Bacterial infections are a common cause of morbidity and mortality worldwide. The early detection of pathogens is crucial to minimize health risks, control spread, and prevent outbreaks. Current detection methods are either cultivation-based and time-consuming or rely on sophisticated, costly assays that require specialist equipment, making them unsuitable for rapid and cost-efficient screening, e.g., in food safety. Here, we investigated antimicrobial peptides (AMPs) conjugated to HRP-mimicking DNAzymes as a potentially novel class of broad-spectrum bacterial biosensors. AMPs bind to bacterial cells, and the DNAzyme catalyzes a peroxidase reaction exploitable for sensing applications. We established and optimized a synthesis protocol to prepare AMP-DNAzyme conjugates, synthesized several AMP/DNAzyme combinations, and characterized them. Molecular dynamics simulations of selected conjugates were conducted to gain insight into their binding capacity. We also investigated the potential of these constructs for the proof-of-concept detection of a clinicalEscherichia coliisolate on two low-cost detection platforms: a syringe microfilter system and an ELISA-like assay. On the syringe microfilter, we detected 108 cells/mL within 30 min, while the ELISA-like assay achieved a limit of detection below 5 cells/mL, resulting in 1-2 orders of magnitude improved sensitivity compared to similar AMP-based biosensors. This highly stable and cost-efficient biosensor can have potential applications in fields such as environmental monitoring, food safety, and quality control, where a broad approach is used to detect the presence of bacterial contamination.

Nickel(0)‐catalyzed [2 + 2 + 2] cycloadditions of linked bisdiynes with diarylacetylenes were performed using the Ni(II) precursor complex [(TMEDA)Ni(o‐tolyl)Cl] in the presence of PPh3 ligands to generate the active Ni(0) species. Optimal reactivity was observed when electron‐rich bisdiynes reacted with electron‐deficient diarylacetylenes, affording air‐stable, approximately C2v‐symmetric D‐π‐A‐π‐D bis(arylethynyl)indane‐based dyes containing o‐terphenyl cores. Spectroscopic analyses revealed strong solvatochromic emission with high quantum yields in solution, except for the nonemissive bis‐N‐methylpyridinium derivative 4,4′‐(4,7‐bis((4‐(bis(4‐methoxyphenyl)amino)phenyl)ethynyl)−2,3‐dihydro‐1H‐indene‐5,6‐diyl)bis(1‐methylpyridin‐1‐ium) ditriflate, 10, pointing toward the decisive impact of pyridine methylation on the nonradiative relaxation of the fluorophore. Vibrational studies of the triarylamine dyes displayed a prominent alkyne stretching band in both Raman and IR spectra, applicable for use as a probe. Biorelevant studies revealed that the synthesized dyes bind strongly to double‐stranded DNA and RNA, most likely through groove insertion, leading to significant fluorescence quenching. Only the dicationic derivative 10 showed a characteristic CD response, attributed to additional electrostatic interactions that orient the dye within the DNA minor or RNA major groove. The dyes had minimal effects on polynucleotide thermal stability, suggesting that hydrophobic and van der Waals forces dominate the binding, except for derivative 10, which also involves electrostatic interactions. All compounds were found to be nontoxic toward human cell lines, indicating their promise as multimodal fluorescence, CD, and Raman probes for intracellular and extracellular bioimaging applications.

We report a series of uracil-triazole-pyrene peptidomimetics (2-4) designed to achieve modular geometric control through peptide linkers of varying length and flexibility. All three compounds exhibited submicromolar affinity for ds-DNA, while compound 4 also showed strong binding to ds-RNA, demonstrating the advantage of combining a uracil recognition unit with an extended pyrene aromatic surface compared to the Pyr-Trp reference ligand and the phenanthridine analog 4 '. Structural variations strongly affected Cu(II) coordination. Although Pyr-Trp bound Cu(II) approximately 1000-fold more strongly than the short, rigid compound 2, elongation and increased linker flexibility restored high affinity in 4. Notably, replacing phenanthridine (4 ') with pyrene (4) enhanced Cu(II) binding by nearly 100-fold, highlighting the superior coordination properties of the pyrene scaffold. Cu(II) complexation significantly enhanced nucleic acid binding exclusively for compound 2, increasing its DNA/RNA affinity 15-fold. The resulting 2-Cu(II) complex displayed exceptional selectivity for poly rA-poly rU, exceeding that of the Pyr-Trp-Cu(II) analog by more than 10-fold, consistent with uracil-mediated Hoogsteen-type recognition. Membrane interactions with POPC MLVs were linker-dependent, producing fluorescence enhancements of up to 1400% (2), 650% (3), and 100% (4). Together with negligible cytotoxicity, these findings indicate that compounds 2-4 represent promising multifunctional platforms for Cu2+ sensing and photoinduced therapeutic applications.

