RU2810089C2 - Method of using binary system consisting of agents of various natures to develop new class of antibacterial medicinal products - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of using a binary system to increase the effectiveness of an antibacterial agent. The method involves exposing a bacterial cell in a random order to a binary system, which consists of an antibacterial agent capable of binding to the active center of bacterial RNA polymerase (bRNAP) and an agent that increases the expression and/or activity of the elongation factor in the bacterium.

EFFECT: obtaining agents with antibacterial effect based on enhancing the action of the bRNAP inhibitor while increasing the expression and/or activity of the bRNAP elongation factor.

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Description

TECHNICAL FIELD

[001] The present invention relates to the fields of biochemistry, molecular biology and medicine. More specifically, the present invention also provides methods for the preparation and use of binary transcription inhibition systems. The new methods for inhibiting transcription according to the present invention can be used in medicine, in particular to eliminate bacterial and viral pathogens, and also, for example, in biotechnology.

BACKGROUND OF THE ART

[002] The use of antibiotics is the main approach to eliminating infections. Widespread use of antibiotics in turn leads to the emergence and spread of multidrug resistance in pathogenic gram-positive (G+) (for example, Staphylococcus aureus, Enterococcus spp., Mycobacteriaceae spp and Streptococcus pneumoniae) and gram-negative (G-) pathogenic bacteria (for example, Escherichia coli/ , Enterobacter spp., Salmonella spp., Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa), as well as pathogenic fungi such as Candida spp. Accordingly, with the emergence of antibiotic resistance in existing pathogens and the discovery of new pathogens, there is a need to develop new classes of antimicrobials, particularly antibacterial and antiviral drugs, with new mechanisms of action to complement and replace currently available agents. In addition, there is a need to optimize the action of known drugs in terms of increasing their effectiveness and reducing undesirable side effects.

[003] RNA polymerase is a promising target for inhibition for the control and elimination of unwanted pathogens. Several classes of agents are known that are capable of inhibiting the action of bacterial RNA polymerase, including naturally occurring substances and synthesized molecules that are capable of binding to RNA polymerase and inhibiting various stages of the transcription process. Many of these agents also demonstrate inhibitory effects on RNA polymerases of viruses and fungi. New approaches to transcription inhibition can also be adapted to produce new and increase the effectiveness of existing antibacterial and antiviral agents, as well as for applications in biotechnology and medicine.

[004] The object of the present invention is to address the need for new approaches to inhibiting the activity of RNA polymerases and to overcome at least one of the disadvantages of the prior art.

BRIEF DESCRIPTION OF THE INVENTION

[005] This application provides new approaches for inhibiting RNA polymerase. These approaches can be used to inhibit transcription in bacteria, eukaryotes and inhibit the activity of RNA polymerases that viruses use.

[006] The present invention is generally based on the fact that the inhibitory effect of a compound that binds to RNA polymerase on transcription is exerted or enhanced by increasing the amount or activity of the elongation factor (transcription factor) that is used by said RNA polymerase (RNAP).

[007] Accordingly, the present invention relates to the use of agents that increase the expression and/or activity of the RNAP elongation factor to enhance or cause transcriptional inhibition by a compound that is capable of binding to RNAP.

[008] This application also provides methods for preparing drugs that may be compositions and combinations for inhibiting RNAP.

[009] In one embodiment, the method of the present invention includes providing a compound that is capable of binding to RNAP; providing an agent that increases the expression and/or activity of an elongation factor of said RNAP; contacting an effective amount of said compound and an effective amount of said agent with RNAP or a cell in which RNAP is present, under conditions that allow said compound to bind to RNAP and increase the expression and/or activity of the elongation factor of said RNAP under the influence of said agent, and determining the effect that the combination of said compound and agent has on the activity of the RNAP, wherein the combination is selected as the agent for inhibiting transcription if the effect of the combination on the activity is negative (inhibitory) and stronger (pronounced) than the effect of the specified compound alone on the activity of the specified RNAP.

[0010] In one embodiment of the present invention, the transcription elongation step is inhibited.

[0011] In some embodiments, the agent is an agent or effect.

[0012] In some embodiments, the RNAP is bacterial RNAP (bRNAP), the elongation factors are bRNAP elongation factors, and the combinations of the present invention have an antibacterial effect, such as a negative effect on the growth and/or viability of the bacterium .

[0013] In some embodiments, the RNAP is viral RNAP (vRNAP), the elongation factors are elongation factors used by vRNAP, such as viral or eukaryotic cell proteins, and the inventive combinations are antiviral.

[0014] In some embodiments, the RNAP is fungal RNAP (gRNAP), the elongation factors are gRNAP elongation factors, and the combinations of the present invention have an antimycotic effect, such as a negative effect on the growth and/or viability of the fungus. .

[0015] In some embodiments of the present invention, these combinations are prepared in the form of preparations, for example, pharmaceutical compositions, which also contain pharmaceutically acceptable carriers and excipients. Examples of carriers are known in the art and are described herein. In some embodiments of the present invention, the drug or pharmaceutical composition is adapted to deliver the composition to a subject in an area of presence of an unwanted microorganism (area of infection), for example, in a specific organ, tissue, area of the body, membrane, area of injury, wound.

[0016] Also provided herein are screening methods to find new agents for inhibiting RNA polymerase and/or transcription. These methods include determining the inhibitory effect of a plurality of substances, for example, small molecules, (poly)peptides, nucleotides having the ability to bind to RNAP, when the inhibitory effect of these substances is determined in combination with exposure to an effective amount of an agent that increases the expression and/or activity of the factor elongation (transcription factor) of the specified RNAP in accordance with the present invention. Those substances from the specified set of substances, the inhibitory effect of which is manifested or enhanced by increasing the expression and/or activity of the RNAP elongation factor, are used as RNAP inhibitors in combination with the specified agent.

[0017] In accordance with one embodiment of the present invention, screening methods for finding new antibacterial agents are also provided. These methods involve determining the antibacterial effect of a variety of substances, for example, small molecules, (poly)peptides, nucleotides having the ability to bind to bRNAP, when the inhibitory effect of these substances is determined in combination with an agent that increases the expression and/or activity of the bRNAP elongation factor (bacterial transcription factor) in accordance with the present invention. Those substances from the specified set of substances, the antibacterial effect of which appears or increases with an increase in the expression and/or activity of the bRNAP elongation factor, are used as antibacterial drugs (agents) in combination with the specified agent.

[0018] In accordance with one embodiment of the present invention, screening methods for finding new antiviral agents are also provided. These methods involve determining the antiviral effect of multiple substances, for example, small molecules, (poly)peptides, nucleotides, having the ability to bind to vRNAP, when the inhibitory effect of these substances is determined in combination with an agent that increases the expression and/or activity of the elongation factor of vRNAP (for example eukaryotic transcription factor) in accordance with the present invention. Those substances from the specified set of substances, the inhibitory effect of which on vRNAP appears or increases with an increase in the expression and/or activity of the vRNAP elongation factor, are used as antiviral drugs (agents) in combination with the specified agent.

[0019] In some of its aspects, the present invention discloses approaches to identifying bRNAP elongation factors, as well as their analogues, homologues, mutants and variants, the activity of which, in combination with a compound that is capable of binding to bRNAP, provides or enhances the antibacterial effect of the specified compound and/or such a combination. In other embodiments, the present invention discloses approaches to identifying vRNAP elongation factors, as well as analogs, homologs, mutants, and variants thereof, the activity of which, in combination with a compound that is capable of binding to vRNAP, provides the antiviral effect of such combination.

[0020] The present invention also provides transcription inhibitory agents, the effect of which is to enhance or exert an inhibitory effect on transcription of a compound that binds to an RNA polymerase by increasing the amount or activity of an elongation factor (transcription factor) that is used by said RNA polymerase (RNAP), including those obtained using the methods of the present invention.

[0021] The RNAP inhibition approaches underlying the present invention have a variety of applications, including the elimination of unwanted pathogens, including bacteria, fungi and viruses, as well as the inhibition of their development, the control of expression systems in biotechnology, and the modulation of transcription and RNA synthesis in eukarytic systems.

[0022] All technical and/or scientific terms used in this specification have the meaning commonly accepted by those skilled in the art to which the invention relates. In some cases, additional definitions are given to the terms used, which may also be used in interpreting the spirit of the present invention. Although methods and materials similar or equivalent to those described herein may be used in implementing or exploring embodiments of the invention, some exemplary methods and/or materials are described below. In case of conflict, the description of this application, including definitions, should be considered. In addition, all materials, methods and examples presented herein are purely illustrative and do not impose any restrictions on the inventive concept of the authors of the present invention.

[0023] Additional embodiments and the full scope of the present invention will become apparent upon examination of the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are provided for illustration purposes only, as various changes and modifications without departing from the spirit and scope of the invention will be apparent to those skilled in the art. field of technology after reading the description of this application.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A shows a diagram of the experimental setup and microfluidic chip with a piezoelectric element for an experiment using ACC: 1 - DNA molecule, 2 - polystyrene microsphere, 3 - reference microsphere, 4 - AFS chip, 5 - lens, 6 - beam splitter, 7 - LED, 8 - photosensitive matrix, 9 - piezoelectric element, 10 - glass, 11 - mirror, 12 - liquid. Figure 1B shows reference and free microspheres, scale bar 5 µm.

[0025] Figure 2A is an illustration of the experimental design. Elongation profiles of individual RNAP transcription complexes were recorded using a commercial AFS setup (Lumicks B.V., Amsterdam). The digoxigenin-modified end of the DNA molecule was attached to the anti-digoxigenin-coated surface of the AFS chip. A polystyrene bead coated with streptavidin was attached to a stalled biotin-modified RNA polymerase. A constant tension was maintained with a force of 4–6 pN by applying voltage to the piezoelectric element. RNA elongation under the influence of RNAP was resumed by applying a buffer containing ribonucleotides to the AFS chip, and the dynamics of RNAP translocation were recorded by tracking the position of the microsphere in 3D; 1 - DNA molecule, 2 - polystyrene microsphere, 3 - reference microsphere, 9 - piezoelectric element, 10 - glass, 13 - RNA molecule, 14 - bRNAP; Figure 2B shows typical elongation profiles of single RNAPs in the absence of the influence of inhibitory factors.

[0026] Figure 3A shows a typical histogram of instantaneous velocities during ACC; Figure 3B shows the Gaussian function approximation of the velocity distribution after subtracting the noise signal, corresponding to the true instantaneous velocity distribution.

[0027] Figure 4 shows that streptolidigin induces long pauses in RNA synthesis: FIG. 4A. Typical elongation profiles are shown for individual RNAP transcribing complexes in the presence of 1 mM NTP in the absence (15) and with the addition (16) of 10 μM streptolidigin; Fig. 4B. Instantaneous and average elongation rates for individual RNAP molecules at 1 mM NTP in the absence (unshaded bars) and with the addition (shaded bars) of 10 μM streptolidigin p-value: *=≤0.001; n.s.=unreliable.

[0028] Figure 5 shows that streptolidigin affects the length of intermediates synthesized by RNAP: Typical elongation profiles for individual RNAP transcription complexes are shown: FIG. 5A - elongation profiles under control conditions: 1 mM NTP (17), 200 μM ITP with 1 mM NTP (18), 400 μM NTP (19); Fig. 5B - Representative elongation profiles for individual RNAPs in the presence of 10 μM streptolidigin: 1 mM NTP (20), 200 μM ITP with 1 mM NTP (21), 400 μM NTP (22); Fig. 5B - Instantaneous and average elongation rates: (23) - 1 mM NTP, (24) - 10 µM STL, 1 mM NTP, (25) - 1 mM NTP, 200 µM ITP, (26) - 10 µM STL, 1 mM NTP, 200 µM ITP, (27) - 400 µM NTP, (28) - 10 µM STL, 400 µM NTP. Fig. 5G - Number of long pauses (>20 s) per lengthening profile (elongation); Fig. 5D. Number of short pauses (2.5 s - 20 s) per lengthening profile (elongation); for all cases p-value: *=≤0.001; **=≤0.005; ***=≤0.01; ****=≤0.05; n.s.=unreliable.

[0029] Figure 6 shows typical elongation profiles for individual RNAP transcription complexes. Fig. 6A. elongation profiles under control conditions: no streptolidigin: 1 mM NTP (29), 1 mM NTP in the presence of transcription factors 5 μM GreA (30) or 5 μM GreB (31); Fig. 6B. elongation profiles in the presence of 10 µM streptolidigin: 1 mM NTP (32), 1 mM NTP + 5 µM GreA (33), 1 mM NTP + 5 µM GreB (34); Fig. 6B. Instantaneous and average elongation rates: (35) - 1 mM NTP, (36) - 10 μM STL, 1 mM NTP, (37) - 1 mM NTP, 200 μM ITP, (38) - 10 μM STL, 1 mM NTP, 200 µM ITP, (39) - 400 µM NTP, (40) - 10 µM STL, 400 µM NTP; Fig. 6G. Number of long pauses (>20 s) per lengthening profile. For all cases p-value: *=≤0.001; **=≤0.005; ***=≤0.01; ****=≤0.05; n.s.=unreliable.

[0030] Figure 7 shows graphs of optical density versus time for bacterial cell cultures supplemented with 100 μM streptolidigin and induced expression of GreB, as well as the corresponding controls: (41) - - polymexine, (42) - + polymexine, (43) - -polymexin, +STL, (44) - +polymexin, +STL, (45) - +polymexin, +GreB, (46) - +polymexin, +GreB, +STL.

[0031] Figure 8 provides examples of the effect of a number of antibiotics on elongation profiles for individual RNAP transcription complexes. The curves correspond to elongation profiles at different concentrations and types of antimicrobial agents (microcin J25, klebsidin and acinetodin) and in the absence of inhibitors: (47) - 0 µM, (48) - 5 µM acinetodin, (49) - 25 µM acinetodin, (50) - 2.5 µM klebsidin, (51) - 2.5 µM microcin J25, (47) - 5 µM klebsidin, (47) - 2.5 µM microcin J25.

[0032] Figure 9 shows the inhibition of cell growth by klebsidin and acinetodin: (54) - Arabinose control -, (55) - Acinetodin gene, arabinose -, (56) - Klebsidin gene, arabinose -, (57) - Arabinose control +, (58) - Acinetodin gene, arabinose+, (59) - Klebsidin gene, arabinose+.

DETAILED DESCRIPTION OF THE INVENTION

[0033] Abbreviations and abbreviations used in this application may include:

BD - Becton Dickinson

BSA - bovine serum albumin

RNAP - DNA-dependent RNA polymerase; RNA polymerase

bRNAP - bacterial RNA polymerase

vRNAP - viral RNA polymerase

gRNAP - fungal RNA polymerase

FET - transcription elongation factor

STL - streptolidigin

NTP - nucleotide triphosphate

CDS - coding sequence

CFU - colony forming units

PFU - plaque forming units

µM - microMol

mm - milliMol

ACC - acoustic force spectroscopy

PCR - polymerase chain reaction

pN - piconewtons

E. coli - Escherichia coli

ATP, GTP, CTP - adenine-5'-, guanosine-5'-, cyotsine-5'-triphosphate

AfU - adenine phosphouracil

ITP - inosine triphosphate

DMSO - dimethyl sulfoxide

NTP - nucleoside triphosphate

TP - trigger loop

VN-α - spiral bridge

BSA - bovine serum albumin

ME - international unit

NCBI - National Center for Biotechnology Information

NIBSC - National Institute of Biological Standards and Control

PBS - phosphate buffered saline

PCR - polymerase chain reaction

PFA - paraformaldehyde

WHO - World Health Organization

[0034] It should be noted that as used in this application and in the accompanying claims, the singular forms also include the plural forms unless otherwise indicated. Thus, for example, a reference to the term “reagent” includes one or more different reagents, and a reference to the term “method” includes equivalent steps and methods known to those skilled in the art, which may be modified or substituted for the methods described herein. application. Likewise, the term "cell" includes many cells, including mixtures thereof. Likewise, the use of a “compound” of an “agent”, “action” or “means” to inhibit, provide or obtain the results described in the description herein includes the use of one or more compounds, agents, effects or means according to the present invention to provide the specified result or results, unless otherwise clearly evident from the context.

