Tsvetkov, Vladimir B

Iudin M.S., Khodarovich Y.M., Varizhuk A.M., Tsvetkov V.B., Severov V.V.

Bromodomain and extra-terminal domain (BET) proteins are epigenetic readers that recognize the histone acetylation code and play a critical role in regulating gene transcription. Dysregulation of BET proteins is associated with a number of pathologies, including cancer, inflammation-related metabolic disorders, etc. BET proteins can also be hijacked by some viruses and mediate latent viral infections, making BET proteins promising targets for therapeutic intervention. Research in this area has mainly focused on bromodomain inhibition, with less attention paid to other domains. Bromodomain inhibitors have great potential as anticancer and anti-inflammatory drug candidates. However, their broad-spectrum impact on transcription and potential cross-reactivity with non-BET bromodomain-containing proteins raise concerns about unforeseen side effects. Non-bromodomain BET inhibitors hold promise for gaining better control over the expression of host and viral genes by targeting different stages of BET-dependent transcriptional regulation. In this review, we discuss recent advances in the development of non-bromodomain BET inhibitors, as well as their potential applications, advantages, and perspectives.

Tsvetkov V., Mir B., Alieva R., Arutyunyan A., Oleynikov I., Novikov R., Boravleva E., Kamzeeva P., Zatsepin T., Aralov A., González C., Zavyalova E.

Abstract Non-canonical nucleic acid structures play significant roles in cellular processes through selective interactions with proteins. While both natural and artificial G-quadruplexes have been extensively studied, the functions of i-motifs remain less understood. This study investigates the artificial aptamer BV42, which binds strongly to influenza A virus hemagglutinin and unexpectedly retains its i-motif structure even at neutral pH. However, BV42 conformational heterogeneity hinders detailed structural analysis. Molecular dynamics simulations and chemical modifications of BV42 helped us to identify a potential binding site, allowing for aptamer redesign to eliminate the conformational diversity while retaining binding affinity. Nuclear magnetic resonance spectroscopy confirmed the i-motif/duplex junction with the three-cytosine loop nearby. This study highlights the unique structural features of the functional i-motif and its role in molecular recognition of the target.

Litvinova V.A., Tsvetkov V.B., Volodina Y.L., Dezhenkova L.G., Markova A.A., Nguyen M.T., Tikhomirov A.S., Shchekotikhin A.E.

Fisunov G.Y., Tsvetkov V.B., Tsoy E.A., Evsyutina D.V., Vedyaykin A.D., Garanina I.A., Semashko T.A., Manuvera V.A., Varizhuk A.M., Kovalchuk S.I., Zubov A.I., Barinov N.A., Pobeguts O.V., Govorun V.M.

IntroductionWhiA is a conserved protein found in numerous bacteria. It consists of an HTH DNA-binding domain linked with a homing endonuclease (HEN) domain. WhiA is one of the most conserved transcription factors in reduced bacteria of the class Mollicutes. Its function in Mollicutes is unknown, while it is well-characterized in Streptomyces. Here, we focused on WhiA protein from Mycoplasma gallisepticum.MethodsWe used a combination molecular dynamics, EMSA, MST and AFM to study the DNA-binding and ATP-binding properties of WhiA from M. gallisepticum. The transcriptional repressor function of WhiA was demonstrated using gene knockdown, reporter constructs and proteome analysis.ResultsWe demonstrate that WhiA homolog from M. gallisepticum binds a conserved sequence of the GAYACRCY core (Y = C or T, R = A or G), which is located in the promoter of an operon coding for ribosomal proteins and adenylate kinase (rpsJ operon). We show that WhiA in M. gallisepticum is a repressor of rpsJ operon and a sensor of ATP. HTH domain binds to the core motif and HEN domain binds to the auxiliary motif GTTGT that is located downstream to the core motif. We show that binding by both domains to DNA is required to fulfill the transcription repressor function. Knockdown of whiA does not affect actively growing M. gallisepticum, but leads to the growth retardation after freezing.DiscussionWe propose the following model for M. gallisepticum WhiA function. WhiA remains bound to the core motif at any conditions. At low ATP concentrations (starvation) HEN domain binds auxiliary motif and represses rpsJ operon transcription. At high ATP concentrations (nutrient-rich conditions) HEN domain binds ATP and releases auxiliary motif. It leads to the de-repression of rpsJ operon and increased production of ribosomal proteins.

Shtork A., Tsvetkov V., Slushko G., Lushpa V., Severov V., Kamzeeva P., Varizhuk A., Aralov A.

