SARS-CoV-2 Recombination and Coinfection Events Identified in Clinical Samples in Russia.

Recombination is one of the mechanisms of SARS-CoV-2 evolution along with the occurrence of point mutations, insertions, and deletions. Recently, recombinant variants of SARS-CoV-2 have been registered in different countries, and some of them have become circulating forms. In this work, we performed screening of SARS-CoV-2 genomic sequences to identify recombination events and co-infections with various strains of the SARS-CoV-2 virus detected in Russia from February 2020 to March 2022. The study included 9336 genomes of the COVID-19 pathogen obtained as a result of high-throughput sequencing on the Illumina platform. For data analysis, we used an algorithm developed by our group that can identify viral recombination variants and cases of co-infections by estimating the frequencies of characteristic substitutions in raw read alignment files and VCF files. The detected cases of recombination were confirmed by alternative sequencing methods, principal component analysis, and phylogenetic analysis. The suggested approach allowed for the identification of recombinant variants of strains BA.1 and BA.2, among which a new recombinant variant was identified, as well as a previously discovered one. The results obtained are the first evidence of the spread of recombinant variants of SARS-CoV-2 in Russia. In addition to cases of recombination we identified cases of coinfection: eight of them contained the genome of the Omicron line as one of the variants, six of them the genome of the Delta line, and two with the genome of the Alpha line.

Genomic epidemiology of SARS-CoV-2 in Russia reveals recurring cross-border transmission throughout 2020.

In 2020, SARS-CoV-2 has spread rapidly across the globe, with most nations failing to prevent or substantially delay its introduction. While many countries have imposed some limitations on trans-border passenger traffic, the effect of these measures on the global spread of COVID-19 strains remains unclear. Here, we report an analysis of 3206 whole-genome sequences of SARS-CoV-2 samples from 78 regions of Russia covering the period before the spread of variants of concern (between March and November 2020). We describe recurring imports of multiple COVID-19 strains into Russia throughout this period, giving rise to 457 uniquely Russian transmission lineages, as well as repeated cross-border transmissions of local circulating variants out of Russia. While the phylogenetically inferred rate of cross-border transmissions was somewhat reduced during the period of the most stringent border closure, it still remained high, with multiple inferred imports that each led to detectable spread within the country. These results indicate that partial border closure has had little effect on trans-border transmission of variants, which helps explain the rapid global spread of newly arising SARS-CoV-2 variants throughout the pandemic.

Synthesis and Biological Evaluation of Novel Dispiro-Indolinones with Anticancer Activity.

Novel variously substituted thiohydantoin-based dispiro-indolinones were prepared using a regio- and diastereoselective synthetic route from 5-arylidene-2-thiohydantoins, isatines, and sarcosine. The obtained molecules were subsequently evaluated in vitro against the cancer cell lines LNCaP, PC3, HCTwt, and HCT(-/-). Several compounds demonstrated a relatively high cytotoxic activity vs. LNCaP cells (IC50 = 1.2-3.5 µM) and a reasonable selectivity index (SI = 3-10). Confocal microscopy revealed that the conjugate of propargyl-substituted dispiro-indolinone with the fluorescent dye Sulfo-Cy5-azide was mainly localized in the cytoplasm of HEK293 cells. P388-inoculated mice and HCT116-xenograft BALB/c nude mice were used to evaluate the anticancer activity of compound 29 in vivo. Particularly, the TGRI value for the P388 model was 93% at the final control timepoint. No mortality was registered among the population up to day 31 of the study. In the HCT116 xenograft model, the compound (170 mg/kg, i.p., o.d., 10 days) provided a T/C ratio close to 60% on day 8 after the treatment was completed. The therapeutic index-estimated as LD50/ED50-for compound 29 in mice was ≥2.5. Molecular docking studies were carried out to predict the possible binding modes of the examined molecules towards MDM2 as the feasible biological target. However, such a mechanism was not confirmed by Western blot data and, apparently, the synthesized compounds have a different mechanism of cytotoxic action.