[0035] Unless otherwise indicated, the term “at least” preceding a row of elements should be understood to refer to each element in the row. Those skilled in the art will recognize or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be covered by the present invention.

[0036] Throughout the specification and claims that follow, the word “comprise” and variations such as “comprises” and “comprising” are intended to include an integer or a step, or a group of integers or steps, but not to the exclusion of any other integer or stage or group of integers or stages unless the context otherwise requires. When used herein, the term “comprising” may be replaced by the term “consisting of,” or sometimes when used herein, the term “having.”

[0037] When used in this application, the term “consisting of” excludes any element, step or ingredient not specified in the claim. When used in this application, the expression “consisting essentially of” does not exclude materials or steps that do not significantly affect the essential and novel features of the claims. Accordingly, a composition consisting essentially of the elements as defined herein does not exclude the presence of trace amounts of impurities due to the isolation and purification process, and pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives, and the like.

[0038] In this application, on a case-by-case basis, any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced by any of the other two terms. The term “approximately” with respect to a referenced numerical value, as well as its grammatical equivalents, as used herein may include the referenced numerical value itself and values within a range of ±10% of said referenced numerical value. For example, the term "about 10" includes 10 and any quantities from 9 to 11, inclusive. In some cases, the term "about" with respect to a referenced numerical value may also include values in the range of ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of the specified referenced numerical value. In some embodiments, "about" associated with the amount or range measured using a particular method indicates that the specified numerical value includes values determined by the variability of measurements when using the specified method.

[0039] The term "international unit" (ME) is used herein in accordance with its recognized meaning in the art and corresponds to the amount of a substance (eg, a polypeptide). The mass or volume that makes up one international unit varies depending on the substance being measured. The World Health Organization (WHO) provides characteristics of bioactive substance units and polypeptides.

[0040] As used herein, the following terms and related terms preferably have the following meanings unless the context clearly indicates otherwise:

[0041] "Bacteria" as used herein preferably refers to any gram-negative and gram-positive organisms, including, but not limited to, Acinetobacter spp., Citrobacter spp., Escheriachia coli, Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella spp., Morganella morganii, Pseudomonas aeruginosa, Enterobacter spp., Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumonia. Streptococcus pyogenes, Enterococcus faecalis, Clostridium spp. and Bifidobacterium spp., among others, which can cause diseases or adverse conditions in humans and/or animals, as well as have negative impacts on the environment. In particular, gram-positive (G+) (for example, Staphylococcus aureus, Enterococcus spp., Mycobacteriaceae spp. and Streptococcus pneumoniae) or gram-negative (for example, Escherichia coli/, Enterobacter spp., Salmonella spp., Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa), for which there is a clear and urgent need for new drugs and methods for treating infections caused by these bacteria resistant to a number of known antibiotics.

[0042] Within the scope of the present invention, the term “bacteria” also includes various species of probiotic bacteria and bacteria that are part of the normal microflora of humans and animals (for example, Lactococcus, Lactobacillus or Bifidobacterium, Enterococcus, Streptococcus spp.). Reference herein to a particular genus or genus of bacteria does not limit the present invention to a particular species or subspecies; any of the species or subspecies of the stated bacterial genera is intended to be included. Within the scope of the present invention, the term "bacteria" also includes various species of bacteria that have a positive meaning, such as symbionts, or a negative meaning, such as pathogens, in agriculture or the food industry. Additionally, in some embodiments of the present invention, the term "bacteria" refers to bacteria that are genetically modified to contain heterologous nucleic acids to express desired polypeptides or polynucleotides. For example, in some embodiments of the present invention, the term "bacteria" refers to bacteria that are genetically modified to applications in medicine, agriculture, food processing or biotechnology, for example, contain heterologous nucleic acids for the expression of desired polypeptides. In some embodiments of the present invention, the bacteria are resistant to at least one antibiotic. In other embodiments of the present invention, the bacteria are MDR, that is, they exhibit resistance to multiple antibiotics (classes of antibiotics).

[0043] The effectiveness of currently available therapies is limited to highly resistant infectious strains. These resistant bacteria are major causes of morbidity and mortality in patients. Helfand, β-lactams Against Emerging 'Superbugs': Progress and Pitfalls, Expert Rev. Clin. Pharmacol. 1(4): 559-571 (2008). Non-limiting examples of pathogens for which the development of new antibacterial drugs is important include carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, carbapenem-resistant Enterobacteriaceae spp (CRE); vancomycin-resistant Enterococcus faecium (VRE), strains of Pseudomonas aeruginosa, Acinetobacter baumannii, Escherichia coli, Klebsiella pneumonia and other multidrug-resistant (MDR) Enterobacteriaceae; methicillin-resistant Staphylococcus aureus (MRSA), partially resistant and vancomycin-resistant Helicobacter pylori, clarithromycin-resistant Campylobacter spp., fluoroquinolone-resistant Salmonella (Salmonellae spp), cephalosporin- and fluoroquinolone-resistant Neisseria gonorrhoeae, penicillin-non-susceptible Streptococcus pneumonia; ampicillin-resistant Haemophilus influenza; Fluoroquinolone-resistant Shigella spp., MDR Mycobacterium tubercidosis (MDR-TB) and MDR Acinetobacter baumannii.

[0044] "Bactericidal" or "bactericidal activity" refers to the property of causing bacterial death or the ability to kill bacteria to a degree of at least 3-log10 (99.9%) or greater reduction in the initial bacterial population over an 18-24 hour period .

[0045] "Bacteriostatic" or "bacteriostatic activity" refers to the property of inhibiting bacterial growth, including inhibiting the growth of bacterial cells, thereby causing a 2-log10 (99%) or better, and almost 3-log reduction in the original bacterial population within 18-24 hour period.

[0046] "Antibacterial" refers to both bacteriostatic and bactericidal agents.

[0047] "Antibiotic" refers to a compound having properties that have a negative effect on bacteria, such as lethality or decreased growth. An antibiotic may have a negative effect on gram-positive bacteria, gram-negative bacteria, or both. In some cases, an antibiotic is understood as an antiviral agent that inhibits the proliferation (reproduction cycle) of a virus and/or reduces the viral load in a subject.

[0048] “Antiviral” refers to substances and agents that inhibit or prevent the life cycle of a virus, for example, substances and agents that inhibit the synthesis of viral proteins, the synthesis of viral RNA and/or DNA, and the assembly of viral particles.

[0049] "Antimycotic" refers to substances and agents that inhibit or prevent the life cycle of a fungus.

[0050] The term "IC50" or IC50 refers to the inhibitory dose that causes 50% inhibition of a given quantifiable parameter. The indicated quantitative value means how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or chemical parameter (or component of a parameter, i.e. enzyme, cell, cellular receptor or microorganism) by half.

[0051] The term "MIC" means minimum inhibitory concentration (μg/ml), i.e. the minimum concentration of a substance that inhibits the visible growth of the tested strain of microorganism or virus in a standard experiment. The MIC value is the preferred indicator of the sensitivity of a particular microorganism to an antibiotic or antibacterial drug. MIC values for microorganisms can be determined using various techniques. For example, you can use the disk diffusion method or other gradient diffusion methods, the serial dilution method, methods using E-tests or HiComb strips, the boundary concentration method, and others.

[0052] The criterion for sensitivity in the treatment of infections is conventionally the value of the therapeutic index (T): T = MIC/K, where K is the concentration of a given antibiotic (μg/ml) in the site of infection (or in the blood serum) when therapeutic doses of the drug are administered. The microorganism is sensitive, and the antibiotic is usually clinically effective if T is less than 0.3. Experts can find the “K” values in special tables.

[0053] Specialists in the field of technology know various approaches to determining the sensitivity of microorganisms to drugs (see, for example, guidelines MUK 4.2. 1890-04: DETERMINATION OF THE SENSITIVITY OF MICROORGANISMS TO ANTIBACTERIAL DRUGS; https://docs.cntd.ru/document/ 1200038583; Manual for Clinical Laboratory Diagnostics of the Ministry of Health of the Russian Federation, “Determination of the sensitivity of microorganisms to antimicrobial drugs”: http://antimicrob.net/wp-content/uploads/Opredelenie-chuvstvitelnosti-mikroorganizmov-k-antibakterialnym-preparatam-2015.pdf)

[0054] “Drug-resistant” generally refers to pathogens, eg bacteria, fungi, viruses, that are resistant to the antibacterial activity of a drug. When used in certain ways, drug resistance can specifically refer to antibiotic resistance. In some cases, a bacterium that is normally sensitive to a particular antibiotic can develop resistance to the antibiotic, thereby becoming a drug-resistant microbe or strain. A “multidrug-resistant” (“MDR”) pathogen is a pathogen that has developed resistance to at least two classes of antimicrobial drugs, each used as monotherapy. For example, certain strains of S. aureus have been found to be resistant to several antibiotics, including methicillin and/or vancomycin (Antibiotic Resistant Threats in the United States, 2013, U.S. Department of Health and Services, Centers for Disease Control and Prevention). One of ordinary skill in the art can readily determine whether a particular microorganism is resistant to a drug by using routine laboratory techniques that determine the sensitivity or resistance of a microorganism, such as a bacterium or fungus, to a drug or antibiotic.

[0055] In one embodiment, a reduction in viral infection is a reduction in plaque forming units (PFU)/mL, and a reduction in bacterial infection is a reduction in colony forming units (CFU)/mL. "Plaque-forming units" is the number of particles capable of forming plaques, such as virus particles, per unit volume. It is a functional quantitative measure rather than an absolute particle number indicator: viral particles that are defective or unable to infect a target cell will not form a plaque and therefore will not be counted. For example, a solution of influenza virus at a concentration of 1000 PFU/μl indicates that 1 μl of the solution contains enough viral particles to produce 1000 infectious plaques in a cell monolayer. In connection with the present invention, a cell culture treated with an antiviral agent according to the present invention exhibits a reduced number of plaque-forming units in the culture after treatment compared to the culture before treatment with such agent.

[0056] Possible "decrease in plaque forming units (PFU)/mL" is analyzed as follows. First, cultured cells co-infected with influenza virus and bacteria are analyzed for their ability to form plaque-forming units (PFU)/ml, for example, selecting a few cells from a Petri dish and plating them to count the bacterial plaques that will form. This result is then compared with the number of plaque forming units (PFU)/ml produced by cells in the same culture after application of the inhibitor. If the number of plaque-forming units (PFU)/ml after treatment with an inhibitor is reduced compared to the amount formed before application of the inhibitor, a reduction in the number of plaque-forming units has occurred.

[0057] “Colony forming units (CFU)/ml” is a measure of the number of viable bacteria in a sample. There are different ways. For example, to create colony-forming units, a sample (eg, cells cultured in a small volume) is spread on the surface of a nutrient agar plate and allowed to dry before incubation for counting. "Viable bacterium" means a bacterium capable of reproducing by binary fission under controlled conditions. For a colony to be visible, it needs to grow quite a lot - when counting colonies, it is not clear whether the colony arose from one cell or from 1000 cells. Therefore, to reflect this ambiguity, results are expressed in CFU/mL (colony-forming units per milliliter) for liquids and CFU/g (colony-forming units per gram) for solids (rather than cells/mL or cells/g).

[0058] "Colony forming units (CFU)/ml" can be analyzed as follows. First, cultured cells coinfected with influenza virus and a bacterium, or with just one bacterium, are analyzed for their ability to produce colony forming units (CFU)/ml, for example, by selecting a few cells from a Petri dish and plating them for counting. The result obtained is then compared with the number of colony-forming units (CFU)/ml produced by cells of the same culture after application of the inhibitor. If the number of colony forming units (CFU)/ml is reduced to the amount formed before the application of the inhibitor, a reduction in population has occurred.

[0059] Typically, one skilled in the art will be skilled in the use of these well-known techniques for analyzing bacterial and viral infections. The methodology for determining the number of plaque-forming units (PFU)/ml and colony-forming units (CFU)/ml is described in more detail in the literature (Tuchscherr et al. 2011, Hrincius et al. 2010).

[0060] As used herein, the term “bringing into contact” refers to the spatial approach of a cell, virus, fungus, bacterium, or enzyme, such as RNAP, to the specified compound or substance. This may mean, for example, that the inhibitor is added to the medium or environment in which the cell, virus, fungus or bacterium is located, or provides entry/delivery of the specified compound or substance into the cell, virus, fungus or bacterium.

[0061] “Biofilm” refers to bacteria that adhere to surfaces and aggregate into a hydrated polymer matrix, which may contain components derived from bacteria and/or hosts. A biofilm is a group of microorganisms in which cells adhere to each other on a biotic or abiotic surface. Such adherent cells are often embedded in a matrix containing, but not limited to, an extracellular polymeric substance, EPS. CHD biofilm, also called mucus (although not everything described as mucus is biofilm) or plaque, is a polymeric conglomerate typically composed of extracellular DNA, proteins, and polysaccharides.

[0062] “Suitable” in the context of an antibiotic or agent suitable for use against a particular bacterium, virus or fungus refers to an antibiotic that has been found to be effective against that bacterium, virus or fungus, even if resistance has subsequently developed.

[0063] "Effective amount" refers to an amount that, when used or administered at an appropriate frequency or dosage schedule, is sufficient to prevent, reduce, inhibit or eliminate the growth of microorganisms, bacterial growth, viral propagation or bacterial or viral load or prevent, reduce or alleviating the onset, severity, duration or progression of the disorder being treated (in this case, the condition or consequences of infection with bacterial or viral pathogens, fungi), preventing the progression of the disorder being treated, causing regression of the disorder being treated, or enhancing or improving a prophylactic or therapeutic effect(s) of other activity or therapy, such as antibiotic therapy, antiviral therapy, or bacteriostatic therapy.

[0064] "Co-administration" or "Co-exposure" refers to the administration of two agents and/or the implementation of specified effects in a sequential manner, as well as the administration of these agents and/or the implementation of specified effects substantially simultaneously, for example in one mixture/composition or combination . In addition, depending on the application, co-administration or exposure need not be continuous or simultaneous. For example, when using agent A and exposure or agent B, for example, A may be administered only initially and exposure B may be administered or agent B may be administered at subsequent times without further administration of A and vice versa.

