Publications by authors named "Ilya A Osterman"

52 Publications

Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents.

Antibiotics (Basel) 2021 Apr 23;10(5). Epub 2021 Apr 23.

Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1, 119991 Moscow, Russia.

In the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.
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http://dx.doi.org/10.3390/antibiotics10050489DOI Listing
April 2021

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome.

Antibiotics (Basel) 2021 Apr 5;10(4). Epub 2021 Apr 5.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL-CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome-PTC and peptide exit tunnel.
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http://dx.doi.org/10.3390/antibiotics10040390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8066774PMC
April 2021

Antibacterial activity of noscapine analogs.

Bioorg Med Chem Lett 2021 Apr 21;43:128055. Epub 2021 Apr 21.

Department of Chemistry and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia; Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 143026, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 142290, Russia.

The antibacterial properties of close noscapine analogs have not been previously reported. We used our pDualrep2 double-reporter High Throughput Screening (HTS) platform to identify a series of noscapine derivatives with promising antibacterial activity. The platform is based on RPF (SOS-response/DNA damage) and Katushka2S (inhibition of translation) proteins and simultaneously provides information on antibacterial activity and the mechanism of action of small-molecule compounds against E. coli. The most potent compound exhibited an MIC of 13.5 µM(6.25 µg/ml) and a relatively low cytotoxicity against HEK293 cells (CC = 71 µM, selectivity index: ~5.5). Some compounds from this series induced average Katushka2S reporter signals, indicating inhibition of translation machinery in the bacteria; however, these compounds did not attenuate translation in vitro in a luciferase-based translation assay. The most effective compounds did not significantly arrest the mitotic cycle in HEK293 cells, in contrast to the parent compound in a flow cytometry assay. Several molecules showed activity against clinically relevant gram-negative and gram-positive bacterial strains. Compounds from the discovered series can be reasonably regarded as good templates for further development and evaluation.
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http://dx.doi.org/10.1016/j.bmcl.2021.128055DOI Listing
April 2021

Bioprospecting of Soil-Derived Actinobacteria Along the Alar-Hotan Desert Highway in the Taklamakan Desert.

Front Microbiol 2021 15;12:604999. Epub 2021 Mar 15.

Department of Microbial Chemistry, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.

Taklamakan desert is known as the largest dunefield in China and as the second largest shifting sand desert in the world. Although with long history and glorious culture, the Taklamakan desert remains largely unexplored and numerous microorganisms have not been harvested in culture or taxonomically identified yet. The main objective of this study is to explore the diversity, novelty, and pharmacological potential of the cultivable actinomycetes from soil samples at various sites along the Alar-Hotan desert highway in the Taklamakan desert. A total of 590 actinobacterial strains were recovered by the culture-dependent approach. Phylogenetic analysis based on 16S ribosomal RNA (rRNA) gene sequences unveiled a significant level of actinobacterial diversity with 55 genera distributed in 27 families of 12 orders. Thirty-six strains showed relatively low 16S rRNA similarities (<98.65%) with validly described species, among which four strains had already been characterized as novel taxa by our previous research. One hundred and forty-six actinobacterial isolates were selected as representatives to evaluate the antibacterial activities and mechanism of action by the paper-disk diffusion method and a double fluorescent protein reporter "pDualrep2" system, respectively. A total of 61 isolates exhibited antagonistic activity against the tested "ESKAPE" pathogens, among which seven strains could produce bioactive metabolites either to be able to block translation machinery or to induce SOS-response in the pDualrep2 system. Notably, sp. 16Sb2-4, harboring a promising antibacterial potential with the mechanism of interfering with protein translation, was analyzed in detail to gain deeper insights into its bioactive metabolites. Through ultra-performance liquid chromatography (UPLC)-quadrupole time-of-flight (QToF)-MS/MS based molecular networking analysis and databases identification, four families of compounds (-) were putatively identified. Subsequent bioassay-guided separation resulted in purification of four 16-membered macrolide antibiotics, aldgamycin H (), aldgamycin K (), aldgamycin G (), and swalpamycin B (), and their structures were elucidated by HR-electrospray ionization source (ESI)-MS and NMR spectroscopy. All compounds - displayed antibacterial activities by inhibiting protein synthesis in the pDualrep2 system. In conclusion, this work demonstrates that Taklamakan desert is a potentially unique reservoir of versatile actinobacteria, which can be a promising source for discovery of novel species and diverse bioactive compounds.
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http://dx.doi.org/10.3389/fmicb.2021.604999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005632PMC
March 2021

