INTRODUCTION:The indole nucleus is considered a privileged pharmacophore in medicinal chemistry. Indole-2-carboxylic acid derivatives are valued for their antiproliferative and antibacterial properties, including the potential to overcome drug resistance. This study aimed to synthesize novel salicylidene hydrazones of indole-2-carboxylic acid and evaluate their potential as metallo-β-lactamase (NDM-1) inhibitors and antiproliferative agents alongside previously reported indole-2-carboxylic acid derivatives. METHODS:A novel series of salicylidene hydrazones of indole-2-carboxylic acid was synthesized with potential as metallo-β-lactamase inhibitors in mind. All compounds were evaluated for their inhibitory activity against NDM-1 and tested for antiproliferative potency against human chronic myelogenous leukemia K-562 cells and the multidrug-resistant (MDR) subline K- 562/4. Doxorubicin and previously reported indole derivatives (1, 5, 6) were used as reference compounds. RESULTS:Unlike the parent acid 1, the novel derivatives showed weak NDM-1 inhibition (IC50 > 50 μM). In contrast, salicylidene hydrazones 4a-g demonstrated strong antiproliferative activity (IC50 = 0.15-0.80 μM). This effect was particularly pronounced against the MDR subline K- 562/4. Notably, the IC50 values for several derivatives were up to 50-fold lower than that of doxorubicin against resistant cells. Hydrazide 3, hydrazones 4h and 4i, thiosemicarbazide 5, and hydroxamic acids 6a-c showed moderate antiproliferative activity (IC50 = 2.4-13.3 μM). DISCUSSION:The weak NDM-1 inhibition confirms that a free 2-carboxyl group is critical for binding within the enzyme's active site. However, converting this scaffold into salicylidene hydrazones significantly enhances antiproliferative potency. It also effectively enables the circumvention of P-glycoprotein (P-gp) mediated efflux in MDR leukemia cells. CONCLUSION:Indole-2-carboxylic acid salicylidene hydrazones emerged as promising scaffolds for developing new anticancer agents. They exhibit potent activity against leukemia cells, particularly against the P-gp-overexpressing MDR subline K-562/4.

The rapid spread of metallo-β-lactamase (MBL)-mediated resistance, particularly by NDM- and VIM-type enzymes, poses a major threat to the clinical efficacy of β-lactam antibiotics. Building on our previous work, we report the design, synthesis, and evaluation of 4-substituted 3,5-diarylpyrrole-2-carboxylic acids as MBL inhibitors. Structure-activity relationship studies revealed that both the nature and position of substituents in the A and B rings, as well as functionalization at the C4 position, critically influence inhibitory activity. Notably, C4 halogenation, especially bromination, significantly enhanced inhibition of VIM-2 while maintaining activity against NDM-1. Molecular modelling supported these findings, indicating distinct binding modes and coordination patterns within the enzyme active sites. The most active compounds displayed nanomolar potency and effectively restored the activity of β-lactam antibiotics, including meropenem, against NDM- and VIM-producing resistant strains. Importantly, the compounds exhibited no detectable cytotoxicity and lacked intrinsic antibacterial activity. These results identify C4-functionalized pyrrole-2-carboxylic acids as promising dual-target MBL inhibitors for combination antibacterial therapy.

Antimicrobial resistance mediated by metallo-β-lactamases (MBLs) represents a critical challenge for the efficacy of β-lactam antibiotics. Here, we report the design, synthesis, and evaluation of a novel class of 3,5-diaryl-1H-pyrrole-2-carboxylic acids as potent New Delhi metallo-β-lactamase (NDM)-type MBL inhibitors. Guided by molecular modeling and structure-activity relationship studies, the pyrrole scaffold was optimized through systematic modifications of the phenyl rings at position 3 and 5 of pyrrole core, yielding derivatives with low-nanomolar IC50 values against NDM-1. Key interactions involve coordination of the pyrrole carboxyl groups with Zn2+ ions and hydrogen bonding with active-site residues, supporting enhanced binding and inhibitory potency. Lead compounds restored the activity of cefepime and meropenem against NDM-positive E. coli and K. pneumoniae strains, while triple combinations with serine β-lactamase inhibitors further enhanced antimicrobial efficacy. These results highlight 1H-pyrrole-2-carboxylic acids as a versatile scaffold for MBL inhibition and provide a rational framework for the development of therapeutics against multidrug-resistant pathogens.