Three asymmetric cationic diarylethene (DAE) photoswitches—featuring in bis-thiophene analogues varied thiophene attachment positions to central perfluorocyclopentene or replacement of one thiophene with pyrrole-2-carbonitrile—were evaluated for photoswitching under biorelevant conditions. Relative to prior cyclopentene analogues, they offer distinct benefits: a +100 nm red-shift in the open form's UV absorption enables photocyclization near visible light; robust reversible close-open cycling in bio-media; and equivalent efficiency in water versus methanol. Subtle shifts in thiophene-perfluorocyclopentene linkage yield stark visible absorption differences (Δλ = 60 nm), facilitating selective switching in mixtures of two switch molecules. Unlike bis-thiophene analogues, the thiophene-pyrrole variant undergoes irreversible changes post-cyclization. Exclusively closed forms of (DAE) photoswitches notably interact with ds-DNA/RNA, contrasting prior studies, whereby thiophene-perfluorocyclopentene linkage controls GC- or AT-DNA selectivity. Compounds bind strongly to protein (BSA), yielding unique spectrophotometric changes, allowing differentiation between DNA and BSA in mixtures. While open bis-thiophene forms show moderate antiproliferative activity against tumor cells, only the asymmetric bis-thiophene analogue exhibits marked enhancement of bioactivity in its closed form. This suggests potential for reversible, visible-light (500–600 nm)-controlled cytotoxicity, supporting further optimization for therapeutic wavelengths, potency, and intracellular target selectivity.

A water‐soluble and water‐stable 2,5‐bis(arylethynyl)rhodacyclopentadiene ( 4a ) containing terminal 4‐Me 3 N–C 6 H 4 groups has been synthesized for bioimaging and sensing of biomolecules, and its linear and nonlinear optical properties investigated. Complex 4a exhibits fluorescence from the S 1 excited state in both organic and aqueous solutions, with no observable phosphorescence from T 1 at RT. However, competitive intersystem crossing (ISC) to T 1 leads to potent 1 O 2 sensitization. DFT calculations show that the HOMO and LUMO are essentially ligand‐localized with very modest contributions from Rh, leading to slow S 1 → T 1 ISC and competitive fluorescence and triplet state formation, typical of such 2,5‐bis(arylethynyl)rhodacyclopentadienes. Complex 4a shows a two‐photon absorption cross‐section of 290 GM at 680 nm. It binds strongly to DNA, RNA, and protein (BSA) with similar affinities, but gives opposite fluorimetric response, the emission being strongly enhanced for BSA but efficiently quenched by DNA/RNA. Compound 4a efficiently enters living human cells and accumulates preferentially in mitochondrial membranes, being nontoxic even at high (10 µM) concentrations. Although 4a is a potent 1 O 2 sensitizer in a cuvette, exposure of the complex to intense visible light inside human cell results in fast bleaching of 4a with no effect on cell viability, suggesting local consumption of 1 O 2 by the complex.

Protein MntR is a transcription factor regulating Mn2+ homeostasis in Mycobacterium tuberculosis, the causative agent of tuberculosis. A combination of computational (QM, MD, protein-DNA docking) and experimental (AUC, ITC, DSC, EPR, NMR, CD, EMSA) methods was used to characterize wild-type MntR and its selected mutants, as well as its complex with the target DNA sequence. Molecular dynamics simulations revealed that the binding of Mn2+ into the mononuclear metal-binding site is crucial for the distance and orientation of the DNA-binding helices of the protein, which is necessary for adequate DNA binding. The network of noncovalent interactions between the DNA-binding domain and the FeoA-like domain, which is important for protein structural and dynamical properties, was identified. The roles of key amino acid residues of the identified interaction network were further investigated by simulations of in silico prepared mutants. The R167A mutant was also experimentally characterized, and the results showed the importance of the interaction hub between the N-terminal domain and C-terminal domain and its influence on overall MntR properties. A novel potential DNA binding motif for MntR was identified and its interactions with the protein were computationally and experimentally described.