[0065] Bacterial infections: In one embodiment, the terms “infection” and “bacterial infection” refer to an infection in a subject caused by a gram-negative (G-) bacterium, also referred to as a “gram-negative infection.” In one aspect of this embodiment, a gram-negative infection is an infection that is resistant to one or more antibiotics. In one aspect of this embodiment, the gram-negative infection is a multidrug-resistant infection. In certain embodiments, the gram-negative bacterium is Acinetobacter spp. In certain embodiments, the gram-negative bacterium is Acinetobacter spp., such as Acinetobacter baumannii. In certain embodiments, the gram-negative bacterium is Burkholderia spp. In certain embodiments, the gram-negative bacterium is Burkholderia pseudomallei. In certain embodiments, the gram-negative bacterium is Pseudomonas aeruginosa. In certain embodiments, the gram-negative bacterium is Enterobacteriaceae. In any of these embodiments, the gram-negative infection is caused by a pathogen or a pathogen expressing one or more β-lactamases. In any of these embodiments, the gram-negative infection is caused by a pathogen or a pathogen expressing one or more class A, class C, and/or class D β-lactamases. In any of these embodiments, the gram-negative infection is caused by a pathogen or a pathogen expressing one or more β-lactamases class A lactamase. In any of these embodiments, the gram-negative infection is caused by a pathogen or a pathogen expressing one or more class C β-lactamases. In any of these embodiments, the gram-negative infection is caused by a pathogen or pathogen expressing one or more class D β-lactamases.

[0066] Infection caused by "Enterobacteriaceae" refers to any gram-negative bacterium belonging to the specified family of bacteria, including, but not limited to, species such as Salmonella spp., Escherichia coli, Yersinia pestis, Klebsiella spp., Shigella spp. , Proteus spp., Enterobacter spp., Serratia spp. and Citrobacter spp. Thus, a method of treating a bacterial infection caused by "Enterobacteriaceae" includes treating any infection caused by any one or more bacteria constituting the specified family. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one Salmonella spp pathogen is present. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one pathogen Escherichia coli is present. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one pathogen, Yersinia pestis, is present. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one pathogen Klebsiella spp is present. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one pathogen Shigella spp is present. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one pathogen Proteus spp is present. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one pathogen Enterobacter spp is present. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one Serratia spp pathogen is present. In one embodiment, the bacterial infection caused by "Enterobacteriaceae" includes bacterial infections in which at least one pathogen, Citrobacter spp, is present.

[0067] In certain embodiments, the terms “infection” and “bacterial infection” refer to an infection caused by a gram-negative bacterium, where the gram-negative bacterium is an Enterobacteriaceae expressing one or more class A, class B, class C, and/or class β-lactamases D. In one aspect of this embodiment, the gram-negative bacterium is an Enterobacteriaceae expressing at least one class B β-lactamase.

[0068] In certain embodiments, the gram-negative bacterium is Acinetobacter spp. expressing one or more β-lactamases. In one embodiment, the gram-negative bacterium is Acinetobacter baumannii expressing one or more class A, class C, and/or class D β-lactamases. In one embodiment, the gram-negative bacterium is Acinetobacter baumannii expressing one or more class β-lactamases A. In one embodiment, the gram-negative bacterium is Acinetobacter baumannii expressing one or more class C β-lactamases. In one embodiment, the gram-negative bacterium is Acinetobacter baumannii expressing one or more class D β-lactamases. In one embodiment implementation, the gram-negative bacterium is Acinetobacter baumannii, expressing TEM-1 or KPC-2.

[0069] In one aspect, the terms "infection" and in particular "bacterial infection" can refer to any causative agent of a disease or undesirable condition in a subject, in particular to pathogens that cause: respiratory tract infections (RTI), gynecological infections, diseases sexually transmitted infections, urinary tract infections (UTI), including complicated urinary tract infections (cUTI), bacteremia, acute attacks of chronic bronchitis (ACEB), acute otitis media, acute sinusitis, sepsis, community-acquired pneumonia (CAP), hospital-acquired pneumonia (HAP) ), ventilator associated pneumonia (VAP), complicated skin and skin structure infections (cSSSI), acute bacterial skin and skin structure infections (ABSSSI), uncomplicated skin and skin structure infections (SSSI), intra-abdominal infection (IAI), endocarditis, osteomyelitis , febrile neutropenia, cervicitis, urethritis, melioidosis, tularemia, infections in an immunocompromised host such as liver abscess, biliary tract infections and bacteremia.

[0070] All of the above infections can be caused by a variety of bacteria and/or viruses, the growth, reproduction and/or viability of which can potentially be reduced according to the present invention, including the complete elimination (eradication) of these bacterial and viral pathogens.

[0071] “Viruses,” “Viral disease,” or “viral infection,” to which the present invention also applies, may occur as part of a coinfection or may occur as a separate infection present in a host, such as a subject and/or a cell. For the purposes of this application, the term “viruses” includes animal and avian viruses, plant viruses, fungal viruses, archaeal and bacterial viruses (bacteriophages). A viral disease or viral infection may be mediated by a virus; preferably they are mediated by an animal virus such as an RNA virus, for example coronovirus, norovirus, rhabdovirus, enterovirus, influenza virus, picornavirus, arenavirus, Ebola virus, rhabdovirus (including stomatitis and rabies), paramyxovirus, flavivirus, hepadnovirus, preferably hepatitis virus C, HIV or RNA virus that causes intestinal or respiratory tract infections. The preferred viruses that cause respiratory tract infections are rhinoviruses, influenza viruses and coronaviruses. Preferably, the influenza virus infection is mediated by an influenza A virus or an influenza B virus, with influenza A viruses being preferred. Particularly preferred are the influenza A virus subtypes H1N1, H2N2, H3N2, H5N6, H5N8, H6N1, H7N2, H7N7, H7N9, H9N2, H10N7, N10N8 and/or H5N1. Preferably, the coronavirus infection is mediated by SARS-CoV, SARS-CoV2 or MERS-CoV.

[0072] As used herein, the term “fungus” refers to any member of the kingdom Fungi or Mycota. In some embodiments, the term “fungi” refers to members of the kingdom Fungi that have positive or negative (e.g., pathogens) significance in agriculture, industry, such as the food and forestry industries, or other areas of the economy. In some embodiments, the term “fungus(s)” refers to pathogenic fungi, such as fungi that cause mycoses, particularly dermatomycoses, deep mycoses, and mycotoxicoses, in humans or animals. In some embodiments, the term “fungus(s)” refers to phytopathogenic fungi or fungi that cause spoilage of various products or materials, such as wood. Additionally, in some embodiments of the present invention, the term “fungi” refers to fungi, such as yeast, that are genetically modified to contain heterologous nucleic acids to express desired polypeptides or polynucleotides. For example, in some embodiments of the present invention, the term “fungus” refers to fungi that are genetically modified modified for use in medicine, agriculture, food processing or biotechnology, for example, containing heterologous nucleic acids for the expression of desired polypeptides. In some embodiments of the present invention, the fungus is resistant to at least one antibiotic or antimycotic.

[0073] "Treatment" refers to any process, action, application, therapy, etc., wherein medical care is provided to a subject, including a human, for the purpose of curing a disorder, eradicating a pathogen, or improving the subject's condition, either directly or indirectly. Treatment also refers to reducing morbidity, relieving symptoms, eliminating relapses, preventing relapses, preventing morbidity, reducing the risk of morbidity, improving symptoms, improving prognosis, or combinations thereof. “Treatment” may further include reducing the population, growth rate, or virulence of bacteria in a subject and thereby controlling or reducing bacterial infection in the subject, or bacterial contamination of an organ, tissue, or environment. Thus, a “treatment” that reduces disease may, for example, be effective in inhibiting the growth of at least one gram-positive or gram-negative bacterium in a particular environment, whether the subject or the environment. On the other hand, “treating” an established infection refers to depopulation, destruction, growth inhibition, and/or eradication of the gram-positive bacteria responsible for the infection or contamination.

[0074] “Prevention” refers to preventing the incidence, recurrence, spread, occurrence or development of a disorder, such as a bacterial infection. It is not intended that the present invention be limited to completely preventing or preventing the development of infection. In some embodiments, the onset of disease is delayed, or the severity of subsequent disease or the likelihood of infection is reduced, and these constitute examples of prevention.

[0075] "Emerging diseases" refers to diseases manifested by clinical or subclinical symptoms, such as the detection of fever, sepsis or bacteremia, as well as diseases that can be detected by the growth of a bacterial pathogen (for example, in culture), when symptoms associated with Such pathology has not yet manifested itself.

[0076] "Subject" refers to a mammal, plant, lower animal, single-celled organism, or cell culture. For example, the term “subject” is intended to include organisms, such as prokaryotes and eukaryotes, that are susceptible to or affected by bacterial, viral or fungal infections, such as gram-positive bacterial infections or RNA virus infections. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and non-human transgenic animals. In some embodiments, the subject is a human, such as a human, suffering from, at risk of, or susceptible to infection by gram-positive bacteria, fungi, or viruses, whether such infection is systemic, local, or otherwise concentrated or limited to a specific organ or tissue .

[0077] "Polypeptide" refers to a polymer composed of amino acid residues and typically having at least about 30 amino acid residues. The term "polypeptide" is used interchangeably herein with the terms "protein" and "peptide". The term includes not only polypeptides in isolated form, but also active fragments and their derivatives. The term "polypeptide" also includes fusion proteins or fusion polypeptides comprising a transcription elongation factor polypeptide and maintaining, for example, the function or activity of the transcription elongation factor. Depending on the context, the polypeptide or protein or peptide may be a naturally occurring polypeptide or a recombinant, engineered or synthetically produced polypeptide. A particular polypeptide having transcription elongation factor activity, for example, may, for example, be obtained from the native protein by enzymatic or chemical digestion, or may be prepared using conventional peptide synthesis methods (such as solid phase synthesis) or molecular biology techniques (such as those described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989)), or can be strategically truncated or segmented to produce active fragments supporting, for example, factor activity transcription elongation in relation to the same (target) RNAP.

[0078] "Derivative" in the context of a peptide or polypeptide or an active fragment thereof is intended to cover, for example, a polypeptide modified to contain one or more chemical moieties other than an amino acid that do not substantially adversely affect or destroy activity or do not interfere with the function of said polypeptide, such as RNAP elongation factor activity, for example exonuclease activity. The chemical moiety may be covalently linked to the peptide, for example through an amino-terminal amino acid residue, a carboxy-terminal amino acid residue, or through an internal amino acid residue. Such modifications may be natural or non-natural. In some embodiments, the non-natural modification may include adding a protecting or capping group to the reactive moiety, adding a detectable label such as an antibody and/or fluorescent label, adding or modifying glycosylation or adding a bulking group such as PEG (PEGylation), and other changes , known to those skilled in the art. In some embodiments, the non-natural modification may be a capping modification, such as N-terminal acetylation and C-terminal amidation. Exemplary protecting groups that may be added to polypeptides include, but are not limited to, t-Boc and Fmoc. Commonly used proteins for fluorescent labeling include, but are not limited to, green fluorescent protein, GFP, red fluorescent protein, RFP, cyan fluorescent protein, CFP, yellow fluorescent protein, YFP, and mCherry are compact proteins that can be covalently or non-covalently linked to or fused to a polypeptide without interfering with the normal functions of cellular proteins. In some embodiments, a polynucleotide encoding a fluorescent protein is inserted upstream or downstream of the polynucleotide sequence. This will produce a fusion protein (eg FET::ZPB polypeptide) that does not interfere with cellular function or the function of the polypeptide to which it is attached. Conjugation of polyethylene glycol (PEG) to proteins has been used as a method to increase the half-life of many pharmaceutical proteins. Thus, in the context of polypeptide derivatives, the term “derivative” includes polypeptides, such as FET polypeptides, chemically modified by the covalent attachment of one or more PEG molecules. Such pegylated FETs are expected to exhibit a longer half-life compared to non-pegylated polypeptides while maintaining biological and therapeutic activity.

[0079] “Percent amino acid sequence identity” refers to the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a reference polypeptide sequence, after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity and without regard to any conservative substitutions as part of sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways known to those skilled in the art, for example using publicly available software such as Basic Local Alignment Search Tool (BLAST) or commercially available software such as DNASTAR. Two or more polypeptide sequences may be 0-100% identical or any whole number in between. In the context of the present invention, two polypeptides are "substantially identical" if at least 80% of the amino acid residues (typically at least about 85%, at least about 90%, and typically at least about 95%, at least about 98 % or at least 99%) identical. The term "percentage (%) amino acid sequence identity" described herein also applies to peptides. Thus, the term “substantially identical” will include mutated, truncated, fused, or otherwise modified sequence variants of isolated polypeptides and peptides such as those described herein and active fragments thereof, as well as polypeptides with substantially identical sequence (e.g., at least 80%, at least 85%, at least 90%, at least 95% identical, at least 98% identical, or at least 99% identical, as measured, for example, by one or more of the methods specified above) by compared to a reference (wild type or other intact) polypeptide. Two amino acid sequences are "substantially homologous" if at least about 80% of the amino acid residues (typically at least about 85%, at least about 90%, at least about 95%, at least about 98% identity, or at least approximately 99% identity) are identical or represent conservative substitutions. The sequences of the polypeptides of the present invention are substantially homologous if one or more, or up to 10%, or up to 15%, or up to 20%) of the amino acids in the sequence of a polypeptide, such as the FET polypeptides described herein, are replaced by the same or conservative amino acid substitution, and the resulting polypeptide has at least one activity, antibacterial effect and/or bacterial specificity of the reference FET polypeptide.

[0080] As used herein, a “conservative amino acid substitution” is preferably one in which an amino acid residue is replaced by an amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been identified in the field. These families include amino acids with basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine).

[0081] "Fusion polypeptide" refers to an expression product resulting from the fusion of two or more nucleic acid segments, whereby the fused expression product typically has two or more domains or segments that typically have different properties or functionality. In some aspects of the present invention, the term “hybrid polypeptide” also refers to a polypeptide or peptide comprising two or more heterologous polypeptides or peptides covalently linked either directly or through an amino acid or peptide linker. The polypeptides forming the fusion polypeptide are typically C-terminal to N-terminal linked, although they can also be C-terminal to C-terminal, N-terminal to N-terminal, or N-terminal to C-terminal linked. The term "fusion polypeptide" may be used interchangeably with the term "fusion protein". Thus, the open-ended expression “polypeptide comprising” a particular structure implies molecules larger than the specified structure, such as fusion polypeptides.

[0082] The term "nucleotyl" as used herein refers to any nucleotide, natural or synthetic. It includes the usual DNA and RNA bases (A, G, C, T, U), base analogues, for example, inosine, 5-nitroindazole and others, imidazole-4-carboxamide, derivatives of purines or pyrimidines, for example, modified pyrimidine base 6H, 8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (sometimes referred to as the “P” base that binds to A or G) and the modified purine base N6-methoxy-2,6 -diaminopurine (sometimes referred to as the "K" base that binds to C or T), nucleoside analogues such as hypoxanthine, N-4-methyldeoxyguanosine, 4-ethyl-2'deoxycytidine, 4,6-difluorobenzimidazole and 2,4-difluorobenzene, pyrene fluorophore-carrying nucleoside analogs included in probes based on conformation-locked (“locked”) nucleic acids (LNA - locked nucleic acids), deaza- or aza-modified purines and pyrimidines, pyrimidines with substituents at position 5 or 6, and purines with substituents at positions 2, 6 or 8, 2-aminoadenine (nA), 2-thiouracil (sU), 2-amino-6-methylaminopurine, O-6-methylguanine, 4-thio-pyrimidines, 4-aminopyrimidines, 4- dimethylhydrazine-pyrimidines, O-4-alkyl-pyrimidines and hydrophobic base analogues that form duplex DNA without the formation of hydrogen bonds. Bases can be joined through a variety of bonds and conformations, including phosphodiester, thiophosphate or methylphosphonate bonds, and peptide-nucleic acid bonds.