Intercalated DNA motifs (iMs) provide a convenient scaffold for the design of biocompatible pH sensors. Among unimolecular iM-based sensors, only those labeled with conventional fluorophores or fluorophore/quencher pairs at the 3′ and 5′ termini for FRET/quenching upon pH-dependent iM folding have been tested in cells and tended to accumulate in the nuclei. Here, we used cytosine mimics as internal iM labels and synthesized a new phenoxazine-based non-fluorescent nucleoside analog, tC°Azo, which quenches the fluorescence of a known cytosine mimic, tC°. Incorporation of the tC°/tC°Azo pair into a genomic iM-forming sequence C5T resulted in a high-contrast pH sensor with an increased pH transition point and a working range compatible with physiological conditions. Importantly, unlike the known nuclei-specific C5T-based sensors with conventional labels that provide a fluorescent signal in the green/red channels, the new sensor localized mainly in the cytoplasm and allowed pH monitoring based on the tC° signal in the blue channel. As the labeling scheme was the only unique feature of the new sensor, it must account for the unique distribution pattern, i.e., the accumulation of the sensor in the cytoplasm. These findings highlight the importance of the labeling scheme of unimolecular iM-based pH sensors and open the way for multiplexed pH monitoring in different cellular compartments.

Tsvetkov V., Mir B., Alieva R., Arutyunyan A., Oleynikov I., Novikov R., Boravleva E., Kamzeeva P., Zatsepin T., Aralov A., González C., Zavyalova E.

Non-canonical nucleic acid structures have been shown the capacity to selectively interact with proteins, thereby exerting influence over various intracellular processes. Among these structures, both natural and artificial G-quadruplexes have been extensively studied in relation to their structure-activity relationships. In contrast, the role of i-motifs remains incompletely evaluated. In this study the artificial aptamer BV42 possessing a high affinity for hemagglutinin of influenza A virus was proven to contain the i-motif structure even at neutral pH. However, conformational heterogeneity of BV42 poses challenges for in-depth structural investigations. Using molecular dynamics simulations and introducing chemical modifications for molecular probing, a putative binding site in the aptamer was suggested. These findings have enabled us to redesign the aptamer, eliminating the conformational diversity associated with the i-motif while preserving its binding affinity. Subsequent validation through NMR spectroscopy confirms the presence of the i-motif/duplex junction with the 3-cytosine loop located near the junction. This study elucidates a distinctive instance of an unusual nucleic acid architecture involved in molecular recognition, thereby shedding light on the structural peculiarities of functional i-motifs.

Zabrodskaya Y., Tsvetkov V., Shurygina A., Vasyliev K., Shaldzhyan A., Gorshkov A., Kuklin A., Fedorova N., Egorov V.

One of the critical stages of the T-cell immune response is the dimerization of the intramembrane domains of T-cell receptors (TCR). Structural similarities between the immunosuppressive domains of viral proteins and the transmembrane domains of TCR have led several authors to hypothesize the mechanism of immune response suppression by highly pathogenic viruses: viral proteins embed themselves in the membrane and act on the intramembrane domain of the TCRalpha subunit, hindering its functional oligomerization. It has also been suggested that this mechanism is used by influenza A virus in NS1-mediated immunosuppression. We have shown that the peptide corresponding to the primary structure of the potential immunosuppressive domain of NS1 protein (G51) can reduce concanavalin A-induced proliferation of PBMC cells, as well as in vitro, G51 can affect the oligomerization of the core peptide corresponding to the intramembrane domain of TCR, using AFM and small-angle neutron scattering. The results obtained using in cellulo and in vitro model systems suggest the presence of functional interaction between the NS1 fragment and the intramembrane domain of the TCR alpha subunit. We have proposed a possible scheme for such interaction obtained by computer modeling. This suggests the existence of another NS1-mediated mechanism of immunosuppression in influenza.

Tsvetkov V., Mir B., Alieva R., Arutyunyan A., Oleynikov I., Novikov R., Boravleva E., Kamzeeva P., Zatsepin T., Aralov A., González C., Zavyalova E.

