8-Oxoadenine: A «New¼ Player of the Oxidative Stress in Mammals?

Numerous studies have shown that oxidative modifications of guanine (7,8-dihydro-8-oxoguanine, 8-oxoG) can affect cellular functions. 7,8-Dihydro-8-oxoadenine (8-oxoA) is another abundant paradigmatic ambiguous nucleobase but findings reported on the mutagenicity of 8-oxoA in bacterial and eukaryotic cells are incomplete and contradictory. Although several genotoxic studies have demonstrated the mutagenic potential of 8-oxoA in eukaryotic cells, very little biochemical and bioinformatics data about the mechanism of 8-oxoA-induced mutagenesis are available. In this review, we discuss dual coding properties of 8-oxoA, summarize historical and recent genotoxicity and biochemical studies, and address the main protective cellular mechanisms of response to 8-oxoA. We also discuss the available structural data for 8-oxoA bypass by different DNA polymerases as well as the mechanisms of 8-oxoA recognition by DNA repair enzymes.

Synthesis and Biological Evaluation of Benzo [4,5]- and Naphtho[2',1':4,5]imidazo[1,2-c]pyrimidinone Derivatives.

Azacarbazoles have attracted significant interest due to their valuable properties, such as anti-pathogenic and antitumor activity. In this study, a series of structurally related tricyclic benzo[4,5]- and tertacyclic naphtho[2',1':4,5]imidazo[1,2-c]pyrimidinone derivatives with one or two positively charged tethers were synthesized and evaluated for anti-proliferative activity. Lead tetracyclic derivative 5b with two amino-bearing arms inhibited the metabolic activity of A549 lung adenocarcinoma cells with a CC50 value of 3.6 µM, with remarkable selectivity (SI = 17.3) over VA13 immortalized fibroblasts. Cell-cycle assays revealed that 5b triggers G2/M arrest without signs of apoptosis. A study of its interaction with various DNA G4s and duplexes followed by dual luciferase and intercalator displacement assays suggests that intercalation, rather than the modulation of G4-regulated oncogene expression, might contribute to the observed activity. Finally, a water-soluble salt of 5b was shown to cause no acute toxic effects, changes in mice behavior, or any decrease in body weight after a 72 h treatment at concentrations up to 20 mg/kg. Thus, 5b is a promising candidate for studies in vivo; however, further investigations are needed to elucidate its molecular target(s).

Phenotypic Test of Benzo[4,5]imidazo[1,2-c]pyrimidinone-Based Nucleoside and Non-Nucleoside Derivatives against DNA and RNA Viruses, Including Coronaviruses.

Emerging and re-emerging viruses periodically cause outbreaks and epidemics around the world, which ultimately lead to global events such as the COVID-19 pandemic. Thus, the urgent need for new antiviral drugs is obvious. Over more than a century of antiviral development, nucleoside analogs have proven to be promising agents against diversified DNA and RNA viruses. Here, we present the synthesis and evaluation of the antiviral activity of nucleoside analogs and their deglycosylated derivatives based on a hydroxybenzo[4,5]imidazo[1,2-c]pyrimidin-1(2H)-one scaffold. The antiviral activity was evaluated against a panel of structurally and phylogenetically diverse RNA and DNA viruses. The leader compound showed micromolar activity against representatives of the family Coronaviridae, including SARS-CoV-2, as well as against respiratory syncytial virus in a submicromolar range without noticeable toxicity for the host cells. Surprisingly, methylation of the aromatic hydroxyl group of the leader compound resulted in micromolar activity against the varicella-zoster virus without any significant impact on cell viability. The leader compound was shown to be a weak inhibitor of the SARS-CoV-2 RNA-dependent RNA polymerase. It also inhibited biocondensate formation important for SARS-CoV-2 replication. The active compounds may be considered as a good starting point for further structure optimization and mechanistic and preclinical studies.

Recent Advances in Molecular Mechanisms of Nucleoside Antivirals.