Bacterial resistance to β-lactam antibiotics has emerged as a major challenge in healthcare. This form of antibiotic resistance is driven by the ability of pathogens to produce β-lactamases, which are divided into four classes (A-D) according to the Ambler classification. The most concerning are metallo-β-lactamases (MBLs) of class B, with the New Delhi Metallo-β-lactamase (NDM) enzyme family being among the most clinically significant. These zinc-dependent enzymes can inactivate almost all β-lactam antibiotics, and, to date, no effective inhibitors for this type of β-lactamase have been developed. Certain derivatives of indole-2-carboxylic acid and azoles have been shown to inhibit New Delhi metallo-β-lactamase-1 (NDM-1) by coordinating with Zn2+ ions and specifically interacting with key amino acid residues in the active site of the enzyme. However, the antibacterial potential of azolylindoles as metallo-β-lactamase inhibitors remains unexplored. In searches of novel scaffolds for the development of metallo-β-lactamase inhibitors a strategy for modifying a known NDM-1 inhibitor chemotype based on indole-2-carboxylic acid is proposed. This approach leads to the synthesis of previously unreported 2-azolylindoles incorporating triazole, thiadiazole, oxadiazole, tetrazole, and tetrazolylmethyl moieties. Synthetic methodologies for the preparation of intermediates and target compounds were optimized and adapted. Obtained compounds have demonstrated the ability to inhibit NDM-1 across a broad concentration range (IC50 = 40 nM–15 µM), highlighting the significant influence of the azole nuclei structure on in the enzyme inhibition. The docking-predicted binding poses of the most active compounds in active site of NDM-1 closely matched with the experimental ligand orientation and revealed interactions with key amino acid residues and Zn2+ ions. For the most potent lead-compounds, the effect on the activity of meropenem and cefepime against NDM-1-producing strains of E. coli and K. pneumoniae was evaluated, as these antibiotics are highly relevant in antimicrobial therapy. Cytotoxicity of the synthesized series was evaluated using the noncancerous HaCaT keratinocyte cell line. The most active NDM-1 inhibitors, including the paternal acid 1 and the tetrazole analogue 19, demonstrated low cytotoxicity (IC50 > 50 µM), supporting their potential as safe candidates. In contrast, compounds containing triazole, thiadiazole, or oxadiazole moieties showed increased cytotoxicity, which also limits the further development of compounds 25 and 26 as NDM-1 inhibitors. Thus, among all synthesized compounds, only 2-tetrazolylindole derivative was identified as a potential candidate for further development of novel MBL inhibitors.

Antibiotic-resistant bacteria represent a global issue that calls for novel approaches to diagnosis and treatment. Given the variety of genetic factors that determine resistance, multiplex methods hold promise in this area. We developed a novel method to covalently attach oligonucleotide probes to the wells of polystyrene plates using photoactivation with 4-azidotetrafluorobenzaldehyde. Then, it was used to develop the technique of microarrays in the wells. It consists of the following steps: activating polystyrene, hybridizing the probes with biotinylated target DNA, and developing the result using a streptavidin–peroxidase conjugate with colorimetric detection. The first microarray was designed to identify 11 different gene types and 16 single-nucleotide polymorphisms (SNPs) of clinically relevant ESBLs and carbapenemases, which confer Gram-negative bacteria resistance to β-lactam antibiotics. The detection of bla genes in 65 clinical isolates of Enterobacteriaceae demonstrated the high sensitivity and reproducibility of the technique. The highly reproducible spot staining of colorimetric microarrays allowed us to design a second microarray that was intended to quantify four different types of bla mRNAs in order to ascertain their expressions. The combination of reliable performance, high throughput in standard 96-well plates, and inexpensive colorimetric detection makes the microarrays suitable for routine clinical application and for the study of multi-drug resistant bacteria.