Non-canonical nucleic acid structures have been shown the capacity to selectively interact with proteins, thereby exerting influence over various intracellular processes. Among these structures, both natural and artificial G-quadruplexes have been extensively studied in relation to their structure-activity relationships. In contrast, the role of i-motifs remains incompletely evaluated. In this study the artificial aptamer BV42 possessing a high affinity for hemagglutinin of influenza A virus was proven to contain the i-motif structure even at neutral pH. However, conformational heterogeneity of BV42 poses challenges for in-depth structural investigations. Using molecular dynamics simulations and introducing chemical modifications for molecular probing, a putative binding site in the aptamer was suggested. These findings have enabled us to redesign the aptamer, eliminating the conformational diversity associated with the i-motif while preserving its binding affinity. Subsequent validation through NMR spectroscopy confirms the presence of the i-motif/duplex junction with the 3-cytosine loop located near the junction. This study elucidates a distinctive instance of an unusual nucleic acid architecture involved in molecular recognition, thereby shedding light on the structural peculiarities of functional i-motifs.

Zabrodskaya Y., Tsvetkov V.B., Shurygina A., Vasyliev K., Shaldzhyan A., Gorshkov A.N., Kuklin A., Fedorova N., Egorov V.V.

AbstractOne of the critical stages of the T-cell immune response is the dimerization of the intramembrane domains of T-cell receptors (TCR). Structural similarities between the immunosuppressive domains of viral proteins and the transmembrane domains of TCR have led several authors to hypothesize the mechanism of immune response suppression by highly pathogenic viruses: viral proteins embed themselves in the membrane and act on the intramembrane domain of the TCRalpha subunit, hindering its functional oligomerization. It has also been suggested that this mechanism is used by influenza A virus in NS1-mediated immunosuppression. We have shown that the peptide corresponding to the primary structure of the potential immunosuppressive domain of NS1 protein (G51) can reduce concanavalin A-induced proliferation of PBMC cells, as well as in vitro, G51 can affect the oligomerization of the core peptide corresponding to the intramembrane domain of TCR, using AFM and small-angle neutron scattering.The results obtained usingin celluloandin vitromodel systems suggest the presence of functional interaction between the NS1 fragment and the intramembrane domain of the TCR alpha subunit. We have proposed a possible scheme for such interaction obtained by computer modeling.This suggests the existence of another NS1-mediated mechanism of immunosuppression in influenza.Abstract Figure

Kolganova N.A., Tsvetkov V.B., Stomakhin A.A., Surzhikov S.A., Timofeev E.N., Varizhuk I.V.

Modification of DNA aptamers is aimed at increasing their thermodynamic stability, and improving affinity and resistance to biodegradation. G-quadruplex DNA aptamers are a family of affinity ligands that form non-canonical DNA assemblies based on a G-tetrads stack. Modification of the quadruplex core is challenging since it can cause complete loss of affinity of the aptamer. On the other hand, increased thermodynamic stability could be a worthy reward. In the current paper, we developed new three- and four-layer modified analogues of the thrombin binding aptamer with high thermal stability, which retain anticoagulant activity against alpha-thrombin. In the modified aptamers, one or two G-tetrads contained non-natural anti-preferred alpha-deoxyguanosines at specific positions. The use of this nucleotide analogue made it possible to control the topology of the modified structures. Due to the presence of non-natural tetrads, we observed some decrease in the anticoagulant activity of the modified aptamers compared to the natural prototype. This negative effect was completely compensated by conjugation of the aptamers with optimized tripeptide sequences.

Bychenko O., Khrulev A., Svetlova J., Tsvetkov V., Kamzeeva P., Skvortsova Y., Tupertsev B., Ivanov I., Aseev L., Khodarovich Y., Belyaev E., Kozlovskaya L., Zatsepin T., Azhikina T., Varizhuk A., et. al.

Abstract 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.

Varizhuk I.V., Tsvetkov V.B., Toropygin I.Y., Stomakhin A.A., Kolganova N.A., Surzhikov S.A., Timofeev E.N.

Currently, oligonucleotide therapy has emerged as a new paradigm in the treatment of human diseases. In many cases, however, therapeutic oligonucleotides cannot be used directly without modification. Chemical modification or the conjugation of therapeutic oligonucleotides is required to increase their stability or specificity, improve their affinity or inhibitory characteristics, and address delivery issues. Recently, we proposed a conjugation strategy for a 15-nt G-quadruplex thrombin aptamer aimed at extending the recognition interface of the aptamer. In particular, we have prepared a series of designer peptide conjugates of the thrombin aptamer, showing improved anticoagulant activity. Herein, we report a new series of aptamer–peptide conjugates with optimized peptide sequences. The anti-thrombotic activity of aptamer conjugates was notably improved. The lead conjugate, TBA–GLE, was able to inhibit thrombin-induced coagulation approximately six-fold more efficiently than the unmodified aptamer. In terms of its anticoagulant activity, the TBA–GLE conjugate approaches NU172, one of the most potent G-quadruplex thrombin aptamers. Molecular dynamics studies have confirmed that the principles applied to the design of the peptide side chain are efficient instruments for improving aptamer characteristics for the proposed TBA conjugate model.