Marine microalgae are sustainable sources of bioactive compounds and drug delivery platforms that can utilize whole cells, cell fragments, or vesicles. Although extracellular vesicles secreted by microalgae have shown potential, broader application is limited by low yields, instability, complex isolation, and poor standardization. To address these challenges, we previously proposed an alternative strategy in which hypoosmotic stress induces cell disruption, followed by self-assembly of membrane fragments into reconstructed microalgae-derived vesicles. Here, a comprehensive biophysical approach combining top-down and bottom-up strategies provides insight into vesicle structural features relevant to their function as drug delivery platforms. They form a heterogeneous population but can be reduced to the nanometer range. Their pigmented membranes contain chlorophyll degradation products and carotenoids, and provide significant antioxidant activity. The protein-to-lipid ratio of cells and vesicles is maintained mainly during self-assembly, indicating an effective reconstruction process. Vesicles have a balanced fatty acid profile, hydrophilicity, pronounced softness, and structure-dependent permeability. In vitro cytotoxicity studies indicated that vesicles do not exhibit acute cellular toxicity and show only mild, cell line-dependent effects at high concentrations. An in vivo immunogenicity study also demonstrated mild adjuvant activity. Confocal imaging shows that a glycopeptide antibiotic and an oligonucleotide bind via non-covalent interactions with the membrane surface. A lyophilization and rehydration protocol was developed to extend material stability, highlighting the importance of the self-assembly process during which vesicle morphology is preserved. These findings provide proof of concept that reconstructed microalgae-derived vesicles can be used to develop next-generation sustainable and safe drug delivery platforms.

Streptococcus mutans, a bacterium commonly found in the human oral cavity, is considered the primary causative agent of dental caries. A key player in the pathophysiology of S. mutans is SloR, a 25-kDa metalloregulatory protein. SloR plays a crucial role in coordinating the uptake of essential metal ions, particularly manganese, with the transcription of the bacterium's virulence genes. To elucidate the molecular mechanism underlying the enhanced binding affinity of SloR to DNA upon Mn2+ ion binding, a combination of computational (QM and MD) and experimental (ITC, DSC, CD, EPR) methods have been employed. Computational simulations revealed that Mn2+ binding induces a conformational change of SloR, primarily affecting the positioning of its DNA-binding domains, bringing them to an appropriate position for DNA binding. Consequently, the protein's DNA binding affinity is modulated. Additionally, experimental findings indicate that the SloR monomer binds up to three Mn2+ ions and that the thermodynamic stability of SloR increases upon Mn2+ complexation. The presented computational results also suggest that Mn2+ binding at the primary binding sites is sufficient to trigger the observed conformational change in SloR.

Studies of lipidous membranes are of utmost interest in biomedical applications, whereby fluorescent stains of membranes often play an essential role. Only a few stains are of small molecular weight, which could allow easier incorporation in lipid membranes. Here we present the discovery of a new structural entity applicable for membrane staining, 3-hydroxy-3-methyl-2-phenylisoindolin-1-one, whereby a study of a series of analogues (all Mw < 350) revealed their emission is strongly solvatochromic. Detailed studies revealed that fine-tuning of substituents strongly controls lipophilicity and emissive response, the optimum performance showing analogues with substituents on N-aryl moiety, particularly pronounced for p-hydroxy (5), o, p-dimethoxy (6) and p-methyl (7) derivatives, latter showing the highest logP. Chosen p-methyl (7) is characterised by the unique switch-on fluorescence in visible range upon membrane binding, based on very strong bathochromic shift (Delta 7 = +80-100 nm) between water (lambda = 375 nm) and membrane (lambda = 460 nm), in comparison to referent stain Laurdan (R) under the same conditions (Delta lambda = +20 nm). The novel neutral probe 7 prefers incorporation into the non-polar part of the lipid bilayers, not disturbing their properties. Future perspectives aim for the development of a structurally new group of low molecular weight lipid membrane stains, which eventually could be in situ tethered by "click" chemistry to the free propargyl bond of referent building block with a variety of additional functions.

