The impact of counterions in biological activity: case study of antibacterial alkylguanidino ureas.

Trifluoroacetic acid (TFA), due to its strong acidity and low boiling point, is extensively used in protecting groups-based synthetic strategies. Indeed, synthetic compounds bearing basic functions, such as amines or guanidines (commonly found in peptido or peptidomimetic derivatives), developed in the frame of drug discovery programmes, are often isolated as trifluoroacetate (TF-Acetate) salts and their biological activity is assessed as such in in vitro, ex vivo, or in vivo experiments. However, the presence of residual amounts of TFA was reported to potentially affect the accuracy and reproducibility of a broad range of cellular assays (e. g. antimicrobial susceptibility testing, and cytotoxicity assays) limiting the further development of these derivatives. Furthermore, the impact of the counterion on biological activity, including TF-Acetate, is still controversial. Herein, we present a focused case study aiming to evaluate the activity of an antibacterial AlkylGuanidino Urea (AGU) compound obtained as TF-Acetate (1a) and hydrochloride (1b) salt forms to highlight the role of counterions in affecting the biological activity. We also prepared and tested the corresponding free base (1c). The exchange of the counterions applied to polyguanidino compounds represents an unexplored and challenging field, which required significant efforts for the successful optimization of reliable methods of preparation, also reported in this work. In the end, the biological evaluation revealed a quite similar biological profile for the salt derivatives 1a and 1b and a lower potency was found for the free base 1c.

An update on antibacterial AlkylGuanidino Ureas: Design of new derivatives, synergism with colistin and data analysis of the whole library.

Antimicrobial resistance (AMR) represents one of the most challenging global Public Health issues, with an alarmingly increasing rate of attributable mortality. This scenario highlights the urgent need for innovative medicinal strategies showing activity on resistant isolates (especially, carbapenem-resistant Gram-negative bacteria, methicillin-resistant S. aureus, and vancomycin-resistant enterococci) yielding new approaches for the treatment of bacterial infections. We previously reported AlkylGuanidino Ureas (AGUs) with broad-spectrum antibacterial activity and a putative membrane-based mechanism of action. Herein, new tetra- and mono-guanidino derivatives were designed and synthesized to expand the structure-activity relationships (SARs) and, thereby, tested on the same panel of Gram-positive and Gram-negative bacteria. The membrane-active mechanism of selected compounds was then investigated through molecular dynamics (MD) on simulated bacterial membranes. In the end, the newly synthesized series, along with the whole library of compounds (more than 70) developed in the last decade, was tested in combination with subinhibitory concentrations of the last resort antibiotic colistin to assess putative synergistic or additive effects. Moreover, all the AGUs were subjected to cheminformatic and machine learning analyses to gain a deeper knowledge of the key features required for bioactivity.

Antibacterial alkylguanidino ureas: Molecular simplification approach, searching for membrane-based MoA.

The ever-faster rise of antimicrobial resistance (AMR) represents a major global Public Health challenge. New chemical entities with innovative Modes of Action (MoAs) are thus desirable. We recently reported the development of a novel class of broad-spectrum bactericidal agents, the AlkylGuanidino Ureas (AGU). Due to their polycationic structure, they likely target bacterial membranes. In order to better understand their MoA, we synthesized a library of AGU derivatives by structural simplification of selected hit compounds and developed specific assays based on membrane models by means of both analytical and computational techniques. Cell-based assays provided experimental evidence that AGUs disrupt bacterial membranes without showing hemolytic behavior. Hence, we herein report a thorough chemical and biological characterization of a new series of AGUs obtained through molecular simplification, allowing the rational design of potent antibacterial compounds active on antibiotic-resistant strains.