[0083] The term “polynucleotide” as used herein refers to a multimeric compound containing linked nucleotides forming a polymer, including conventional RNA, DNA, locked nucleic acids (LNA), and conformation-locked nucleic acids due to intramolecular bridges. (BNA - bridged nucleic acids), copolymers of all the above compounds and their analogues. The term "nucleic acid" as used herein refers to single-stranded polynucleotides or a duplex of two polynucleotides. Such duplexes are not necessarily renatured throughout their entire length and may contain breaks (gaps) and looping areas.

[0084] “Heterologous” refers to nucleotide or polypeptide sequences that are not contiguous in nature. For example, in the context of the present invention, the term “heterologous” can be used to describe a combination or fusion of two or more polypeptides wherein the fusion polypeptide is not typically found in nature. In addition, the term “heterologous” also means that the specified molecule is not present in a certain environment in nature. For example, the use of the expression “heterologous polynucleotide” in relation to a cell will mean that the specified polynucleotide is not characteristic of the specified cell, for example, it was absent from it in nature, that is, before it was mutated or changed in a targeted way.

[0085] Constructs: In one embodiment, the present invention includes the use of stably transfected cells or organisms, for example, stably transfected microorganisms, in which a heterologous or accessory gene encoding an RNAP elongation factor polypeptide has been integrated into the genome of the host cell or all cells of the organism. Techniques for creating stably transfected (micro)organisms are known in the art. For example, an RNAP elongation factor polypeptide gene can be cloned into a host genome, such as a chromosome, by homologous recombination. In some embodiments, a host essential gene is disrupted using homologous recombination, such as a deletion of the gene, one or more amino acid substitutions resulting in an inactive form of the protein encoded by the essential gene, or a frameshift mutation resulting in a truncated form of the protein encoded by said essential gene. The preferred technique is described, for example, in WO 02/090551, which is incorporated herein by reference in its entirety. Said plasmid may be self-replicating, may carry, for example, one or more genes of interest and one or more resistance markers. Further, the transforming plasmid can be any plasmid, provided that it is not capable of complementing the disrupted essential gene. Alternatively, said plasmid is an integrative plasmid. In the latter case, the integrative plasmid itself can be used to disrupt the essential gene by integrating into the locus of said essential gene. In some cases, the essential gene is replaced by double homologous recombination with a cassette containing the gene or genes of interest flanked by targeting sequences that target the insertion to the essential gene. It should be understood that these targeting sequences are of sufficient length and sufficient homology to allow integration of the gene of interest at the target site.

[0086] In cases where a genetic construct encoding an RNAP elongation factor polypeptide may be integrated into microbial genomic DNA, for example, a bacterial chromosome, one or more copies of the nucleic acid may be integrated; integration can occur at a random chromosomal site or, as described above, at a predetermined chromosomal site. Accordingly, according to an embodiment, said genetic construct may further comprise sequences capable of inserting said genetic construct into the genome of a host cell, for example, into a chromosome.

[0087] In some embodiments, the insertion of said genetic construct into specific sites in the genome, for example, in a host cell chromosome, may be facilitated by homologous recombination. For example, a genetic construct according to the present invention may contain one or more regions of homology to a specified integration site in the genome, for example, in the chromosome of a host cell. The sequence at said genomic, for example chromosomal, site may be natural, i.e. naturally occurring, or may be an exogenous sequence introduced by genetic engineering earlier. For example, the size of the homology region or regions may be at least 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 By. 700 bp, 800 bp, 900 bp, 1000 bp or more.

[0088] According to one embodiment of the present invention, two regions of homology may be included, one flanking the region on each side of the relevant expression units present in the genetic construct of the present invention. Such a configuration may provide favorable insertion of relevant sequences, i.e. at least those sequences that encode and effect expression of the antigen of interest in host cells. Methods for performing homologous recombination, in particular in bacterial hosts, and selecting recombinant hosts are well known in the art.

[0089] As used herein, the term “expressing” a gene, polypeptide or polynucleotide, or “producing” a polypeptide or polynucleotide means the formation of sufficient quantities of the specified polypeptide, polynucleotide or product of the specified gene for the specified purposes, for example, to carry out the present invention and achieve its technical result .

[0090] As used herein, the term “constitutive” in the context of a promoter (or, more broadly, in relation to gene expression or polypeptide secretion) refers to a promoter that allows continuous transcription of its associated gene. A constitutive promoter is contrasted with an “inducible” promoter, the activity of which can be modulated by a specific agent, for example, a polypeptide, peptide, polynucleotide, oligonucleotide, molecule, compound or effect. Various inducible expression systems are known to those skilled in the art.

[0091] Methods for producing genetically modified plant, fungal and animal cells are known to those skilled in the art. In addition, methods for transforming microorganisms, such as, for example, protoplast transformation and electroporation, are known to one skilled in the art.

[0092] High levels of expression can be achieved by using homologous expression and/or secretion signals on expression vectors present in the microorganism. The expression signals will be apparent to one skilled in the art. Said expression vector may be optimized for expression based on the particular microorganism or cell into which it is included. Those skilled in the art know specific expression vectors that are optimized for expression of polypeptides in specific host cells.

[0093] "Active fragment" refers to a portion of a polypeptide or molecule that retains one or more of the functions or biological activities of the isolated polypeptide or molecule from which such fragment was taken, e.g., exonuclease activity, RNAP binding ability, RNAP inhibitory ability.

[0094] “Increase in activity” includes both an increase in specific activity, that is, an increase in the corresponding properties of the molecule, for example, due to its modification, removal of a blocking or inactivating group, or transfer of the molecule to an active conformation or state, and a total increase in activity due to an increase in the overall the number of molecules in a cell or in a particular environment. In the case of polypeptides, such a net increase in activity can, for example, be achieved by increasing the level of their expression, for example, in the case of inducible expression or as a result of transfection or transduction, accumulation of active forms of the polypeptides in the cell, or delivery of the polypeptides into the cell.

[0095] "Decrease" can refer to any change that results in a decrease in a parameter, state, or activity. A substance is also considered to reduce the activity of an enzyme or polypeptide when the amount of product produced by a biochemical reaction involving said enzyme or polypeptide is reduced compared to the product produced without the substance. Also, for example, a decrease may be a change in the symptoms of a disorder such that the symptoms are less severe than previously observed. By “decrease” or other forms of the word, we mean a decrease in an event or characteristic (for example, RNAP activity, CFU, PFU, etc.). It is clear that this usually refers to some standard or expected value, in other words, it is a relative value, but it is not always necessary to refer to the standard or relative value. For example, “reduces transcription efficiency” means a decrease in transcription rate compared to a standard or control.

[0096] The decrease can be any individual, average, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the reduction can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70. 75, 80, 85, 90, 95 or 100%.

[0097] “Inhibit,” “inhibit,” and “reduce” mean a decrease in a measurable amount, such as a decrease in activity, amount, rate, response, condition, disease, or other biological parameter. This may include, but is not limited to, complete elimination of an activity, response, condition or disease. This may also include, for example, a 10% reduction in activity, response, condition or disease compared to the native or control level. Thus, the reduction may be 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any reduction in between, compared to native or control levels.

[0098] By “increasing” the action within the framework of this application is meant both a corresponding increase in the expression of a certain parameter, which can be determined qualitatively or quantitatively, and the acquisition of a certain property or the receipt of a certain effect that was previously absent. An increase in inhibitory effect is understood as a corresponding change, usually a decrease, in the parameter or property that is used to assess inhibition.

[0099] “Binary” means that at least two components or means are used, as a result of the joint action or activity of which the specified effect or result is achieved. Preferably, the two components have different natures and/or mechanisms of action, but when used together provide an additive or synergistic effect.

[00100] “Synergistic” or “superadditive” refers to the beneficial effect caused by two agents in combination that is greater than the sum of the effects of the two agents acting independently. In some embodiments, the synergistic or superadditive effect is significant, i.e. statistically significant, exceeds the sum of the effects of two agents acting independently. One or both active ingredients can be used at subthreshold levels, i.e. at a level at which, if the active substance is used individually, it has no or very limited effect. The effect can be measured using any methods known to specialists.

[00101] For example, to evaluate the effect of using two agents together, additivity index (CI) values based on Leve's additivity model can be used, which can be additive (CI=1), antagonistic (CI>1) or synergistic (CI<1 ) for different concentrations of drugs and levels of effect (total number of CFU, PFU, proportion of dead cells; inhibition of RNAP activity, inhibition of transcription, inhibition of the transcript elongation stage, inhibition of proliferation or viability of bacteria, fungi, inhibition of the dynamics of virus reproduction). CI values can, for example, be calculated from linear regression trend lines using CompuSyn software (ComboSyn Inc., Paramus, NJ, www.combosyn.com), then transforming the hyperbolic and sigmoidal dose response curves into linear form by the method of Chou et al., (Chou TS (2010) Drug combination studies and their synergy quantification using the Chou-Talalay method. Cancer Res 70: 440-6).

[00102] The synergistic effect makes it possible to effectively achieve the desired action or result using reduced quantities (doses) compared to using only one of the agents used (monotherapy). Lower doses provide reduced toxicity without reducing effectiveness. In addition, synergistic effects may result in increased effectiveness. In some cases, a synergistic effect will result in the appearance of a desired property or activity that was not previously present, substantially not present, or present at an undetectable level. Finally, synergy may result in improved disease prevention or relief compared with either monotherapy. The term "additive" refers to the sum of the actions of any two or more agents in combination. As used herein, the term “antagonistic” refers to blocking (eg, reducing or preventing) biological activity.

[00103] The term “synergistic” also refers to a combination of two or more therapeutic agents, interventions, or agents and interventions that is more effective than the additive effect of any two or more individual agents and/or interventions. In some cases, the synergy of pharmacological agents and/or effects can be quantified by calculating additivity index (CI) values.

[00104] In some cases, the cell or bacterium constitutively expresses the RNAP elongation factor polypeptide used in accordance with the present invention. The use of a constitutive promoter avoids the need to add an inducer or other regulatory signal to effect expression. Such expression may preferably occur at an increased level. For example, the specified level of expression may result in the accumulation of the specified expression product at a level of less than about 10% of the cellular protein, about or less than about 5%, for example, about 1-3%. Said promoter may be homologous to the bacterium used, i.e. be a promoter found naturally in said bacterium.

[00105] The term “RNA polymerase” refers to a polypeptide or several polypeptides (subunits) that are capable of synthesizing ribonucleotide polymers. The structure of RNAP and the general mechanism of RNA synthesis are highly conserved and, in their main features, are very similar in bacteria, archaea and eukaryotes. In prokaryotic cells there is only one RNAP, which synthesizes all types of RNA in the cell. There are two forms of RNAP: core and holoenzyme. The core enzyme has a relative molecular weight of about 400 kDa and consists of five subunits: two α, β, β' and ω. The bRNAP holoenzyme consists of a core enzyme and a σ subunit. The core enzyme bRNAP has catalytic activity but is not capable of initiating transcription. Transcription initiation occurs through the σ subunit. Between the β'- and β-subunits there is the main channel of bRNAP, in which DNA and RNA are bound, and the active center of the enzyme is also located inside it. The active center of bRNAP contains two mobile domains: the trigger loop (TL) and the α-helical bridge (AH), both of these domains play an important role in the process of translocation and nucleotide addition. Lateral to the main channel there is a secondary channel, which connects to the main one in the region of the active center. The secondary channel serves as a binding site for many regulatory factors and is the main route for the entry of nucleotides into the active center of RNAP

[00106] The expression “transcription elongation” refers to the process of elongation of an RNA molecule under the action of RNA polymerase. In the case of DNA-dependent RNAP, at the elongation stage, nucleotides complementary to the DNA template strand are added by the core enzyme. The complex of RNAP with DNA and synthesized RNA is called the elongation complex (EC). RNAP moves unevenly along DNA. During elongation, this enzyme can make temporary stops (pauses), stop moving (permanent stop) or terminate RNA synthesis in response to regulatory influences from protein factors and/or signals encoded in DNA and RNA. The core enzyme is also capable of editing errors in RNA chain extension; for this purpose, backtracking is performed. Backtracking is a phenomenon in which RNAP is shifted in the opposite direction along the template DNA chain and, due to exonuclease activity, corrects the error.

[00107] The term “elongation factor” refers to a polypeptide/protein that interacts with RNA polymerase at the transcription elongation stage and helps to increase the efficiency of RNA molecule synthesis, which can be expressed in the formation of longer RNA molecules and faster transcription. When serious errors occur during the operation of the elongation complex, a state of arrest occurs, from which specialized transcription factors (transcription elongation factors), in particular those with exonuclease activity, are required. Examples of elongation factors whose expression or activity can be increased by the practice of the present invention are the GreA and GreB proteins of E. coli (as well as their homologues), which are very conserved transcription factors and the genes encoding them are present in large numbers, if not all , sequenced bacterial genomes (See, for example, search results in the NCBI sequence database. https://www.ncbi.nlm.nih.gov/protein/?term=GreB, https://www.ncbi.nlm.nih. gov/protein/?term=GreA). In particular, the conservative structure and activity of “Gre-factors” has been shown for Deinococcus radiodurans, Thermus aquaticus, Mycobacterium smegmatis, P. aeruginosa, Salmonella enterica serovar Typhimurium, Mycobacterium tuberculosis, clinical isolates of Staphylococcus aureus (MRSA), Francisella tularensis (G-), Thermus thermophiles and many other G- and G+ bacteria.

[00108] These elongation factors have exonuclease activity and are responsible for the processivity of bRNAP, with GreA allowing the cleavage of 2 to 5 nucleotides, while GreB from 10 to 15 nucleotides. Homologs and analogs (including functional analogs) of these factors will be known or can be readily identified by those skilled in the art.

[00109] One skilled in the art will appreciate that the optimal amount of an RNAP elongation factor polypeptide, e.g., GreA or GreB, or an analogue or homolog thereof, that has substantially similar function or activity, in the practice of the present invention will vary, for example, depending the particular RNAP whose activity is inhibited, the microorganism or cell expressing such polypeptide, the genetic constructs, for example, the strength of the promoter used in the genetic constructs, and the particular compound of the present invention that binds to the RNAP and acquires enhanced transcription inhibitory activity and/or an inhibitory effect on the growth and viability of the corresponding microorganism. In all cases of implementation of the present invention, those skilled in the art will be able to determine the effective concentrations and/or amounts of substances and the strength or extent of effect that are required to provide transcriptional inhibition and/or an inhibitory effect on the growth and viability of the corresponding pathogen. Specific values of concentration and parameters do not affect the inventive concept of the authors of the present invention.

[00110] By “polypeptide (elongation factor) analogue” as used herein, we preferably mean a polypeptide that performs a similar function in transcription, but, for example, is derived from a different microorganism or cell, or is of artificial or recombinant origin. In some cases, the analogue exhibits sequence homology to the referenced polypeptide (elongation factor) as defined herein.

[00111] When indicating the use of a “means for” together with the purpose of such a means or the characteristics of the result, change or effect obtained as a result of the application or use of the specified means, within the framework of this application we understand the use of any agent or influence with the help of which a specialist can ensure the achievement of the specified result, change or effect. The means that those skilled in the art may use to carry out the present invention are not part of the present invention, since, given the description of this application and the general knowledge in the field of use of the present invention, those skilled in the relevant fields of technology can choose and use any means and methods of using them in the implementation of the present invention, which do not significantly affect the technical result of the present invention.

