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Aim. The goal of the study is to demonstrate the binding sites for curcumin on the protein carriers — bovine serum albumin and diphtheria toxoid CRM197. BSA was chosen as a potential nonspecific protein carrier because of its widely used in medicine as a drug carrier. Methods. In the investigation, both spectrophotometric and molecular docking methods were used. Results. Two stable binding sites were demonstrated for BSA to bind curcumin. CRM197 was taken as a well-studied carrier protein with its own antitumor activity and has been investigated as a specific carrier with a high affinity for cancer cells with overexpression of epidermal growth factor receptor. Our results showed one possible curcumin binding site, making CRM197 an ideal specific curcumin delivery platform that provides at least an additive effect in anticancer therapies. Conclusions. In conclusion, both studied proteins form stable complexes with curcumin that can lay in base of the commercial drug application. [ABSTRACT FROM AUTHOR]

Intravascular thrombus formation is one of the main causes of such deadly diseases and pathologies as ischemic heart disease, ischemic stroke, pulmonary embolism, atrial fibrillation, and venous thromboembolism [1, 2]. That is why the development of new approaches for preventing intravascular blood coagulation is an important task for biochemistry and biotechnology. Most of biomolecules that target clotting factors. However, in our study we focused on another type of molecules, which are direct inhibitors of fibrin polymerization. As a scaffold, we have used a calix[4]arene molecule because of its tunable diversity at both upper and lower rims, its pre-organized nonpolar cavities, pre-organized ion-bonding sites and well-defined conformations [3]. Previously we have shown that calix[4]arenes are active inhibitors of fibrin polymerization process. Binding to the ‘A’-knob of fibrin desAB molecule calix[4] arenes prevents knob-hole interactions between different fibrin molecules thus suppressing clotting. Recent studies proved the efficacy of sodium salt of 5, 11, 17, 23-bis(dihydroxyphos phoryl)methylcalix[4]arene (C-145) as an effective anticoagulant agent [4, 5]. Molecular dynamic studies of C-145 demonstrated the importance of conformation of the macrocycle for its fibrin-binding activity. In particular calyx[4]arene has two different conformations of its cup (the macrocycle) that can be described as ‘1,3-alternate’ (opened) or ‘conus-conformation’ (fixed). Aim. The purpose of the present study was to develop a method for the fixation of ‘fixed’ conformation for estimation of the impact of calix[4]arene structure on the efficacy of its anticoagulant activity. This was achieved by substitution of the lower rim of C-145 analogue. Metods. Calix[4]arene C-145 was obtained according to the method described in [6] with one additional step that included Mitsunobu reaction in order to obtain disubstituted calixarene C-145F (Fig. 1). Modeling of 3D-structure of calix[4]arene C-145 and its analogue C-145F was performed in Maestro, Schrodinger software. Calix[4]arene C-145F (compound 6) was obtained in 4 steps starting with Duff reaction. Calix[4]arene methylene-bis-phosphonic ester 3 was prepared via addition of diisopropylphosphite in presence of metallic sodium to the parent calix[4]arene aldehyde 2. Further steps included Mitsunobu reaction, that afforded dipropoxycalix[4]arene 5 with rather good yields (80%), following the hydrolysis step that resulted in compound 6 in almost quantitative yield. Results. Using a 2D NMR-NOESY spectroscopy, we can observe a distinct cross-peak between an aromatic singlet with a chemical shift on 7.72 ppm and protons of isopropyl group with a chemical shift on 1.62 ppm, which are moved in the strong field (Fig. 2). This correlation proves us their steric proximity, that can be observed only in conus-conformation (Fig. 3, A), not in the 1,3-alternate. So, in this work we confirmed that disubstituted calix[4]arenes (low-rim modification) have fixed conus conformation. NMR spectroscopy proves us that calix[4]arene C145F exists in fixed conus-conformation. Discussion. Presented experiment represents how easily we can modify calix[4]arene cup in order to obtain molecule with new chemical and physical properties. The fine-tuning and incorporation of different ligating sites in the calix[4]arene scaffold may be used to produce numerous molecules, forming a library of compounds. Comparison of the action of fibrin polymerization of calix[4]arene molecules with fixed and opened conformations will allow to estimate the role of different conformation in fibrin recognition and will support targeted design of even more effective calix[4]arene-based inhibitors of blood clotting in future. Conclusions. The easy method of the fixation of conus conformation of calix[4]arene cup will be useful for synthesis of novel functionally active compounds. We believe that further development and study of different calix[4]arenes will help scientists to obtain bioactive molecules that could be prospective anti-thrombotic drugs. [ABSTRACT FROM AUTHOR]

Background: Fibrinogen is a large polyfunctional plasma protein consisting of a number of structural and functional domains. Among them, two αC-domains, each formed by the amino acid residues Аα392-610, are involved in fibrin polymerization, activation of fibrinolysis, platelet aggregation, and interaction with different cell types. Previous study revealed that each fibrinogen αC-domain consists of the N-terminal and C-terminal sub-domains. The major objections of the present study were to test functional role of these sub-domains in the above mentioned processes., Methods: To achieve these objections, we used specific proteases to prepare two truncated forms of fibrinogen, fibrinogen desAα505-610 and fibrinogen desAα414-610, missing their N-terminal and both N- and C-terminal sub-domains, respectively., Results: Our study with these truncated forms using turbidity measurements and electron microscopy revealed that the N- and C-terminal subdomains both contribute to protofibril formation and their lateral aggregation into fibers during fibrin polymerization process. These two sub-domains also contributed to platelet aggregation with the N-terminal sub-domains playing a more significant role in this process. At the same time, the C-terminal sub-domains make the major contribution to the plasminogen activation process. Further, our experiments revealed that the C-terminal sub-domains are involved in endothelial cell viability and migration of cancer cells., Conclusions: Thus, the results obtained establish the functional role of individual sub-domains of the αC-domains in fibrin polymerization, activation of fibrinolytic system, platelet aggregation, and cellular interactions., General Significance: The present study expands our understanding of the functional role of individual fibrinogen domains and their specific portions in various fibrin(ogen)-dependent processes., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)