V is specific enzymatic activity, V 0 is specific enzymatic activity at the absence of the inhibitor in the incubation medium and І is the inhibitor concentration in the incubation medium.Typical value of the correlation coefficient r was 0.970.99.
Photon-correlation spectroscopy experiments.This research was carried out by O. yu.Chunikhin , r. D. labintseva and О. А. Bevza (Palladin insti tute of Biochemistry, nas of Ukraine).Photon correlation spectroscopy (PCs) experiments were performed on the device Zetasizer3 malvern in strument (great Britain) equipped with the multi computing correlator type 7032 ce.samples were irradiated by the heliumneon laser lgn111 (λ = 633 nm, power 25 mw) and laser radiation scattered at the angle 90° was registered.Computer program PCssize mode v.1.61[40] was used to analyze the experimentally measured autocorrela tion function and calculate the function of dis tribution by hydrodynamic diameter (hDD) for microparticles in the volume of the measuring cell.PCs was used to determine average hDD and the function of distribution by dimensions for particles of myometrium myosin subfragment1 as well as for particles that are formed at interaction of myo sin subfragment1 with calix [4]arene С97 (20, 60 та 100 μm).
Computer simulation of the ligand-receptor interaction in the system "calix [4]arene С-97 -myosin subfragment-1".These experiments were car ried out by О. v. Bevza, r.D. labintseva and О. А. Bevza (Palladin institute of Biochemistry, nas of Ukraine).Computer simulation of the ligandreceptor interaction for the system "ca lix [4]arene С97 -myosin subfragment1" was performed using the softwere mvD 4.1.0.This program allowed rea lizing several algorithms of searching for the optimal ligand position in the active site of the protein (a variant of the evolu tionary algorithm was used) [41].actually, dock ing of the ligand (calix [4]arene С97) into the ligandbinding site (lBs) of myosin subfrag ment1 was realized in conditions of entirely ioni zed methylene bisphosphonic fragment.standard АМBer force field parameters were used for the metal ion (mg 2+ ) [42].When mode ling interac tions between calix [4]arene С97 and myome trium myosin subfragment1, we reali zed so called "semiflexible" docking (only ligand motili ty was taken into account) with selection of a series of complexes having the lowest total energy of the ligand binding .Calculation of the optimal geo metry of the complexes formed and determina tion of energetically most advantageous spatial arrangement of calixarene in the area of myosin molecule were performed taking into account van der Waals, electrostatic and hydrophobic interac tions, hydrogen bonds as well as contribution of desolvation energy. in the investigation, there has been used three dimensional structure of myosin subfragment1 with the identifier 1B7T in rsCB Protein Data Bank [43].molecular dynamics of calix [4]arene С97 interaction with lBs of myosin subfragment1 was investigated with the use of the gromos96 force field [44].The protein model has been "placed" into a virtual cell in the form of cut octahedron in such a manner that spacing from the protein to the cell walls did not exceed 1.5 nm.The cell was "packed" with water molecule models (solvent) -sPC (single Point Charge).The water molecules were partially substituted for na and Cl ions to neutralize the system charge and simulate the physiological ionic strength (0.1 m)."Counter balancing" of the solvent molecules was carried out during 0.5 ns with the protein atoms "attached" to their initial coordinates.The temperature and the pressure were 298 К and 1 atm, accordingly (Beren dsen's method was used) [45].following the solvent mole cule counterbalan cing, additional s.v. KOmisarenKO, s.O. KOsTerin, e. v. lUgOvsKOy, v. i. KalChenKO minimization of the system energy has been per formed.actually, mD simulation method was rea lized using the same parameters as at the solvent "counterbalancing" except for limiting the protein atom motility.The coordinates were recorded into the initial trajectory file every 10 picoseconds.motility of the calix [4]arene С97 molecule in the course of molecular dynamics was visualized with the use of the program UCsf Chimera 1.5.3 [46].