The review analyzes recent advances, challenges, and practical applications in the field of enzymes within the framework of chemical enzymology and enzyme engineering. The achievements in the fundamental understanding of molecular mechanisms of the catalytic cycle of enzymatic reactions made using quantum mechanics/molecular mechanics methods with supercomputer technologies and bioinformatic approaches are considered. The design of protein biocatalysts with new properties is a fundamentally significant methodology of the bioengineering approach to solving practical problems, which is demonstrated by a number of examples. The increasing role of biocatalysis in medicine and biomedical research is illustrated by addressing the problems of antibiotic synthesis and overcoming antibiotic resistance of bacteria, mechanisms of neurodegenerative diseases and development of drugs to treat Alzheimer's disease, biocatalytic processes of DNA repair and the role of mechanisms of functioning of heme peroxidases in the human body. The use of enzymes to degrade endogenous and exogenous toxicants has been greatly developed in recent decades. The advances and problems of using enzymes in therapy and drug delivery are analyzed. The fundamental role of enzymes in modern analysis and diagnosis is noted. The review considers a new trend in the development of bioanalytical methods using aptamers, multi-analysis systems on biochips, surface-enhanced Raman scattering systems, and bioelectroanalysis. The bibliography includes 460 references.

Resistance to β-lactam antibiotics caused by β-lactamases such as New-Delhi lactamase (NDM-1) has become one of the major challenges in the current antimicrobial therapy. Pyridine-2,6-dicarboxylic acid (DPA) derivatives have been demonstrated to inhibit NDM-1 in a due to the interactions with Zn ion and amino acid residues of the enzyme’s active site. In this study, a series of new 4-substituted DPA derivatives was synthesized. The SAR study has proven that the presence of a substituent at the 4-position of pyridine-2,6-dicarboxylic acid had a certain impact on the NDM-1 inhibitory. Some representatives, e.g., 4e exhibited IC50 values against NDM-1 close to the previously reported hit-compound 4-(3-aminophenyl)pyridine-2,6-dicarboxylic acid. The microdilution broth test confirmed an ability of derivative 4e to increase susceptibility of NDM-1-producing E. coli strain and did not demonstrate cytotoxicity to eukaryotic cells. However, NDM-1 inhibition by 4-substituted derivatives dramatically dropped when Zn2+ was added. We observed a strong complexation of 4-modified derivatives with Zn2+ similar to unsubstituted pyridine-2,6-dicarboxylic acid. Taken together, a complexation mode of NDM-1 inhibition leading to potential off-target action on other metalloenzymes and low efficiency of structure optimization make DPA derivatives an unproductive scaffold for future development of clinically relevant metallo-β-lactamase inhibitors.

X-ray imaging of virus particles at the European XFEL could eventually allow their complete structures to be solved, potentially approaching the resolution of other structural virology methods. To achieve this ambitious goal with today's technologies, about 1 ml of purified virus suspension containing at least 10 12 particles per millilitre is required. Such large amounts of concentrated suspension have never before been obtained for enveloped viruses. Tick-borne encephalitis virus (TBEV) represents an attractive model system for the development of enveloped virus purification and concentration protocols, given the availability of large amounts of inactivated virus material provided by vaccine-manufacturing facilities. Here, the development of a TBEV vaccine purification and concentration scheme is presented combined with a quality-control protocol that allows substantial amounts of highly concentrated non-aggregated suspension to be obtained. Preliminary single-particle imaging experiments were performed for this sample at the European XFEL, showing distinct diffraction patterns.