[00112] As used herein, "agent" means any substance, compound, molecule, such as a small molecule ion, complex or composition, peptide, polynucleotide, lipid complex, genetic and/or protein construct that, when brought into contact with appropriate and suitable target ensures the desired result is achieved. By “impact” we mean any influence that changes the state of the environment, substances, cell or its environment, compartment or microenvironment in the cell, which leads to a change in their physical, chemical or physicochemical characteristics. Non-limiting examples of effects include heating, irradiation, including visible, UV or IR irradiation, application of a magnetic or electric field, changes in pH, tonicity or concentrations of substances (molecules), phase separation, for example, phase separation in a liquid. Various examples of influences are known in the art that affect the state and activity of biomolecular complexes and gene expression, which include, but are not limited to: changes in temperature in the case of thermally inducible promoters, mechanical effects in the case of mechanosensitive ion channels, changes in ion concentrations in the case of ion channels, changes in the concentration of free radicals in the case of oxidative stress, changes in the concentration of oxygen in the case of hypoxia, heat shock, cooling, gamma irradiation and high-frequency irradiation, which lead to the induction of sos responses.

[00113] In some embodiments of the present invention, an agent that increases the expression and/or activity of an RNAP elongation factor, such as bRNAP, vRNAP, or gRNAP, may be an agent that is, for example, selected from: a genetic construct that causes expression of an RNAP elongation factor , a virus, in particular a bacteriophage, which delivers into a cell or bacterium a genetic construct that ensures the expression of the RNAP elongation factor, a small molecule, nucleotide, peptide or polypeptide that ensures the induction or increase in the expression of the RNAP elongation factor or the transition of the RNAP elongation factor to an active form and/or said agent provides an increase in the amount of RNAP elongation factor in the cell. In some embodiments of the present invention, the agent that increases the expression and/or activity of the RNAP elongation factor may be an effect that increases the expression and/or activity of the RNAP elongation factor, for example, a change in temperature, radiation, including optical irradiation and high frequency radiation, a change physicochemical characteristics of the medium or liquid in the area or environment in which the cell, in particular a bacterium, is located.

[00114] Within the framework of the present invention, “delivery” of genetic constructs or polypeptides into a cell can be accomplished in any manner known to those skilled in the art. In the case of delivery of polynucleotides, this may be transduction or transfection, for example, transfection using salts, for example, calcium phosphate transfection, transfection using lipid complexes, including liposomes, transfection using positively charged polymers, for example, DEAE-dextran or polyethylenimines, electroporation , infection with appropriate viruses, for example adenovirus in the case of eukaryotes and bacteriophage in the case of bacteria, the use of membrane-penetrating peptides or channel-forming agents.

[00115] To ensure an increased level of activity and/or expression of a certain protein, for example, a bRNAP elongation factor such as GreA or GreB, in bacteria, one can, for example, use activation or increase in the level of expression of the corresponding genes, transformation of bacteria with the introduction of an appropriate genetic construct under control constitutive or inducible promoter or delivery of the corresponding polypeptides into cells. Activation of elongation factor genes, for example, greA and/or greB or their homologs, present in the genome of a bacterium, for example, in Escherichia coli, can be carried out in a specific and non-specific manner. In the first case, the promoters of a limited number of genes or only the promoters of target genes are activated or increased. In the second case, the effect on the cell leads to activation and increase in the level of activity of the promoters of a wide range of genes, which also include target genes/promoters. An example of such nonspecific gene activation is heat shock, high-frequency irradiation, and osmotic shock.

[00116] Bacteriophages and agents based on them can be used to deliver genetic constructs and polynucleotides to bacteria. As part of this approach, it is possible to use temperate bacteriophages or their derivatives (for example, phagemids), capable of integrating into the genome of a bacterium and thus delivering the required gene to the bacterium, for example, the greA and/or greB genes or their homolog, or a polynucleotide or expression construct encoding a desired transcription elongation factor or its functional analogue for the purpose of increasing their expression level. Non-limiting examples of such delivery systems are disclosed, for example, in: Principi N. Et al., 2019, https://doi.org/10.3389/fphar.2019.00513; Westwater S. et al., 2003, doi: 10.1128/AAC.47.4.1301-1307.2003; Karimi M et al., 2017, doi: 10.1016/i.addr.2016.03.003. In the case of bacteria, methods for transferring genetic constructs from a donor to a recipient in vivo are also known, for example, those described in Ronda et al., 2019 (doi: 10.1038/s41592-018-0301-y).

[00117] In addition, those skilled in the art will know methods for delivering the transcription elongation factor polypeptides themselves, such as GreA and/or GreB, into cells. Non-limiting examples of such nanoparticle delivery systems are disclosed, for example, in Gao W. et al., 2014, doi: 10.1002/wnan.l282. Non-limiting examples of such delivery systems using (poly)peptides are disclosed, for example, in: Yakimov A.P. et al., ACTA NATURAE | VOLUME 8 No. 4 (31), 2016; Rajarao, G.K. et al., 2002, DOI: 10.1111/j.1574-6968.2002.tb11401.x; Lee, H.-M., et al., 2021, https://doi.org/10.1038/s42003-021-01726-w.

[00118] The term "Exonuclease activity" means an enzymatic activity that causes the cleavage of multiple consecutive nucleotides from the 3' end of a polynucleotide molecule, preferably an RNA molecule.

[00119] By indicating “substantially similar function/activity” of any two or more agents or effects, we mean their interchangeability within the framework of some embodiment of the present invention, in which the choice of any of these agents or effects does not affect the ability to achieve a technical result inventions.

[00120] As used herein, a first compound (eg, an RNAP inhibitor) is considered to “bind” to a second compound (eg, RNAP) if it has a dissociation constant Kd for said second compound of 1 mM or less, preferably 100 μM or less, preferably 50 μM or less, preferably 10 μM or less, preferably 5 μM or less, more preferably 1 μM or less, more preferably 500 nM or less, more preferably 200 nM or less, even more preferably 50 nM or less.

[00121] The expression “conditions that enable” means providing the specified result or activity, but does not affect the technical result of the invention. For example, the phrase “providing contact of compound X with RNAP under conditions that allow inhibition of RNAP activity” would mean providing delivery of compound X to RNAP in such quantity/concentration and under other conditions, for example, native conditions for RNAP, and physicochemical parameters of the environment that RNAP activity will be inhibited.

[00122] Similarly, the phrase: “under conditions that allow an increase in the expression and/or activity of the RNAP elongation factor by Y” would mean that Y is provided in the right place and amount so that the expression and/or activity of the factor increases as a result elongation of RNAP. Those skilled in the art will be aware of methods and means by which it is possible to provide “conditions that enable” a particular result to be achieved, and such methods and means can be anything and are not part of the present invention.

[00123] The terms "effective" or "therapeutically effective" refer to an effect sufficient to initiate the desired biochemical or biological response. Those skilled in the art will appreciate that the effect of the combination of the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the agents being delivered, the RNAP to be inhibited, the particular cell, bacterium or virus in which transcription is to be inhibited, the disease being targeted. treatment, route of administration and specific patient. Treatment is usually “effective” if one or more symptoms or clinical markers improve. Alternatively, a treatment is “effective” if the progression of a disease, disorder, or medical condition is slowed or stopped.

[00124] By “effective concentration” we mean the amount of a substance per unit volume that produces the desired effect or result. Preferably, by effective concentration is meant local concentration, that is, the amount of active substance in the place or volume in which it is desired to provide its effect.

[00125] Effective Amount: The inhibitor or inhibitors are preferably administered in a therapeutically effective amount. The "therapeutically effective amount" for each active compound/inhibitor may vary depending on factors including, but not limited to: the potency of the compound used, the stability of the active compound in the patient, the severity of the condition to be alleviated, the total weight of the patient being treated, route of administration, ease of absorption, distribution and excretion of the compound by the body, age and sensitivity of the patient to be treated, adverse events, etc., which should be obvious to a person skilled in the art. The amount administered may be adjusted as various factors change over time. The inhibitors, methods and applications described in this application are applicable both for human treatment and for use in veterinary medicine.

[00126] In the case of compounds that bind RNAP in accordance with the present invention and provide transcription inhibition by increasing the expression and/or activity of the RNAP elongation factor, the effective amount may correspond to the MIC or IC50 in the region of transcription inhibition or in the target cell. In some embodiments of the present invention, the compound is present in the region of transcription inhibition or in the target cell in an amount or concentration that is effective to significantly or completely inhibit transcription while increasing the expression and/or activity of the elongation factor RNAP. By significant inhibition of transcription can, for example, be understood as such inhibition of transcription that reduces the growth, reproduction and/or viability of the corresponding pathogen or unwanted organism (cell) or prevents the growth and/or reproduction of the pathogen or unwanted organism. In some embodiments of the present invention, significant inhibition of transcription means slowing down the transcription process to a desired value or level. In some embodiments of the present invention, the effective amount of a compound that binds to and/or inhibits RNAP when RNAP elongation factor expression and/or activity is increased is substantially less, for example, two, five, ten, one hundred, two hundred, five hundred, one thousand, or more times less than the amount of the specified compound, which provides transcription inhibition in the case when the expression and/or activity of the elongation factor RNAP is not increased.

[00127] Those skilled in the art will appreciate that the effective amount of compounds that are used in accordance with the present invention may vary depending on the specific RNAP, the specific microorganism, whether it is in a biofilm or any compartment, cell, tissue, microenvironment, or additional substances, molecules or influences that are present at the site or cell where inhibition of RNAP activity and/or inhibition of transcription occurs. For example, the MIC of streptolidigin is approximately 2-3 micrograms per milliliter for E. coli, approximately 1.6 and 6.25 milligrams per liter for Streptococcus salivarius and Mycobacterium smegmatis, respectively, and approximately 100 milligrams per liter for Mycobacterium tuberculosis. The addition of divalent cations, for example MgCl2, can lead to a decrease in MIC values by 10-20 times. The MICs of various tetramic acid derivatives towards methicillin-sensitive Staphylococcus aureus 8325 (MSSA), Enterococcus faecalis ATCC 33186 (EF) and Bacillus anthracis Sterne 34F2 (BA) differ significantly and range from 0.4 to more than 200 micrograms per milliliter (see " for example, Tables 2.1, 2.2 in Wilson, Jason W., 2008, http://dx.doi.org/10.21007/etd.cghs.2008.0354). Effective concentrations of various tetramic acid derivatives against certain G+ bacteria, for example, are also disclosed in Yendapally R et al., 2008, https://doi.org/10.1021/jm701356q. The effect of routericycline derivatives on MDR Staphylococcus aureus located in biofilm is disclosed, for example, in Hurdle et al., 2009, DOI: https://doi.org/10.1128/AAC.00457-09. In each particular case of implementation of the present invention, one skilled in the art will be able to determine the effective amount of the compound that binds to RNAP.

[00128] The term “impact” means a change or changes that occurred during the implementation of specified actions or as a result of the mentioned event, combination of parameters or process, and which would not have occurred in the absence of at least part of the specified actions or the absence of such event, combination parameters or process. Within the scope of the present invention, the result of a particular operation, series of operations, influence/impact, or degree of change can be determined qualitatively or quantitatively based on the analysis of specific measurable characteristics. In most cases, those skilled in the art will know means and methods for measuring such characteristics, including characteristics and parameters by which one can judge whether the technical result of the present invention has been achieved. However, in some cases it is possible to make a qualitative assessment of the outcome of a particular operation, set of operations, impact/impact or degree of change. Non-limiting examples of qualitative assessment of influence by specialists or subjects, which they can carry out in addition to or instead of a quantitative assessment, include determining the presence of changes according to the “yes/no” principle, judgments about changes in taste, smell, color, and other tangible parameters.

[00129] The term "carrier" refers to a solvent, additive, excipient, dispersion medium, solubilizing agent, coating, preservative, isotonic and absorption retarding agent, surfactant, propellant, diluent, base, and the like, with which the active compound is administered. Such carriers may be sterile liquids such as water, saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, and oils, including petroleum, animal, vegetable, or synthetic oils such as peanut oil, soybean oil, mineral oil, sesame oil, and things like that.

[00130] As used herein, the phrase “pharmaceutically acceptable” refers to compounds, materials, compositions and/or dosage forms that, based on careful medical evaluation, are suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic response or other problem or complication, and have an acceptable benefit/risk ratio. Thus, "pharmaceutically acceptable carrier" refers to any and all solvents, additives, excipients, dispersion media, solubilizing agents, coatings, preservatives, isotonic and absorption retarding agents, surfactants, propellants, diluents, bases and the like , which is physiologically compatible. The carrier(s) must be "acceptable" in the sense of not causing harm to the subject being treated, in amounts typically used in medications. Pharmaceutically acceptable carriers are compatible with the other ingredients of the composition without rendering the composition unsuitable for its intended purpose. In addition, pharmaceutically acceptable carriers are suitable for use in subjects as provided herein without undue adverse side effects (such as toxicity, irritation and allergic reaction). Side effects are “excessive” if their risk outweighs the benefit provided by the composition. Non-limiting examples of pharmaceutically acceptable carriers or excipients include any of the standard pharmaceutical carriers such as phosphate buffered saline solutions, water and emulsions such as oil/water emulsions and microemulsions. Suitable pharmaceutical carriers are described, for example, in Remington's Pharmaceutical Sciences, E.W. Martin, 18th Edition. The pharmaceutically acceptable carrier may be a carrier that does not exist in nature.

[00131] For purposes of the present invention, an active compound as defined herein also includes a salt thereof, such as pharmaceutically acceptable salt(s). As used herein, the phrase “pharmaceutically or cosmetically acceptable salt(s)” means salts of the compounds of the present invention that are safe and effective in the desired form of administration. Pharmaceutically acceptable salts include salts formed from anions, for example, salts derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and salts formed from cations, for example, salts of sodium, potassium, ammonium, calcium, iron hydroxides , isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[00132] The term "pharmaceutically acceptable salts" as used herein includes salts of the active compounds derived from relatively non-toxic acids or bases, depending on which substituent groups are present in the particular compounds described herein. If the compounds of the present invention contain functional groups that give them relatively acidic properties, their salts can be prepared by addition of bases by contacting the neutral form of such compounds with a sufficient amount of the required base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts, or the like. If the compounds of the present invention contain functional groups that give them relatively basic properties, their acid addition salts may be prepared by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either pure or diluted in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monocarbonic, phosphoric, monophosphoric, diphosphoric, sulfuric, monosulfuric, hydroiodic or phosphoric acids and the like. as well as salts of relatively non-toxic organic acids, such as acetic, propionic, isobutyric, oxalic, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, etc. Also included are salts of amino acids such as arginate and the like, and salts of organic acids such as glucuronic or galacturonic acids and the like. (See, for example, Berge S.M. et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Some specific compounds of the present invention contain both basic and acid functional groups, which allows the formation of salts of these compounds by addition of both bases and acids.

[00133] As used herein, an “RNAP inhibitor” refers to any compound that is capable of binding to RNAP and inhibiting or completely stopping RNA synthesis. Several classes of agents are known to bind to and inhibit the action of bacterial RNA polymerase, including naturally occurring and synthesized molecules such as the ansamycins (e.g., streptovaricins, rifamycins), sorangicin, and the ripostatins, streptolydigin. and other tetramic acid derivatives, microcins and bacterial lasso-peptides (such as, for example, acinetodin and klebsidin, see Fig. 6), pyronins (for example, myxopyronin, corallopyronin) as well as numerous synthetic molecules (for example analogs of CBR703, GE23077, SB2 (phenyl-furanyl-rhodanines), ureidothiophene, which are able to bind to RNA polymerase and inhibit various stages of the transcription process.