ІІ.1. investigation by photon correlation spectroscopy of calix[4]arene С-97 effect on the effective hydrodynamic diameter (hdd) of isolated myosin subfragment-1
PCs method gives distribution of different molecules and supramolecular structures in solu tion by their sizes.To determine hDD by PCs method , stokeseinstein formula is used that is valid only for spherical particles [47,48].Therefore, molecular diameters determined by this method will be close to the real ones only for globular pro teins.When a protein is far from spherical form, calculated hDD is a rather conventional value.
myosin head, main functional unit of myo sin [49], is rounded or pearshaped [16]; therefore, the preparation of isolated myometrium myosin subfragment1 fits rather well for studying by PCs method the effect of calix [4]arene on contractile proteins.There were determined average subfrag ment1 hDD and the function of particle size distribution.isolated myosin subfragment1 had narrow spectrum of particles in the interval from approximately 10 to 50 nm (fig.4).The size of most frequently registered particles (≈ 50%) was close to 20 nm.There were also present in small quantity particles with diameter close to 70 nm and particles less then 20 nm. it was shown that the average (most probable) hDD of myosin sub fragment1 was 22 ± 3 nm (М ± m; n = 15).This value fits to the literature data [50,51].
Thus, calix [4]arene С97 at a concentration of 100 μm caused a significant (vs.control) increase of myosin subfragment1 hDD, which indicated the formation of a complex between calix [4]arene and myosin head.
Of interest is the fact that using photon cor relation spectroscopy with other supramolecular effector (activator) of myosin subfragment1 aTP hydrolase (calix [4]arene С107), we could ob serve time dependent changes of myosin subfrag
Using linearized graphs at analysis of con centration dependence of subfragment1 aTPase activi ty on calix [4]arene С97, we also calculated hill coefficient n h , which amounts to 1.3 ± 0.5 (fig.7).Therefore, this value of hill coefficient most probably suggests that myosin head may bind only one molecule of calix [4]arene С97.
Therefore, calix [4]arene С97 effect on aTPase of contractile complex may follow from its ability to bind with myosin subfragment1; con sequently, myosin head is supposed to be one of targets by means of which calix [4]arene С97 may influence the contractile complex (based on the above value of hill coefficient n h , the stoichiomet ry of binding is 1 : 1).

ІІ.3. Using the computer simulation method for examining the structural basis of molecular interactions between calix[4]arene С-97 and myometrium myosin subfragment-1
To understand the molecular mechanism of calix [4]arene С97 inhibi ting effect on myosin sub fragment1 АТРase, it needs to have information on threedimensional structure of the enzyme in complexes with substrate or/and inhibitor.With this purpose, we carried out the computer simula tion for the interaction of calix [4]arene С97 (as a separate ligand or coupled with aTP) with myo metrium myosin subfragment1 in the presence of

Fig. 6. catalytic titration of myosin subfragment-1 atPase activity with calix[4]arene С-97 (М ± m; n=5)
Miosin subfragment-1 ATPase activity, %  an analysis of docking of calix [4]arene С97 as a separate ligand into the ligand binding site of myosin subfragment1 suggests that residues asp 320 , asn 321 , leu 676 and gln 678 may be involved in fixa tion of calix [4]arene phosphonate groups (the data are not shown in the figure).Calixa rene fragments are oriented in the space formed by residues ile 322 , asn 238 and lys 677 on one side, and by Tyr 12 6, arg 127 and arg 128 on the other.Besides, positively charged nitrogen atom of lys 677 residue is located next to oxygen atoms of methylene bisphosphonate frag ment (distance between nitrogen atom of lys 677 residue and ionized oxygen atoms of the phos phonate group is 0.31 nm).There were indentified bonds taking part in calix [4]arene binding to a site close to the active centre of myosin s1; they are: hydrogen bonds, π-πstacking interactions between aromatic fragments of calix [4]arene bowl and Tyr 126 of benzene ring in myosin head as well as elec trostatic interactions with involvement of arg 127 , arg 128 , asp 320 and lys 677 .