Multifaceted Mechanism of Amicoumacin A Inhibition of Bacterial Translation.

Front Microbiol 2021 12;12:618857. Epub 2021 Feb 12.

Petersburg Nuclear Physics Institute named by B. P. Konstantinov, NRC "Kurchatov Institute", Gatchina, Russia.

Amicoumacin A (Ami) halts bacterial growth by inhibiting the ribosome during translation. The Ami binding site locates in the vicinity of the E-site codon of mRNA. However, Ami does not clash with mRNA, rather stabilizes it, which is relatively unusual and implies a unique way of translation inhibition. In this work, we performed a kinetic and thermodynamic investigation of Ami influence on the main steps of polypeptide synthesis. We show that Ami reduces the rate of the functional canonical 70S initiation complex (IC) formation by 30-fold. Additionally, our results indicate that Ami promotes the formation of erroneous 30S ICs; however, IF3 prevents them from progressing towards translation initiation. During early elongation steps, Ami does not compromise EF-Tu-dependent A-site binding or peptide bond formation. On the other hand, Ami reduces the rate of peptidyl-tRNA movement from the A to the P site and significantly decreases the amount of the ribosomes capable of polypeptide synthesis. Our data indicate that Ami progressively decreases the activity of translating ribosomes that may appear to be the main inhibitory mechanism of Ami. Indeed, the use of EF-G mutants that confer resistance to Ami (G542V, G581A, or ins544V) leads to a complete restoration of the ribosome functionality. It is possible that the changes in translocation induced by EF-G mutants compensate for the activity loss caused by Ami.
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http://dx.doi.org/10.3389/fmicb.2021.618857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907450PMC
February 2021

Nybomycin inhibits both types of DNA gyrase - fluoroquinolone-sensitive and fluoroquinolone-resistant.

Antimicrob Agents Chemother 2021 Feb 16. Epub 2021 Feb 16.

Department of Chemistry, Faculty of Bioengineering and Bioinformatics and Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119992, Russia.

Bacterial type II topoisomerases, DNA gyrase and topoisomerase IV, are targets of many antibiotics including fluoroquinolones (FQs). Unfortunately, a number of bacterial species easily acquire resistance to FQs by mutations in either DNA gyrase or topoisomerase IV genes. The emergence of resistant pathogenic strains is a global problem in healthcare, therefore, identifying alternative pathways to thwart their persistence is the current frontier in drug discovery. An attractive class of compounds is nybomycins, reported to be "reverse antibiotics" that selectively inhibit growth of some Gram-positive FQ-resistant bacteria by targeting the mutant form of DNA gyrase, while being inactive against wild-type strains with FQ-sensitive gyrases. The strong "reverse" effect was demonstrated only for a few Gram-positive organisms resistant to FQs due to the S83L/I mutation in GyrA subunit of DNA gyrase. However, the activity of nybomycins has not been extensively explored among Gram-negative species. Here, we observed that in Gram-negative ΔtolC strain with enhanced permeability, wild-type gyrase and GyrA S83L mutant, resistant to fluoroquinolones, are both similarly sensitive to nybomycin.
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http://dx.doi.org/10.1128/AAC.00777-20DOI Listing
February 2021

Beilunmycin, a new virginiamycins antibiotic from mangrove-derived sp. 2BBP-J2 and the antibacterial activity by inhibiting protein translation.

J Asian Nat Prod Res 2020 Sep 14:1-9. Epub 2020 Sep 14.