Petrunina N.A., Shtork A.S., Lukina M.M., Tsvetkov V.B., Khodarovich Y.M., Feofanov A.V., Moysenovich A.M., Maksimov E.G., Shipunova V.O., Zatsepin T.S., Bogomazova A.N., Shender V.O., Aralov A.V., Lagarkova M.A., Varizhuk A.M.

Pavlova I., Barinov N., Novikov R., Severov V., Iudin M., Vedekhina T., Larin A., Babenko V., Aralov A., Gnuchikh E., Sardushkin M., Klinov D., Tsvetkov V., Varizhuk A.

G-quadruplexes (G4s) are gaining increasing attention as possible regulators of chromatin packaging, and robust approaches to their studies in pseudo-native context are much needed. Here, we designed a simple in vitro model of G4-prone genomic DNA and employed it to elucidate the impact of G4s and G4-stabilizing ligands on nucleosome occupancy. We obtained two 226-bp dsDNA constructs composed of the strong nucleosome positioning sequence and an internucleosomal DNA-imitating tail. The tail was G4-free in the control construct and harbored a "strong" (stable) G4 motif in the construct of interest. An additional "weak" (semi-stable) G4 motif was found within the canonical nucleosome positioning sequence. Both G4s were confirmed by optical methods and 1H NMR spectroscopy. Electrophoretic mobility assays showed that the weak G4 motif did not obstruct nucleosome assembly, while the strong G4 motif in the tail sequence diminished nucleosome yield. Atomic force microscopy data and molecular modeling confirmed that the strong G4 was maintained in the tail of the correctly assembled nucleosome structure. Using both in vitro and in silico models, we probed three known G4 ligands and detected nucleosome-disrupting effects of the least selective ligand. Our results are in line with the negative correlation between stable G4s and nucleosome density, support G4 tolerance between regularly positioned nucleosomes, and highlight the importance of considering chromatin context when targeting genomic G4s.

Svetlova J., Knizhnik E., Manuvera V., Severov V., Shirokov D., Grafskaia E., Bobrovsky P., Matyugina E., Khandazhinskaya A., Kozlovskaya L., Miropolskaya N., Aralov A., Khodarovich Y., Tsvetkov V., Kochetkov S., et. al.

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.

Pradhan T., Gupta O., Chawla G.

Kumar S., Arora A., Deo G., Tiwari M.K., Maity J., Singh B.K.

Zhang J., Cao M., Hou T., Li B., Gu C., Han Z., Yu R., Xia Y., Gao W.

Zhang S., Chen J., Wang Y., Sun W., Lv C.

Ryazantsev D.Y., Meshcheryakova N.F., Alferova V.A., Kamzeeva P.N., Ryabukhina E.V., Zatsepin T.S., Zavyalova E.G., Aralov A.V.

Vangala V.N., Uversky V.N.

Wu M., Liu Y., Zhu X., Zhang X., Kong Q., Lu W., Yuan X., Liu Y., Lu K., Dai Y., Zhang B.

Galambos N.S., Crocker O.J., Schneider B.K., Allerton K.S., Gross K.E., Schneider A.E., Lynch J., Yukselen O., Kucukural A., Conine C.C.

AbstractThe environment encountered by an organism can modulate epigenetic information in gametes to transmit non-genetically inherited phenotypes to offspring. In mouse models, the diet of males regulates specific tRNA-derived RNAs (tDRs) in sperm. After fertilization, tDRs regulate embryonic gene expression and generate metabolic phenotypes in adult offspring through uncharacterized changes during development. Here we demonstrate that inC. elegans,tDRs also accumulate in sperm and can similarly transmit epigenetically inherited phenotypes to offspring. We identify the RNaseT2 enzyme,rnst-2, as a regulator ofC. eleganstDR accumulation. RNST- 2 processes or degrades tRNA-halves, leading to short <30 nt fragments. Thisrnst-2dependent regulation of tDR length distribution modulates specific tDRs in sperm which, after fertilization, regulate early embryonic and developmental gene expression, leading to adaptive phenotypes in progeny. Our findings establish tDRs as a deeply conserved carrier of intergenerational epigenetic information and position the worm as a model for dissecting paternal non-genetic inheritance mechanistically.