The paper reports an example of a successful upgrade of the JEOL JEM-2100 analytical transmission electron microscope to a low-resolution cryo-electron microscope, which can be used to optimize sample preparation and to assess preparation quality. As a result of the instrument upgrade, it is possible to obtain subnanometer resolution of protein molecule reconstructions (within 8 Å). The role of graphene and amorphous carbon substrates in preventing the effect of preferential orientation of protein particles in a frozen sample is discussed.

Serine β-lactamase TEM-1 is the first β-lactamase discovered and is still common in Gram-negative pathogens resistant to β-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a “global suppressor”, has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of β-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the β-lactamase to be reactivated.

The main approach to preventing tick-borne encephalitis (TBE) is vaccination. Formaldehyde-inactivated TBE vaccines have a proven record of safety and efficiency but have never been characterized structurally with atomic resolution. We report a cryoelectron microscopy (cryo-EM) structure of the formaldehyde-inactivated TBE virus (TBEV) of Sofjin-Chumakov strain representing the Far-Eastern subtype. A 3.8 angstrom resolution reconstruction reveals the structural integrity of the envelope E proteins, specifically the E protein ectodomains. The comparative study shows a high structural similarity to the previously published structures of the TBEV European subtype strains Hypr and Kuutsalo-14. A fraction of inactivated virions exhibits asymmetric features including the deformations of the membrane profile. We propose that the heterogeneity is caused by inactivation and perform a local variability analysis on the small parts of the envelope protein shell to reveal membrane curvature features possibly induced by the inactivation. The results of this study will have implications for the design of novel vaccines against diseases caused by flaviviruses.

In the system of sanitary, anti-epidemic and anti-epizootic measures that ensure the well-being of the country in terms of infectious diseases, increasing animal productivity and the sanitary quality of products, raw materials and feed of animal origin, disinfection occupies one of the important places. Disinfection is understood as the destruction of objects or the removal from them of pathogenic and conditionally pathogenic microorganisms. The main purpose of disinfection is to break the epizootic chain by influencing its important link - the factor of transmission of the pathogen from the source of infection to the susceptible organism. Given the variety of existing disinfectants and their constituent components, preparations with high bacterio- and virusstatic activity are very limited, which does not allow effective disinfection of contaminated surfaces, especially those contaminated with organic substances. The problem of introducing new highly effective disinfectants has become particularly relevant in connection with the spread of highly pathogenic microorganisms throughout the country.

The increasing antibiotic resistance is a clinical problem worldwide. Numerous Gram-negative bacteria have already become resistant to the most widely used class of antibacterial drugs, β-lactams. One of the main mechanisms is inactivation of β-lactam antibiotics by bacterial β-lactamases. Appearance and spread of these enzymes represent a continuous challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. Drug repurposing is a prospective approach for finding new targets for drugs already approved for use. We describe here the inhibitory potency of known detoxifying antidote 2,3-dimercaptopropane-1-sulfonate (unithiol) against metallo-β-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-β-lactamase NDM-1 with the KI of 16.7 µM. It is an order of magnitude lower than the KI for l-captopril, the inhibitor of angiotensin-converting enzyme approved as a drug for the treatment of hypertension. Phenotypic methods demonstrate that the unithiol inhibits natural metallo-β-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant K. pneumoniae and P. aeruginosa bacterial strains. The 3D full atom structures of unithiol complexes with NDM-1 and VIM-2 are obtained using QM/MM modeling. The thiol group is located between zinc cations of the active site occupying the same place as the catalytic hydroxide anion in the enzyme–substrate complex. The sulfate group forms both a coordination bond with a zinc cation and hydrogen bonds with the positively charged residue, lysine or arginine, responsible for proper orientation of antibiotics upon binding to the active site prior to hydrolysis. Thus, we demonstrate both experimentally and theoretically that the unithiol is a prospective competitive inhibitor of metallo-β-lactamases and it can be utilized in complex therapy together with the known β-lactam antibiotics.