Fig. 7. averaged curve linearized in hill coordinates for catalytic titration of myosin subfragment-1 atPase activity with calix[4]arene С-97
simultaneous docking of the paired ligand "calix [4]arene С97 + aTP" into the ligand binding centre of myosin subfragment1 in the presen ce of mg 2+ revealed the possible formation of a complex between calix[4]arene С97 and aTP, which binds with involvement of amino acid resi dues to a site of myosin head close to the active

A B
centre; this is somewhat different from results ob tained with docking of calix [4]arene С97 alone.in addition, spatial orientation of calix [4]arene С97 in the ligand binding centre of myosin head differs: methyle ne bisphosphonate fragment of ca lix [4]arene at its interaction with АТР mole cule be comes oriented towards the active centre.Binding of the complex "calix [4]arene С97 + АТР" to a site close to the myosin s1 active centre is realized with participation of hydrogen bonds, π-πstacking interactions between aromatic rings of calix [4] arene bowl and benzene rings of Tyr 126 and Tyr 132 in myosin head as well as electrostatic interac tions with participation of arg 127 .residues asn 238 and asn 321 , which are rather close to oxygen atoms of phosphonate groups, may form hydrogen bonds at interaction with ligand phosphonate groups.it is also obvious that hydrophobic platform of func tionalized calix [4]arene С97 may contribute to the binding due to the interaction with hydrophobic sites in the centre of substrate binding.Table 1 shows amino acid residues of the ac tive centre of myometrium myosin s1, which are involved in interactions with calix [4]arene С97 and the complex "calix [4]arene С97 + АТР".ac cording to literature, residues Tyr 126 , arg 127 , leu 676 and lys 677 , enabled in fixation of calix [4]arene С97 phosphonate groups, are incorporated into the 7stranded βsheet structure of myosin sub fragment1, which is joined with polypeptide sites switch 1 and switch 2. These polypeptides partici pate directly in the АТРase active centre forma tion [14].Therefore, it is obvious that interac tion of calix [4]arene С97 with myosin subfragment1 in the site located near the АТРase centre of myo sin will influence this centre conformation and, accordin gly, the process of АТР hydrolysis cata lyzed by myosin subfragment1.
Therefore, the results obtained by the method of molecular docking indicate that: 1) calix [4] arene С97 may bind with myosin subfragment1 to the site close to the active centre, which leads to conformational changes in the last; 2) this calix [4] arene may form a complex with aTP prior to the interaction with the protein, which complicate ac cess of nucleoside triphosphate to the aTP binding site. it may well be that both these variants of ca lix [4]arene С97 interaction with subfragment1 may cause their inhibiting effect on aTPase activi ty of myometrium myosin subfragment1.never theless, it should be noted that the last variant can not explain considerable differences (almost three orders of magnitude) between affini ty towards ca lix [4]arene С97 for na + ,K + aTPase and myosin subfragment1 aTPase (values of inhibition coef ficient І 0.5 is equal 100 nm [29] and 8090 μm, respectively).
note that the docking method fails to take into account motility of myosin s1 and ligands, which may occur in real conditions.Therefore, to verify results obtained by the docking method, we investigated interaction of calix [4]arene С97 with the ligand binding centre of myosin subfragment1 by the method of molecular dynamics taking into consideration the motility of both the receptor (myosin subfragment1) and the ligand.
The dynamic analysis of the calix [4]arene С97 molecule position in the ligand binding site of myosin subfragment1 allowed to determine time dependent deflections of the active site atoms from their initial state; this deflections were characteri zed by changes of distances (in nm) between ca lix [4]arene centre of mass and the nearest amino acid residue (asn 321 ).accordingly, there was plot ted (at intervals of 4 ns) time dependence of С97 position in the ligand binding site of myosin sub fragment1.analysis of the deflections showed that initial calix [4]arene location was changing noticea bly starting approximately from 1 ns: calix [4]arene conformational mobility increased and afterwards again decreased, which correlated with the change of distance between calix [4]arene and asn 321 cent ers of mass.Therefore, the interaction of calix [4] arene С97 with myosin head is accompanied by time variation of calix [4]arene location.in the pro cess of molecular dynamics, the total energy of the system "calix [4]arene С97 -myosin subfrag ment1" becomes somewhat lower; therefore, ca lix [4]arene С97 apparently takes up a more energy advantageous position in the ligand binding site of myosin subfragment1.it was shown that calix [4] arene С97 molecule shifts relative to amino acid residue asn 320 by about 1.81 Å and that arrange ment of calix [4]arene С97 in the final binding location is stable energetically.
The analysis of hydrogen bonds during 4 ns interval between calix [4]arene С97 and amino acid residues of the subfragment1 ligand binding site (fig.9) shows that the number of hydrogen bonds enabled in the interaction between ca lix [4]arene С97 and myosin head remains on the average unchanged.Consequently, calix [4] arene location changes in the course of molecular dynamics should be most probably caused by opti mization of hydrophobic and electrostatic interac tions.