Chernikova E.Y., Ustimova M.A., Fedorov Y.V., Chernikova P.A., Godovikov I.A., Chmelyuk N.S., Abakumov M.A., Fedorova O.A.

AbstractThis paper focused on the DNA‐binding properties of novel dicationic cyanine dyes, in which pyridinium centers are linked by bridges of different functionalities. We found that dye 1, bearing a flexible butyl‐4‐methylpyridinium terminal fragment, has the ability to bind to the DNA groove. The attachment of a quite rigid para‐xylylene‐4‐methylpyridinium unit as a terminal group in dye 2 contributed to dual DNA‐binding modes: intercalation and groove binding. We tested the encapsulation ability and the effect on the dye‐DNA binding mode by using a synthetic molecular container such as CB[7]. Unexpectedly, monitoring dye‐DNA interaction in the presence of CB[7] revealed that dye molecules partially remain in DNA frameworks, even though they had a higher affinity towards CB[7]. In the case of dye 1, the transformation of dimeric forms to the left‐handed aggregates to yield a ternary system CB[7]‐dye‐DNA occurred. As for dye 2, reversibility of intercalation and a slight right‐handed aggregation templated by DNA are observed. A cytotoxicity and ability of dyes to stain the living cells in their free and encapsulated forms have been investigated. These findings provide useful information about ligand–DNA interactions, which are valuable for the rational design of drug delivery systems and platforms for cellular imaging and therapy.

Hiu J.J., Tan H.S., Yap M.K.

Ren S., Zhao X., Geng S., Wang X., Ye T., Chen L.

Perevozchikova P.S., Chernikova E.Y., Fedorov Y.V., Fedorova O.A.

Travagliante G., Gaeta M., Purrello R., D’Urso A.

Porphyrins are highly conjugated macrocyclic compounds that possess exceptional photophysical and chemical properties, progressively establishing themselves as versatile tools in the structural investigation of biomolecules. This review explores their role as chiroptical conformational probes, focusing on their interactions with DNA and RNA. The planar electron rich structure of porphyrin macrocycle that promote π–π interactions, their easy functionalization at the meso positions, and their capacity to coordinate metal ions enable their use in probing nucleic acid structures with high sensitivity. Emphasis is placed on their induced circular dichroism (ICD) signals in the Soret region, which provide precise diagnostic insights into binding mechanisms and molecular interactions. The review examines the interactions of porphyrins with various DNA structures, including B-, Z-, and A-DNA, single-stranded DNA, and G-quadruplex DNA, as well as less common structures like I-motif and E-motif DNA. The last part highlights recent advancements in the use of porphyrins to probe RNA structures, emphasizing binding behaviors and chiroptical signals observed with RNA G-quadruplexes, as well as the challenges in interpreting ICD signals with other RNA motifs due to their inherent structural complexity.

Liu Y., Yang Y., Zhang Y., Yi J., Wan L., Wang Y., Gao Q., Xu D., Guo P., Han D.

Comprehensive SummaryRNA‐protein interactions are crucial for regulating various cellular processes such as gene expression, RNA modification and translation. In contrast, undesirable RNA‐protein interactions often cause dysregulated cellular activities associated with many human diseases. The RNA containing expanded GGGGCC repeats forms secondary structures that sequester various RNA binding proteins (RBPs), leading to the development of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, a gap persists in understanding the structural basis for GGGGCC repeat RNA binding to RBPs. Here, we resolve the first solution NMR structure of a natural GGGGCC repeat RNA containing a 2 × 2 GG/GG internal loop, and perform MD simulations and site‐directed mutagenesis to elucidate the mechanism for GGGGCC repeat RNA binding to SRSF2, a splicing factor and key marker of nuclear speckles. We reveal that the R47/T51/R61 residues in RNA recognition motif of SRSF2 and the 2 × 2 GG/GG internal loop in GGGGCC repeat RNA are essential for binding. This work furnishes a valuable high‐resolution structural basis for understanding the binding mechanism for GGGGCC repeat RNA and RBPs, and steers RNA structure‐based drug design.

Vidal Ceballos A., Geissmann A., Favaro D.C., Deshpande P., Elbaum-Garfinkle S.

Fornelli C., Sofia Cento A., Nevi L., Mastrocola R., Ferreira Alves G., Caretti G., Collino M., Penna F.

