Properties and Activity of Peptide Derivatives of ACE2 Cellular Receptor and Their Interaction with SARS-CoV-2 S Protein Receptor-Binding Domain.

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.

Peptide Inhibitors of the Interaction of the SARS-CoV-2 Receptor-Binding Domain with the ACE2 Cell Receptor.

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin-converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which terminal cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of each precursor peptide molecule. The precursors h1 and h2 mimic amino acid sequences of α1- and α2-helices of the ACE2 extracellular peptidase domain, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their constituent peptides with RBD (particularly in dependence of the middle and terminal methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified to 95-97% purity, and characterized by HPLC and MALDI-TOF mass spectrometry. Binding of these peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. The original RBD of the Chinese variant bound to three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant. The antiviral activity of the proposed peptides was evaluated in Vero cell line.

[Peptide inhibitors of the interaction of the SARS-CoV-2 receptor-binding domain with the ACE2 cell receptor]. / Peptidnye ingibitory vzaimodeistviia retseptor-sviazyvaiushchego domena virusa SARS-CoV-2 s kletochnym retseptorom ASE2.

Computer simulation has been used to identify peptides that mimic the natural target of the SARS-CoV-2 coronavirus spike (S) protein, the angiotensin converting enzyme type 2 (ACE2) cell receptor. Based on the structure of the complex of the protein S receptor-binding domain (RBD) and ACE2, the design of chimeric molecules consisting of two 22-23-mer peptides linked to each other by disulfide bonds was carried out. The chimeric molecule X1 was a disulfide dimer, in which edge cysteine residues in the precursor molecules h1 and h2 were connected by the S-S bond. In the chimeric molecule X2, the disulfide bond was located in the middle of the molecule of each of the precursor peptides. The precursors h1 and h2 modelled amino acid sequences of α1- and α2-helices of the extracellular peptidase domain of ACE2, respectively, keeping intact most of the amino acid residues involved in the interaction with RBD. The aim of the work was to evaluate the binding efficiency of chimeric molecules and their RBD-peptides (particularly in dependence of the middle and edge methods of fixing the initial peptides h1 and h2). The proposed polypeptides and chimeric molecules were synthesized by chemical methods, purified (to 95-97% purity), and characterized by HPLC and MALDI-TOF mass spectrometry. The binding of the peptides to the SARS-CoV-2 RBD was evaluated by microthermophoresis with recombinant domains corresponding in sequence to the original Chinese (GenBank ID NC_045512.2) and the British (B. 1.1.7, GISAID EPI_ISL_683466) variants. Binding to the original RBD of the Chinese variant was detected in three synthesized peptides: linear h2 and both chimeric variants. Chimeric peptides were also bound to the RBD of the British variant with micromolar constants. The antiviral activity of the proposed peptides in Vero cell culture was also evaluated.

Overcoming Antibiotic Resistance in Microorganisms: Molecular Mechanisms.

This issue of the Biochemistry (Moscow) journal presents reviews and experimental articles on the new strategies for solving the problem of antibiotic resistance and on the search for novel antimicrobial preparations using the methods of molecular biology, genetics, and nanotechnology. A wide variety of scientific approaches and successful (as a rule) research results give hope for overcoming microbial antibiotic resistance in the fight against infectious diseases.

Inhibitors of ß-Lactamases. New Life of ß-Lactam Antibiotics.

ß-Lactam antibiotics account for about 60% of all produced antibiotics. Due to a high activity and minimal side effects, they are the most commonly used class of antibacterial drugs for the treatment of various infectious diseases of humans and animals, including severe hospital infections. However, the emergence of bacteria resistant to ß-lactams has led to the clinical inefficiency of these antibiotics, and as a result, their use in medicine has been limited. The search for new effective ways for overcoming the resistance to ß-lactam antibiotics is an essential task. The major mechanism of bacterial resistance is the synthesis of ß-lactamases (BLs) that break the antibiotic ß-lactam ring. Here, we review specific inhibitors of serine ß-lactamases and metallo-ß-lactamases and discuss approaches for creating new inhibitors that would prolong the "life" of ß-lactams.

[Scaffold hopping computational approach for searching novel ß-lactamase inhibitors]. / Virtual'nyi skrining s ispol'zovaniem izmeneniia strukturnogo ostova ligandov dlia poiska novykh ingibitorov ß-laktamaz.

