The Ukrainian Biochemical Journal (Dec 2013)
Calixarene methylene bisphosphonic acids as promising effectors of biochemical processes
Abstract
This interdisciplinary study, performed with participation of research workers of Palladin Institute of Biochemistry and Institute of Organic Chemistry 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.
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