Obesity, one of the most frequent health problems in the adult population, is a condition characterized by excessive white adipose tissue accumulation and accompanied by the increased risk to develop other disorders such as type II diabetes, cardiovascular disorders, physical disability, frailty and sarcopenia. Total fat mass frequently increases during aging, often coexisting with sarcopenia, thus resulting in an emerging condition defined sarcopenic obesity (SO). Our previous data demonstrated the relevant role of the bromo and extra-terminal domain (BET) proteins inhibitor JQ1 in attenuating inflammation and fibrosis in sarcopenic mice. Moreover, we preliminarily observed that JQ1 administration markedly reduces white adipose tissue mass, suggesting a potential role of BET proteins on visceral fat deposition during aging. Starting from those observations, the aim of this study was to investigate the ability of JQ1 to reduce adiposity in a chronic diet-induced obesity (DIO) mouse model mimicking the human metabolic syndrome. Male C57BL/6J mice were divided in subgroups, either fed a standard diet or a high fat diet for 22 or 12 weeks, treated over the last 14 days with JQ1 or with vehicle. The results showed that JQ1 administration reduces fat mass, preserving skeletal muscle mass and function. A direct JQ1 lipolytic effect was demonstrated on mature adipocyte cultures. JQ1-mediated loss of adipose tissue mass was not associated with systemic inflammation or with lipid accumulation in muscle and liver. JQ1 administration did not impinge on skeletal muscle metabolism and oxidative capability, as shown by the lack of significant impact on mitochondrial mass and biogenesis. In conclusion, the current data highlight a potential benefit of JQ1 administration to counteract obesity, suggesting epigenetic modulation as a prospective target in the treatment of obesity and sarcopenic obesity, despite the underlying multiorgan molecular mechanism is still not completely elucidated.

Litvinova V. A., Tikhomirov Alexander S, Shchekotikhin Andrey

Anthraquinone derivatives (anthracene-9,10-dione) have found applications in medicine, chemical technology, and analytical chemistry, and are also promising for the creation of new materials. The development of methodologies for introducing functional groups into anthraquinones is fundamental for the targeted synthesis of new compounds. The structural features of anthraquinones, particularly the presence of electron-withdrawing carbonyl groups, significantly affect their chemical properties, making the modification of such derivatives often a non-trivial task. This review examines and analyzes methods for forming the most important bonds (C–C, C–N, C–O, C–S, C–Hal, C–Se, C–B, C–P) with anthraquinone, as presented in the scientific literature since 2000. The review of methods for modifying anthraquinone derivatives aims to systematize and evaluate the synthetic possibilities of constructing these bonds. The analysis of both new and classical methods for transforming this molecular scaffold will help to expand its structural diversity, identify new areas for studying its chemical properties, and facilitate the task of targeted synthesis of anthraquinone derivatives. The bibliography includes 224 references.

Mohammed A., Churion K., Danda A., Philips S.J., Ansari A.Z.

Yi X., Wang Q., Zhang M., Shu Q., Zhu J.

Doxorubicin (DOX), a commonly used chemotherapy drug, is hindered due to its tendency to induce cardiotoxicity (DIC). Ferroptosis, a novel mode of programmed cell death, has received substantial attention for its involvement in DIC. Recently, natural product-derived ferroptosis regulator emerged as a potential strategy for treating DIC. In this review, a comprehensive search was conducted across PubMed, Web of Science, Google Scholar, and ScienceDirect databases to gather relevant articles on the use of natural products for treating DIC in relation to ferroptosis. The available papers were carefully reviewed to summarize the therapeutic effects and underlying mechanisms of natural products in modulating ferroptosis for DIC treatment. It was found that ferroptosis plays an important role in DIC pathogenesis, with dysregulated expression of ferroptosis-related proteins strongly implicated in the condition. Natural products, such as flavonoids, polyphenols, terpenoids, and quinones can act as GPX4 activators, Nrf2 agonists, and lipid peroxidation inhibitors, thereby enhancing cell viability, attenuating myocardial fibrosis, improving cardiac function, and suppressing ferroptosis in both in vitro and in vivo models of DIC. This review demonstrates a strong correlation between DOX-induced cardiac ferroptosis and key proteins, such as GPX4, Keap1, Nrf2, AMPK, and HMOX1. Natural products are likely to exert therapeutic effects against DIC by modulating the activity of these proteins.

Lee Y., Nam K., Kim Y.M., Yang K., Kim Y., Oh J., Roh Y.H.

