Agnieszka Chylewska

In this chapter, we present the possibility of using ultraviolet (UV) and circular dichroism (CD) spectroscopies to analyze RNA–RNA interactions as an alternative or complementary technique to the classical RNA gel mobility shift assay. We present relevant, detailed protocols and the results obtained for a specific biological model to investigate all three methods. In this model, we studied the binding of small noncoding sRNA molecules of bacteriophage origin to fragments of bacterial mRNAs that were selected based on in silico analysis. We conclude that spectroscopic methods can be especially useful in analyzing the interactions of small RNAs with target transcripts and in the experimental validation of their target sites of action.

This study extends previous research, particularly focusing on patented scientific objects No. ID: PL 240 353 B1, investigating the physicochemical properties of the methyl 3-azido- and 3-amino-2,3-dideoxysaccharides with a nucleoside scaffold similar to 3'-azidothymidine (AZT). The study utilizes multiwavelength spectrophotometric and potentiometric methods to evaluate the ionization of the saccharide units in aqueous solutions. pK_a values, obtained from two independent methods, reveal significant sugar ionization effects on UV spectra with varying pH levels. Stability constants for divalent metal ion complexes (Cu²⁺ and Ni²⁺) with the saccharide isomers indicate that complex stoichiometries and stabilities are highly dependent on the configuration of sugar ring substituents. Spectrophotometric results show a descending order of CT-DNA-binding affinity: BRNH₂OMe > BRN₃OMe > ARN₃OMe > ARNH₂OMe, suggesting varied interaction strengths. Molecular docking of models of synthesized O-glycosides confirmed their potential as reverse transcriptase inhibitors. Among the derivatives tested, the compound BRN₃OMe displays the highest interaction with the enzyme active site residues and DNA, suggesting it may possess the greatest efficacy. Our reported results highlight the promising inhibitory properties of novel O-glycosides against HIV reverse transcriptase, supporting their potential development as antiviral agents.

Organometallic half-sandwich complexes [(η⁵-Cp)IrCl(L)]PF₆ (1) and [(η⁵-Cp)RhCl(L)]PF₆ (2) were prepared using pentamethylcyclopentadienyl chloride dimers of iridium(III) or rhodium(III) with the 4-amino-N-(2,2'-bipyridin-5-yl)benzenesulfonamide ligand (L) and ammonium hexafluorophosphate. The crystal structures of L, 1, and 2 were analyzed in detail. The coordination reactions of the ligand with the central ions were confirmed using various spectroscopic techniques. Additionally, the interactions between sulfaligand, Ir(III), and Rh(III) complexes with carbonic anhydrase (CA), human serum albumin (HSA), and CT-DNA were investigated. The iridium(III) complex (1) did not show any antiproliferative properties against four different cancer cell lines, i.e., nonsmall cell lung cancer A549, colon cancer HCT-116, breast cancer MCF7, lymphoblastic leukemia Nalm-6, and a nonmalignant human embryonic kidney cell line HEK293, due to high binding affinity to GSH. The sulfonamide ligand (L) and rhodium(III) complex (2) were further studied. L showed competitive inhibition toward CA, while complexes 1 and 2, uncompetitive. All compounds interacted with HSA, causing a conformational change in the protein's α-helical structure, suggesting the induction of a more open conformation in HSA, reducing its biological activity. Both L and 2 were found to induce cell death through a caspase-dependent pathway. These findings position L and 2 as potential starting compounds for pharmaceutical, therapeutic, or medicinal research.

The measurement of oxygen consumption is an important element in the understanding of an organism’s metabolic state. Oxygen is also a phosphorescence quencher, which allows the evaluation of phosphorescence emitted by oxygen sensors. Two Ru(II)-based oxygen-sensitive sensors were used to study the effect of chemical compounds [(1) = [CoCl₂(dap)₂]Cl, and (2) = [CoCl₂(en)₂]Cl (AmB = amphotericin B) against reference and clinical strains of Candida albicans. The tris-[(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)] chloride ([Ru(DPP)₃]Cl₂) (Box) adsorbed onto the Davisil^TM silica gel was embedded in the silicone rubber Lactite NuvaSil^® 5091 and the coating on the bottom of 96-well plates. The water-soluble oxygen sensor (BsOx = tris-[(4,7-diphenyl-1,10-phenanthrolinedisulphonic acid disodium)ruthenium(II)] chloride ‘x’ hydrate = {Ru[DPP(SO₃Na)₂]₃}Cl₂ = water molecules were omitted in the BsOx formula) was synthesized and characterized by RP-UHPLC, LCMS, MALDI, elemental analysis, ATR, UV-Vis, ¹H NMR, and TG/IR techniques. The microbiological studies were performed in the environment of RPMI broth and blood serum. Both Ru(II)-based sensors turned out to be useful in the study of the activity of Co(III) complexes and the commercial antifungal drug amphotericin B. In addition, a new activity of the oxygen sensor, the soluble Ru(II) complex BsOx, was demonstrated, which is a mixture with amphotericin B that caused a significant increase in its antifungal activity. Thus, it is also possible to demonstrate the synergistic effect of compounds active against the microorganisms under study.