Department of Microbial Chemistry, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.

One new virginiamycin derivative, 'beilunmycin' (), and three known virginiamycin antibiotics, 16-hydroxy-virginiamycin M1 (), virginiamycin M2 (), and virginiamycin M1 (), were isolated from the culture of a mangrove-derived endophytic sp. 2BBP-J2. The structures were characterized on the basis of their spectroscopic data, and the absolute configuration of was established by ECD calculations. Compounds - exhibited antibacterial activities against Gram-positive bacteria, with MIC values in the range of 0.5-16 μg/ml. All the compounds demonstrated strong protein translation-stalling activity, with minimal concentrations detected with pDualrep2 in the range of 1.9-5.9 nmol.
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http://dx.doi.org/10.1080/10286020.2020.1810669DOI Listing
September 2020

A kinase bioscavenger provides antibiotic resistance by extremely tight substrate binding.

Sci Adv 2020 Jun 24;6(26):eaaz9861. Epub 2020 Jun 24.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia.

Microbial communities are self-controlled by repertoires of lethal agents, the antibiotics. In their turn, these antibiotics are regulated by bioscavengers that are selected in the course of evolution. Kinase-mediated phosphorylation represents one of the general strategies for the emergence of antibiotic resistance. A new subfamily of AmiN-like kinases, isolated from the Siberian bear microbiome, inactivates antibiotic amicoumacin by phosphorylation. The nanomolar substrate affinity defines AmiN as a phosphotransferase with a unique catalytic efficiency proximal to the diffusion limit. Crystallographic analysis and multiscale simulations revealed a catalytically perfect mechanism providing phosphorylation exclusively in the case of a closed active site that counteracts substrate promiscuity. AmiN kinase is a member of the previously unknown subfamily representing the first evidence of a specialized phosphotransferase bioscavenger.
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http://dx.doi.org/10.1126/sciadv.aaz9861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314540PMC
June 2020

Tetracenomycin X inhibits translation by binding within the ribosomal exit tunnel.

Nat Chem Biol 2020 10 29;16(10):1071-1077. Epub 2020 Jun 29.

Center of Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo, Russia.

The increase in multi-drug resistant pathogenic bacteria is making our current arsenal of clinically used antibiotics obsolete, highlighting the urgent need for new lead compounds with distinct target binding sites to avoid cross-resistance. Here we report that the aromatic polyketide antibiotic tetracenomycin (TcmX) is a potent inhibitor of protein synthesis, and does not induce DNA damage as previously thought. Despite the structural similarity to the well-known translation inhibitor tetracycline, we show that TcmX does not interact with the small ribosomal subunit, but rather binds to the large subunit, within the polypeptide exit tunnel. This previously unappreciated binding site is located adjacent to the macrolide-binding site, where TcmX stacks on the noncanonical basepair formed by U1782 and U2586 of the 23S ribosomal RNA. Although the binding site is distinct from the macrolide antibiotics, our results indicate that like macrolides, TcmX allows translation of short oligopeptides before further translation is blocked.
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http://dx.doi.org/10.1038/s41589-020-0578-xDOI Listing
October 2020

Mechanism of translation inhibition by type II GNAT toxin AtaT2.

Nucleic Acids Res 2020 09;48(15):8617-8625

Centre for Life Sciences, Skolkovo Institute of Science and Technology, Skolkovo 143025, Russia.

Type II toxin-antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin-antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin-antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specificaly target other if not all individual aminoacyl tRNAs.
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http://dx.doi.org/10.1093/nar/gkaa551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7470980PMC
September 2020

Translation at first sight: the influence of leading codons.

Nucleic Acids Res 2020 07;48(12):6931-6942

Skolkovo Institute of Science and Technology, Skolkovo, Moscow region 143025, Russia.