Targeted and stimuli-responsive drug delivery enhances therapeutic efficacy and minimizes undesirable side effects of cancer treatment. Although cellulose nanocrystals (CNCs) are used as drug carriers because of their robustness, spindle shape, biocompatibility, renewability, and nontoxicity, the lack of programmability and functionality of CNCs-based platforms hampers their application. Thus, high adaptability and the capacity to form dynamic 3D nanostructures of DNA may be advantageous, as they can provide functionalities such as target-specific and stimuli-responsive drug release. Using DNA nanotechnology, the functional polymeric form of DNA nanostructures can be replicated using rolling circle amplification (RCA), and the biologically and physiologically stable DNA nanostructures may overcome the challenges of CNCs. In this study, multifunctional polymeric DNAs produced with RCA were strongly complexed with surface-modified CNCs via electrostatic interactions to form polymeric DNA-decorated CNCs (pDCs). Particle size, polydispersity, zeta potential, and biostability of the nanocomplexes were analyzed. As a proof of concept, the dynamic structural functionalities of DNA nanostructures were verified by observing cancer-targeted intracellular delivery and pH-responsive drug release. pDCs showed anticancer properties without side effects in vitro, owing to their aptamer and i-motif functionalities. In conclusion, pDCs exhibited multifunctional anticancer activities, demonstrating their potential as a promising hybrid nanocomplex platform for targeted cancer therapy.

Charushin Valery N., Verbitskiy Egor V., Chupakhin Oleg N., Vorobyeva Daria V., Gribanov Pavel S., Osipov Sergey N., Ivanov Andrey V., Martynovskaya Svetlana V., Sagitova Elena F., Dyachenko Vladimir D., Dyachenko Ivan V., Krivokolysko Sergey G., Dotsenko Viktor V., Aksenov Aleksandr V., Aksenov Dmitrii A., et. al.

The chemistry of heterocyclic compounds has traditionally been and remains a bright area of chemical science in Russia. This is due to the fact that many heterocycles find the widest application. These compounds are the key structural fragments of most drugs, plant protection agents. Many natural compounds are also derivatives of heterocycles. At present, more than half of the hundreds of millions of known chemical compounds are heterocycles. This collective review is devoted to the achievements of Russian chemists in this field over the last 15–20 years. The review presents the achievements of leading heterocyclists representing both RAS institutes and university science. It is worth noting the wide scope of the review, both in terms of the geography of author teams, covering the whole of our large country, and in terms of the diversity of research areas. Practically all major types of heterocycles are represented in the review. The special attention is focused on the practical applications of heterocycles in the design of new drugs and biologically active compounds, high-energy molecules, materials for organic electronics and photovoltaics, new ligands for coordination chemistry, and many other rapidly developing areas. These practical advances would not be possible without the development of new fundamental transformations in heterocyclic chemistry.The bibliography includes 2237 references.

Lara-Ureña N., Gómez-Marín E., Pozuelo-Sánchez I., Reyes J.C., García-Domínguez M.

AbstractBromodomain and extra-terminal (BET) proteins are relevant chromatin adaptors involved in the transcriptional control of thousands of genes. Two tandem N-terminal bromodomains are essential for chromatin attachment through acetyl-histone recognition. Recently, the BET proteins members BRD2 and BRD4 were found to interact with the SARS-CoV-2 envelope (E) protein, raising the question of whether the interaction constitutes a virus hijacking mechanism for transcription alteration in the host cell. To shed light on this question, we have compared the transcriptome of cells overexpressing E with that of cells treated with the BET inhibitor JQ1. Notably, E overexpression leads to a strong upregulation of natural immunity- and interferon response-related genes. However, BET inhibition results in the downregulation of most of these genes, indicating that these two conditions, far from causing a significant overlap of the altered transcriptomes, course with quite different outputs. Concerning the interaction of E protein with BET members, and differing from previous reports indicating that it occurs through BET bromodomains, we find that it relies on SEED and SEED-like domains, BET regions rich in Ser, Asp, and Glu residues. By taking advantage of this specific interaction, we have been able to direct selective degradation of E protein through a PROTAC system involving a dTAG-SEED fusion, highlighting the possible therapeutic use of this peptide for targeted degradation of a viral essential protein.

Shtork A., Tsvetkov V., Slushko G., Lushpa V., Severov V., Kamzeeva P., Varizhuk A., Aralov A.