Genomic Variability of SARS-CoV-2 Omicron Variant Circulating in the Russian Federation during Early December 2021 and Late January 2022.

Analysis of genomic variability of pathogens associated with heightened public health concerns is an opportunity to track transmission routes of the disease and helps to develop more effective vaccines and specific diagnostic tests. We present the findings of a detailed genomic analysis of the genomic variability of the SARS-CoV-2 Omicron variant that spread in Russia between 8 December 2021 and 30 January 2022. We performed phylogenetic analysis of Omicron viral isolates collected in Moscow (n = 589) and downloaded from GISAID (n = 397), and identified that the BA.1 lineage was predominant in Russia during this period. The BA.2 lineage was also identified early in December 2021. We identified three cases of BA.1/BA.2 coinfections and one case of Delta/Omicron coinfection. A comparative genomic analysis of SARS-CoV-2 viral variants that spread in other countries allowed us to identify possible cases of transmission. We also found that some mutations that are quite rare in the Global Omicron dataset have a higher incidence rate, and identified genetic markers that could be associated with ways of Omicron transmission in Russia. We give the genomic variability of single nucleotide variations across the genome and give a characteristic of haplotype variability of Omicron strains in both Russia and around the world, and we also identify them.

Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents.

BACKGROUND: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. METHODS: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. RESULTS: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 µg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. CONCLUSION: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

Identification of pyrrolo-pyridine derivatives as novel class of antibacterials.

A series of 5-oxo-4H-pyrrolo[3,2-b]pyridine derivatives was identified as novel class of highly potent antibacterial agents during an extensive large-scale high-throughput screening (HTS) program utilizing a unique double-reporter system-pDualrep2. The construction of the reporter system allows us to perform visual inspection of the underlying mechanism of action due to two genes-Katushka2S and RFP-which encode the proteins with different imaging signatures. Antibacterial activity of the compounds was evaluated during the initial HTS round and subsequent rescreen procedure. The most active molecule demonstrated a MIC value of 3.35 µg/mL against E. coli with some signs of translation blockage (low Katushka2S signal) and no SOS response. The compound did not demonstrate cytotoxicity in standard cell viability assay. Subsequent structural morphing and follow-up synthesis may result in novel compounds with a meaningful antibacterial potency which can be reasonably regarded as an attractive starting point for further in vivo investigation and optimization.

Substituted Furanocoumarins as Novel Class of Antibacterial Translation Inhibitors.

INTRODUCTION: A variety of organic compounds has been reported to have antibacterial activity. However, antimicrobial resistance is one of the main problems of current anti-infective therapy, and the development of novel antibacterials is one of the main challenges of current drug discovery. METHODS: Using our previously developed dual-reporter High-Throughput Screening (HTS) platform, we identified a series of furanocoumarins as having high antibacterial activity. The construction of the reporter system allows us to differentiate three mechanisms of action for the active compounds: inhibition of protein synthesis (induction of Katushka2S), DNA damaging (induction of RFP) or other (inhibition of bacterial growth without reporter induction). RESULTS: Two primary hit-molecules of furanocoumarin series demonstrated relatively low MIC values comparable to that observed for Erythromycin (Ery) against E. coli and weakly induced both reporters. Dose-dependent translation inhibition was shown using in vitro luciferase assay, however it was not confirmed using C14-test. A series of close structure analogs of the identified hits was obtained and investigated using the same screening platform. Compound 19 was found to have slightly lower MIC value (15.18 µM) and higher induction of Katushka2S reporter in contrast to the parent structures. Moreover, translation blockage was clearly identified using both in vitro luciferase assay and C14 test. The standard cytotoxicity test revealed a relatively low cytotoxicity of the most active molecules. CONCLUSION: High antibacterial activity in combination with low cytotoxicity was demonstrated for a series of furanocoumarins. Further optimization of the described structures may result in novel and attractive lead compounds with promising antibacterial efficiency.