BACKGROUND:Calixarene derivatives have been widely recognized as promising fluorescent sensors when suitably functionalized with fluorophoric groups, as it has been believed that these compounds are not enough intrinsically fluorescent. In this work, we investigated the intrinsic fluorescence of three calix[4]arenes lacking additional fluorescent moieties and explored the possibility of quantitative fluorimetric examination of their cation-binding processes taking place in different solvents (water, methanol, and acetonitrile). RESULTS:The obtained results clearly showed that, although the fluorescence of free calixarenes in solutions was relatively low, it substantially increased, and was blue shifted to some extent, upon alkali and alkaline earth metal cation binding. For comparison, fluorescence of a monomeric compound which constitutes tertiary-amide calixarene derivative and the corresponding complexation-induced spectral changes were studied as well. The excited-state lifetimes of the free ligands and their cation complexes were measured and discussed. The detailed quantum chemical calculations provided an insight into the origin of the observed luminescence. SIGNIFICANCE:Overall, the results of this comprehensive and integrated investigation indicate that, contrary to current opinion, spectrofluorimetry can be successfully employed as an effective and sensitive technique for the quantitative monitoring of complexation reactions involving calixarene derivatives and related compounds, regardless of whether additional fluorophores are present in their structures.

The development of mitochondria-targeting fluorescent compounds with theranostic potential for tumor cells remains a topic of great interest. Inspired by previously reported delocalized lipophilic cations (DLCs) based on styryl dye framework, we introduce a series of seventeen novel styrene dyes, several of which exhibit enhanced optical properties compared to their parent compounds. Most dyes display strong Stokes shifts (104-112 nm), primarily due to the indolyl chromophore, with minimal influence from other substituents. These dyes bind noncovalently to the grooves of ds-DNA/RNA with moderate affinity, responding by significantly increased fluorescence. The selected derivatives effectively penetrate living human cells, accumulating primarily in mitochondria and becoming highly fluorescent. However, their bioactivity is strongly influenced by subtle structural modifications. Notably, the introduction of a bromo-substituent to the indole ring converts a non-cytotoxic dye into the highly cytotoxic analogues 10a and 10g. Interestingly, while A549 tumor cells treated with 10a and 10g exhibit similar cytotoxic responses, the underlying mechanisms differ between normal and cancerous cells. In WI-38 cells, toxicity appeared to result from mitochondrial hyperactivation and oxidative stress, whereas in A549 cells, it is driven by mitochondrial dysfunction and metabolic collapse. This distinction underscores key differences in mitochondrial metabolism between normal and cancer cells, positioning 10a and 10g as promising lead compounds for further development as theranostic agents targeting mitochondrial vulnerabilities in cancer.

Aiming toward a novel, noninvasive technique, with a real-time potential application in the monitoring of the complexation of steroidal neuromuscular blocker drugs Vecuronium (Vec) and Rocuronium (Roc) with sugammadex (SDX, medication for the reversal of neuromuscular blockade induced by Vec or Roc in general anesthesia), we developed proof-of-principle methodology based on surface-enhanced Raman spectroscopy (SERS). Silver nanoparticles prepared by the reduction of silver ions with hydroxylamine hydrochloride were used as SERS-active substrates, additionally aggregated with calcium nitrate as needed. The Vec and Roc SERS spectra were obtained within the biorelevant 5 × 10−7–1 × 10−4 M range, as well as the SERS of SDX, though the latter was observed only in the presence of the aggregating agent. SDX/drug complexes at a 1/1 molar ratio revealed significant spectral changes in the vibrational bands of the SDX glucose rings and the drug steroid rings, implying that the insertion of Vec and Roc molecules into the SDX cavity was not only driven by attractive electrostatic interactions between the positively charged cyclic unit of the drug and the negative carboxylate groups of cyclodextrin but also supported by hydrophobic interactions between the host cyclodextrin and the guest drug molecule. The observed changes in SERS signals are applicable in biorelevant conditions and support further studies of SDX/drug complexes in vivo.

