New Biocides Based on N4-Alkylcytidines: Effects on Microorganisms and Application for the Protection of Cultural Heritage Objects of Painting.

The rapid increase in the antibiotic resistance of microorganisms, capable of causing diseases in humans as destroying cultural heritage sites, is a great challenge for modern science. In this regard, it is necessary to develop fundamentally novel and highly active compounds. In this study, a series of N4-alkylcytidines, including 5- and 6-methylcytidine derivatives, with extended alkyl substituents, were obtained in order to develop a new generation of antibacterial and antifungal biocides based on nucleoside derivatives. It has been shown that N4-alkyl 5- or 6-methylcytidines effectively inhibit the growth of molds, isolated from the paintings in the halls of the Ancient Russian Paintings of the State Tretyakov Gallery, Russia, Moscow. The novel compounds showed activity similar to antiseptics commonly used to protect works of art, such as benzalkonium chloride, to which a number of microorganisms have acquired resistance. It was also shown that the activity of N4-alkylcytidines is comparable to that of some antibiotics used in medicine to fight Gram-positive bacteria, including resistant strains of Staphylococcus aureus and Mycobacterium smegmatis. N4-dodecyl-5- and 6-methylcytidines turned out to be the best. This compound seems promising for expanding the palette of antiseptics used in painting, since quite often the destruction of painting materials is caused by joint fungi and bacteria infection.

New Flexible Analogues of 8-Aza-7-deazapurine Nucleosides as Potential Antibacterial Agents.

A variety of ribo-, 2'-deoxyribo-, and 5'-norcarbocyclic derivatives of the 8-aza-7-deazahypoxanthine fleximer scaffolds were designed, synthesized, and screened for antibacterial activity. Both chemical and chemoenzymatic methods of synthesis for the 8-aza-7-deazainosine fleximers were compared. In the case of the 8-aza-7-deazahypoxanthine fleximer, the transglycosylation reaction proceeded with the formation of side products. In the case of the protected fleximer base, 1-(4-benzyloxypyrimidin-5-yl)pyrazole, the reaction proceeded selectively with formation of only one product. However, both synthetic routes to realize the fleximer ribonucleoside (3) worked with equal efficiency. The new compounds, as well as some 8-aza-7-deazapurine nucleosides synthesized previously, were studied against Gram-positive and Gram-negative bacteria and M. tuberculosis. It was shown that 1-(ß-D-ribofuranosyl)-4-(2-aminopyridin-3-yl)pyrazole (19) and 1-(2',3',4'-trihydroxycyclopent-1'-yl)-4-(pyrimidin-4(3H)-on-5-yl)pyrazole (9) were able to inhibit the growth of M. smegmatis mc2 155 by 99% at concentrations (MIC99) of 50 and 13 µg/mL, respectively. Antimycobacterial activities were revealed for 4-(4-aminopyridin-3-yl)-1H-pyrazol (10) and 1-(4'-hydroxy-2'-cyclopenten-1'-yl)-4-(4-benzyloxypyrimidin-5-yl)pyrazole (6). At concentrations (MIC99) of 40 and 20 µg/mL, respectively, the compounds resulted in 99% inhibition of M. tuberculosis growth.

A Desmethylphosphinothricin Dipeptide Derivative Effectively Inhibits Escherichia coli and Bacillus subtilis Growth.

New antibiotics are unquestionably needed to fight the emergence and spread of multidrug-resistant bacteria. To date, antibiotics targeting bacterial central metabolism have been poorly investigated. By determining the minimal inhibitory concentration (MIC) of desmethylphosphinothricin (Glu-γ-PH), an analogue of glutamate with a phosphinic moiety replacing the γ-carboxyl group, we previously showed its promising antibacterial activity on Escherichia coli. Herein, we synthetized and determined the growth inhibition exerted on E. coli by an L-Leu dipeptide derivative of Glu-γ-PH (L-Leu-D,L-Glu-γ-PH). Furthermore, we compared the growth inhibition obtained with this dipeptide with that exerted by the free amino acid, i.e., Glu-γ-PH, and by their phosphonic and non-desmethylated analogues. All the tested compounds were more effective when assayed in a chemically-defined minimal medium. The dipeptide L-Leu-D,L-Glu-γ-PH had a significantly improved antibacterial activity (2 µg/mL), at a concentration between the non-desmethytaled (0.1 µg/mL) and the phosphonic (80 µg/mL) analogues. Also, in Bacillus subtilis, the dipeptide L-Leu-D,L-Glu-γ-PH displayed an activity comparable to that of the antibiotic amoxicillin. This work highlights the antibacterial relevance of the phosphinic pharmacophore and proposes new avenues for the development of novel antimicrobial drugs containing the phosphinic moiety.