[00134] Preferred compounds within the context of the present invention are compounds that bind to RNAP, in particular RNA polymerase inhibitors that bind to the active site of RNAP, for example entering the active site through a secondary channel. In the case of bRNAP, compounds whose binding region to bRNAP at least partially overlaps with the binding region of streptolidigin are preferred.

[00135] Tetramic acid derivatives: By “tetramic acid derivative” is meant any compound that contains a pyrrolidine-2,4-dione (tetramic acid) group.

[00136] Various tetramic acid derivatives can be isolated from natural sources or synthesized. Methods for the synthesis of such derivatives are known to those skilled in the art, see, for example, Isao Mizota et al., Org. Lett. 2020, https://doi.org/10.1021/acs.orglett.0c00824; Jeong & Moloney, Synlett 2009(15): 2487-2491, doi: 10.1055/s-0029-1217745; Singh et al., 2006, Belastein Journal of Organic Chemistry, doi: 10.1186/1860-5397-2-24; Wilson, Jason W., "Synthesis and Evaluation of Tetramic Acids as Antimicrobial Agents" (2008). Theses and Dissertations (ETD). http://dx.doi.org/10.21007/etd.cghs.2008.0354

[00137] Non-limiting examples of tetramic acid derivatives include:

[00138] Tenuazonic acid Org Biomol Chem, 2, 2004, 3524; Reutericyclin Synthesis, 2006, 3902; Melophlin B Tetrahedron, 61, 2005, 2301; macrocidin A (Macrocidin A) Org Lett, 18, 2016, 6352; Ravenic acid Chem Eur J, 16, 2010, 2599; epicoccamide D Chem Eur J, 19, 2013, 10619; penicillenol C1 (Penicillenol C ₁ ) J Org Chem, 78, 2013, 2455; Torrubiellone D Org Lett, 18, 2016, 1136; Aurantoside G Angew Chem IE, 55, 2016, 10122; Methiosetin J Org Chem, 81, 2016, 7336; rigidiusculamide B Org Biomol Chem, 14, 2016, 9262; F-14329* Chem Eur J, 23, 2017, 5692; Spiroscytalin J Org Chem, 82, 2017, 7791; Ancorinoside A Chem Eur J, 23, 2017, DOI: 10.1002/chem.201704379; Cladosin C Org Biomol Chem, 15, 2017, 7672, derivatives of these compounds, as well as compounds disclosed in the publications: Jiang et al., Mar Drugs, 2020 18(2): 114, (doi: 10.3390/md18020114) ; US 4,169,096, 09/25/1979; WO 01/51465 A1, 07/19/2001; US 6,962,943 B2, 8.11.2005; US 6,599,930 B2 of 07/29/2003; WO 2014/001775 A1, 01/03/2014, Yendapally R et al., 2008, https://doi.org/10.1021/jm701356q; Hurdle et al., 2009, DOI: https://doi.org/10.1128/AAC.00457-09, including the contents of sources cited by reference in these publications.

[00139] By analogue of a reference compound or small molecule can be understood a structural, chemical or functional analogue thereof. Thus, the term covers compounds whose chemical or dimensional structure differs from the referenced compound, but such differences do not affect or significantly affect the function or activity that enables the present invention. Such analogues may include isomers, chemically modified derivatives or mimetics of the title compound. For example, an analogue of a compound that binds to RNAP can be a molecule or substance of any nature that binds to the same parts of the RNAP molecule and, when bound, has the same effect on the structure or activity of RNAP as the referenced compound.

[00140] As used herein, the term “prodrug” refers to any compound that, when introduced into a biological system, forms a compound as described herein that inhibits the activity of HCV (an “active inhibitory compound”). A compound may be formed from a prodrug by: (i) spontaneous chemical reaction(s), (ii) enzyme-catalyzed chemical reaction(s), (iii) photolysis, and/or (iv) ) metabolic chemical reaction(s).

[00141] The term “prodrug moiety” refers to a labile functional group that is separated from the active inhibitory compound by metabolism, in the body, within a cell, by hydrolysis, enzymatic degradation, or other process (Bundgaard, Hans, “Design and Application of Prodrugs" in A Textbook of Drug Design and Development (1991), P. Krogsgaard-Larsen and H. Bundgaard, Eds. Harwood Academic Publishers, pp. 113-191). Enzymes capable of enzymatic activation of prodrugs of the compounds herein include, but are not limited to, amidases, esterases, microbial enzymes, phospholipases, cholinesterases, and phosphases. Prodrug moieties are capable of increasing solubility, absorption, and lipophilicity to optimize drug delivery, bioavailability, and efficacy. The prodrug moiety may contain an active metabolite or the drug itself.

[00142] Solvates: Additionally, the compounds useful in the present invention, salts or prodrugs of such compounds, may exist in hydrated or non-hydrated (anhydrous) form or as solvates with other solvent molecules. Non-limiting examples of hydrates include monohydrates, dihydrates, etc. Non-limiting examples of solvates include ethanol solvates, acetone solvates, etc.

Detailed Disclosure of the Nature and Aspects of the Present Invention

[00143] In accordance with the present invention, new approaches to inhibiting RNA polymerase activity, as well as corresponding means and methods for inhibiting transcription, are proposed, which can be used, in particular, to obtain new classes of antibacterial, antimycotic and antiviral agents.

[00144] In connection with the present invention, we have discovered that transcription factors that are responsible for RNA polymerase processivity unexpectedly provide or enhance the inhibitory effect of compounds that bind to RNA polymerase on the transcription process.

[00145] Thus, the present invention is generally based on the fact that the inhibitory effect of a compound that binds to RNA polymerase on transcription is exerted or enhanced by increasing the amount and/or activity of the elongation factor (transcription factor) that is used by said RNA polymerase (RNAP). Preferably, said manifestation or enhancement of the inhibitory effect on RNAP is synergistic.

[00146] Accordingly, the present invention relates to the use of agents that increase the expression and/or activity of the RNAP elongation factor to enhance or cause transcriptional inhibition by a compound that is capable of binding to RNAP.

[00147] As a technical result of the present invention, for example, one can consider the enhancement or manifestation of the inhibitory effect that a compound that binds in RNAP has on RNA synthesis. As another technical result of the present invention, one can consider expanding the range of agents (drugs) for inhibiting transcription. As another technical result of the present invention, one can consider increasing the effectiveness of existing transcription inhibition agents.

[00148] In the case where effective transcription inhibition is provided for medical applications, for example, to eliminate bacterial, yeast and/or viral pathogens, as another technical result of the present invention, it can be considered to reduce or eliminate the negative consequences and symptoms of infection with these bacteria, fungi and/or viruses, and reducing the undesirable side effects of the antibacterial, antimycotic or antiviral agents used, for example, by reducing their quantity or duration of action, which are required to ensure the desired result.

[00149] In the case of implementing the screening methods according to the present invention, the technical result of the present invention may be to identify a series (library) of candidate substances/compounds (promising, potentially useful substances) for inhibiting transcription (inhibiting RNAP activity) in combination with an agent in combination with an agent , which increases the expression and/or activity of the RNAP elongation factor. The technical result of the screening methods of the present invention may also be to exclude a number of substances or classes of substances/compounds as suitable for use in inhibiting transcription in accordance with the present invention.

[00150] In the case of bRNAP, inhibition of RNA synthesis in the bacterium will result in suppressed growth and/or decreased viability of the bacterium. In the case of vRNAP, inhibition of viral RNA synthesis will lead to suppression of viral reproduction or development cycle. In the case of gRNAP, inhibition of the RNA synthesis of a fungus, such as yeast or mold, will result in inhibition of the reproduction or developmental cycle of the fungus itself or the fungal virus. In the case of eRNAP, inhibition of RNA synthesis can be used to modulate transcription, such as suppressing the synthesis of unwanted RNA or total RNA in the cell.

[00151] This application also discloses methods for preparing drugs, which may be compositions and combinations for inhibiting RNAP that include at least two components. In cases where these drugs have an inhibitory effect on bacterial RNA polymerases (bRNAP), they can be used as antibacterial agents. In cases where these drugs have an inhibitory effect on fungal RNA polymerases (gRNAP), they can be used as antifungal agents. In cases where these drugs have an inhibitory effect on RNA polymerases that viruses use (vRNAP), they can be used as antiviral agents.

[00152] Those skilled in the art will understand that the present invention is not limited only to those compounds that are capable of binding and preferentially inhibiting RNAP, and only to those means that provide an increase in the amount and/or activity of the elongation factor (transcription factor) used by the specified RNAP, which are directly disclosed in this application, and also applies to those compounds and agents whose mutual influence on RNAP specialists can detect after reading this application, for example, using the methods of preparation and screening disclosed in this description. Accordingly, the specific embodiments of the present invention and the targets of the agents(s) that can be prepared in accordance with the present inventions will depend on the origin (e.g., a particular bacterium, virus, fungus, or cell) of the particular RNAP that will be inhibited. the effect of the compound according to the present invention by increasing the activity and/or amount of the corresponding elongation factor (transcription factor RNAP.

[00153] Accordingly, compounds that have been shown to bind to RNAP and/or inhibit RNAP activity and their use in inhibiting RNAP, particularly the compounds disclosed herein, may be considered analogues of the present invention.

Summary of the present invention

[00154] Thus, in general, the present invention relates to the use of an agent that increases the expression and/or activity of an RNAP elongation factor to enhance or provide an inhibitory effect of a compound that is capable of binding to said RNAP.

[00155] In some embodiments of the present invention, the compound that binds (interacts) with the RNA polymerase is a small molecule. In some embodiments of the present invention, a compound that binds (interacts) with RNA polymerase and that is used in accordance with the present invention increases the lifetime of one of the RNAP conformations and/or helps to increase the duration of the transition state between RNAP conformations (the lifetime of the intermediate RNAP conformations) and/or mediates the emergence of new structural conformations of RNAP during the transcription process. In some embodiments of the present invention, the compound that binds (interacts) with the RNA polymerase is an RNA polymerase inhibitor.

[00156] In some embodiments of the present invention, a compound that binds (interacts) with RNA polymerase and that is used in accordance with the present invention acquires the property of inhibiting transcription elongation of RNA polymerase when the expression and/or activity of the elongation factor RNAP is increased.

[00157] In some embodiments of the present invention, the RNA polymerase inhibitor is an inhibitor of transcription at the initiation, elongation, or termination stages of transcription.

[00158] In some embodiments, the RNA polymerase is a bacterial RNA polymerase, a fungal RNA polymerase, such as a yeast, or a viral RNA polymerase. In some aspects of the present invention, the RNA polymerase may be a eukaryotic RNA polymerase.

[00159] In some embodiments of the present invention, the RNA polymerase is a viral RNA polymerase from an RNA virus, for example, corona virus, norovirus, rhabdovirus, enterovirus, influenza virus, picornavirus, arenavirus, Ebola virus, rhabdovirus, paramyxovirus, flavivirus, hepadnovirus, for example , RNA polymerase from SARS-Cov-2, RNA polymerase from the influenza virus, or RNA polymerase used by the hepatitis C virus or HIV.

[00160] In some embodiments of the present invention, the RNA polymerase inhibitor does not, or does not substantially inhibit, eukaryotic RNA polymerase.

[00161] In some embodiments of the present invention, the RNA polymerase inhibitor is a tetramic acid derivative. In some embodiments, the RNA polymerase inhibitor is a tetramic acid derivative that is capable of binding to bRNAP and preferably has antibacterial activity, or a solvate, salt, prodrug, or derivative of said tetramic acid derivative that has similar properties and/or antibacterial activity

[00162] In some embodiments of the present invention, the tetramic acid derivative is streptolidigin

[00163] In some embodiments of the present invention, the agent that increases the expression and/or activity of the RNAP elongation factor is a genetic construct that causes the expression of the RNAP elongation factor in a cell. In some embodiments of the present invention, the agent that increases the expression and/or activity of the RNAP elongation factor is a virus that contains a genetic construct that causes the expression of the RNAP elongation factor in the cell. In some embodiments of the present invention, the agent that increases the expression and/or activity of the RNAP elongation factor is a genetic construct that provides expression of the RNAP elongation factor and is integrated into the genome of the cell. In some embodiments of the present invention, the construct is under the control of an inducible promoter.

[00164] In some embodiments of the present invention, the agent that increases the expression and/or activity of the RNAP elongation factor is a small molecule that induces the expression of the RNAP elongation factor. In some embodiments, the agent that increases the expression and/or activity of the RNAP elongation factor is a small molecule, nucleotide, peptide, or polypeptide that causes the expression of the RNAP elongation factor or the conversion of the RNAP elongation factor to an active form.

[00165] In some embodiments of the present invention, the agent that increases the expression and/or activity of the elongation factor RNAP is a bacteriophage that delivers to the bacterium a genetic construct that causes expression of the elongation factor bRNAP.

[00166] In some embodiments of the present invention, an agent or treatment that increases the expression and/or activity of RNAP elongation factor provides an increase in the amount of RNAP elongation factor in the cell. In some embodiments of the present invention, increased RNAP elongation factor activity is achieved by increasing the amount of RNAP elongation factor in the cell. In some embodiments of the present invention, increased RNAP elongation factor activity is achieved by delivering RNAP elongation factor into the cell.

[00167] In some embodiments of the present invention, the effect that increases the expression and/or activity of the elongation factor RNAP is a change in temperature, irradiation, including optical and high frequency irradiation, a change in the physical and/or chemical characteristics of the medium or fluid in the area or environment, in which the cell is located.

[00168] In some embodiments of the present invention, the expression and/or activity of the RNAP elongation factor is increased by an agent or effect that increases the expression and/or activity of the RNAP elongation factor, for example, by activating the promoter of an RNAP elongation factor gene already present in the genome the specified cell; transformation or transduction of said cell with the introduction into the cell of a heterologous RNAP elongation factor gene; delivery of FET proteins to the specified cell.

[00169] In some embodiments of the present invention, the agent that increases the expression and/or activity of the RNAP elongation factor is selected from: a genetic construct that provides expression of the RNAP elongation factor, a virus that delivers into the cell a genetic construct that provides expression of the RNAP elongation factor, a small molecule, nucleotide, peptide or polypeptide that provides expression of the RNAP elongation factor or the transition of the RNAP elongation factor to an active form and/or the specified agent ensures an increase in the amount of RNAP elongation factor in the cell; and/or an effect that increases the expression and/or activity of the elongation factor RNAP is a change in temperature, irradiation, including optical irradiation, a change in the physicochemical characteristics of the medium or fluid in the area or environment in which the specified cell is located.

[00170] In some embodiments of the present invention, the cell is a bacterial or fungal cell. In some embodiments, the RNAP is a bacterial RNAP, a eukaryotic RNAP, such as a fungal RNAP, or an RNAP that is used by a virus, such as a virus-encoded vRNAP.

[00171] In some embodiments of the present invention, the RNAP elongation factor is an RNAP elongation factor or transcription factor that has exonuclease activity. In some embodiments of the present invention, the RNAP elongation factor causes the cleavage of at least about 2-3, or at least up to about 10-15 ribonucleotides from the end of the RNA molecule that is synthesized by the RNA polymerase.

[00172] In some embodiments of the present invention, the RNAP elongation factor is GreA or GreB from E. coli, or an analogue or homologue thereof that has substantially similar function or activity.