Identification of Novel Antibacterials Using Machine Learning Techniques.

Many pharmaceutical companies are avoiding the development of novel antibacterials due to a range of rational reasons and the high risk of failure. However, there is an urgent need for novel antibiotics especially against resistant bacterial strains. Available in silico models suffer from many drawbacks and, therefore, are not applicable for scoring novel molecules with high structural diversity by their antibacterial potency. Considering this, the overall aim of this study was to develop an efficient in silico model able to find compounds that have plenty of chances to exhibit antibacterial activity. Based on a proprietary screening campaign, we have accumulated a representative dataset of more than 140,000 molecules with antibacterial activity against Escherichia coli assessed in the same assay and under the same conditions. This intriguing set has no analogue in the scientific literature. We applied six in silico techniques to mine these data. For external validation, we used 5,000 compounds with low similarity towards training samples. The antibacterial activity of the selected molecules against E. coli was assessed using a comprehensive biological study. Kohonen-based nonlinear mapping was used for the first time and provided the best predictive power (av. 75.5%). Several compounds showed an outstanding antibacterial potency and were identified as translation machinery inhibitors in vitro and in vivo. For the best compounds, MIC and CC50 values were determined to allow us to estimate a selectivity index (SI). Many active compounds have a robust IP position.

2-Pyrazol-1-yl-thiazole derivatives as novel highly potent antibacterials.

The present report describes our efforts to identify new structural classes of compounds having promising antibacterial activity using previously published double-reporter system pDualrep2. This semi-automated high-throughput screening (HTS) platform has been applied to perform a large-scale screen of a diverse small-molecule compound library. We have selected a set of more than 125,000 molecules and evaluated them for their antibacterial activity. On the basis of HTS results, eight compounds containing 2-pyrazol-1-yl-thiazole scaffold exhibited moderate-to-high activity against ΔTolC Escherichia coli. Minimum inhibitory concentration (MIC) values for these molecules were in the range of 0.037-8 µg ml-1. The most active compound 8 demonstrated high antibacterial potency (MIC = 0.037 µg ml-1), that significantly exceed that measured for erythromycin (MIC = 2.5 µg ml-1) and was comparable with the activity of levofloxacin (MIC = 0.016 µg ml-1). Unfortunately, this compound showed only moderate selectivity toward HEK293 eukaryotic cell line. On the contrary, compound 7 was less potent (MIC = 0.8 µg ml-1) but displayed only slight cytotoxicity. Thus, 2-pyrazol-1-yl-thiazoles can be considered as a valuable starting point for subsequent optimization and morphing.

Development and Evaluation of a One-Step Quantitative RT-PCR Assay for Detection of Lassa Virus.

Lassa fever is a severe viral hemorrhagic illness caused by Lassa virus. Based on estimates, the number of LASV infections ranges from 300,000 to 500,000 cases in endemic areas with a fatality rate of 1%. Development of fast and sensitive tools for the control and prevention of Lassa virus infection as well as for clinical diagnostics of Lassa fever are crucial. Here we reported development and evaluation of a one-step quantitative RT-qPCR assay for the Lassa virus detection - LASV-Fl. This assay is suitable for the detection of lineages I-IV of Lassa virus. The limit of detection of the assay ranged from 103 copies/ml to 105 copies/ml and has 96.4% diagnostic sensitivity, whereas analytical and diagnostic specificities both were 100%. Serum, whole blood and tissue are suitable for use with the assay. The assay contains all the necessary components to perform the analysis, including an armored positive control (ARC+) and an armored internal control (IC). The study was done during the mission of specialized anti-epidemic team of the Russian Federation (SAET) in the Republic of Guinea in 2015-2018. Based on sequencing data, LASV-specific assay was developed using synthetic MS2-phage-based armored RNA particles, RNA from Lassa virus strain Josiah, and further, evaluated in field conditions using samples from patients and Mastomys natalensis rodents.