The search for new drugs has been greatly accelerated by the emergence of new viruses and drug-resistant strains of known pathogens. Nucleoside analogues (NAs) are a prospective class of antivirals due to known safety profiles, which are important for rapid repurposing in the fight against emerging pathogens. Recent improvements in research methods have revealed new unexpected details in the mechanisms of action of NAs that can pave the way for new approaches for the further development of effective drugs. This review accounts advanced techniques in viral polymerase targeting, new viral and host enzyme targeting approaches, and prodrug-based strategies for the development of antiviral NAs.

Red light-emitting short Mango-based system enables tracking a mycobacterial small noncoding RNA in infected macrophages.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

Hydrophobic Rose Bengal Derivatives Exhibit Submicromolar-to-Subnanomolar Activity against Enveloped Viruses.

Rose Bengal (RB) is an anionic xanthene dye with multiple useful biological features, including photosensitization properties. RB was studied extensively as a photosensitizer, mostly for antibacterial and antitumor photodynamic therapy (PDT). The application of RB to virus inactivation is rather understudied, and no RB derivatives have been developed as antivirals. In this work, we used a synthetic approach based on a successful design of photosensitizing antivirals to produce RB derivatives for virus photoinactivation. A series of n-alkyl-substituted RB derivatives was synthesized and evaluated as antiviral photosensitizers. The compounds exhibited similar 1O2 generation rate and efficiency, but drastically different activities against SARS-CoV-2, CHIKV, and HIV; with comparable cytotoxicity for different cell lines. Submicromolar-to-subnanomolar activities and high selectivity indices were detected for compounds with C4-6 alkyl (SARS-CoV-2) and C6-8 alkyl (CHIKV) chains. Spectrophotometric assessment demonstrates low aqueous solubility for C8-10 congeners and a significant aggregation tendency for the C12 derivative, possibly influencing its antiviral efficacy. Initial evaluation of the synthesized compounds makes them promising for further study as viral inactivators for vaccine preparations.

Cationic Perylene Antivirals with Aqueous Solubility for Studies In Vivo.

Perylene-based compounds are attracting significant attention due to their high broad-spectrum antiviral activity against enveloped viruses. Despite unambiguous results of in vitro studies and high selectivity index, the poor water solubility of these compounds prevented in vivo evaluation of their antiviral properties. In this work, we synthesized a series of compounds with a perylene pharmacophore bearing positively charged substituents to improve the aqueous solubility of this unique type of antivirals. Three types of charged groups were introduced: (1) quaternary morpholinium salts (3a-b); (2) a 2'-O-l-valinyl-uridine hydrochloride residue (8), and (3) a 3-methylbenzothiazolium cation (10). The synthesized compounds were evaluated based both on antiviral properties in vitro (CHIKV, SARS-CoV-2, and IAV) and on solubility in aqueous media. Compound 10 has the greatest aqueous solubility, making it preferable for pre-evaluation by intragastrical administration in a mouse model of lethal influenza pneumonia. The results indicate that the introduction of a positively charged group is a viable strategy for the design of drug candidates with a perylene scaffold for in vivo studies.

SERS-based biosensor with Raman-active external responsive element for rapid determination of adenosine monophosphate.

Phosphorylated adenosine derivatives are important biological molecules with diverse biological functions connected with the energetic balance of the cell, biosynthesis of cell components and regulation of protein activity. Measurement of these compounds provides information about the cell signalling in the body as well as the quantity of microorganisms in the environment. Surface-enhanced Raman spectroscopy (SERS) is an optical method that provides a unique spectrum of a substance at low concentrations. Specificity and limit of detection of SERS-based sensors can be increased drastically using nucleic acid aptamers and Raman-active dyes, respectively. Here we describe an adenosine monophosphate (AMP) biosensor based on AMP-dependent interaction between the well-known DNA aptamer for AMP and a novel Raman-active dye. The SERS intensity of novel Black Hole Quencher-2 (BHQ-2) derivatives was shown to be proportional to the charge of the molecule indicating electrostatic interactions with negatively charged colloidal silver nanoparticles. The novel derivative of BHQ-2 with two amine groups, BHQ-2-(NH2)2, binds an unpaired guanine stacked between guanine-guanine and guanine-adenine mismatches in DNA aptamer-AMP complex with KD = 26 nM as shown by 1H nuclear magnetic resonance, molecular docking and biolayer interferometry. The aptamer is pre-structured by AMP being folded in the conformation favorable for the interaction with BHQ-2-(NH2)2. This specific mechanism of the interaction allows designing of a SERS-based aptasensor with a limit of detection being as low as 3.4 nM of AMP and the dynamic range of nearly 5 orders - from 3.4 nM to 200 µM. The results illustrate a new approach to biosensors where DNA-interacting ligands act as external responsive elements providing an analyte-dependent SERS signal.