We present a novel computational ligand-based virtual screening approach with scaffold hopping capabilities for the identification of novel inhibitors of ß-lactamases which confer bacterial resistance to ß-lactam antibiotics. The structures of known ß-lactamase inhibitors were used as query ligands, and a virtual in silico screening a database of 8 million drug-like compounds was performed in order to select the ligands with similar shape and charge distribution. A set of numerical descriptors was used such as chirality, eigen spectrum of matrices of interatomic distances and connectivity together with higher order moment invariants that showed their efficiency in the field of pattern recognition but have not yet been employed in drug discovery. The developed scaffold-hopping approach was applied for the discovery of analogues of four allosteric inhibitors of serine ß-lactamases. After a virtual in silico screening, the effect of two selected ligands on the activity of TEM type ß-lactamase was studied experimentally. New non-ß-lactam inhibitors were found that showed more effective inhibition of ß-lactamases compared to query ligands.

Bacterial Enzymes and Antibiotic Resistance.

The resistance of microorganisms to antibiotics has been developing for more than 2 billion years and is widely distributed among various representatives of the microbiological world. Bacterial enzymes play a key role in the emergence of resistance. Classification of these enzymes is based on their participation in various biochemical mechanisms: modification of the enzymes that act as antibiotic targets, enzymatic modification of intracellular targets, enzymatic transformation of antibiotics, and the implementation of cellular metabolism reactions. The main mechanisms of resistance development are associated with the evolution of superfamilies of bacterial enzymes due to the variability of the genes encoding them. The collection of all antibiotic resistance genes is known as the resistome. Tens of thousands of enzymes and their mutants that implement various mechanisms of resistance form a new community that is called "the enzystome." Analysis of the structure and functional characteristics of enzymes, which are the targets for different classes of antibiotics, will allow us to develop new strategies for overcoming the resistance.

[Bacterial TEM-type serine beta-lactamases: structure and analysis of mutations]. / Bakterial'nye serinovye beta-laktamazy TEM-tipa: struktura i analiz mutatsii.

Beta-lactamases (EC 3.5.2.6) represent a superfamily containing more than 2,000 members: it includes genetically and functionally different bacterial enzymes capable to destroy the beta-lactam antibiotics. The most common are beta-lactamases of molecular class A with serine in the active center. Among them, TEM-type beta-lactamases are of particular interest from the viewpoint of studying the mechanisms of the evolution of resistance due to their broad polymorphism. To date, more than 200 sequences of TEM-type beta-lactamases have been described and more than 60 structures of different mutant forms have been presented in Protein Data Bank. We have considered the main structural features of the enzymes of this type with particular attention to the analysis of key drug resistance and the secondary mutations, their location relative to the active center and the surface of the protein globule. We have developed the BlaSIDB database (www.blasidb.org) which is an open information resource combining available data on 3D structures, amino acid sequences and nomenclature of the corresponding forms of beta-lactamases.

Novel non-ß-lactam inhibitor of ß-lactamase TEM-171 based on acylated phenoxyaniline.

The microbial resistance to antibiotics is a genuine global threat. Consequently, a search of new inhibitors remains of acute importance due to the increasing spread of multidrug resistance. Here we present a new type of non-ß-lactam ß-lactamase inhibitor PA-34 based on natural phenoxyaniline, identified using computer-assisted screening of scaffolds related to those of known low-affinity inhibitors. The compound displays reversible competitive inhibition of bacterial ß-lactamase TEM-171, with a Ki of 88 µM. Using enzyme kinetics, infra-red spectroscopy, fluorescence quenching and computer docking, we propose that the inhibitor binds at the entrance to the enzyme active site. This is a novel inhibition mechanism compared to binding covalently to the catalytic serine in the active site or non-covalently to the allosteric site. The residues involved in binding the inhibitor are conserved among molecular class A ß-lactamases. The identified compound and its proposed binding mode may have a potential for a regulation of the catalytic activity of a wide range of class A ß-lactamases. We also hypothesise that the presented route for finding non-ß-lactam compounds may be an effective and durable approach for combating bacterial antibiotic resistance.

[Investigation the role of mutations M182T and Q39K in structure of beta-lactamase TEM-72 by molecular dynamics method]. / Izuchenie roli mutatsii M182T i Q39K v strukture beta-laktamazy TEM-72 metodom molekuliarnoi dinamiki.