First triplets of mRNA coding region affect the yield of translation. We have applied the flowseq method to analyze >30 000 variants of the codons 2-11 of the fluorescent protein reporter to identify factors affecting the protein synthesis. While the negative influence of mRNA secondary structure on translation has been confirmed, a positive role of rare codons at the beginning of a coding sequence for gene expression has not been observed. The identity of triplets proximal to the start codon contributes more to the protein yield then more distant ones. Additional in-frame start codons enhance translation, while Shine-Dalgarno-like motifs downstream the initiation codon are inhibitory. The metabolic cost of amino acids affects the yield of protein in the poor medium. The most efficient translation was observed for variants with features resembling those of native Escherichia coli genes.
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http://dx.doi.org/10.1093/nar/gkaa430DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337518PMC
July 2020

Influence of the spacer region between the Shine-Dalgarno box and the start codon for fine-tuning of the translation efficiency in Escherichia coli.

Microb Biotechnol 2020 07 23;13(4):1254-1261. Epub 2020 Mar 23.

Skolkovo Institute of Science and Technology, Moscow, 143025, Russia.

Translation efficiency contributes several orders of magnitude difference in the overall yield of exogenous gene expression in bacteria. In diverse bacteria, the translation initiation site, whose sequence is the primary determinant of the translation performance, is comprised of the start codon and the Shine-Dalgarno box located upstream. Here, we have examined how the sequence of a spacer between these main components of the translation initiation site contributes to the yield of synthesized protein. We have created a library of reporter constructs with the randomized spacer region, performed fluorescently activated cell sorting and applied next-generation sequencing analysis (the FlowSeq protocol). As a result, we have identified sequence motifs for the spacer region between the Shine-Dalgarno box and AUG start codon that may modulate the translation efficiency in a 100-fold range.
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http://dx.doi.org/10.1111/1751-7915.13561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7264876PMC
July 2020

Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the 70S ribosome.

RNA 2020 06 6;26(6):715-723. Epub 2020 Mar 6.

Petersburg Nuclear Physics Institute named by B.P. Konstantinov of NRC "Kurchatov Institute," Gatchina, 188300, Russia.

Macrolides are one of the most successful and widely used classes of antibacterials, which kill or stop the growth of pathogenic bacteria by binding near the active site of the ribosome and interfering with protein synthesis. Dirithromycin is a derivative of the prototype macrolide erythromycin with additional hydrophobic side chain. In our recent study, we have discovered that the side chain of dirithromycin forms lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4 of the 70S ribosome. In the current work, we found that neither the presence of the side chain, nor the additional contact with the ribosome, improve the binding affinity of dirithromycin to the ribosome. Nevertheless, we found that dirithromycin is a more potent inhibitor of in vitro protein synthesis in comparison with its parent compound, erythromycin. Using high-resolution cryo-electron microscopy, we determined the structure of the dirithromycin bound to the translating 70S ribosome, which suggests that the better inhibitory properties of the drug could be rationalized by the side chain of dirithromycin pointing into the lumen of the nascent peptide exit tunnel, where it can interfere with the normal passage of the growing polypeptide chain.
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http://dx.doi.org/10.1261/rna.073817.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7266154PMC
June 2020

Exploitation of Potentially New Antibiotics from Mangrove Actinobacteria in Maowei Sea by Combination of Multiple Discovery Strategies.

Antibiotics (Basel) 2019 Nov 27;8(4). Epub 2019 Nov 27.

Department of Microbial Chemistry, Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.