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform.

AIM AND OBJECTIVE: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. MATERIALS AND METHODS: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. RESULTS: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. CONCLUSION: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products.

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method's application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the "non-amplifiable" samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

Sequencing and genetic characterization of two strains Paramushir virus obtained from the Tyuleniy Island in the Okhotsk Sea (2015).

Paramushir virus belongs to Sakhalin virus genogroup within Orthonairovirus genus and is one of the poorly studied viruses with unknown pathogenicity. At the moment, only one nearly complete sequence of Paramushir virus genome, isolated in 1972, is available. Two new strains of PARV were isolated in 2015 from a sample collected at the Tyuleniy Island in the Okhotsk Sea and sequenced using a combination of high throughput sequencing and specific multiplex PCR. Both strains are closely related to the early sequenced PARV strain LEIV-1149 K. The signs of intersegment reassortment and probable recombination were revealed, which point to a high variability potential of Paramushir virus and may lead to the formation of strains with novel properties, different from those of the predecessors. The new data regarding Paramushir virus can promote a better understanding of the diversity and relations within Orthonairovirus genus and help define intragenic demarcation criteria, which have not yet been established.

The Study of Viral RNA Diversity in Bird Samples Using De Novo Designed Multiplex Genus-Specific Primer Panels.

Advances in the next generation sequencing (NGS) technologies have significantly increased our ability to detect new viral pathogens and systematically determine the spectrum of viruses prevalent in various biological samples. In addition, this approach has also helped in establishing the associations of viromes with many diseases. However, unlike the metagenomic studies using 16S rRNA for the detection of bacteria, it is impossible to create universal oligonucleotides to target all known and novel viruses, owing to their genomic diversity and variability. On the other hand, sequencing the entire genome is still expensive and has relatively low sensitivity for such applications. The existing approaches for the design of oligonucleotides for targeted enrichment are usually involved in the development of primers for the PCR-based detection of particular viral species or genera, but not for families or higher taxonomic orders. In this study, we have developed a computational pipeline for designing the oligonucleotides capable of covering a significant number of known viruses within various taxonomic orders, as well as their novel variants. We have subsequently designed a genus-specific oligonucleotide panel for targeted enrichment of viral nucleic acids in biological material and demonstrated the possibility of its application for virus detection in bird samples. We have tested our panel using a number of collected samples and have observed superior efficiency in the detection and identification of viral pathogens. Since a reliable, bioinformatics-based analytical method for the rapid identification of the sequences was crucial, an NGS-based data analysis module was developed in this study, and its functionality in the detection of novel viruses and analysis of virome diversity was demonstrated.

Small-molecule inhibitors of hepatitis C virus (HCV) non-structural protein 5A (NS5A): a patent review (2010-2015).

INTRODUCTION: Non-structural 5A (NS5A) protein has achieved a considerable attention as an attractive target for the treatment of hepatitis C (HCV). A number of novel NS5A inhibitors have been reported to date. Several drugs having favorable ADME properties and mild side effects were launched into the pharmaceutical market. For instance, daclatasvir was launched in 2014, elbasvir is currently undergoing registration, ledipasvir was launched in 2014 as a fixed-dose combination with sofosbuvir (NS5B inhibitor). Areas covered: Thomson integrity database and SciFinder database were used as a valuable source to collect the patents on small-molecule NS5A inhibitors. All the structures were ranked by the date of priority. Patent holder and antiviral activity for each scaffold claimed were summarized and presented in a convenient manner. A particular focus was placed on the best-in-class bis-pyrrolidine-containing NS5A inhibitors. Expert opinion: Several first generation NS5A inhibitors have recently progressed into advanced clinical trials and showed superior efficacy in reducing viral load in infected subjects. Therapy schemes of using these agents in combination with other established antiviral drugs with complementary mechanisms of action can address the emergence of resistance and poor therapeutic outcome frequently attributed to antiviral drugs.