Peroxidases are essential elements in many biotechnological applications. An especially interesting concept involves split enzymes, where the enzyme is separated into two smaller and inactive proteins that can dimerize into a fully active enzyme. Such split forms were developed for the horseradish peroxidase (HRP) and ascorbate peroxidase (APX) already. Both peroxidases have a high potential for biotechnology applications. In the present study, we performed biophysical comparisons of these two peroxidases and their split analogues. The active site availability is similar for all four structures. The split enzymes are comparable in stability with their native analogues, meaning that they can be used for further biotechnology applications. Also, the tertiary structures of the two peroxidases are similar. However, differences that might help in choosing one system over another for biotechnology applications were noticed. The main difference between the two systems is glycosylation which is not present in the case of APX/sAPEX2, while it has a high impact on the HRP/sHRP stability. Further differences are calcium ions and cysteine bridges that are present only in the case of HRP/sHRP. Finally, computational results identified sAPEX2 as the systems with the smallest structural variations during molecular dynamics simulations showing its dominant stability comparing to other simulated proteins. Taken all together, the sAPEX2 system has a high potential for biotechnological applications due to the lack of glycans and cysteines, as well as due to high stability.

Triazoles and triazolium salts are very common subunits in the structures of various drugs. Medicaments with a characteristic 1,2,3-triazole core are also being developed to treat neurodegenerative disorders associated with cholinesterase enzyme activity. Several naphtho- and thienobenzo-triazoles from our previous research emerged as being particularly promising in that sense. For this reason, in this research, new naphtho- and thienobenzo-triazoles 23–34, as well as 1,2,3-triazolium salts 44–51, were synthesized and tested. Triazolium salts 44–46 showed excellent activity while salts 47 and 49 showed very good inhibition toward both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) enzymes. In contrast, neutral photoproducts were shown to be selective towards BChE but with very good inhibition potential as molecules 24–27. The representative of newly prepared compounds, 45 and 50, were stable in aqueous solution and revealed intriguing fluorimetric properties, characterized by a strong Stokes shift of >160 nm. Despite their condensed polycyclic structure shaped similarly to well-known DNA-intercalator ethidium bromide, the studied compounds did not show any interaction with ds-DNA, likely due to the unfavorable steric hindrance of substituents. However, the studied dyes bind proteins, particularly showing very diverse inhibition properties toward AChE and BChE. In contrast, neutral photoproducts were shown to be selective towards a certain enzyme but with moderate inhibition potential. The molecular docking of the best-performing candidates to cholinesterases’ active sites identified cation–π interactions as the most responsible for the stability of the enzyme–ligand complexes. As genotoxicity studies are crucial when developing new active substances and finished drug forms, in silico studies for all the compounds synthesized have been performed.

Herein, the presented results show that previously studied DNA/RNA-interacting bis-imidazole-calix[4]arene systems can, in aqueous solutions, efficiently bind a series of biorelevant transition metal cations by coordination with the two imidazole arms at the small rim of their macrocyclic basket. The SCXRD and NMR results structurally characterised the complexes formed by referent bis-imidazole-calix[4]arene with Cu2+ and Zn2+. In solid-state (crystal), the bis-anilino derivative/Cu2+ complex, only upon exposure to the air, undergoes intramolecular dehydrogenative coupling of two neighbouring aniline units, yielding an azo bridge at the large rim of the calix[4]arene basket. In the biorelevant aqueous solution, the comparison of fluorometric titrations of referent calix[4]arene, with its analogues having one or two pyrene units grafted at the opposite (large) rim, revealed moderate-to-strong affinity towards transition metal cations, and, more importantly, a strong impact of pyrene on the binding affinity towards some cations. The pyrene arm(s) significantly diminished the affinity of the calix[4]arene-imidazole ligand towards Cu+ and strongly increased the affinity towards divalent Co2+ and Cd2+ cations. Moreover, the fluorometric response of some studied derivatives was strappingly sensitive to cation type. Since the counter-anion plays only a marginal role, such a change in selectivity is attributed to the intramolecular interaction of pyrene(s) with the calix[4]arene-imidazole system, sterically controlling the metal cation binding site.