Rediscovery of known antibiotics from actinobacteria, especially , has become a bottleneck issue. Nowadays, more specific identification and dereplication could be acquired by a combination of modern analytic techniques with various databases. In this study, 261 actinobacterial strains were isolated from 8 mangrove soil samples by culture-dependent method. A total of 83 strains were selected to evaluate antibacterial activities and mechanisms by disc diffusion method and a unique double fluorescent protein reporter system (pDualrep2), respectively. Thirty-two strains exhibited antagonistic activity against at least one of the "ESKAPE" pathogens. Four strains (B475, B486, B353, and B98) showed strong inhibitory activity against Gram-positive bacteria and induced DNA damage SOS response. One strain (B704) exhibited inhibitory activity against several pathogens and induced attenuation-based translational inhibitors reporter. Seven members of quinoxaline-type antibiotics including quinomycin A, quinomycin monosulfoxide, and other five putative new analogues were found from the culture broth of strain B475 by a combination of anti-MRSA guide, HPTLC, HPLC-UV, and UPLC-UV-HRESIMS/MS analysis, Chemspider searching, and MS/MS-based molecular networking analysis. In conclusion, this study not only demonstrated that mangrove is a rich source of actinobacteria with the potentially new antibiotics but showed rapid dereplication of known antibiotics in the early stage can improve efficiency for the discovery of new antibiotics.
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http://dx.doi.org/10.3390/antibiotics8040236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6963217PMC
November 2019

Structure of ribosome-bound azole-modified peptide phazolicin rationalizes its species-specific mode of bacterial translation inhibition.

Nat Commun 2019 10 8;10(1):4563. Epub 2019 Oct 8.

Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia.

Ribosome-synthesized post-translationally modified peptides (RiPPs) represent a rapidly expanding class of natural products with various biological activities. Linear azol(in)e-containing peptides (LAPs) comprise a subclass of RiPPs that display outstanding diversity of mechanisms of action while sharing common structural features. Here, we report the discovery of a new LAP biosynthetic gene cluster in the genome of Rhizobium Pop5, which encodes the precursor peptide and modification machinery of phazolicin (PHZ) - an extensively modified peptide exhibiting narrow-spectrum antibacterial activity against some symbiotic bacteria of leguminous plants. The cryo-EM structure of the Escherichia coli 70S-PHZ complex reveals that the drug interacts with the 23S rRNA and uL4/uL22 proteins and obstructs ribosomal exit tunnel in a way that is distinct from other compounds. We show that the uL4 loop sequence determines the species-specificity of antibiotic action. PHZ expands the known diversity of LAPs and may be used in the future as biocontrol agent for agricultural needs.
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http://dx.doi.org/10.1038/s41467-019-12589-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783444PMC
October 2019

Substituted Furanocoumarins as Novel Class of Antibacterial Translation Inhibitors.

Comb Chem High Throughput Screen 2019 ;22(6):400-410

Lomonosov Moscow State University, Chemistry Dept, Leninskie Gory, Building 1/3, GSP-1, Moscow 119991, Russian Federation.

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.
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http://dx.doi.org/10.2174/1386207322666190723110539DOI Listing
August 2020

Oligoglutamylation of E. coli ribosomal protein S6 is under growth phase control.

Biochimie 2019 Dec 11;167:61-67. Epub 2019 Sep 11.

Lomonosov Moscow State University, Department of Chemistry, A.N. Belozersky Institute of Physico-Chemical Biology, Moscow, 119992, Russia; Skolkovo Institute for Science and Technology, Moscow, 143025, Russia. Electronic address:

Ribosomal protein S6 in Escherichia coli is modified by ATP-dependent glutamate ligase RimK. Up to four glutamate residues are added to the C-terminus of S6 protein. In this work we demonstrated that unlike the majority of ribosome modifications in E. coli, oligoglutamylation of S6 protein is regulated and happens only in the stationary phase of bacterial culture. Only S6 protein incorporated into assembled small ribosomal subunits, but not newly made free S6 protein is a substrate for RimK protein. Overexpression of the rimK gene leads to the modification of S6 protein even in the exponential phase of bacterial culture. Thus, it is unlikely that any stationary phase specific factor is needed for the modification. We propose a model that S6 modification is regulated solely via the rate of ribosome biosynthesis at limiting concentration of RimK enzyme.
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http://dx.doi.org/10.1016/j.biochi.2019.09.008DOI Listing
December 2019

Identification of Novel Antibacterials Using Machine Learning Techniques.

Front Pharmacol 2019 27;10:913. Epub 2019 Aug 27.

Department of Chemistry, Lomonosov Moscow State University, Moscow, Russia.

