Uridine phosphorylases are used for biotechnological synthesis of pyrimidine derivatives and, moreover, their substrates and inhibitors are used in medicine. Therefore, studies of the mechanisms of the chemical reaction catalyzed by the enzyme and its specificity for various substrates are relevant. The research into the enzymatic reaction main stage-nucleophilic substitution of the nitrogenous base in uridine with an orthophosphate or orthovanadate group by hybrid QM/MM methods-was carried out. A comparison of various levels of theory and calculation schemes showed that preliminary optimization of the reactants's geometry, as well as calculation of the initial trajectory of the minimum energy path, can be achieved by semi-empirical methods. At the same time, for the minimum energy path clarification, transition state geometry optimization, and calculation of the thermochemical parameters, it is preferable to use density functional theory in combination with modern ab initio methods. In comparison with the calculations of the activation barrier carried out in a solvent without an enzyme, differences in the kinetics of the enzymatic reaction due to the orientation and concentration actions of amino acid residues of the enzyme were revealed. This led to lowering the activation barrier by 20 kcal/mol and contributed to the reaction under physiologically acceptable conditions. It was shown that the free activation energy during the nucleophilic attack for uridine with hydrovanadate ion is 2 kcal/mol lower than for the hydrophosphate ion and this is consistent with the literature data.

Influenza A virus pandemics still remain a threat to global health. One class of antiviral drugs, namely, inhibitors of the specific viral enzyme neuraminidase, is predominantly used in the fight against these pandemics. These antivirals include zanamivir (Relenza™) and oseltamivir (Tamiflu™). The viral resistance to this class of compounds steadily increases. The M2 proton channel of influenza A virus is an alternative clinically proven target for antiviral therapy. However, many circulating virus strains bear amino acid mutations in the M2 protein, causing resistance to drugs of the adamantane series, M2 blockers, such as rimantadine and amantadine. Consequently, inhibitors targeting mutants of the M2 channel are urgently needed for public biosafety and health. This review is devoted to structural-functional interactions used in practice and mediated by the action of experimental drugs on the protein target, the transmembrane domain of the influenza virus M2 proton channel. An analysis of the experimental and model structural data available in open access is presented.

Searching for a specific inhibitor of pyrimidine nucleoside phosphorylase (PyNP) was a central objective of the work. The search was carried out by modeling protein–ligand complexes via molecular docking and molecular dynamics (MD), which make it possible to calculate the protein–ligand binding energy Δ G bind . The following compounds were selected as possible inhibitors: 2',3'-didehydro-3'-deoxythymidine (d4T), 1-(2-deoxy-2-fluoro-β- D -arabinofuranosyl)-5-ioduracil (fiauridine, FIAU), 1-(2-deoxy-2-fluoro-β- D -arabinofuranosyl)-5-uracil (FAU), and 2-pyrimidin-2-yl-1 H -imidazole-4-carboxylic acid (PIA). A preliminary estimate of the binding energy was obtained by the linear interaction energy (LIE) method, and a more accurate calculation was carried out by the free energy perturbation (FEP) method in the GROMACS software package. The calculation results showed that PIA and d4T bind to the active site of Bacillus subtilis PyNP ( Bs PyNP) with higher affinities as compared with the other putative inhibitors. PIA binds less strongly to human thymidine phosphorylase (hTP). This was assumed to minimize the possible side effects of this compound used for therapeutic purposes.

The calculations of the free energy of ligand binding with a receptor (most often, protein) are widely used in the rational design of new bioactive compounds. The gold standard of such calculations is the equilibrium methods based on molecular dynamics and the “alchemical” transformations, including the thermodynamic integration and Bennett method. Technically, these methods call for introduction of additional restraints on the mutual arrangement of atoms in the ligand–receptor system. A plugin to the PyMOL molecular graphics system has been developed to facilitate the introduction of required restraints and analytical calculation of correction when computing the free energy of ligand‒receptor (protein) binding.

Uridine phosphorylases are known as key targets for the development of new anticancer and antiparasitic agents. Crystals of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae were grown in microgravity by the capillary counter-diffusion method on board of the International Space Station . The three-dimensional structure of this enzyme was determined at atomic (1.04 Å) resolution (RCSB PDB ID: 6Z9Z). Alternative conformations of long fragments (β-strands and adjacent loops) of the protein molecule were found for the first time in the three-dimensional structure of uridine phosphorylase in the absence of specific bound ligands. Apparently, these alternative conformations are related to the enzyme function. Conformational analysis with Markov state models demonstrated that conformational rearrangements can occur in the ligand-free state of the enzyme.