[00173] In some embodiments of the present invention, a GreA or GreB homologue or derivative that retains the activity of GreA or GreB has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least % sequence identity with GreA or GreB from E. coli.

Methods of obtaining

[00174] This application also provides methods for preparing agents that may be compositions and combinations for inhibiting RNAP.

[00175] In one embodiment, the method of the present invention includes providing a compound that is capable of binding to RNAP; providing an agent that increases the expression and/or activity of an elongation factor of said RNAP; contacting an effective amount of said compound and an effective amount of said agent with RNAP or a cell in which RNAP is present, under conditions that allow said compound to bind to RNAP and increase the expression and/or activity of the elongation factor of said RNAP under the influence of said agent, and determining the effect that the combination of the specified compound and agent has on the activity of RNAP, and the combination is selected as a means for inhibiting transcription if the effect of the combination on the activity of RNAP is negative (inhibitory) and stronger (pronounced) than the effect of the specified compound alone on the activity of the specified RNAP.

[00176] In one embodiment of the present invention, the transcription elongation step is inhibited.

[00177] In some embodiments, the agent that increases the expression and/or activity of the RNAP elongation factor is an agent or effect.

[00178] In some embodiments, the RNAP is bacterial RNAP (bRNAP), the elongation factors are bRNAP elongation factors, and the compositions or combinations of the present invention have an antibacterial effect, such as a negative effect on the growth and/or viability of the bacterium . In embodiments where the bRNAP is used by a virus (phage), these combinations prevent or inhibit the development of the virus in the bacterial cell.

[00179] In other embodiments, the RNAP is viral RNAP (vRNAP), the elongation factors are elongation factors used by vRNAP, such as eukaryotic cell proteins, and the compositions or combinations of the present invention have an antiviral effect, such as preventing or inhibit the development of the virus in the cell.

[00180] In some embodiments, the RNAP is fungal RNAP (gRNAP), the elongation factors are elongation factors that gRNAP uses, and the compositions or combinations of the present invention have antifungal effects, such as growth and/or viability of the specified fungus. In embodiments where the bRNAP is used by a virus (phage), these combinations prevent or inhibit the development of the virus in the fungal cell.

Screening methods

[00181] Also provided herein are screening methods to find new RNA polymerase inhibitors for use in combination with an agent that increases elongation factor expression and/or activity, or to identify existing RNAP inhibitors whose effectiveness is enhanced by such combination. These methods involve determining the inhibitory effect of a plurality of substances, for example, small molecules, (poly)peptides, nucleotides, having the ability to bind to RNAP in combination with exposure to an effective amount of an agent that increases the expression and/or activity of the elongation factor (transcription factor) of the specified RNAP in accordance with the present invention. Those substances from the specified set of substances, the inhibitory effect of which is manifested or enhanced by increasing the expression and/or activity of the RNAP elongation factor, are selected for use as RNAP inhibitors in combination with the specified agent.

[00182] In one embodiment, screening methods are provided to find new antibacterial agents. These methods include determining the antibacterial effect of a variety of substances, for example, small molecules, (poly)peptides, nucleotides, having the ability to bind to bRNAP in combination with an agent that increases the expression and/or activity of the bRNAP elongation factor (bacterial transcription factor) in accordance with the present invention. Those substances from the specified set of substances, the antibacterial effect of which appears or increases with an increase in the expression and/or activity of the bRNAP elongation factor, are selected for use as antibacterial drugs (agents) in combination with the specified agent.

[00183] In one embodiment, screening methods are provided to find new antiviral agents. These methods involve determining the antiviral effect of a variety of substances, for example, small molecules, (poly)peptides, nucleotides, having the ability to bind to vRNAP or RNAP used by the virus, in combination with an agent that increases the expression and/or activity of the vRNAP elongation factor or factor elongation or RNAP that a virus uses (eg a eukaryotic transcription factor) in accordance with the present invention. Those substances from the specified set of substances, the inhibitory effect of which on vRNAP or RNAP that the virus uses appears or increases with an increase in the expression and/or activity of the elongation factor that is used by the specified RNAP, an increase in the expression and/or activity of the elongation factor vRNAP is selected for use as antiviral drugs (agents) in combination with the specified agent.

[00184] In one embodiment, screening methods are provided to find new antifungal agents. These methods include determining the antimycotic effect of a variety of substances, for example, small molecules, (poly)peptides, nucleotides, having the ability to bind to gRNAP in combination with an agent that increases the expression and/or activity of the gRNAP elongation factor in accordance with the present invention. Those substances from the specified set of substances, the inhibitory effect of which on gRNAP appears or increases with an increase in the expression and/or activity of the gRNAP elongation factor, are selected for use as antifungal drugs (agents) in combination with the specified agent.

[00185] In some embodiments of the screening methods of the present invention, specified substances from a specified plurality of substances, the inhibitory effect of which on the corresponding RNAP appears or is enhanced by increasing the expression and/or activity of the elongation factor that the specified RNAP uses, are initially selected as candidate substances (promising, potentially suitable substances) for use in combination with the specified agent for inhibiting the corresponding RNAP. Once a population of such candidate substances is determined, in some embodiments they are further screened for the presence of a desired property, e.g., pharmaceutical acceptability, bioavailability, stability, non-toxicity, etc., and/or modified to ensure the presence of said desired property, and those substances are selected that have specified desired property.

[00186] In some embodiments, modifications, modified forms, derivatives, or analogs of a portion or all of such candidate substances are prepared to modulate their effects on RNAP, such as enhancing their binding to or inhibiting effects on RNAP. After selecting and/or producing modifications, modified forms, derivatives or analogs of at least a portion of the initially selected population of candidate substances, the screening methods or production methods of the present invention can be applied to such selected, modified, derivative or similar substances.

[00187] In some embodiments, the present invention relates to a library of substances that are identified by applying the screening methods of the present invention. Accordingly, in some embodiments, the screening methods of the present invention are methods of obtaining a library of substances for use in inhibiting RNAP in accordance with the present invention.

[00188] In some aspects, the present invention discloses methods for determining (identifying) RNAP elongation factors, and/or their analogs, homologues, mutants and variants, the activity of which, in combination with a compound that is capable of binding to RNAP, provides or enhances the inhibitory effect of such combination on RNAP or the transcription process. RNAP, in relation to the elongation factors of which these methods are carried out, is selected from bRNAP, vRNAP, gRNAP, or RNAP, which is used by viruses. Thus, in some embodiments, the present invention discloses methods for determining vRNAP elongation factors or elongation factors that vRNAP uses, as well as analogues, homologues, mutants and variants thereof, the activity of which, in combination with a compound that is capable of binding to vRNAP, provides antiviral activity such combinations. In certain aspects, the present invention discloses methods for determining gRNAP elongation factors, as well as analogs, homologues, mutants and variants thereof, the activity of which, in combination with a compound that is capable of binding to gRNAP, provides or enhances the antimycotic effect of such combination.

[00189] These methods determine the inhibitory effect of a substance, for example, a small molecule, (poly)peptide, nucleotide, having the ability to bind to RNAP in combination with exposure to an effective amount of an agent that increases the expression and/or activity of an elongation factor (transcription factor ) the specified RNAP, and the specified stage of determining the inhibitory effect is carried out for several (several) RNAP elongation factors, and/or their analogs, homologs, mutants or variants, and those RNAP elongation factors, and/or their analogs, homologues, mutants or variants, the expression and/or activity of which in combination with the specified substance provides the manifestation or enhancement of an inhibitory effect on RNAP or the transcription process is selected to enhance their expression and/or activity when inhibited by the use of the specified substance of the means of increasing expression or activity according to the present invention.

[00190] In some embodiments of the present invention, these analogs or variants are hybrid polypeptides or RNAP elongation factors that are heterologous to a particular RNAP or cell. In some embodiments of the present invention, these analogs or variants are polypeptides that contain an additional non-peptide group.

Agents and drugs for transcription inhibition

[00191] The present invention also covers transcription inhibitory agents that include, in an effective amount, a compound that is capable of interacting with RNA polymerase (RNAP), wherein said compound is defined herein; and an agent that increases the expression and/or activity of an RNAP elongation factor that the specified RNAP uses or may use, wherein the combination of the specified compound and the RNAP agent provides a decrease in the transcription efficiency of the specified RNAP, which is greater than the decrease in the transcription efficiency under the influence of the specified RNAP alone connections.

[00192] In some embodiments, the compound by itself does not reduce or significantly reduce the efficiency of transcription.

[00193] In some embodiments, the agents of the present invention may be a composition or combination of two substances, one of which is a compound that is capable of interacting with RNA polymerase (RNAP), where the specified compound is defined in this application, and the second is a compound that a molecule, genetic construct, protein construct or substance that provides an increase in the expression and/or activity of the elongation factor of the specified RNAP.

[00194] Non-limiting examples of the specified compound, molecule, genetic construct, protein construct, lipid complex or substance can, for example, serve as a polynucleotide, vector, virus, liposome, which includes a polynucleotide encoding the specified elongation factor and is adapted for its transmission and expression by PET in target cell. Other examples of such compounds, molecules, genetic constructs, protein constructs, or substances include, for example, various small molecules, polypeptides, peptides, oligos, and polynucleotides that are inducers of the expression of corresponding PETs in a cell, for example, PETs encoded by heterologous polynucleotides.

[00195] In some embodiments, the agents of the present invention may be a combination of a substance that is a compound that is capable of interacting with RNA polymerase (RNAP), wherein said compound is defined herein, and an effect that provides an increase in expression and/or activity elongation factor of the specified RNAP.

[00196] Non-limiting examples of such effects are temperature and irradiation, which can for example be used in the case of thermo- or photo-inducible expression of PET.

[00197] In some embodiments, the agents of the present invention may be a composition or combination of two substances, one of which is a compound that is capable of interacting with RNA polymerase (RNAP), where the specified compound is defined in this application, the second is a compound, a molecule , a genetic construct, a protein construct or a substance that provides an increase in the expression and/or activity of the elongation factor of the specified RNAP, in combination with an effect that.

[00198] In some embodiments, the transcription inhibitory agents of the present invention are formulated as formulations, such as disinfectants or pharmaceutical compositions, that also contain pharmaceutically acceptable carriers and excipients. Examples of carriers are known in the art and are described herein. The compositions of the present invention may contain at least one transcription inhibitory agent of the present invention in combination with a pharmaceutically acceptable carrier. Examples of such carriers and drug manufacturing techniques can be found in Remington: The Science and Practice of Pharmacy (20th Ed., Lippincott, Williams & Wilkins, Daniel Limmer, editor) and are generally well understood by one skilled in the art. In some embodiments of the present invention, the drug or pharmaceutical composition is adapted to deliver the composition to a subject in an area of presence of an unwanted microorganism (area of infection), for example, in a specific organ, tissue, area of the body, membrane, area of injury, wound.

[00199] To formulate a drug or pharmaceutically acceptable composition suitable for administration or application to a desired area, the compositions must contain an effective amount of the active compound or compounds. For the purposes of the present invention, an effective amount of an active compound is an amount of an active compound that is sufficient to provide inhibition of RNAP-mediated transcription in the event of an increase in the activity and/or expression of a transcription elongation factor that the RNAP uses. When the effective amount provides a positive therapeutic result, the term "therapeutically effective amount" is also used.

Additional Aspects of the Present Invention

[00200] Those skilled in the art will appreciate that the compositions and agents for inhibiting transcription in accordance with the present invention can be used in medicine and various fields of technology. The objectives that can be achieved using the transcription inhibitory agents of the present invention may depend on the particular RNAP whose transcription efficiency is reduced (RNAP that is inhibited). For example, the preparations and agents of the present invention can be adapted and used in methods of treating unwanted infections or eliminating unwanted microorganisms (eg, pathogens) in subjects in need thereof. In some cases, transcription/RNAP inhibition approaches and RNAP binding compounds can be used to selectively inhibit the growth and elimination of transformed mammalian cells, such as cancer cells. In addition, approaches to inhibit RNAP and/or inhibit transcription may be useful in agriculture, food processing, and biotechnology.

[00201] In addition to inhibiting and/or eliminating unwanted pathogens that harm plants, fungi and farm animals, approaches to inhibit RNAP activity may be useful in biotechnology, for example, in production organisms that are genetically modified to provide constitutive or inducible expression of the RNAP elongation factor . In particular, modulation of transcription rate may be useful for optimizing the level of gene expression in the biotechnological production of recombinant proteins; regulation of the expression of certain enzymes in artificial bioreactors; optimization of the processes of synthesis and assembly of recombinant viral particles. Likewise, specialists will understand other options for using agents to inhibit vRNAP, gRNAP, and eRNAP.

[00202] One skilled in the art will appreciate that as technology advances, the inventive spirit of the present invention may be implemented in a variety of ways. The invention and embodiments thereof are not limited to the specific embodiments directly disclosed herein, but may vary within the scope of the claims.

EXAMPLES

[00203] The following examples are provided to demonstrate the principles underlying the present invention and to further illustrate certain aspects of the present invention and should not be construed as limiting the scope of the present specification or claims.

Acoustic force spectroscopy technique

[00204] The results of in situ experiments were obtained using the technique of studying transcription at the single-molecule level and subsequent analysis of transcription elongation profiles using acoustic force spectroscopy (AFS). To understand that the data presented in the examples were obtained using an adequate experimental model for studying the transcription and activity of RNAP, below is a detailed description of the main sections of the ACC methodology.

Equipment and materials used

[00205] Purification of RNAP and GreA/GreB: Biotinylated RNAP was purified as previously described (see, for example, Metelev, M. et al., 2017). To purify recombinant GreA and GreB proteins, E. coli Rosetta cells containing plasmids encoding GreA or GreB were grown to 0.6 OD, then protein expression was induced by adding 1 mM IPTG and the cells were incubated at 37°C for 5 hours. Proteins were purified using Me-affinity (HisTrap Hp, Cytivia) and SEC (Superdex 75 GE) chromatography.

[00206] Acoustic force spectroscopy (ACC): ACC is the most recent approach to studying the synthesis of polynucleotides at the single-molecule level. This method was first presented at the end of 2014. When implementing this technique in the examples, the Lumicks AFS installation was used. The essence of the method is as follows: DNA molecules are fixed between the surface of the glass and the microspheres (Figure 1A). The force, which is of an acoustic nature, allows the microspheres to be manipulated. The experiment is carried out using a special AFS chip, which is a cell delimited by two glass plates with liquid between them. A piezo generator located on the AFS chip, when voltage is applied, creates a plane acoustic wave acting on polymer microspheres, their displacement is recorded using an inverted microscope and camera.

[00207] An acoustic wave propagating inside the AFS chip creates a force acting on the microsphere. The magnitude of this force depends on the size of the microsphere, the frequency of the acoustic wave and its amplitude, which in turn depends on the voltage applied to the piezo element.

ρ - sound pressure

ν - speed of sound

- ratio of microsphere and medium densities

- ratio of the elasticity coefficients of the microsphere and the medium

[00208] As a rule, the range of forces when using this method ranges from units to tens and even hundreds of pN, which makes it possible to apply it to studies of biological objects. To study transcription, the microsphere must interact with RNA polymerase (see Fig. 2A). Using this method, RNAP elongation profiles are recorded, which are a graphical interpretation of the characteristic movement of single RNAP complexes that carry out transcription elongation, presented in the form of a dependence of the transcribed length of DNA (in nanometers or nucleotides) from time (seconds). Elongation profiles reflect the dynamic characteristics of a single RNAP, such as speed, the presence of stops/pauses and their duration. Typical types of RNAP elongation profiles that were obtained in the absence of the influence of inhibitory factors are shown in Figure 2B.