5-Substituted Uridines with Activity against Gram-Positive Bacteria.

The emergence of drug-resistant strains of pathogenic microorganisms necessitates the creation of new drugs. A series of uridine derivatives containing an extended substituent at the C-5 position as well as C-5 alkyloxymethyl, alkylthiomethyl, alkyltriazolylmethyl, alkylsulfinylmethyl and alkylsulfonylmethyl uridines were obtained in order to explore their antimicrobial properties and solubility. It has been shown that new ribonucleoside derivatives have an order of magnitude better solubility in water compared to their 2'-deoxy analogues and effectively inhibit the growth of a number of Gram-positive bacteria, including resistant strains of Mycobacterium smegmatis (MIC=15-200 µg/mL) and Staphylococcus aureus (MIC=25-100 µg/mL). Their activity is comparable to that of some antibiotics used in medicine.

C-Methylated Spermidine Derivatives: Convenient Syntheses and Antizyme-Related Effects.

The biogenic polyamines, spermidine (Spd) and spermine (Spm), are present at millimolar concentrations in all eukaryotic cells, where they participate in the regulation of vitally important cellular functions. Polyamine analogs and derivatives are a traditional and important instrument for the investigation of the cellular functions of polyamines, enzymes of their metabolism, and the regulation of the biosynthesis of antizyme-a key downregulator of polyamine homeostasis. Here, we describe convenient gram-scale syntheses of a set of C-methylated analogs of Spd. The biochemical properties of these compounds and the possibility for the regulation of their activity by moving a methyl group along the polyamine backbone and by changing the stereochemistry of the chiral center(s) are discussed.

Transcriptome Analysis of Redox Systems and Polyamine Metabolic Pathway in Hepatoma and Non-Tumor Hepatocyte-like Cells.

Reactive oxygen species (ROS) play a major role in the regulation of various processes in the cell. The increase in their production is a factor contributing to the development of numerous pathologies, including inflammation, fibrosis, and cancer. Accordingly, the study of ROS production and neutralization, as well as redox-dependent processes and the post-translational modifications of proteins, is warranted. Here, we present a transcriptomic analysis of the gene expression of various redox systems and related metabolic processes, such as polyamine and proline metabolism and the urea cycle in Huh7.5 hepatoma cells and the HepaRG liver progenitor cell line, that are widely used in hepatitis research. In addition, changes in response to the activation of polyamine catabolism that contribute to oxidative stress were studied. In particular, differences in the gene expression of various ROS-producing and ROS-neutralizing proteins, the enzymes of polyamine metabolisms and proline and urea cycles, as well as calcium ion transporters between cell lines, are shown. The data obtained are important for understanding the redox biology of viral hepatitis and elucidating the influence of the laboratory models used.

Synthesis of the Indole-Based Inhibitors of Bacterial Cystathionine γ-Lyase NL1-NL3.

Bacterial cystathionine γ-lyase (bCSE) is the main producer of H2S in pathogenic bacteria such as Staphylococcus aureus, Pseudomonas aeruginosa, etc. The suppression of bCSE activity considerably enhances the sensitivity of bacteria to antibiotics. Convenient methods for the efficient synthesis of gram quantities of two selective indole-based bCSE inhibitors, namely (2-(6-bromo-1H-indol-1-yl)acetyl)glycine (NL1), 5-((6-bromo-1H-indol-1-yl)methyl)- 2-methylfuran-3-carboxylic acid (NL2), as well as a synthetic method for preparation 3-((6-(7-chlorobenzo[b]thiophen-2-yl)-1H-indol-1-yl)methyl)- 1H-pyrazole-5-carboxylic acid (NL3), have been developed. The syntheses are based on the use of 6-bromoindole as the main building block for all three inhibitors (NL1, NL2, and NL3), and the designed residues are assembled at the nitrogen atom of the 6-bromoindole core or by the substitution of the bromine atom in the case of NL3 using Pd-catalyzed cross-coupling. The developed and refined synthetic methods would be significant for the further biological screening of NL-series bCSE inhibitors and their derivatives.