Benzothiazole-substituted 1,3-diaza-2-oxophenoxazine as a luminescent nucleobase surrogate for silver(I)-mediated base pairing.

A benzothiazole-substituted derivative (X) of 1,3-diaza-2-oxophenoxazine was evaluated with respect to its ability to engage in Ag(I)-mediated homo base pair formation in two different DNA duplexes. The metal binding was determined by a combination of temperature-dependent UV spectroscopy, CD spectroscopy, and fluorescence spectroscopy, indicating the incorporation of two Ag(I) ions to generate a dinuclear X-Ag(I)2-X base pair. Interestingly, a luminescence increase was observed upon metal binding. Theoretical luminescence spectra were calculated using time-dependent density functional theory (TDDFT) for all possible Ag(I)-mediated X : X base pair geometries to identify the species responsible for the increase in luminescence. The study shows that even bulky non-planar artificial nucleobases can be applied to form stabilizing metal-mediated base pairs.

Anticancer activity of G4-targeting phenoxazine derivatives in vitro.

G4-stabilizing ligands are now being considered as anticancer, antiviral and antibacterial agents. Phenoxazine is a promising scaffold for the development of G4 ligands. Here, we profiled two known phenoxazine-based nucleoside analogs and five new nucleoside and non-nucleoside derivatives against G4 targets from telomere repeats and the KIT promoter region. Leading new derivatives exhibited remarkably high G4-stabilizing effects (comparable or superior to the effects of the commonly used selective G4 ligands PDS and NMM) and selectivity toward G4s over duplex (superior to BRACO-19). All phenoxazine-based ligands inhibited cellular metabolic activity. The phenoxazine derivatives were particularly toxic for lung adenocarcinoma cells A549' and human liver cancer cells HepG2 (CC50 of the nucleoside analogues in the nanomolar range), but also affected breast cancer cells MCF7, as well as immortalized fibroblasts VA13 and embryonic kidney cells HEK293t (CC50 in the micromolar range). Importantly, the CC50 values varied mostly in accordance with G4-binding affinities and G4-stabilizing effects, and the phenoxazine derivatives localized in the cell nuclei, which corroborates G4-mediated mechanisms of action.

Probing GFP Chromophore Analogs as Anti-HIV Agents Targeting LTR-III G-Quadruplex.

Green fluorescent protein (GFP) chromophore and its congeners draw significant attention mostly for bioimaging purposes. In this work we probed these compounds as antiviral agents. We have chosen LTR-III DNA G4, the major G-quadruplex (G4) present in the long terminal repeat (LTR) promoter region of the human immunodeficiency virus-1 (HIV-1), as the target for primary screening and designing antiviral drug candidates. The stabilization of this G4 was previously shown to suppress viral gene expression and replication. FRET-based high-throughput screening (HTS) of 449 GFP chromophore-like compounds revealed a number of hits, sharing some general structural features. Structure-activity relationships (SAR) for the most effective stabilizers allowed us to establish structural fragments, important for G4 binding. Synthetic compounds, developed on the basis of SAR analysis, exhibited high LTR-III G4 stabilization level. NMR spectroscopy and molecular modeling revealed the possible formation of LTR-III G4-ligand complex with one of the lead selective derivative ZS260.1 positioned within the cavity, thus supporting the LTR-III G4 attractiveness for drug targeting. Selected compounds showed moderate activity against HIV-I (EC50 1.78-7.7 µM) in vitro, but the activity was accompanied by pronounced cytotoxicity.