Synthesis of b-lactamases is one of the common mechanisms of bacterial resistance to b-lactam antibiotics including penicillins and cephalosporins. The widespread use of antibiotics results in appearance of numerous extended-spectrum b-lactamase variants or resistance to inhibitors. Mutations of 92 residues of TEM type were found. Several mutations are the key mutations that determine the extension of spectrum of substrates. However, roles of the most associated mutations, located far from active site, remain unknown. We have investigated the role of associated mutations in structure of b-lactamase TEM-72, which contain two key mutation (G238S, E240K) and two associated mutations (Q39K, M182T) by means of simulation of molecular dynamics. The key mutation lead to destabilization of the protein globule, characterized by increased mobility of amino acid residues at high temperature of modelling. Mutation M182T lead to stabilization protein, whereas mutation Q39K is destabilizing mutation. It seems that the last mutation serves for optimization of conformational mobility of b-lactamase and may influence on enzyme activity.

The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

Recombinant horseradish peroxidase: production and analytical applications.

Horseradish peroxidase is a key enzyme in bio- and immunochemical analysis. New approaches in functional expression of the peroxidase gene in E. coli cells and the subsequent refolding of the resulting protein yield a recombinant enzyme that is comparable in its spectral and catalytic characteristics to the native plant peroxidase. Genetic engineering approaches allow production of recombinant peroxidase conjugates with both protein antigens and Fab antibody fragments. The present article reviews the use of recombinant horseradish peroxidase as the marker enzyme in ELISA procedures as well as in amperometric sensors based on direct electron transfer.

[Conjugates of streptavidin with gold nanoparticles for the visualization of dna single interactions on the silicon surface].

The potential of the method of scanning electron microscopy (SEM) to visualize the results of individual acts of DNA and oligonucleotides hybridization using gold nanoparticles as label was investigated. Molecule of biotin was introduced into DNA or oligonucleotide, and then it was detected in DNA duplex using a conjugate of streptavidin with gold nanoparticles. Effective imaging of DNA duplexes was possible using a conjugate prepared by covalent binding.. The detection limit of the model oligonucleotide of 19 bases was 20 pg.

[Implementation of scanning probe microscopy for the solution of molecular diagnostics tasks].

We present new approaches to improve the efficiency of DNA by scanning probe microscopy using a highly specific hybridization on affine surfaces and nanostructures of gold as a labels. Scanning probe microscopy allows to register of individual acts of hybridization by the detection of gold labels on the surface affinity followed by automatic calculation of the total.

Multiplex PCR for joint amplification of carbapenemase genes of molecular classes A, B, and D.

Here we present a method for joint amplification of genes of carbapenemases of molecular classes A, B, and D for hybridization analysis on DNA microarrays. Using new-generation DNA polymerase KAPA2G Fast (KAPA Biosystems, USA) together with optimization of the conditions for the multiplex PCR with 20 primer pairs allowed us to carry out joint amplification of full-length genes of seven different types of carbapenemases (KPC, VIM, IMP, SPM, SIM, GIM, and OXA) with simultaneous inclusion of biotin as a label. Yield of the labeled PCR product sufficient for further analysis by microarray hybridization was achieved 40 min after the start of the reaction. This reduced the total duration of DNA identification techniques, including sample preparation stage, to 4 h. The method for gene identification by DNA microarrays with the improved stage of amplification of specific carbapenemase genes was tested with clinical strains of gram-negative bacteria Pseudomonas aeruginosa, Acinetobacter baumannii, and Enterobacteriaceae spp. with different sensitivity towards carbapenems according to phenotyping tests. All clinical strains of A. baumannii resistant to carbapenems were found to have genes of OXA-type carbapenemases (subtypes OXA-51, OXA-23, OXA-40, and OXA-58), and clinical strains of P. aeruginosa resistant to carbapenems were found to possess the gene of VIM-type metallo-beta-lactamase (subtype VIM-2). When testing clinical strains sensitive to carbapenems, carbapenemase genes were not detected. Thus, the method of identifying carbapenemase genes on DNA microarrays is characterized by high accuracy and can be used in clinical microbiology laboratories for express diagnostics of resistance to carbapenems.

[Enzyme-linked immunosorbent assay for determination of cyclosporin A in whole blood].

A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range 60-1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radio-immunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.983, respectively. The developed test system is stable for at least 9 months when stored at 4 degrees C and can be used in clinical practice.

Recombinant Production of Horseradish Peroxidase Conjugates with Fab Antibodies in Pichia pastoris for Analytical Applications.