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 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 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 assessed in the same assay and under the same conditions. This intriguing set has no analogue in the scientific literature. We applied six 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 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 and . For the best compounds, MIC and CC values were determined to allow us to estimate a selectivity index (SI). Many active compounds have a robust IP position.
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http://dx.doi.org/10.3389/fphar.2019.00913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719509PMC
August 2019

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

J Antibiot (Tokyo) 2019 11 29;72(11):827-833. Epub 2019 Jul 29.

Lomonosov Moscow State University, Chemistry Department, Leninskie Gory, Building 1/3, GSP-1, 119991, Moscow, Russia.

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. The most active compound 8 demonstrated high antibacterial potency (MIC = 0.037 μg ml), that significantly exceed that measured for erythromycin (MIC = 2.5 μg ml) and was comparable with the activity of levofloxacin (MIC = 0.016 μg ml). 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) but displayed only slight cytotoxicity. Thus, 2-pyrazol-1-yl-thiazoles can be considered as a valuable starting point for subsequent optimization and morphing.
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http://dx.doi.org/10.1038/s41429-019-0211-yDOI Listing
November 2019

Studies on Antibacterial Activity and Diversity of Cultivable Actinobacteria Isolated from Mangrove Soil in Futian and Maoweihai of China.

Evid Based Complement Alternat Med 2019 9;2019:3476567. Epub 2019 Jun 9.

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China.

Mangrove is a rich and underexploited ecosystem with great microbial diversity for discovery of novel and chemically diverse antimicrobial compounds. The goal of the study was to explore the pharmaceutical actinobacterial resources from mangrove soil and gain insight into the diversity and novelty of cultivable actinobacteria. Consequently, 10 mangrove soil samples were collected from Futian and Maoweihai of China, and the culture-dependent method was employed to obtain actinobacteria. A total of 539 cultivable actinobacteria were isolated and distributed in 39 genera affiliated to 18 families of 8 orders by comparison analysis of partial 16S rRNA gene sequences. The dominant genus was (16.0 %), followed by (14.5 %), (14.3 %), and (11.9 %). Other 35 rare actinobacterial genera accounted for minor proportions. Notably, 11 strains showed relatively low 16S rRNA gene sequence similarities (< 98.65 %) with validly described species. Based on genotypic analyses and phenotypic characteristics, 115 out of the 539 actinobacterial strains were chosen as representative strains to test their antibacterial activities against "ESKAPE" bacteria by agar well diffusion method and antibacterial mechanism by the double fluorescent protein reporter system. Fifty-four strains in 23 genera, including 2 potential new species, displayed antagonistic activity in antibacterial assay. Meanwhile, 5 strains in 3 genera exhibited inhibitory activity on protein biosynthesis due to ribosome stalling. These results demonstrate that cultivable actinobacteria from mangrove soil are potentially rich sources for discovery of new antibacterial metabolites and new actinobacterial taxa.
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http://dx.doi.org/10.1155/2019/3476567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590530PMC
June 2019

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

Curr Drug Discov Technol 2020 ;17(5):716-724

Lomonosov Moscow State University, Chemistry Dept, Leninskie gory, Building 1/3, GSP-1, Moscow, 119991, Russian Federation.

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.
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http://dx.doi.org/10.2174/1570163816666190603095521DOI Listing
January 2020

eIF4G2 balances its own mRNA translation via a PCBP2-based feedback loop.

RNA 2019 07 22;25(7):757-767. Epub 2019 Apr 22.

Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Leninskie Gory, Moscow 119992, Russia.

