Institute of Bioorganic Chemistry

Jasmonic and salicylic acids are key hormones involved in plant responses to pests and pathogens. Existing fluorescence-based approaches to imaging plant defence hormones are constrained by the need for external illumination and by autofluorescence of plant tissues, while luminescence-based ones require exogenous substrates. Here, we use jasmonate- and salicylate-responsive promoters to engineer autoluminescent plants that report hormone signalling activity with up to a 53-fold contrast. Using consumer-grade cameras, we image reporter Arabidopsis thaliana and Nicotiana benthamiana plants throughout normal development and in response to pest and pathogen attacks, visualising local and systemic responses. Because the luminescence is self-sustained, these reporters enable non-invasive, substrate-free imaging of defence signalling over extended time courses without specialised equipment.

In this work, we analyzed the ability to incorporate 8-oxo-dATP by several human DNA polymerases: replicative Pol ε (exo-) from Family B; base excision repair (BER) enzymes Pol β and Pol λ from Family X; and translesion Pol η, Pol ι, and Pol κ from Family Y. We demonstrated that human DNA polymerases differ in their abilities to discriminate against 8-oxo-dATP. Among the tested DNA polymerases, Pol λ exhibited the worst ability to discriminate against 8-oxo-dATP opposite template T on DNA substrates with a protruding single-stranded 5'-end and a double-stranded DNA with a 1 nt gap. Pol β and DNA polymerases of Family Y showed relatively high accuracy. Pol η demonstrated the most effective discrimination against 8-oxo-dATP on templates T and G. Pol ι exclusively incorporated 8-oxo-dATP opposite template G but not T. Unexpectedly, the catalytic subunit of high-fidelity Pol ε (exo-) incorporated 8-oxo-dATP opposite templates T and G with higher efficiency compared with the error-prone polymerases of Family Y and Pol β. While the structures of human polymerases with incoming 8-oxo-dATP are not available, we speculate on a possible mechanism of 8-oxo-dATP discrimination.

ABSTRACT Tuberculosis persists as a major global health threat, significantly exacerbated by the rise of drug-resistant strains. Cytochrome P450 of 124 family CYP124 from Mycobacterium tuberculosis (CYP124), implicated in host sterol metabolism and bacterial virulence, represents an emerging and promising therapeutic target. While its precise physiological role was previously debated, CYP124’s confirmed ability to metabolize immunomodulatory host sterols underscores its pharmacological relevance. Utilizing surface plasmon resonance binding assays and UV-Vis spectral titration screening, we identified nine novel non-azole ligands for CYP124 from a library of 32 plant-derived and marine natural compounds. Among these hits, (25S)-5α-cholestane-3β,4β,6α,7α,8,15β,16β,26-octaol (termed 15β-octaol) and henricioside H 2 (HD-4) induced characteristic difference spectra and formed long-lived inhibitory complexes with CYP124, exhibiting dissociation half-lives of 181 min and 65 min, respectively. However, their inhibitory potency was moderate, with IC 50 values of approximately 86 μM for 15β-octaol and exceeding 100 μM for HD-4. Complementary in silico molecular docking and analysis identified key conserved hydrophobic residues within the CYP124 active site crucial for ligand binding, suggesting a shared pharmacophore. Furthermore, structural similarity analysis revealed that 37 human endogenous metabolites, including known immunoregulatory sterols, bear resemblance to the identified CYP124 ligands. This finding points towards a potential sterol-mediated interplay at the host-pathogen interface. Collectively, these results provide a foundation for the future development of mechanism-based CYP124 inhibitors as therapeutics against multidrug-resistant tuberculosis.

Quinoxaline represents an important class of bicyclic nitrogen-containing fused heterocyclic compounds. A combination of benzene and pyrazine rings makes a stable structure, with broad popularity in material science, catalysis, and microbiology. A number of various synthetic methods have emerged for their synthesis, driven by their versatile biological importance. However, most of these approaches are promoted by transition metal catalysts. In recent years, the pursuit of transition-metal-free green synthetic strategies has heightened. In this review, we aimed to highlight the most critical aspects of these synthetic protocols, mechanisms, capabilities, and limitations are summarized. Maximalization of the resource efficiency in the synthetic technique, promotion of chemical process sustainability, energy saving, low toxicity of both reagents and product, in addition to hazards, pollution, cost, and purification challenges minimization, are the key advantages of utilizing the transition metal-free catalysts. In summary, we highlight quinoxaline green and metal-free synthetic methods, with a focus on advancements of the last ten years and the potential pharmacological applications in the future.

Abstract Chemically modified nucleic acids have become a powerful platform for basic research and applied technologies. Universal nucleobases are used in PCR,sequencing, and the design of nanodevices and aptamers. Fluorescent universal nucleobases have an even wider range of applications, including the development of nucleic acid-based sensors, switches, and relay logic gates. However, few such nucleobases have been proposed to date, and most of them have suboptimal optical properties. Here, we propose an adenine-based molecular rotor, 7,8-dihydro-8-oxo-6-(3-methylbenzo[d]thiazol-2(3H)-ylidene)adenine ( oxo-Ade BZT ), as a new, remarkably bright and potent fluorescent universal nucleobase. Its brightness in both oligodeoxyribonucleotides (ODNs) and DNA duplexes (4200 - 10000 M -1 × cm -1 ) originates from a high molar extinction coefficient (averaged ε 368 37000 M -1 × cm -1 ), provided by the appended 3-methylbenzo[d]thiazolyl moiety, and a relatively high quantum yield (0.11 – 0.27). Melting temperature variations observed upon the incorporation of oxo-Ade BZT opposite native nucleobases in a duplex context did not exceed 10%. The basis of these universal hybridizing properties was unveiled using computational methods. According to molecular dynamics simulations, oxo-Ade BZT pushes the opposite nucleobase out of the DNA double helix and forms multiple hydrophobic contacts with the flanking base pairs. At the same time, the rotational mobility of the bonds between the oxo-Ade BZT -constituting heterobicycles decreases, and oxo-Ade BZT adopts a planar conformation in both ODNs and their duplexes, resulting in the light-up effect. These properties make oxo-Ade BZT a promising molecular tool for analytical, biophysical and biochemical studies.