Intercalated DNA motifs (iMs) provide a convenient scaffold for the design of biocompatible pH sensors. Among unimolecular iM-based sensors, only those labeled with conventional fluorophores or fluorophore/quencher pairs at the 3′ and 5′ termini for FRET/quenching upon pH-dependent iM folding have been tested in cells and tended to accumulate in the nuclei. Here, we used cytosine mimics as internal iM labels and synthesized a new phenoxazine-based non-fluorescent nucleoside analog, tC°Azo, which quenches the fluorescence of a known cytosine mimic, tC°. Incorporation of the tC°/tC°Azo pair into a genomic iM-forming sequence C5T resulted in a high-contrast pH sensor with an increased pH transition point and a working range compatible with physiological conditions. Importantly, unlike the known nuclei-specific C5T-based sensors with conventional labels that provide a fluorescent signal in the green/red channels, the new sensor localized mainly in the cytoplasm and allowed pH monitoring based on the tC° signal in the blue channel. As the labeling scheme was the only unique feature of the new sensor, it must account for the unique distribution pattern, i.e., the accumulation of the sensor in the cytoplasm. These findings highlight the importance of the labeling scheme of unimolecular iM-based pH sensors and open the way for multiplexed pH monitoring in different cellular compartments.

Strom A.R., Eeftens J.M., Polyachenko Y., Weaver C.J., Watanabe H., Bracha D., Orlovsky N.D., Jumper C.C., Jacobs W.M., Brangwynne C.P.

Nuclear compartments form via biomolecular phase separation, mediated through multivalent properties of biomolecules concentrated within condensates. Certain compartments are associated with specific chromatin regions, including transcriptional initiation condensates, which are composed of transcription factors and transcriptional machinery, and form at acetylated regions including enhancer and promoter loci. While protein self-interactions, especially within low-complexity and intrinsically disordered regions, are known to mediate condensation, the role of substrate-binding interactions in regulating the formation and function of biomolecular condensates is under-explored. Here, utilizing live-cell experiments in parallel with coarse-grained simulations, we investigate how chromatin interaction of the transcriptional activator BRD4 modulates its condensate formation. We find that both kinetic and thermodynamic properties of BRD4 condensation are affected by chromatin binding: nucleation rate is sensitive to BRD4-chromatin interactions, providing an explanation for the selective formation of BRD4 condensates at acetylated chromatin regions, and thermodynamically, multivalent acetylated chromatin sites provide a platform for BRD4 clustering below the concentration required for off-chromatin condensation. This provides a molecular and physical explanation of the relationship between nuclear condensates and epigenetically modified chromatin that results in their mutual spatiotemporal regulation, suggesting that epigenetic modulation is an important mechanism by which the cell targets transcriptional condensates to specific chromatin loci. [Media: see text]

Huang Q., Fan D., Zheng Z., Ran T., Bai A., Xiao R., Hu G., Liu W.

Wang S., Lei K., Lai H., Liu T., Du L., Wu S., Ye X., Chiang C., Li M.

Guo Y., Tong Z., Huang Y., Tang J., Xue X., Yang D., Yao C.

Víšková P., Ištvánková E., Ryneš J., Džatko Š., Loja T., Živković M.L., Rigo R., El-Khoury R., Serrano-Chacón I., Damha M.J., González C., Mergny J., Foldynová-Trantírková S., Trantírek L.

AbstractI-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pHT < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pHT > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states.

Xin B., Huang L., Yuan L., Liu N., Li H., Ai X., Lei D., Hou X., Rety S., Xi X.

Abstract RNA helicases function as versatile enzymes primarily responsible for remodeling RNA secondary structures and organizing ribonucleoprotein complexes. In our study, we conducted a systematic analysis of the helicase-related activities of Escherichia coli HrpA and presented the structures of both its apo form and its complex bound with both conventional and non-canonical DNAs. Our findings reveal that HrpA exhibits NTP hydrolysis activity and binds to ssDNA and ssRNA in distinct sequence-dependent manners. While the helicase core plays an essential role in unwinding RNA/RNA and RNA/DNA duplexes, the N-terminal extension in HrpA, consisting of three helices referred to as the APHB domain, is crucial for ssDNA binding and RNA/DNA duplex unwinding. Importantly, the APHB domain is implicated in binding to non-canonical DNA structures such as G-quadruplex and i-motif, and this report presents the first solved i-motif-helicase complex. This research not only provides comprehensive insights into the multifaceted roles of HrpA as an RNA helicase but also establishes a foundation for further investigations into the recognition and functional implications of i-motif DNA structures in various biological processes.

Narum S., Deal B., Ogasawara H., Mancuso J.N., Zhang J., Salaita K.