The high-resolution three-dimensional structure of uridine phosphorylase from the pathogenic bacterium Vibrio cholerae in complex with the competitive inhibitor 2,2'-anhydrouridine was determined by X-ray diffraction (RCSBPDB ID: 6RCA). The three-dimensional structure of this complex is compared with the previously determined structures of V. cholerae uridine phosphorylase in complex with the substrate (uridine) and S. typhimurium uridine phosphorylase in complex with 2,2'-anhydrouridine. The protein–inhibitor and protein–substrate binding free energies were calculated by the free-energy perturbation method. The number of stable hydrogen bonds between the 2,2'-anhydrouridine molecule and the active site of the enzyme is smaller and these bonds are longer compared to the natural substrate of the enzyme (uridine). However, calculations taking into account solvation energy of the molecule and the entropy effects showed that the binding of the inhibitor (2,2'-anhydrouridine) at the active site of the protein is energetically more favorable than the binding of the native substrate (uridine). These results may be useful in the design of new inhibitors with a higher selectivity for the binding sites of uridine phosphorylases.

Purpose: The aminoadamantane derivative of L-histidyl-1-adamantayl ethylamine hydrochloride (HCl*H-His-Rim) has showed a high inhibition level against influenza A virus strains in vitro. The aim of this work is to search and establish evidence of the direct effect of the drug on influenza A virus proton channel M2. Methods: The compound HCl*H-His-Rim was obtained by classical peptide synthesis methods. Influenza A virus mutants of A/PuertoRico/8/34(H1N1) strain were obtained by reverse genetics methods. The mutant samples of the virus were cultured on chicken embryos with a virus titer in the hemagglutination test. ELISA was carried out on Madin-Darby canine kidney (MDCK) monolayer cells when multiplying the virus 10-4-10-6. The binding stability of HCl*H-His-Rim was compared to those of M2 (S31N) and M2 (S31N_A30T) channels by molecular dynamic (MD) modeling. The calculation was performed taking into account the interaction with the model lipid bilayer (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) in the presence of water molecules in accordance with the three-center model. Results: It was found that HCl*H-His-Rim is a direct action drug against influenza A. The most likely conformation of drug binding to target protein has been shown. It has been found that the A30T mutation reduces the binding energy of the drug, and the results obtained in vitro have confirmed the data calculated in silico. Conclusion: The mechanism of action of HCl*H-His-Rim is directly related to the suppression of the function of the proton channel M2 of influenza A virus.

Hydrophobic cores and hydrophobic clusters play a key role in globular protein folding, providing a framework for functionally important amino acid residues of proteins and enzymes. A program was developed for clustering amino acid residues in protein structures, which takes into account their hydrophobicity and utilizes the DBSCAN algorithm. The program is described and its scope and applicability are outlined.

The structure of bacterial uridine phosphorylase (UPh) belonging to the NP-I family in complex with 6-methyluracil was determined for the first time at 1.17 Å resolution. The structural features of bacterial UPh from the bacterium Vibrio cholerae ( Vch UPh) responsible for selectivity toward 6-methyluracil acting as a pseudosubstrate were revealed. The repulsion between the hydrophilic hydroxyl group of the active-site residue Thr93 of Vch UPh and the hydrophobic methyl group of 6-methyluracil prevents the oxygen atom O4' of the ribose moiety and the phosphate oxygen atom O3P of ribose 1-phosphate from forming hydrogen bonds with OG1_Thr93, which are essential for the enzymatic reaction. This, apparently, makes Vch UPh inactive in the enzymatic synthesis of 6-methyluridine from 6-methyluracil. Hence, Thr93 is the residue, the modification of which will allow Vch UPh to catalyze the biotechnologically important synthesis of 6-methyluridine from 6-methyluracil. Taking into account high structural homology of the functionally significant regions of bacterial UPhs, this conclusion is also true for other bacterial UPhs. It was demonstrated that bacterial thymidine phosphorylases of the NP-II family cannot bind 6-methyluracil in a proper conformation required for the catalysis because of a close contact between the 6-methyl group and Phe210.