Rational search of a ligand for a specific receptor is a cornerstone of a typical drug discovery process. However, to make it more “rational” one would appreciate having detailed information on the functional groups involved in ligand-receptor interaction. Typically, the 3D structure of a ligand-receptor complex can be built on the basis of time-consuming X-ray crystallography data. Here, a combination of FTIR and fluorescence methods, together with appropriate processing, yields valuable information about the functional groups of both the ligand and receptor involved in the interaction, with the simplicity of conventional spectrophotometry. We have synthesized the “molecular containers” based on cyclodextrins, polyethyleneimines (PEI) or spermine with mannose-rich side-chains of different molecular architecture (reticulated, star-shaped and branched) with variable parameters to facilitate delivery to alveolar macrophages. We have shown that synthetic mannose-rich conjugates are highly affine to the model mannose receptor ConA: Kd ≈ 10−5–10−7 M vs. natural ligand trimannoside (10−5 M). Further, it was shown that molecular containers effectively load levofloxacin (dissociation constants are 5·10−4–5·10−6 M) and the eugenol adjuvant (up to 15–80 drug molecules for each conjugate molecule) by including them in the cyclodextrins cavities, as well as by interacting with polymer chains. Promising formulations of levofloxacin and its enhancer (eugenol) in star-shaped and polymer conjugates of high capacity were obtained. UV spectroscopy demonstrated a doubling of the release time of levofloxacin into the external solution from the complexes with conjugates, and the effective action time (time of 80% release) was increased from 0.5 to 20–70 h. The synergy effect of antibacterial activity of levofloxacin and its adjuvants eugenol and apiol on Escherichia coli was demonstrated: the minimum effective concentration of the antibiotic was approximately halved.

Computer modeling of complexation of mono- and oligosaccharide ligands with the main (fourth) carbohydrate-binding domain of the mannose receptor CD206 (CRD4), as well as with the model receptor concanavalin A (ConA), was carried out for the first time, using methods of molecular dynamics and neural network analysis. ConA was shown to be a relevant model of CD206 (CRD4) due to similarity of the structural organization of the binding sites and high correlation of the values of free energies of complexation between the literature data and computer modeling ( r > 0.9). Role of the main factors affecting affinity of the ligand–receptor interactions is discussed: the number and nature of carbohydrate residues, presence of Me-group in the O1 position, type of the glycoside bond in dimannose. Complexation of ConA and CD206 with ligands is shown to be energetically caused by electrostatic interactions (E) of the charged residues (Asn, Asp, Arg) with oxygen and hydrogen atoms in carbohydrates; contributions of hydrophobic and van der Waals components is lower. Possibility of the additional stabilization of complexes due to the CH–π stacking interactions of Tyr with the Man plane is discussed. The role of calcium and manganese ions in binding ligands has been studied. The values of free energies of complexation calculated in the course of molecular dynamics simulation correlate with experimental data (published for the model ConA): correlation coefficient r = 0.68. The Pafnucy neural network was trained based on the set of PDBbind2020 ligand–receptor complexes, which significantly increased accuracy of the energy predictions to r = 0.8 and 0.82 for CD206 and ConA receptors, respectively. A model of normalization of the complexation energy values for calculating the relevant values of ΔG bind , K d is proposed. Based on the developed technique, values of the dissociation constants of a series of CD206 complexes with nine carbohydrate ligands of different structures were determined, which were not previously known. The obtained data open up possibilities for using computer modeling for the development of optimal drug carriers capable of active macrophage targeting, and also determine the limits of applicability of using ConA as a relevant model for studying parameters of the CD206 binding to various carbohydrate ligands. Electronic supplementary material The online version contains supplementary material available at 10.1134/S0006297922010059.

The main factors that determine the low effectiveness of chemotherapy are the low target bioavailability of antitumor drugs and the efflux process. In attempts to overcome this problem, several approaches are proposed here. Firstly, the development of polymeric micellar systems based on chitosan grafted by fatty acids (different types to optimize their properties), which, on the one hand, increase the solubility and bioavailability of cytostatics and, on the other hand, effectively interact with tumor cells due to the polycationic properties of chitosan, allowing for more effective penetration of cytostatic drugs into the cells. Secondly, the use of adjuvants—synergists of cytostatics (such as eugenol) included in the same micellar formulation—that selectively enhance the accumulation and retention of cytostatics in the tumor cells. pH- and temperature-sensitive polymeric micelles developed show high entrapment efficiency for both cytostatics and eugenol (EG) > 60% and release the drug in a prolonged manner for 40 h in a weakly acidic medium corresponding to the microenvironment of tumors. In a slightly alkaline environment, the drug circulates longer (more than 60 h). The thermal sensitivity of micelles is realized due to an increase in the molecular mobility of chitosan, which undergoes a phase transition at 32–37 °C. The effect of the cytostatic drug doxorubicin (Dox) on cancerous A549 cells and model healthy cells of human embryonic renal epithelium (HEK293T) was studied by FTIR spectroscopy and fluorescence microscopy. Micellar Dox penetrates into cancer cells 2–3 times more efficiently when using EG adjuvant, which inhibits efflux, as demonstrated by a significant increase in the ratio of intra- and extracellular concentrations of the cytostatic. However, here it is worth remembering about healthy cells that they should not be damaged: according to changes in the FTIR and fluorescence spectra, the penetration of Dox into HEK293T when using micelles in combination with EG is reduced by 20–30% compared to a simple cytostatic. Thus, experimental developments of combined micellar cytostatic drugs have been proposed to increase the effectiveness of cancer treatment and overcome multiple drug resistance.

Allylpolyalkoxybenzenes (APABs) and terpenoids from plant essential oils exhibit a range of remarkable biological effects, including analgesic, antibacterial, anti-inflammatory, antioxidant, and others. Synergistic activity with antibiotics of different classes has been reported, with inhibition of P-glycoprotein and impairment of bacterial cell membrane claimed as probable mechanisms. Clearly, a more detailed understanding of APABs’ biological activity could help in the development of improved therapeutic options for a range of diseases. However, APABs’ poor solubility in water solutions has been a limiting factor for such research. Here, we found that complex formation with β-cyclodextrins (CD) is an efficient way to transform the APABs into a water-soluble form. Using a combination of spectroscopic (FTIR, NMR, UV) methods, we have estimated the binding constants, loading capacity, and the functional groups of both APABs and monoterpenes involved in complex formation with CD: ethylene, aromatic, methoxy and hydroxy groups. In the presence of a molar excess of CD (up to 5 fold) it was possible to achieve the complete dissolution of APABs and terpenoids in an aqueous medium (at 90–98% encapsulation) higher by 10–1000 times. Further, we have demonstrated that CD-APABs, if used in combination with levofloxacin (Lev), can be antagonistic, indifferent, additive, or synergistic, mostly depending on the concentration ratio: at high Lev concentration with the addition of APAB is typically neutral or even antagonistic; while at a Lev concentration below MIC, the addition of CD-APAB is either additive or synergistic (according to FICI criteria). An over three-fold increase in Lev antibacterial activity was observed in combination with eugenol (EG), as per the growth inhibition diameter measurement in agar. Interestingly, a synergistic effect could be observed with both Gram-positive and Gram-negative bacteria. So, obviously, the APAB-CD and terpenoid-CD mechanism of action is not limited to their interaction with the bacterial membrane, which has been shown earlier for CDs. Further research may open new prospects for the development of adjuvants to improve the therapeutic regimens with existing, as well as with new anti-infective drugs.

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