Three new phenanthridine peptide derivatives (19, 22, and 23) were synthesized to explore their potential as spectrophotometric probes for DNA and RNA. UV/Vis and circular dichroism (CD) spectra, mass spectroscopy, and computational analysis confirmed the presence of intramolecular interactions in all three compounds. Computational analysis revealed that compounds alternate between bent and open conformations, highlighting the latter’s crucial influence on successful polynucleotide recognition. Substituting one glycine with lysine in two regioisomers (22, 23) resulted in stronger binding interactions with DNA and RNA than for a compound containing two glycines (19), thus emphasizing the importance of lysine. The regioisomer with lysine closer to the phenanthridine ring (23) exhibited a dual and selective fluorimetric response with non-alternating AT and ATT polynucleotides and induction of triplex formation from the AT duplex. The best binding constant (K) with a value of 2.5 × 107 M−1 was obtained for the interaction with AT and ATT polynucleotides. Furthermore, apart from distinguishing between different types of ds-DNA and ds-RNA, the same compound could recognize GC-rich DNA through distinct induced CD signals.

Although cancer and malaria are not etiologically nor pathophysiologically connected, due to their similarities successful repurposing of antimalarial drugs for cancer and vice-versa is known and used in clinical settings and drug research and discovery. With the growing resistance of cancer cells and Plasmodium to the known drugs, there is an urgent need to discover new chemotypes and enrich anticancer and antimalarial drug portfolios. In this paper, we present the design and synthesis of harmiprims, hybrids composed of harmine, an alkaloid of the beta-carboline type bearing anticancer and antiplasmodial activities, and primaquine, 8-aminoquinoline antimalarial drug with low antiproliferative activity, covalently bound via triazole or urea. Evaluation of their antiproliferative activities in vitro revealed that N-9 substituted triazole-type harmiprime was the most selective compound against MCF-7, whereas C1-substituted ureido-type hybrid was the most active compound against all cell lines tested. On the other hand, dimeric harmiprime was not toxic at all. Although spectrophotometric studies and thermal denaturation experiments indicated binding of harmiprims to the ds-DNA groove, cell localization showed that harmiprims do not enter cell nucleus nor mitochondria, thus no inhibition of DNArelated processes can be expected. Cell cycle analysis revealed that C1-substituted ureido-type hybrid induced a G1 arrest and reduced the number of cells in the S phase after 24 h, persisting at 48 h, albeit with a less significant increase in G1, possibly due to adaptive cellular responses. In contrast, N-9 substituted triazole-type harmiprime exhibited less pronounced effects on the cell cycle, particularly after 48 h, which is consistent with its moderate activity against the MCF-7 cell line. On the other hand, screening of their antiplasmodial activities against the erythrocytic, hepatic, and gametocytic stages of the Plasmodium life cycle showed that dimeric harmiprime exerts powerful triple-stage antiplasmodial activity, while computational analysis showed its binding within the ATP binding site of PfHsp90.

The wide use of mono- or bis-styryl fluorophores in biomedical applications prompted the presented design and study of a series of trimeric and tetrameric homo-analogues, styryl moieties arranged around a central aromatic core. The interactions with the most common biorelevant targets, ds-DNA and ds-RNA, were studied by a set of spectrophotometric methods (UV-VIS, fluorescence, circular dichroism, thermal denaturation). All studied dyes showed strong light absorption in the 350–420 nm range and strongly Stokes-shifted (+100–160 nm) emission with quantum yields (Φf) up to 0.57, whereby the mentioned properties were finely tuned by the type of the terminal cationic substituent and number of styryl components (tetramers being red-shifted in respect to trimers). All studied dyes strongly interacted with ds-DNA and ds-RNA with 1–10 nM−1 affinity, with dye emission being strongly quenched. The tetrameric analogues did not show any particular selectivity between ds-DNA or ds-RNA due to large size and consequent partial, non-selective insertion into DNA/RNA grooves. However, smaller trimeric styryl series showed size-dependent selective stabilization of ds-DNA vs. ds-RNA against thermal denaturation and highly selective or even specific recognition of several particular ds-DNA or ds-RNA structures by induced circular dichroism (ICD) bands. The chiral (ICD) selectivity was controlled by the size of a terminal cationic substituent. All dyes entered efficiently live human cells with negligible cytotoxic activity. Further prospects in the transfer of ICD-based selectivity into fluorescence-chiral methods (FDCD and CPL) is proposed, along with the development of new analogues with red-shifted absorbance properties.