Toll-like receptor 13 (TLR13) is a critical innate immune sensor that recognizes a conserved RNA sequence, RNA15 (2054–2068, ACG GAA AGA CCC CGU), within bacterial 23S rRNA, thereby initiating a proinflammatory response. While the alarmin high mobility group box 1 (HMGB1) is known to modulate various TLR pathways, its influence on TLR13 signaling has remained unexplored. Here, we reveal that HMGB1 directly binds RNA15 with high affinity and profoundly disrupts its hairpin structure, which is essential for TLR13 recognition. Using a combination of fluorescence anisotropy, FRET assays, NMR spectroscopy, and enhanced-sampling molecular dynamics simulations, we demonstrate that HMGB1 binding remodels RNA15 into a stem-open conformation, making it thermodynamically unfavorable for receptor activation. Functionally, HMGB1 significantly inhibits RNA15-induced TLR13 activation, leading to a dose-dependent reduction in inflammatory mediators. These findings uncover a novel “ligand remodeling” mechanism, whereby HMGB1 acts as a negative regulator of TLR13 signaling by structurally altering the RNA ligand rather than directly blocking the receptor. This work provides new insights into host–pathogen interactions and suggests important implications for the design and immunogenicity of RNA-based therapeutics and vaccines.

C2H2 proteins, characterized by DNA-binding C2H2-type zinc finger domains, constitute the largest group of transcription factors. In addition to binding DNA, C2H2 domains can mediate protein–protein interactions, facilitating the oligomerization of C2H2 proteins. In this study, we identified eight C2H2 proteins in the Drosophila genome that feature a unique single C2H2 domain containing a conserved “CGxΦ” motif. Yeast two-hybrid assays, size-exclusion chromatography coupled with multi-angle light scattering, and chemical cross-linking experiments revealed a strong propensity of these domains to form dimers. Using NMR spectroscopy, we determined the solution structure of the dimeric C2H2 domain from the IMZF (Immune-mediated Zinc Finger) protein, providing structural evidence for the dimerization of C2H2 domains. Dimerization is mediated by the interface between the core C2H2 fold and the adjacent β-strand containing the CGxΦ motif, which was further validated by structure-guided mutagenesis. A bioinformatic survey showed that “CGxΦ”-type C2H2 domains are specific to Diptera. Finally, our predictions demonstrate that dimerizing C2H2 domains containing additional structural elements could be widespread among eukaryotic taxa, with the highest prevalence in insects. These findings establish that single C2H2 domains can mediate self-association and identify the CGxΦ-type C2H2 domains as a distinct structural subclass specific to dipteran insects.

The problem of hunger has plagued humanity throughout its history. It may seem that in the 21st century, due to the development of science, agriculture, international trade, and transportation systems, it should already be solved. However, this does not happen, and millions of people still do not have access to nutritious and safe food. The world is still far from solving this task, and achieving the second Sustainable Development Goal of the United Nations (UN), which is to eliminate hunger by 2030 (the first goal is elimination of poverty), seems difficult. There are many declarations and decisions of international organizations, starting with the United Nations, which state that food should not be used “as an instrument of political and economic pressure” and “as a method of warfare”, but they are not respected. It is becoming increasingly clear that elimination of hunger requires large-scale, innovative approaches at a high intergovernmental level, as well as the prevention of its deliberate usage as a means of solving political problems. At the same time, economic approaches should include not only making decisions about investments in agricultural technologies leading to higher crop yields, increased efficiency of cattle and poultry farming, primarily in underdeveloped regions, but also the creation of more advanced methods of food storage and transportation to minimize waste and losses. But it is equally important to make and implement informed policy decisions that would contribute to a fair distribution of the resources needed to provide food to the people of countries in need, without exposing them to starvation. Examples of the use of famine to wage war are discussed in the article – from the siege of Leningrad during World War II up to the present-day events related to the Israeli-Palestinian conflict and the situation surrounding the special military operation in Ukraine. This paper provides a detailed analysis of the data available on a wide range of issues related to hunger and food shortages on our planet, as well as actions taken to address these issues at the international level.

MicroRNAs (miRNAs) and their isoforms, known as isomiRs, are important regulators of tumorigenesis that act as post-transcriptional modulators of gene expression. Among these, 5'-isomiRs-generated through imprecise cleavage during miRNA biogenesis-exhibit altered seed regions compared to their canonical counterparts, potentially leading to distinct targetomes. Consequently, 5'-isomiRs may exert biological functions that differ substantially from those of the corresponding canonical miRNAs. Despite growing recognition of their potential significance, the functional roles of 5'-isomiRs remain largely uncharacterized for most miRNAs. In this study, we investigated the targetome divergence between canonical miRNAs and their 5'-isomiRs, focusing on hsa-miR-93-5p, a miRNA with a well-established oncogenic role in prostate adenocarcinoma. Target transcripts of the 5'-isomiRs were identified using a shRNA-based overexpression system. Bioinformatic analysis revealed a substantial overlap between the targets of the 5'-isomiRs and the canonical miRNA. This overlap was attributed to the co-occurrence of both canonical and shifted seed motifs within the same mRNA targets. Notably, hsa-miR-93-5p ranked among the top miRNAs with a relatively high number of targets transcripts containing both seed motifs, suggesting a unique dual-targeting capacity.