Hepatitis Delta Virus Antigens Trigger Oxidative Stress, Activate Antioxidant Nrf2/ARE Pathway, and Induce Unfolded Protein Response.

Hepatitis delta virus (HDV) is a viroid-like satellite that may co-infect individuals together with hepatitis B virus (HBV), as well as cause superinfection by infecting patients with chronic hepatitis B (CHB). Being a defective virus, HDV requires HBV structural proteins for virion production. Although the virus encodes just two forms of its single antigen, it enhances the progression of liver disease to cirrhosis in CHB patients and increases the incidence of hepatocellular carcinoma. HDV pathogenesis so far has been attributed to virus-induced humoral and cellular immune responses, while other factors have been neglected. Here, we evaluated the impact of the virus on the redox status of hepatocytes, as oxidative stress is believed to contribute to the pathogenesis of various viruses, including HBV and hepatitis C virus (HCV). We show that the overexpression of large HDV antigen (L-HDAg) or autonomous replication of the viral genome in cells leads to increased production of reactive oxygen species (ROS). It also leads to the upregulated expression of NADPH oxidases 1 and 4, cytochrome P450 2E1, and ER oxidoreductin 1α, which have previously been shown to mediate oxidative stress induced by HCV. Both HDV antigens also activated the Nrf2/ARE pathway, which controls the expression of a spectrum of antioxidant enzymes. Finally, HDV and its large antigen also induced endoplasmic reticulum (ER) stress and the concomitant unfolded protein response (UPR). In conclusion, HDV may enhance oxidative and ER stress induced by HBV, thus aggravating HBV-associated pathologies, including inflammation, liver fibrosis, and the development of cirrhosis and hepatocellular carcinoma.

Design and Synthesis of New Modified Flexible Purine Bases as Potential Inhibitors of Human PNP.

The great interest in studying the structure of human purine nucleoside phosphorylase (hPNP) and the continued search for effective inhibitors is due to the importance of the enzyme as a target in the therapy of T-cell proliferative diseases. In addition, hPNP inhibitors are used in organ transplant surgeries to provide immunodeficiency during and after the procedure. Previously, we showed that members of the well-known fleximer class of nucleosides are substrates of E. coli PNP. Fleximers have great promise as they have exhibited significant biological activity against a number of viruses of pandemic concern. Herein, we describe the synthesis and inhibition studies of a series of new fleximer compounds against hPNP and discuss their possible binding mode with the enzyme. At a concentration of 2 mM for the flex-7-deazapurines 1-4, a decrease in enzymatic activity by more than 50% was observed. 4-Amino-5-(1H-pyrrol-3-yl)pyridine 2 was the best inhibitor, with a Ki = 0.70 mM. Docking experiments have shown that ligand 2 is localized in the selected binding pocket Glu201, Asn243 and Phe200. The ability of the pyridine and pyrrole fragments to undergo rotation around the C-C bond allows for multiple binding modes in the active site of hPNP, which could provide several plausible bioactive conformations.

Antibacterial Activity of Peptide Derivatives of Phosphinothricin against Multidrug-Resistant Klebsiella pneumoniae.