Poly(rC)-binding protein 2 (PCBP2, hnRNP E2) is one of the most abundant RNA-binding proteins in mammalian cells. In humans, it exists in seven isoforms, which are assumed to play similar roles in cells. The protein is shown to bind 3'-untranslated regions (3'-UTRs) of many mRNAs and regulate their translation and/or stability, but nothing is known about the functional consequences of PCBP2 binding to 5'-UTRs. Here we show that the PCBP2 isoform f interacts with the 5'-UTRs of mRNAs encoding eIF4G2 (a translation initiation factor with a yet unknown mechanism of action, also known as DAP5) and Cyclin I, and inhibits their translation in vitro and in cultured cells, while the PCBP2 isoform e only affects Cyclin I translation. Furthermore, eIF4G2 participates in a cap-dependent translation of the PCBP2 mRNA. Thus, PCBP2 and eIF4G2 seem to regulate one another's expression via a novel type of feedback loop formed by the translation initiation factor and the RNA-binding protein.
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http://dx.doi.org/10.1261/rna.065623.118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6573783PMC
July 2019

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

Comb Chem High Throughput Screen 2019 08;22(5):346-354

Lomonosov Moscow State University, Chemistry Dept, Leninskie gory, Building 1/3, GSP-1, Moscow, 119991, Russian Federation.

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.
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http://dx.doi.org/10.2174/1386207322666190412165316DOI Listing
August 2019

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

Mol Divers 2020 Feb 4;24(1):233-239. Epub 2019 Apr 4.

Chemistry Department, Lomonosov Moscow State University, Leninskie Gory, Building 1/3 GSP-1, Moscow, Russian Federation, 119991.

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.
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http://dx.doi.org/10.1007/s11030-019-09946-3DOI Listing
February 2020

Structure of Dirithromycin Bound to the Bacterial Ribosome Suggests New Ways for Rational Improvement of Macrolides.

Antimicrob Agents Chemother 2019 06 24;63(6). Epub 2019 May 24.

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, USA

Although macrolides are known as excellent antibacterials, their medical use has been significantly limited due to the spread of bacterial drug resistance. Therefore, it is necessary to develop new potent macrolides to combat the emergence of drug-resistant pathogens. One of the key steps in rational drug design is the identification of chemical groups that mediate binding of the drug to its target and their subsequent derivatization to strengthen drug-target interactions. In the case of macrolides, a few groups are known to be important for drug binding to the ribosome, such as desosamine. Search for new chemical moieties that improve the interactions of a macrolide with the 70S ribosome might be of crucial importance for the invention of new macrolides. For this purpose, here we studied a classic macrolide, dirithromycin, which has an extended (2-methoxyethoxy)-methyl side chain attached to the C-9/C-11 atoms of the macrolactone ring that can account for strong binding of dirithromycin to the 70S ribosome. By solving the crystal structure of the 70S ribosome in complex with dirithromycin, we found that its side chain interacts with the wall of the nascent peptide exit tunnel in an idiosyncratic fashion: its side chain forms a lone pair-π stacking interaction with the aromatic imidazole ring of the His69 residue in ribosomal protein uL4. To our knowledge, the ability of this side chain to form a contact in the macrolide binding pocket has not been reported previously and potentially can open new avenues for further exploration by medicinal chemists developing next-generation macrolide antibiotics active against resistant pathogens.
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http://dx.doi.org/10.1128/AAC.02266-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6535518PMC
June 2019

Nybomycin-producing Streptomyces isolated from carpenter ant Camponotus vagus.

Biochimie 2019 May 21;160:93-99. Epub 2019 Feb 21.

Skolkovo Institute of Science and Technology, Skolkovo, Moscow Region, 143025, Russia; Department of Chemistry and A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119992, Russia. Electronic address:

A novel strain of Actinomycetes was isolated from the body of an ant (Camponotus vagus Scopoli) and its genetic and morphological properties were characterized. The 16S rDNA gene sequence analysis of the isolate revealed its high phylogenetic relationship with type strains of Streptomyces violaceochromogenes NBRC 13100. As a result of antimicrobial activity assessment, it was found that the fermentation broth of the isolated strain both inhibited the growth and induced the SOS response in E. coli BW25113 ΔtolC strain cells. Using bioassay-guided fractionation, mass spectrometric and NMR analyses we identified the active compound to be nybomycin, a previously described antibiotic. Here we report for the first time Streptomyces producer of nybomycin in association with carpenter ants and demonstrate cytotoxic activity of nybomycin against human cell lines.
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http://dx.doi.org/10.1016/j.biochi.2019.02.010DOI Listing
May 2019

Synthesis and evaluation of biological activity for dual-acting antibiotics on the basis of azithromycin and glycopeptides.