The method of 4 ns molecular dynamics was also used to identify amino acid residues taking part in calix [4]arene binding to the ligand binding centre of myometrium myosin subfragment1 (fig.10).They include Tyr 126 , arg 127 , leu 676 and lys 677 .These residues take part in fixation of ca lix [4]arene phosphonate groups that are located, according to literature [14], next to the aTPase active centre.Overall, these results correlate well with the data obtained by the docking method (see above).
The computer simulation results broadened out our notions about the structural basis of in termolecular interactions between calix [4]arene t a b l e 1. amino acid residues of the substrate binding domain of myosin subfragment-1, which take part in binding of calix [4]arene С-97 in the case of its docking as a separate ligand and when docking is performed for a couple of ligands "С-97 + АТР" in the presence of mg 2+  С97 and myometrium myosin subfragment1.in particu lar, there was elucidated the role of hydro phobic, electrostatic and π-πstacking interactions between calix [4]arene and amino acid residues of myosin subfragment1, some of which are close to aTPase active centre.Thus, calix [4]arene С97 inhibits effec tively aTPase activity of myosin subfragment1

. Dotted lines show Н-bonds between amino acid residues of subfragment-1 and the inhibitor. Interactions of С-97 with myosin head include also π-π stacking interactions and electrostatic bonds
BiOChemisTry anD BiOTeChnOlOgy fOr mODern meDiCine (І 0.5 = 83 ± 7 μm).This substance causes sta tistically significant (vs.control) increase of the myosin head hydrodynamic diameter, which is in dicative of a complex formation between calixarene and myosin head.The use of the computer simu lation method resulted in identification of amino acid residues, hydrophobic and electrostatic forces that take part in interactions between calix [4]arene and myometrium myosin subfragment1.The re sults obtained suggest that myosin head is one of targets for calixarene effect on contractile complex of smooth muscle.

ІІІ. Calix[4]arene methylene bisphosphonic acids as inhibitors of fibrin polymerization
The present study aimed to investigate the anticoagulant properties of phosphorus contained calix[n]arenes [24,53] in the last two steps of blood coagulation: thrombin+fibrinogen reaction and fibrin monomer polymerization, which lead to formation of fibrin network of thrombus.fibrino gen is a glycoprotein (mW ≈ 344 kDa) composed of two monomeric units connected by disulfide bonds.each monomer consists of three noniden tical polypeptide chains aα, Bβ and γ, also con nected by disulfide bridges [54].The nh 2 termi nal ends of all six polypeptide chains are situated in the central region of fibrinogen known as the eregion.Two peripheral regions of the molecule historically are called the Dregion.
When blood coagulation system is activated, thrombin is formed from prothrombin and attacks fibrinogen, splitting off two fibrinopeptides a (aα1-16), and thereby exposing fibrin polymeriza tion sites 'a' (aα17-19) [55].removal of fibrino peptides a leads to a form of fibrinogen deemed 'desa', which polymerizes spontaneously to form twomolecule thick protofibrils.removal of fi brinopeptides B ('desaB') encourages lateral as sociations that lead to fibrils [56,57].The fibrils continue to associate, branching and forming a 3D network: the framework of the blood throm bus [58]. it is widely accepted that the initial step of fibrin polymerization (protofibril formation) is carried out by the intermolecular pairing of 'a' and 'a' polymerization sites of fibrin monomers.site 'a' is a cavity ('hole') that includes amino acid resi dues γgln 329 , γasp 330 , γhis 340 and γasp 364 , and is located in the γCdomain of the fibrinogen/fibrin molecule [59].
in particular, we have focused on compounds in which the molecular scaffold is decorated with methylenebisphosphonic acid groups.One of these, calix [4]arenetetrakismethylenebisphos phonic acid (C192), has four such substituent groups (fig.2).
The experimental data presented in the fol lowing part of the article were carried out by the research workers of the Department of Protein structure and function (Palladin institute of Bio chemistry, nas of Ukraine) and the Department of Phosphorane Chemistry (institute of Organic Chemistry, nas of Ukraine).