The fast spread of bacteria that are resistant to many classes of antibiotics (multidrug resistant) is a global threat to human and animal health with a worrisome scenario ahead. Novel therapeutical strategies are of crucial importance to combat this phenomenon. For this purpose, we investigated the antimicrobial properties of the naturally occurring tripeptide Bialaphos and a dipeptide L-leucyl-L-phosphinoithricin, the synthesis and diastereomers separation of which are herein described. We demonstrate that these compounds are effective on clinical isolates of the human pathogen Klebsiella pneumoniae, causing hospital-acquired and community-acquired infections. The tested isolates were remarkable for their resistance to more than 20 commercial antibiotics of different classes. Based on previous literature data and our experiments consisting of glutamine supplementation, we suggest that both compounds release phosphinothricin-a well-known nanomolar inhibitor of glutamine synthetase-after their penetration in the bacterial cells; and, in this way, exert their antibacterial effect by negatively affecting nitrogen assimilation in this pathogen.

Nucleoside Analogs That Inhibit SARS-CoV-2 Replication by Blocking Interaction of Virus Polymerase with RNA.

The SARS-CoV-2 betacoronavirus pandemic has claimed more than 6.5 million lives and, despite the development and use of COVID-19 vaccines, remains a major global public health problem. The development of specific drugs for the treatment of this disease remains a very urgent task. In the context of a repurposing strategy, we previously screened a library of nucleoside analogs showing different types of biological activity against the SARS-CoV-2 virus. The screening revealed compounds capable of inhibiting the reproduction of SARS-CoV-2 with EC50 values in the range of 20-50 µM. Here we present the design and synthesis of various analogs of the leader compounds, the evaluation of their cytotoxicity and antiviral activity against SARS-CoV-2 in cell cultures, as well as experimental data on RNA-dependent RNA polymerase inhibition. Several compounds have been shown to prevent the interaction between the SARS-CoV-2 RNA-dependent RNA polymerase and the RNA substrate, likely inhibiting virus replication. Three of the synthesized compounds have also been shown to inhibit influenza virus. The structures of these compounds can be used for further optimization in order to develop an antiviral drug.

Effects of natural polymorphisms in SARS-CoV-2 RNA-dependent RNA polymerase on its activity and sensitivity to inhibitors in vitro.

SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) is the key enzyme required for viral replication and mRNA synthesis. RdRp is one of the most conserved viral proteins and a promising target for antiviral drugs and inhibitors. At the same time, analysis of public databases reveals multiple variants of SARS-CoV-2 genomes with substitutions in the catalytic RdRp subunit nsp12. Structural mapping of these mutations suggests that some of them may affect the interactions of nsp12 with its cofactors nsp7/nsp8 as well as with RNA substrates. We have obtained several mutations of these types and demonstrated that some of them decrease specific activity of RdRp in vitro, possibly by changing RdRp assembly and/or its interactions with RNA. Therefore, natural polymorphisms in RdRp may potentially affect viral replication. Furthermore, we have synthesized a series of polyphenol and diketoacid derivatives based on previously studied inhibitors of hepatitis C virus RdRp and found that several of them can inhibit SARS-CoV-2 RdRp. Tested mutations in RdRp do not have strong effects on the efficiency of inhibition. Further development of more efficient non-nucleoside inhibitors of SARS-CoV-2 RdRp should take into account the existence of multiple polymorphic variants of RdRp.

Nucleoside Analogs and Perylene Derivatives Modulate Phase Separation of SARS-CoV-2 N Protein and Genomic RNA In Vitro.

The life cycle of severe acute respiratory syndrome coronavirus 2 includes several steps that are supposedly mediated by liquid-liquid phase separation (LLPS) of the viral nucleocapsid protein (N) and genomic RNA. To facilitate the rational design of LLPS-targeting therapeutics, we modeled N-RNA biomolecular condensates in vitro and analyzed their sensitivity to several small-molecule antivirals. The model condensates were obtained and visualized under physiological conditions using an optimized RNA sequence enriched with N-binding motifs. The antivirals were selected based on their presumed ability to compete with RNA for specific N sites or interfere with non-specific pi-pi/cation-pi interactions. The set of antivirals included fleximers, 5'-norcarbocyclic nucleoside analogs, and perylene-harboring nucleoside analogs as well as non-nucleoside amphiphilic and hydrophobic perylene derivatives. Most of these antivirals enhanced the formation of N-RNA condensates. Hydrophobic perylene derivatives and 5'-norcarbocyclic derivatives caused up to 50-fold and 15-fold enhancement, respectively. Molecular modeling data argue that hydrophobic compounds do not hamper specific N-RNA interactions and may promote non-specific ones. These findings shed light on the determinants of potent small-molecule modulators of viral LLPS.