Recombinant immunoconjugates of marker enzymes with antigens or antibodies present considerably more advantages than those obtained by conventional methods of chemical synthesis; i.e. they are homogeneous, have a strictly determined stoichiometry, and retain the functional activity of both a marker protein and an antigen/antibody. Based on the pPICZαB shuttle vector, we first managed to obtain a recombinant conjugate of key marker enzyme horseradish peroxidase (HRP) withFabfragments of antibodies against atrazine. The resulting genetic construction allows us to switch to any other antibody sequence, via the simple re-cloning of variable parts and an additional reporter enzyme. Conjugates were successfully produced in thePichia pastorismethylotrophic yeast expression system. The target activity of the conjugates (both enzymatic and antigen-binding) has been demonstrated by ELISA method.

Multiparametric determination of genes and their point mutations for identification of beta-lactamases.

More than half of all currently used antibiotics belong to the beta-lactam group, but their clinical effectiveness is severely limited by antibiotic resistance of microorganisms that are the causative agents of infectious diseases. Several mechanisms for the resistance of Enterobacteriaceae have been established, but the main one is the enzymatic hydrolysis of the antibiotic by specific enzymes called beta-lactamases. Beta-lactamases represent a large group of genetically and functionally different enzymes of which extended-spectrum beta-lactamases (ESBLs) pose the greatest threat. Due to the plasmid localization of the encoded genes, the distribution of these enzymes among the pathogens increases every year. Among ESBLs the most widespread and clinically relevant are class A ESBLs of TEM, SHV, and CTX-M types. TEM and SHV type ESBLs are derived from penicillinases TEM-1, TEM-2, and SHV-1 and are characterized by several single amino acid substitutions. The extended spectrum of substrate specificity for CTX-M beta-lactamases is also associated with the emergence of single mutations in the coding genes. The present review describes various molecular-biological methods used to identify determinants of antibiotic resistance. Particular attention is given to the method of hybridization analysis on microarrays, which allows simultaneous multiparametric determination of many genes and point mutations in them. A separate chapter deals with the use of hybridization analysis on microarrays for genotyping of the major clinically significant ESBLs. Specificity of mutation detection by means of hybridization analysis with different detection techniques is compared.

[Spreading and mechanisms of antimicrobial resistance of microorganisms, producing beta-lactamases. Phenotyping of potential AmpC-beta-lactamases producers of Enterobacteriaceae and molecular mechanisms of resistance to beta-lactams of Enterobacter cloacae strains, isolated in cases of nosocomial infections].

Susceptibility of nosocomial potential AmpC-beta-lactamases producers strains (n=128), isolated from patients admitted to 30 medical centers of 15 various regions of Russia has been investigated. The susceptibility testing was performed by the broth microdilution method. The most active antibacterial agents acting to the investigated strains remained carbapenems (imipenem and meropenem). PCR-based detection of beta-lactamase genes (TEM, SHV, CTX) was investigated in 51 E. cloacae strains. Alone or in various combinations TEM type beta-lactamases have been found in 31 (60.8%) isolates, SHV in 22 (43.1%), and CTX--in 22 (43.1%). There were negative results of TEM, SHV, CTX beta-lactamases genotyping in 13 (25.5%) E. cloacae suspect strains.

[Spreading and mechanisms of antibiotic resistance of microorganisms, producing beta-lactamases. Molecular mechanisms of resistance to beta-lactams of Klebsiella spp. strains, isolated in cases of nosocomial infections].

Antibiotic sensivity of nosocomial Klebsiella spp. strains (n = 212), isolated from patients treated in 30 medical centers of 15 various regions of Russia was investigated. The Klebsiella genus was represented by the following species: Klebsiella pneumoniae ss. pneumoniae--182 (85.8%), Klebsiella pneumoniae ss. ozaenae--1 (0.5%), Klebsiella oxytoca--29 (13.7%) isolates. The most active antibacterial agents against the investigated strains were carbapenems (imipenem and meropenem). Among 3rd generation cephalosporine the lowest MICs were observed for ceftazidime/clavulanic acid (MIC50--0.25 microg/ml, MIC90--64 microg/ml) and cefoperazone/sulbactam (MIC50--16 microg/ml, MIC90--64 microg/ml). Beta-lactamase genes (TEM, SHV, CTX) were detected in 42 Klebsiella pneumoniae ss. pneumoniae strains by PCR. Alone or in various combinations TEM type beta-lactamases have been found in 16 (38.1%) isolates, SHV--in 29 (69%), and CTX--in 27 (64.3%). Combinations of 2 different determinants were detected in 23.8% of the isolates, 3--in 26.2%. There were not isolates producing MBL class B among resistant to carbapenems nosocomial Klebsiella spp. strains.