Increasing resistance of fungi, especially Candida spp., has successively reduced the already short list of effective antifungal drugs used in clinical therapy. Thus there is an urgent need for new, non-toxic antifungals with novel modes of action. A very interesting and attractive group of compounds seems to be complexes of Co(III) with diamine chelate ligands due to their therapeutic uses as antiviral, antibacterial, antifungal, antiparasitic, or antitumor agents. Two Co(III) complexes with diamine chelate ligands ([CoCl₂(dap)₂]Cl (1) and [CoCl₂(en)₂]Cl (2)) (where dap = 1,3-diaminopropane, en = ethylenediamine) were synthesized and characterized using elemental analysis, an ATR technique, and a scan method and sequentially tested to evaluate the mode of action. The assessment of cell damage by the newly synthetized photosensitive fluorescein-labelled complex 1 ([Co(dap)₂FLU]Cl₂) was performed using fluorescent microscopy, which indicated cell membrane permeability and altered cell morphology. The study of the C. albicans survival in blood showed a slight reduction in the number of viable, colonizing cells in the sample compared to the results obtained for the substances with confirmed antifungal activity – ketoconazole. DNA cleavage ability of the Co(III) complexes using agarose gel electrophoresis against genomic DNA isolated from a C. albicans cells showed nuclease activity for both complexes. This study provides new data on potential antifungal drugs, especially against Candida species.

Isothermal titration calorimetry, circular dichroism (CD) techniques, and in silico analysis were used to determine potential metal binding sites in human cationic antimicrobial protein (hCAP) corresponding to overlapping the dodecapeptide sequences of hCAP(134-170) referred to as LL-37. The correct antibacterial action of LL-37 is closely related to its established unique structure. Disturbances in the LL-37 structure (e.g., unwanted presence of metal ions) lead to a radical change in its biological functions. Five fragments of the LL-37 [hCAP(134-170)], namely, hCAP(134-145) (A1), hCAP(140-151) (A2), hCAP(146-157) (A3), hCAP(152-163) (A4), and hCAP(159-170) (A5), were taken into account and their affinity to Mn(II) and Zn(II) ions was rigorously assessed. We prove that only three of the investigated peptides (A1, A2, and A5) are capable of forming thermodynamically stable complexes with metal ions. Additionally, based on density functional theory (DFT) calculations, we propose the most likely coordination modes of metal(II) to peptides as well as discuss the chemical nature of the interactions. Finally, we present the structural features of the strongest binding peptide, hCAP(159-170), responsible for the metal binding. The presented results provide important structural and thermodynamic information to understand the influence of some metal ions on the activity of hCAP(134-170).

Pyrazine and its derivatives are a large group of compounds that exhibit broad biological activity, the changes of which can be easily detected by a substituent effect or a change in the functional group. The present studies combined theoretical research with the density functional theory (DFT) approach (B3LYP/6-311+G**) and experimental (potentiometric and spectrophotometric) analysis for a thorough understanding of the structure of chlorohydrazinopyrazine, its physicochemical and cytotoxic properties, and the site and nature of interaction with DNA. The obtained results indicated that 2-chloro-3-hydrazinopyrazine (2Cl3HP) displayed the highest affinity to DNA. Cytotoxicity studies revealed that the compound did not exhibit toxicity toward human dermal keratinocytes, which supported the potential application of 2Cl3HP in clinical use. The study also attempted to establish the possible equilibria occurring in the aqueous solution and, using both theoretical and experimental methods, clearly showed the hydrophilic nature of the compound. The experimental and theoretical results of the study confirmed the quality of the compound, as well as the appropriateness of the selected set of methods for similar research.

Treatment with pyrazine derivatives—antituberculosis pyrazinamide (PZA), anticancer bortezomib (BZM), and antifungal pyrazine-2-amidoxime (PAOX) and pyrazine-2-thiocarboxamide (PTCA)—is associated with side effects, as observed in the case of other therapeutic drugs. To prevent the side effects of pyrazine derivatives, researchers are working to develop a universal method that will detect these compounds in body fluids. There is a lack of literature data about voltammetric measurements with poly-L-amino acid-modified GCEs surfaces. The available reports describe the application of various modifications of these electrodes for the detection of different active substances of drugs; however, they do not indicate one particular method for the detection of drugs with a pyrazine skeleton. This research aimed to prepare three types of glassy carbon electrodes (GCEs) with modified surfaces by electropolymerization using 1, 10, and 100 mM solutions of L-glycine (Gly), L-alanine (Ala), L-lysine (Lys), respectively. The poly-amino acid coatings applied on GCE surfaces were analyzed in detail under a three-dimensional (3D) microscope and were used as chemosensors of four pyrazine drugs in stoichiometric tests. The results were compared with the measurements made on an unmodified GCE. To obtain reliable results, the linearity of measurements was also verified in the concentration gradient and appropriate scanning speed was chosen to achieve the most accurate measurements.