Preparation of fibrinogen, fibrin desAB.These studies were performed by g.K. gogolinska and T. a. Pozniak (Koshel) (Palladin institute of Bio chemistry, nas of Ukraine).human fibrinogen was prepared by sodium sulphate precipitation from human plasma [60] DesaB fibrin monomer was prepared as described previously [61].
Turbidity analysis of fibrin polymerization.These experiments were carried out by T. a. Pozniak (Koshel), P. g. gritsenko and e. v. lugovskoy (Palladin institute of Biochemis try, nas of Ukraine).The effects of compounds on fibrin polymerization were studied spectro photometrically at 350 nm as described previous ly [62].The curve of increasing turbidity during fibrin clotting shows the parameters: τ, lag time, which corresponds to the time of protofibril forma tion; V max , maximum rate of fibrin polyme rization, which was defined by graphic calculation of the angle of the tangent to the turbidity increase curve at the point of maximum steepness; and Δh, fi nal turbidity of fibrin clots.The polymerization of desaB fibrin was studied at its final concentration 0.1 mg•ml 1 in the polymerization medium con taining 0.05 m ammonium acetate (ph 7.4) with 0.1 m naCl and 1•10 4 m CaCl 2 .The polymeriza tion of fibrin formed in the fibrinogen + throm bin reaction was investigated at a final concentra tion of fibrinogen of 0.1 mg•ml 1 and thrombin of 0.4 nih units•ml 1 in the same polyme rization medium.
Electron microscopy.These experiments were done by T. a. Pozniak (Koshel), P. g. gritsenko and v. i. Chernishov (Palladin institute of Bio chemistry, nas of Ukraine).The samples of po lymerizing fibrin produced in the thrombin-fi brinogen reaction in the absence or presence of calixarene C192 (10 5 m) were taken out of the reaction medium at various times, placed on a car boncoated grid for 2 min and then stained with 1% (w/v) uranyl acetate for 1 min.Transmission electron microscopy was performed with an h600 electron microscope (hitachi, Tokyo, Japan) oper ated at 75 kv.electron micrographs were obtained at ×50 000 magnification.
The determination of association constants by the RP-HPLC method.These experiments were done by s.O. Cherenok, O. i. Kalchenko and v. i Kalchenko (institute of Organic Chemis s. v. KOmisarenKO, s.O. KOsTerin, e. v. lUgOvsKOy, v. i. KalChenKO try, nas of Ukraine).The hPlC consisted of a highpressure pump (type hPP 4001) (laborator ni Pristroje, Prague, Czech republic) connected to a rheodyne sample 7120 injector (rheodyne, Berkeley, Ca, Usa) and an ultravioletvisible de tector (type lCD 2563) (laboratorni Pristroje).The column (150•3.3mm inner diameter) was packed with separon sgX Cn (lachema, Prague, Czech republic).The mobile phase was a mix ture of methanol-water in the ratio 50 : 50 (v/v), with the calixareneC192additive at a concentra tion in the range 1.48•10 4 to 5•10 4 m.The flow rate was 0.6 ml•min 1 .The concentration of the guests/analytes in solution for analysis was 10 5 m.The solvent was identical to the mobile phase composition.The amount of the sample injected was 0.5l l. each of the samples was analyzed five times.all chromatograms were obtained at 20 °C.association constants of the calixarene complexes with amino acids gly, Pro, arg and tetrapeptyde glyProargPro (280-3395 m 1 ) were calculated from the dependence of the 1/k′ value versus the calixa rene concentration [Ca] in the mobile phase by eqn (1) (Table 2): .Where k 0 ′ and k′ are the capacity factors determined in the absence and presence of the calixa rene in the mobile phase and [Ca] is the calixa rene concentration in the mobile phase.
PT and APTT assays.These experiments were carried out by T. a. Pozniak (Koshel) and P. g. gritsenko (Palladin institute of Biochemis try, nas of Ukraine).The effects of calixarene C192 on the coa gulation of human blood plasma were studied using a coagulometer (CT 2410 'so lar', minsk, Belarus).reagents from renam (mos cow, russia) were used to calculate PT and aPTT.PT and aPTT ratios were calculated using the for mula: t c /t o , where t o is the time of clot formation in blood plasma without calixarene C192 and t c is the time of clot formation in blood plasma with calixarene C192.