2-Deoxyglucose, an Inhibitor of Glycolysis, Enhances the Oncolytic Effect of Coxsackievirus.

Glioblastoma multiforme (GBM) is one of the most common types of brain tumor. Despite intensive research, patients with GBM have a poor prognosis due to a very high rate of relapse and significant side effects of the treatment, with a median survival of 14.6 months. Oncolytic viruses are considered a promising strategy to eliminate GBM and other types of cancer, and several viruses have already been introduced into clinical practice. However, identification of the factors that underly the sensitivity of tumor species to oncolytic viruses or that modulate their clinical efficacy remains an important target. Here, we show that Coxsackievirus B5 (CVB5) demonstrates high oncolytic potential towards GBM primary cell species and cell lines. Moreover, 2-deoxyglucose (2DG), an inhibitor of glycolysis, potentiates the cytopathic effects of CVB5 in most of the cancer cell lines tested. The cells in which the inhibition of glycolysis enhanced oncolysis are characterized by high mitochondrial respiratory activity and glycolytic capacity, as determined by Seahorse analysis. Thus, 2-deoxyglucose and other analogs should be considered as adjuvants for oncolytic therapy of glioblastoma multiforme.

Synthetic Optimizations for Gram-Scale Preparation of 1-O-Methyl d-Glycero-α-d-gluco-heptoside 7-Phosphate from d-Glucose.

Heptose phosphates-unique linkers between endotoxic lipid A and O-antigen in the bacterial membrane-are pathogen-associated molecular patterns recognized by the receptors of the innate immune system. Understanding the mechanisms of immune system activation is important for the development of therapeutic agents to combat infectious diseases and overcome antibiotic resistance. However, in practice, it is difficult to obtain a substantial amount of heptose phosphates for biological studies due to the narrow scope of the reported synthetic procedures. We have optimized and developed an inexpensive and convenient synthesis for the first performed gram-scale production of 1-O-methyl d-glycero-α-d-gluco-heptoside 7-phosphate from readily available d-glucose. Scaling up to such amounts of the product, we have increased the efficiency of the synthesis and reduced the number of steps of the classical route through the direct phosphorylation of the O6,O7-unprotected heptose. The refined method could be of practical value for further biological screening of heptose phosphate derivatives.

Analogues of Pyrimidine Nucleosides as Mycobacteria Growth Inhibitors.

Tuberculosis (TB) is the oldest human infection disease. Mortality from TB significantly decreased in the 20th century, because of vaccination and the widespread use of antibiotics. However, about a third of the world's population is currently infected with Mycobacterium tuberculosis (Mtb) and the death rate from TB is about 1.4-2 million people per year. In the second half of the 20th century, new extensively multidrug-resistant strains of Mtb were identified, which are steadily increasing among TB patients. Therefore, there is an urgent need to develop new anti-TB drugs, which remains one of the priorities of pharmacology and medicinal chemistry. The antimycobacterial activity of nucleoside derivatives and analogues was revealed not so long ago, and a lot of studies on their antibacterial properties have been published. Despite the fact that there are no clinically used drugs based on nucleoside analogues, some progress has been made in this area. This review summarizes current research in the field of the design and study of inhibitors of mycobacteria, primarily Mtb.

Synthesis of New 5'-Norcarbocyclic Aza/Deaza Purine Fleximers - Noncompetitive Inhibitors of E.coli Purine Nucleoside Phosphorylase.

A new series of flexible 5'-norcarbocyclic aza/deaza-purine nucleoside analogs were synthesized from 6-oxybicyclo[3.1.0.]hex-2-ene and pyrazole-containing fleximer analogs of heterocyclic bases using the Trost procedure. The compounds were evaluated as potential inhibitors of E. coli purine nucleoside phosphorylase. Analog 1-3 were found to be noncompetitive inhibitors with inhibition constants of 14-24 mM. From the data obtained, it can be assumed that the new 5'-norcarbocyclic nucleoside analogs interact with the active site of the PNP like natural heterocyclic bases. But at the same time the presence of a cyclopentyl moiety with 2' and 3' hydroxyls is necessary for the inhibitory properties, since compounds 8-10, without those groups did not exhibit an inhibitory effect under the experimental conditions.