Construction of the shutdown complex

[00209] To study the elongation profiles of RNAP under control conditions in the absence of additional chemical agents, a DNA substrate (4400 bp) was used, which was a construct consisting of the T7A1 promoter, a region of the rpoB gene, two terminators rrnB T1 and rrnB T2. To attach the construct to the surface coated with the appropriate antibodies, modified nucleotides containing digoxigenin were included at the end opposite to the promoter. The transcription enzyme, RNA polymerase (RNAP) of E. coli, was modified with biotin for further attachment to the streptavidin-coated microsphere. To form a stopped transcription complex, 25 nM RNAP and 5 nM template DNA were incubated at 37°C in the presence of the ribonucleotides ATP, GTP, CTP, as well as the diribonucleotide AfU in transcription buffer A (40 mM Tris-HCl, 40 mM KCl, 0.5 mM DTT, 5 mM MgCl ₂ ). Under these conditions, transcription initiation and elongation occurred over a distance of 20 nucleotides from the transcription start point to the position where uracil triphosphate (UTP) ribonucleotides are required to continue elongation. After several successive stages of passivation of the AFS chip, the formed structures with the stopped complex were fixed on its surface. Next, a suspension of polystyrene microspheres with a diameter of 2.1 μm coated with streptavidin (Spherotech) was fed into the AFS chip, which contacted the biotin-modified RNAP “stopped” on the DNA template. The chamber was washed with transcription buffer B (40 mM Tris-HCl, 80 mM KCl, 0.5 mM DTT, 10 mM MgCl2, 0.02% pluronic, 0.02% casein, 50 μg/ml heparin, 1% DMSO) to remove microspheres that did not contact RNAP.

[00210] Using special software, the points of attachment of the resulting structures to the surface were determined and all necessary calibrations were performed. During the experiment, a minimum constant force of about 5 pN was used, the magnitude of which was insufficient to influence the transcription process. In the control experiment, after introducing a mixture of ribonucleotides with a concentration of 1 mM dissolved in buffer B into the research chamber, the elongation process was resumed. The movement of single RNAPs was recorded by changes in the positions of the microspheres. As a result of a series of experiments, elongation profiles of single RNAP molecules were obtained under control conditions, according to which the following transcription parameters were assessed: average speed (the ratio of the path traversed by the polymerase (length of the elongation profile) to the total time spent by RNAP on its passage); instantaneous speed (speed between pauses lasting more than 2.5 s); duration of pauses (time the RNA polymerase remains motionless); and the number of pauses during the passage of 500 nm through the DNA template after the start of transcription.

Data processing algorithm

[00211] Recognition of pauses: After averaging and re-smoothing the noise-containing elongation profiles with a Savitsky-Golay filter, limiting speeds were set, and all time intervals in which the speed values were less than the threshold were considered as pauses. These pauses were sorted into “long” pauses, which lasted more than 20 seconds, and “short” pauses, which lasted from 2.5 seconds to 20 seconds. The threshold value was chosen equal to 0.5σ, where σ is the standard deviation of the mean obtained from the Gaussian distribution for instantaneous velocities (see below). The obtained values were averaged over the entire ensemble of pauses and presented as the mean ± s.e.m. The Mann-Whitney test was used as a criterion for statistical significance.

[00212] Determination of instantaneous speeds: As a result of processing with the selected maximum speed threshold, a distribution of instantaneous speeds was formed. Based on this distribution, a histogram of instantaneous velocities was constructed in OriginPro software (Figure 3A).

[00213] In general, the velocity distribution will contain signals corresponding to the derivatives of the elongation profiles from the noise and useful signals. The noise component is determined by the parameters of the experimental setup and its spectrum corresponds to the distribution of derivatives of those sections of the elongation profile when the movement of the polymerase is stopped, i.e. when it is in paused states. The noise derivative has a symmetric distribution relative to zero velocity, and to take into account its contribution, we used its negative part of the distribution, on which the useful signal is not superimposed. Since the positive values of the noise signal are equal in absolute value to its negative values, the true distribution of instantaneous velocities was obtained by subtracting the absolute values of the noise signal from the total velocity distribution.

[00214] The histogram shown in Figure 3A has a characteristic bimodal distribution. Expansion of this distribution using Gaussian functions allows us to identify a distribution near zero (left peak) corresponding to the noise component of the signal and the true instantaneous speed (right peak).

[00215] To refine the instantaneous speed parameters, the above procedure for subtracting the noise signal was used. Figure 3B shows the Gaussian function approximation of the velocity distribution after subtracting the noise signal. As can be seen from the approximation results, the value of the instantaneous speed with this treatment is slightly higher, which is closer to the true values of the instantaneous speed of RNA polymerase. After carrying out these manipulations for each obtained elongation profile, the data for all profiles corresponding to the given experimental conditions were averaged and presented as the mean value ± standard error of the mean (s.e.m.).

[00216] Pn Determination of average speeds: The value of the average transcription speed for each RNA polymerase is equal to the ratio of the path traveled by the polymerase (length of the elongation profile) to the total time spent on its passage. The obtained values were averaged over the entire ensemble of velocities for each of the experimental conditions and presented as mean ± s.e.m. The Mann-Whitney test was used as a criterion for statistical significance.

EXAMPLE 1

Streptolidigin induces long pauses

[00217] Inhibition of E. coli RNA polymerase by streptolidigin (STL) was studied using the ACC Method. The results of measurements were compared under control conditions without STL in transcription buffer B with the addition of a one-molar concentration of NTP ribonucleotides (ATP, GTP, CTP, UTP) and in the presence of 10 μM STL in the control mixture. Representative elongation profiles for both cases are shown in Figure 4A, B. Processing of the measurement results showed that in the presence of STL, the average transcription elongation rate significantly decreases and rare long-lived pauses appear lasting more than 20 seconds in Figure 4A, B. The average duration of such pauses is 192 ± 44 seconds (mean value ± (SE)), frequency of their occurrence 1 ± 0.2 per elongation profile

EXAMPLE 2

Streptolidigin interacts with RNAP intermediates

[00218] The dependence of the efficiency of STL inhibition on such intermediate states of RNA polymerase as “backtracking” and elementary pauses, the number of which varied by changing the composition of ribonucleotides, was studied. The addition of artificial inosine nucleotide (ITN) stimulated backtracking pauses, since this nucleotide is atypical for RNAP, and a decrease in the concentration of ribonucleotides stimulated an increase in elementary pauses.

[00219] In the presence of 200 μM inosine and 1 mM ribonucleotides, long pauses of more than 20 seconds were observed (Figure 5A). Addition of 10 μM STL to this medium did not change the instantaneous speed and the number of short pauses, but decreased the average speed mainly due to an increase in the number of long pauses. The number of long pauses did not exceed the sum of pauses formed separately by STL and inosine. (Figure 5B, C).

[00220] When the NTP concentration was decreased from 1 mM to 400 μM, a noticeable decrease in both instantaneous and average speed was observed due to a significant increase in the number of short elementary pauses (Figure 5A, B, D). With the addition of STL, a sharp increase in the number and duration of long pauses was recorded (Figure 5B, D).

EXAMPLE 3

The inhibitory effect of streptolidigin is enhanced in the presence of the GreA and GreB factors.

[00221] The effect of STL on RNA polymerase transcription elongation in the presence of GreA or GreB transcription factors was studied. To do this, we compared the results of measuring elongation profiles in a medium containing a one-molar concentration of NTP and 5 µM GreA or GreB and when 10 µM STL was added to these media. Instantaneous transcription rates measured in the absence of STL were 18.4 ± 1.4 nt/s and 9.1 ± 0.7 nt/s with the addition of GreA and GreB, respectively (Figure 6A,B). In the presence of GreA, the instantaneous speed did not decrease significantly compared to control conditions, and the average speed was close to instantaneous. In the presence of GreB, the instantaneous speed was much lower compared to the control conditions and its value approached the value of the average speed. These results indicate that GreA and GreB induce very short pauses of less than 2.5 seconds, which are not detected by the ACC technique. The instantaneous rate of transcription elongation in a medium containing 5 μM GreB and 1 mM NTP remained virtually unchanged with the addition of 10 μM STL, while the average rate decreased sharply due to the appearance of a large number of long pauses, the average duration of which reaches ~400 s (Figure 6C, D) , and in some cases, a complete stop (arrest) of RNA polymerase was observed, indicating the most pronounced effect of the synergy of STL and GreB. A slightly less pronounced effect was observed in the case of GreA, when the average speed in the presence of STL decreased by 2-3 times (Figure 6B)

EXAMPLE 4

Inhibition of bacterial transcription by a binary complex consisting of streptolidigin and GreB in vivo

[00222] In connection with data obtained from single-molecule experiments indicating a synergistic effect of the antibiotic streptolidigin and the transcription factor GreB, it was decided to study the effect of their combined action on bacterial cells of E. coli. in vivo. Experiments to study streptolidigin and the GreB transcription factor on the viability of E. coli bacterial cells in vivo were carried out using a multimodal tablet reader CLARIOstar plus (BMG LABTECH), which allows you to work with small volumes of cell cultures.

[00223] E. coli cells of the Rosetta strain containing the plasmid pET15b-GB were applied to the plate to express GreB under the control of an inducible promoter. When the inducer IPTG was added, GreB synthesis began. Next, 100 micromoles of streptolidigin and 700 nanomoles of polymyxin were added to the wells of the plate. In control experiments, the optical density of cells was varied by adding an inducer, conditions without induction, as well as conditions without polymyxin and without streptolidigin were used. The plate with cells filled with samples was incubated in the reader for 17 hours at 37°C with periodic stirring on a vortex mixer (Vortex). Every 10 min, the optical density of cells was determined at a wavelength of 600 nm. At the end of incubation, graphs of the optical density of the samples versus time were generated.

[00224] From the graphs in FIG. Figure 7 shows that streptolidigin (both with and without polymyxin) does not have a significant effect on the growth of bacterial cells. Induction of GreB expression and no changes in optical density were also observed, indicating that GreB did not alter bacterial growth characteristics. Upon induction of GreB expression and the addition of streptolidigin - brown curve, the optical density of the sample begins to fall over time, which indicates cell death and an increase in the antibacterial effect of STL with an increase in the overall activity of GreB.

EXAMPLE 5

Effect of microcins and lasso peptides on transcription in vitro

[00225] The inhibitory effect of microcin J25 and the lasso peptides klebsidin and acinetodin on transcription elongation of E. coli RNAP was studied. Figure 8 shows the results of experiments with the addition of various concentrations of lasso peptides obtained using the ACC technique.

[00226] The average transcription rate in the absence of peptides was 19.9 ± 3.9 nt/sec (mean ± standard deviation). The addition of microcin J25, klebsidin, and acinetodin caused a concentration-dependent increase in the frequency and duration of the observed pauses and an unchanged instantaneous rate of RNAP elongation. The activity of klebsidin was comparable to that of microcin J25, while acinetodin was less active. The average transcription rates were 2.9 ± 1.4 nt/sec and 1.2 ± 0.4 nt/sec for 2.5 μM and 5 μM microcin J25, respectively. These values for 2.5 µM and 5 µM klebsidin were 6±2.4 nt/sec and 1.9±0.7 nt/sec and 10.7±4.2 nt/sec and 4.6±1.9 nt/sec for 5 µM and 25 µM acinetodin. The nature of the dependences shown in Figure 9, despite the fact that the numerical values differ markedly, appears to be very similar, which indicates a common mechanism of their action by binding NTFR in the secondary RNAP channel, through which substrates enter the catalytic center.

[00227] Klebsidin is active against K. pneumoniae strains at high concentrations with an MIC of 256 μM in M9 medium. Figure 9 shows data on the intracellular toxicity of klebsidin and acinetodin upon accumulation in E. coli cells (Figure 9). Growth curves show an inhibitory effect on cell growth upon accumulation of acinetodin and klebsidin in response to the expression of the plasmid genes aciABC and kleABC.

INCLUSION BY REFERENCE

[00228] All publications, patents and patent applications herein are incorporated by reference to the same extent as if each individual publication, patent or patent application had been specifically and individually identified as being incorporated by reference. In the event of a conflict between a term in this application and a term in a source incorporated by reference, the term in this application controls.

Claims (12)

1. A method of using a binary system to increase the effectiveness of an antibacterial agent, in which the bacterial cell is affected in a random order, ensuring the simultaneous presence of an effective amount of compound 1 and an effective amount of agent 2, forming a binary system 3, to ensure a synergistic inhibitory effect on elongation bRNAP transcription and the viability of bacterial cells, wherein compound 1 is an antibacterial agent capable of binding to the active site of bacterial RNA polymerase (bRNAP) inside the bacterium at the stage of transcription elongation, and agent 2 increases the expression and/or activity of the elongation factor in the bacterium. 2. Binary system 3, designed to inhibit bRNAP, which includes two components in an effective amount: A) Compound 1, which is an antibacterial agent that is capable of binding to the active center of bRNAP at the stage of transcription elongation; And B) Agent 2, which increases the expression and/or activity of the bRNAP elongation factor, wherein the combined effect of A) and B) on the bacterium reduces the growth and/or viability of said bacterium. 3. Binary system 3 according to claim 2, according to which compound 1 is a small molecule. 4. Binary system 3 according to any one of paragraphs. 2, 3, according to which compound 1 is a derivative of tetramic acid. 5. Binary system 3 according to any one of paragraphs. 2-4, wherein Compound 1 is a tetramic acid derivative that is capable of binding to bRNAP and inhibiting bRNAP, preferably, said Compound 1 is straegolidigin, or a solvate, salt, prodrug or derivative of said tetramic acid derivative, which has substantially similar properties for bRNAP inhibition and/or antibacterial activity. 6. Binary system 3 according to any one of paragraphs. 2-5, according to which the specified bRNAP elongation factor has exonuclease activity. 7. Binary system 3 according to any one of paragraphs. 2-6, wherein said bRNAP elongation factor is GreA or GreB from E. coli or an analogue or homolog of said factors that has substantially similar function or activity. 8. Binary system 3 according to any one of paragraphs. 2-7, according to which the specified agent 2 increases the expression and/or activity of the bRNAP elongation factor, provides: activation of the promoter of the bRNAP elongation factor gene present in the genome of the specified bacterium; transformation of the bacterium with the introduction of a heterologous bRNAP elongation factor gene; delivery of GreA and/or GreB proteins into the bacterial cell. 9. Binary system 3 according to any one of paragraphs. 2-8, according to which the specified agent 2 increases the expression and/or activity of the bRNAP elongation factor, preferably, the specified agent 2 is selected from: a genetic construct that ensures the expression of the RNAP elongation factor, a bacteriophage that delivers into the bacterium a genetic construct that ensures the expression of the factor elongation of RNAP, small molecule, nucleotide, peptide or polypeptide, which ensures the expression of the RNAP elongation factor or the transition of the RNAP elongation factor to an active form and/or the specified agent 2 provides an increase in the amount of RNAP elongation factor in the cell; and/or said agent 2 is an effect that increases the expression and/or activity of the elongation factor RNAP, where said effect is preferably a change in temperature, irradiation, including optical irradiation and high-frequency irradiation, a change in the physicochemical characteristics of the environment or fluid in the area or in an environment containing bRNAP or bacteria.