Calix[4]arene methylenebisphosphonic acids as inhibitors of fibrin polymerization
C192 was studied with respect to its ef fects on fibrin polymerization in two kinds of as say. in the first assay, the formation of fibrin fol lowed directly after the addition of thrombin.in the second assay, previously prepared fibrin was dispersed and then allowed to repolymerize un der appropriate conditions. in both cases, fibrin formation was gauged by turbidity measurements.Turbidity analysis showed that the compound de creased the maximum rate of fibrin polymerization in the thrombin-fibrinogen reaction by 50% at a molar ratio of compound to starting fibrinogen of 1.7 : 1 (fig.11).The final turbidity of clots was decreased by 50% at a molar ratio of 4.3 : 1 (com pound : starting fibrinogen) (fig.11, c).The lag time was also increased strongly in the presence of C192, indicating either a decrease of the rate of protofibril formation or, conceivably, an increase of protofibril critical length (fig.11, B). similar results were obtained when calixarene C192 in hibited the reassociation of dispersed desaB fibrin (fig.12, a-c); in this case, iC 50 = 1.26•10 6 m. such a strong and specific inhibition by ca lixarene C192 of both the thrombin-fibrinogen reac tion and the reassociation of fibrin desaB indicates that calixarene retards clotting not as a result of the inhibition of thrombin, but entirely because of the blocking of fibrin polymerization sites.
We also performed electron microscopy studies to determine the stage of fibrin polymeri zation that was inhibited by C192.Transmission electron microscopy of the thrombin + fibrinogen media showed that fibrin remained in monomer state in the presence of calixarene C192 at its fi nal concentration of 10 5 m, whereas, at the same time, mature fibrils were formed in the absence of C192 (fig.13).
The results of turbidity analysis and electron microscopy indicate that the inhibition by C192 occurs at the first stage of fibrin polymerization, presumably by blocking one of the sites of protofi bril formation.
We also investigated the inhibitory effects of two structural fragments of C192: parahydroxy phenylmethylenebisphosphonic acid (1) and methylenebisphosphonic acid (2) (fig.14  it is of interest that the inhibitory activity of C98, which has the two methylenebisphos phonic acid substituents, is much less (Table 2) (iC 50 = 1.31•10 4 m), indicating that the calixarene scaffold and the number of phosphoryl groups in the molecule play a crucial role in the inhibitory effect.
furthermore, calixarene C192 doubles both the prothrombin time (PT) and aPTT in nor mal human blood plasma at concentrations of 7.13⋅10 5 m and 1.10⋅10 5 m, respectively (fig.15).The molar ratios of C192 to plasma fibrinogen were approximately 21 : 1 and 3.3 : 1 for the PT and aPTT assays, respectively.The delays in clot ting times in the blood plasma coagulation experi ments are what would be expected by the inhibi tion of the fibrin polymerization from fibrinogen after the activation of the blood coagulation sys tem.electron microscopy confirmed that C192 inhibits the first stage of fibrin polymerization (i.e. the formation of protofibrils).
Because this stage is fulfilled through the in termolecular binding of the complementary sites 'a'-'a', it appeared to be certain that this inhibi tion is a result of the blocking of site 'a' (aa1719, glyProarg) by the calixarene in a 'knobhole' manner.To confirm that this was the case, we em ployed hPlC to study complex formation between C192 and the synthetic peptide glyProargPro, a synthetic analogue of the a knob; the free amino acids gly, Pro and arg were used as controls.as sociation constants of calixarene C192 complexes with amino acids gly, Pro, arg and tetrapeptide glyProargPro in methanol-water mobile phase (50 : 50, v/v) were determined as previously de scribed [17,18].The addition of calixarene C192 to the mobile phase decreased the capacity factor, k′ , of the guest molecules (Table 3).This confirms s. v. KOmisarenKO, s.O. KOsTerin, e. v. lUgOvsKOy, v. i. KalChenKO the formation of inclusion host-guest complexes.