Role of Polyamine-Induced Dimerization of Antizyme in Its Cellular Functions.

The polyamines, spermine (Spm) and spermidine (Spd), are important for cell growth and function. Their homeostasis is strictly controlled, and a key downregulator of the polyamine pool is the polyamine-inducible protein, antizyme 1 (OAZ1). OAZ1 inhibits polyamine uptake and targets ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine biosynthesis, for proteasomal degradation. Here we report, for the first time, that polyamines induce dimerization of mouse recombinant full-length OAZ1, forming an (OAZ1)2-Polyamine complex. Dimerization could be modulated by functionally active C-methylated spermidine mimetics (MeSpds) by changing the position of the methyl group along the Spd backbone-2-MeSpd was a poor inducer as opposed to 1-MeSpd, 3-MeSpd, and Spd, which were good inducers. Importantly, the ability of compounds to inhibit polyamine uptake correlated with the efficiency of the (OAZ1)2-Polyamine complex formation. Thus, the (OAZ1)2-Polyamine complex may be needed to inhibit polyamine uptake. The efficiency of polyamine-induced ribosomal +1 frameshifting of OAZ1 mRNA could also be differentially modulated by MeSpds-2-MeSpd was a poor inducer of OAZ1 biosynthesis and hence a poor downregulator of ODC activity unlike the other MeSpds. These findings offer new insight into the OAZ1-mediated regulation of polyamine homeostasis and provide the chemical tools to study it.

Selective Inhibition of HDAC Class I Sensitizes Leukemia and Neuroblastoma Cells to Anticancer Drugs.

The acquired resistance of neuroblastoma (NB) and leukemia cells to anticancer therapy remains the major challenge in the treatment of patients with these diseases. Although targeted therapy, such as receptor tyrosine kinase (RTK) inhibitors, has been introduced into clinical practice, its efficacy is limited to patients harboring mutant kinases. Through the analysis of transcriptomic data of 701 leukemia and NB patient samples and cell lines, we revealed that the expression of RTK, such as KIT, FLT3, AXL, FGFR3, and NTRK1, is linked with HDAC class I. Although HDAC inhibitors have antitumor activity, they also have high whole-body toxicity. We developed a novel belinostat derivative named hydrazostat, which targets HDAC class I with limited off-target effects. We compared the toxicity of these drugs within the panel of leukemia and NB cell lines. Next, we revealed that HDAC inhibition with hydrazostat reactivates NTRK1, FGFR3, ROR2, KIT, and FLT3 expression. Based on this finding, we tested the efficacy of hydrazostat in combination with RTK inhibitor imatinib. Additionally, we show the ability of hydrazostat to enhance venetoclax-induced apoptosis. Thus, we reveal the connection between HDACs and RTK and describe a useful strategy to overcome the complications of single-agent therapies.

Dual-targeted anti-CMV/anti-HIV-1 heterodimers.

Despite the development of efficient anti-human immunodeficiency virus-1 (HIV-1) therapy, HIV-1 associated pathogens remain a major clinical problem. Human cytomegalovirus (CMV) is among the most common HIV-1 copathogens and one of the main causes of persistent immune activation associated with dysregulation of the immune system, cerebrovascular and cardiovascular pathologies, and premature aging. Here, we report on the development of dual-targeted drugs with activity against both HIV-1 and CMV. We synthesized seven compounds that constitute conjugates of molecules that suppress both pathogens. We showed that all seven compounds exhibit low cytotoxicity and efficiently inhibited both viruses in cell lines. Furthermore, we chose a representative compound and demonstrated that it efficiently suppressed replication of HIV-1 and CMV in human lymphoid tissue ex vivo coinfected with both viruses. Further development of such compounds may lead to the development of dual-targeted anti-CMV/HIV-1 drugs.