Tick-borne encephalitis virus (TBEV) is an enveloped RNA virus, a member of the genus Flavivirus (family Flaviviridae). Here, we provide a detailed analysis of the size and structure of the inactivated TBEV vaccine strain Sofjin-Chumakov. Four analytical methods were used to analyze individual TBEV particles—negative staining TEM, cryo-EM, atomic force microscopy (AFM), and nanoparticle tracking analysis (NTA). All methods confirmed that the particles were monodisperse and that their mean size was ~50 nm. Cryo-EM data allowed us to obtain a 3D electron density model of the virus with clearly distinguishable E protein molecules. STEM-EELS analysis detected phosphorus in the particles, which was interpreted as an indicator of RNA presence. Altogether, the described analytical procedures can be valuable for the characterization of inactivated vaccine virus samples.

The aim of this work was to design and characterize peptides based on the α-helices h1 and h2 of the ACE2 receptor, forming the interaction interface between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and the cellular ACE2 receptor. Monomeric and heterodimeric peptides connected by disulfide bonds at different positions were synthesized. Solubility, RBD-binding affinity, and peptide helicity were experimentally measured, and molecular dynamics simulation was performed in various solvents. It was established that the preservation of the helical conformation is a necessary condition for the binding of peptides to RBD. The peptides have a low degree of helicity and low affinity for RBD in water. Dimeric peptides have a higher degree of helicity than monomeric ones, probably due to the mutual influence of helices. The degree of helicity of the peptides in trifluoroethanol is the highest; however, for in vitro studies, the most suitable solvent is a water-ethanol mixture.

The resistance of bacteria to β-lactam antibiotics is primarily caused by the production of β-lactamases. Here, novel crystal structures of the native β-lactamase TEM-171 and two complexes with the widely used inhibitor tazobactam are presented, alongside complementary data from UV spectroscopy and fluorescence quenching. The six chemically identical β-lactamase molecules in the crystallographic asymmetric unit displayed different degrees of disorder. The tazobactam intermediate was covalently bound to the catalytic Ser70 in the trans-enamine configuration. While the conformation of tazobactam in the first complex resembled that in published β-lactamase-tazobactam structures, in the second complex, which was obtained after longer soaking of the native crystals in the inhibitor solution, a new and previously unreported tazobactam conformation was observed. It is proposed that the two complexes correspond to different stages along the deacylation path of the acyl-enzyme intermediate. The results provide a novel structural basis for the rational design of new β-lactamase inhibitors.

To improve the effectiveness of the viral infection diagnostic, we offer a new approach of immunochemical biosensors to determine single viral particles by specific antibodies. The antibodies are immobilized on the electrodes in a three-dimensional polymer matrix with several layers of polyelectrolytes on the screen-printed carbon electrode. Non-covalent immobilization of antibodies in successive layers of positively charged polyethyleneimine (PEI) and negatively charged polystyrene sulfonate (PSS) achieves the effect of macromolecular crowding. Such an immobilization approach promotes the preservation of the optimal conformation and antibody active center mobility for interaction with large virion particles. We established an electrochemical biosensor for tick-borne encephalitis virus (TBEV) detection to demonstrate the method's applicability. Under the optimized architecture of the 3D-matrix, including a combination of two layers of a positively charged PEI with antibodies and the last layer of a negatively charged PSS, the assay is characterized by an extremely low limit of detection (LOD). This LOD could be as few as five viral particles in a sample volume of 5 mu l, which is two orders of magnitude lower than conventional ELISA with the same reagents. The advantage of the biosensor is also a wide linear range of detection from 10(3) to 10(9) viral particles/ml. The proposed principle for determining virion particles is well suited to novel express diagnostics and Point-of-Care viral infections detection.