Antimicrobial macrozones interact with biological macromolecules via two-site binding mode of action: Fluorimetric, NMR and docking studies.

Macrozones are novel conjugates of azithromycin and thiosemicarbazones, which exhibit very good in vitro antibacterial activities against susceptible and some resistant bacterial strains thus showing a potential for further development. A combination of spectrometric (fluorimetry, STD and WaterLOGSY NMR) and molecular docking studies provided insights into atomic details of interactions between selected macrozones and biological receptors such as E. coli ribosome and bovine serum albumin. Fluorimetric measurements revealed binding constants in the micro-molar range while NMR experiments provided data on binding epitopes. It has been demonstrated that both STD and WaterLOGSY gave comparable and consistent results unveiling atoms in intimate contacts with biological receptors. Docking studies pointed towards main interactions between macrozones and E. coli ribosome which included specific π - π stacking and hydrogen bonding interactions with thiosemicarbazone part extending down the ribosome exit tunnel. The results of the docking experiments were in fine correlation with those obtained by NMR and fluorimetry. Our investigation pointed towards a two-site binding mechanism of interactions between macrozones and E. coli ribosome which is the most probable reason for their activity against azithromycin-resistant strains. Much better activity of macrozone-nickel coordinated compound against E. coli ribosome compared to other macrozones has been attributed to the higher polarity which enabled better bacterial membrane penetration and binding of the two thiosemicarbazone units thus additionally contributing to the overall binding energy. The knowledge gained in this study should play an important role in anti-infective macrolide design in the future.

Triarylborane-"Click" Fluorescent Tag for Orthogonal Amino Acid Labelling, Interactions with DNA, Protein, and Cyclodextrins.

The innovative design of a triarylborane (TB)-dye with one NMe2-alkylated (propargylated) group and one NMe2 group yielded a system that is both an NMe2 π-donor and an inductive NMe2-alkyl cationic acceptor. Consequently, the new TB-dye was highly sensitive to a "click" reaction with an azide-substituted lysine side chain (yielding TB-lysine), resulting in a bathochromic shift of emission of 100 nm. In addition, fluorene attached to the lysine C-terminus showed FRET with the TB-chromophore, also sensitive to interactions with targets. Both the TB-dye and TB-lysine showed high affinities towards both DNA and proteins, reporting binding by an opposite fluorimetric response for DNA/RNA (quenching) vs. BSA (increase). Thus, the novel TB-dye is an ideal fluorimetric probe for orthogonal incorporation into bio-targets by "click" reactions due to fluorescence reporting of the progress of the "click" reaction and further sensing of the binding site composition. The TB-dye is moderately toxic to human cell lines after 2-3 days of exposure, but efficiently enters cells in 90 min, being non-toxic at short exposure. The most important product of the "click" reaction, TB-lysine, was non-toxic to cells and showed equal distribution between mitochondria and lysosomes. Further studies would focus particularly on the very convenient monitoring of the progress of "click" conjugation of the TB-dye with biorelevant targets inside living cells by confocal microscopy.

Novel Tripodal Polyamine Tris-Pyrene: DNA/RNA Binding and Photodynamic Antiproliferative Activity.

A novel tri-pyrene polyamine (TAL3PYR) bearing net five positive charges at biorelevant conditions revealed strong intramolecular interactions in aqueous medium between pyrenes, characterised by pronounced excimer fluorescence. A novel compound revealed strong binding to ds-DNA and ds-RNA, along with pronounced thermal stabilisation of DNA/RNA and extensive changes in DNA/RNA structure, as evidenced by circular dichroism. New dye caused pronounced ds-DNA or ds-RNA condensation, which was attributed to a combination of electrostatic interactions between 5+ charge of dye and negatively charged polynucleotide backbone, accompanied by aromatic and hydrophobic interactions of pyrenes within polynucleotide grooves. New dye also showed intriguing antiproliferative activity, strongly enhanced upon photo-induced activation of pyrenes, and is thus a promising lead compound for theranostic applications on ds-RNA or ds-DNA targets, applicable as a new strategy in cancer and gene therapy.

Photoluminescent Gold/BSA Nanoclusters (AuNC@BSA) as Sensors for Red-Fluorescence Detection of Mycotoxins.

The BSA-encapsulated gold nanoclusters (AuNC@BSA) have drawn considerable interest and demonstrated applications as biological sensors. In this study, we demonstrated that the red-emitting AuNC@BSA prepared using a modified procedure fully retained the binding of standard BSA-ligands (small molecule drugs), significantly improving fluorescence detection in some cases due to the red-emission property. Further, we showed that AuNC@BSA efficiently bind a series of aflatoxin-related mycotoxins as well as the aliphatic mycotoxin FB1, reporting interactions in the nanomolar range by instantaneous emission change at 680 nm. Such red emission detection is advantageous over current detection strategies for the same mycotoxins, based on complex mass spectrometry procedures or, eventually (upon chemical modification of the mycotoxin), by fluorescence detection in the UV range (<400 nm). The later technique yields fluorescence strongly overlapping with the intrinsic absorption and emission of biorelevant mixtures in which mycotoxins appear. Thus, here we present a new approach using the AuNC@BSA red fluorescence reporter for mycotoxins as a fast, cheap, and simple detection technique that offers significant advantages over currently available methods.