There is linear dependence of 1/k′ versus the con centration of C192 in the mobile phase (fig.16); the correlation coefficient is 0.980.99,indicating a 1 : 1 ratio of calixarene to glyProargPro in the complex.in accordance with the molecular model ling data (fig.17   ring.hydrophobic forces can additionally stabilize the complex in a water solution.Thus, we have shown for the first time that C192 is a powerful and specific inhibitor of the final step of blood coagulation, fibrin polyme rization, and can be used as the basis for the design of new class of antithrombotic agents.We have found that the mechanism of C192 inhibition in volves its effect on the first step of fibrin polymeri protofibril formation, which is carried out via intermolecular interactions of complementary polymerization sites 'a' and 'a' of fibrin molecules. We have also shown that C192 forms com plex with synthetic peptide glyProargPro, which imitates polymerization site 'a' (aa17 gly Proarg), suggesting that the inhibitory effect of C192 may be a result of the blocking of site 'a' by this calixarene.
The previous scientific experiments with healthy adult mice have shown that the calixa rene C192 is median toxic compound (lD 50 is 780 mg/kg, perorally).These experiments demonstrate that calix [4] arene C192 (calixarene tetrakismethylene bispho sphonic acid) is a specific inhibitor of fibrin po lymerization and blood coagulation can be used for the design of a new class of antithrombotic agents.

Fig. 4 .Fig. 5 .
Fig. 4. the hydrodynamic diameter (hDD) distribution for myosin subfragment-1 particles.Photoncorrelation spectroscopy method.the quantity of particles, equivalent hDD of which corresponded to the values in the interval from hDD min to hDD max with the accuracy 0.1% (according to the characteristics of the laser-correlation spectrophotometer), was taken for 100%.In the Figure, a typical experimental result is shown Diameter, nm 0 5 10 50 100 500 1000 [4]arene C-97], M . KOmisarenKO, s.O. KOsTerin, e. v. lUgOvsKOy, v. i. KalChenKO mg 2+ as a cofactor (fig.8,А, B). mg cation takes part in binding of АТР to the active site and in the process of its hydrolysis.

Fig. 11 .Fig. 12 .
Fig. 11.turbidity analysis of the influence of c-192 on fibrin polymerization in the fibrinogen + thrombin reaction.the dependence on calixarene c-192 concentration is shown for (A) the maximum rate of fibrin polymerization V max , (B) the lag-time τ and (C) the final turbidity of fibrin clots Δh

Fig. 13 .
Fig. 13.electron micrographs of fibrinogen + thrombin reaction media in the absence of c-192 (A, B), as well as in its presence (C, D).Scale bar = 100 nm , a, B), there are two modes of C192-glyProargPro complexation.in the first mode (a), cooperative electrostatic interac tions of two proximal negativelycharged phospho nyl groups with the gly αamino terminal group and the arg guanidinium residue play a principal role in complex formation.most of the tetrapeptide molecule is exposed outside the calixarene cavity. in the second mode (B), the hydrophobic part of glyProargPro molecule is deeply embedded into the calixarene cavity.The complex is stabi lized by PO ...h 3 n + electrostatic interactions of the phosphonyl group with the gly amino acid res idue, as well as by Chπ interactions of Ch 2 group in the Pro residue with the calixarene aromatic t a b l e 3. Values 1/k′ of the guests and association constants Ka (m -1 ) for their complexes with calixarene c-192.rSD, relative standard deviation guest Calixarene concentration K a , m

Fig. 15 .
Fig. 15. the dependence of the Pt and aPtt ratios on the calixarene c-192 concentration Fig. 16.Dependence of 1/k′ for gly, Pro, arg and gly-Pro-arg-Pro on thec-192 concentration in the mobile phase level of aTP dependent actinmyosin interaction and can be used for the design of a new class of antithrombotic agents.the authors express their gratitude to r. D. Labintseva, О. А. Bevza and О. V. Bevza, t. a. Pozniak (Koshel), P. g. gritsenko (Palladin Institute of Biochemistry, NaS of ukraine) and S. О. tcherenok, S. o.cherenok (Institute of organic chemistry, NaS of ukraine) for their active participation in experimental investigations as well as o.yu.tchunikhin (Palladin Institute of Biochemistry, NaS of ukraine) for assistance in performing experiments on photon-correlation spectrophotometerBiOChemisTry anD BiOTeChnOlOgy fOr mODern meDiCine and chernishov V. I. for assistance in performing experiments on electron microscopy.