Bioorg Med Chem Lett 2019 01 20;29(2):276-280. Epub 2018 Nov 20.

Department of Organic Chemistry, University of Debrecen, Egyetem ter 1, Debrecen, 4032, Hungary.

One of the promising directions of the combined approach is the design of dual-acting antibiotics - heterodimeric structures on the basis of antimicrobial agents of different classes. In this study a novel series of azithromycin-glycopeptide conjugates were designed and synthesized. The structures of the obtained compounds were confirmed using NMR spectroscopy and mass spectrometry data including MS/MS analysis. All novel hybrid antibiotics were found to be either as active as azithromycin and vancomycin against Gram-positive bacterial strains or have superior activity in comparison with their parent antibiotics. One compound, eremomycin-azithromycin conjugate 16, demonstrated moderate activity against Enterococcus faecium and Enterococcus faecalis strains resistant to vancomycin, and equal to vancomycin's activity for the treatment of mice with Staphylococcus aureus sepsis.
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http://dx.doi.org/10.1016/j.bmcl.2018.11.038DOI Listing
January 2019

Synthesis and evaluation of biological activity of benzoxaborole derivatives of azithromycin.

J Antibiot (Tokyo) 2019 01 12;72(1):22-33. Epub 2018 Oct 12.

Department of Chemistry and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Leninskie Gory, Moscow, Russia.

Novel benzoxaborole derivatives of azithromycin in which benzoxaborole residue is attached to the 4″-hydroxy-group of azithromycin have been synthesized. Antibacterial activity of synthesized derivatives in comparison with azithromycin was tested on a panel of Gram-positive and Gram-negative bacterial strains. All the investigated compounds demonstrated broad spectrum of antibacterial activity being in general more active against Gram-positive strains. New benzoxaborole derivatives of azithromycin demonstrated high activity against Streptococcus pyogenes ATCC 19615 and Propionibacterium acnes ATCC 6919 strains. Some of the new compounds were more active than azithromycin against Streptococcus pneumoniae ATCC 49619 strain or Enterococcus faecium strains. Using a reporter construct created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon pRFPCER-TrpL2A it has been demonstrated that the mechanism of action of azithromycin analogs is blocking nascent peptide in ribosome tunnel.
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http://dx.doi.org/10.1038/s41429-018-0107-2DOI Listing
January 2019

Ultrahigh-throughput functional profiling of microbiota communities.

Proc Natl Acad Sci U S A 2018 09 4;115(38):9551-9556. Epub 2018 Sep 4.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520;

Microbiome spectra serve as critical clues to elucidate the evolutionary biology pathways, potential pathologies, and even behavioral patterns of the host organisms. Furthermore, exotic sources of microbiota represent an unexplored niche to discover microbial secondary metabolites. However, establishing the bacterial functionality is complicated by an intricate web of interactions inside the microbiome. Here we apply an ultrahigh-throughput (uHT) microfluidic droplet platform for activity profiling of the entire oral microbial community of the Siberian bear to isolate strains demonstrating antimicrobial activity against Genome mining allowed us to identify antibiotic amicoumacin A (Ami) as responsible for inhibiting the growth of Proteomics and metabolomics revealed a unique mechanism of self-resistance to Ami, based on a subtle equilibrium of its deactivation and activation by kinase AmiN and phosphatase AmiO, respectively. We developed uHT quantitative single-cell analysis to estimate antibiotic efficacy toward different microbiomes and used it to determine the activity spectra of Ami toward human and Siberian bear microbiota. Thus, uHT microfluidic droplet platform activity profiling is a powerful tool for discovering antibiotics and quantifying external influences on a microbiome.
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http://dx.doi.org/10.1073/pnas.1811250115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156654PMC
September 2018