Design, synthesis and optimization of inhibitors of enzymes involved in the sulfur assimilation pathway in Gram negative Bacteria

Abstract

Worldwide emergence of resistant bacteria is endangering the efficacy of antibiotics, which have saved millions of lives and transformed medicine. After years of success in treating bacterial infections, the lack of new drug development in the field poses bacteria once again as life threatening conditions. ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) and in particular their Gram negative representatives have been causing high morbidity and mortality among critically ill patients and those requiring invasive devices or surgeries. The lack of new antibacterials and the restless spread of antibacterial resistance represent one of the most challenging public health threats of the XXI century, and make urgent to find new ways to fight bacterial infections. Reduction of virulence and decrease in the onset of antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, in an attempt to reduce bacterial fitness and infectivity, we have explored the approach of shutting-down cysteine biosynthesis in bacteria through inhibition of the enzymes that catalyze the last steps of the biosynthetic pathway: O-Acetylserine sulfhydrylase (OASS) and Serine Acetyltransferase (SAT). I have taken the cue from 1-(4-methylbenzyl)-2-phenylcyclopropanecarboxylic acid, an previously reported OASS inhibitor, which, despite binding the enzyme at nanomolar concentration, failed to show any antibacterial activity likely due to permeability issues. Thus four different approaches were taken: (i) a medicinal chemistry campaign aiming at increase the permeability of the previously identified hit was started; (ii) a scaffold hopping was performed in order to skip the cyclopropane scaffold and identify a compound able to cross Gram negative cell wall; (iii) explore the closed conformation of the enzyme through funnel metadynamics and STD NMR to explore tolerability to derivatization and obtain analogues with diverse pharmacokinetic properties; (iv) incorporate in the structure of the nanomolar enzyme binder a siderophore to try to promote uptake trough a trojan-horse strategy. The aim of our medicinal chemistry campaign was to obtain several derivatives with different pharmacokinetic properties from the parent compound. The design was based on the available literature, being aware that established rules to enhance permeability in Gram negative bacteria have not been defined yet. Even though major changes in the lipophilic character of the inhibitors were performed, a few derivatives were able to mantain the nanomolar binding affinity to the enzyme. Scaffold hopping approach resulted in the identification of a fragment that offers noticeable space for further chemical optimization and that presents better pharmacological properties than the original hit. Funnel metadynamics combined with STD NMR allowed identification of a point of derivatization of the hit that presents high tolerability towards lipophilic and hydrophilic groups. This way, expansion of the series towards compounds with very different physicochemical characteristics can be done without impairing the activity of the parent compound. The result of inclusion of a siderophore in the parent compound is still under investigation. To identify SAT inhibitors, a virtual screening of an in house and three commercial focused chemical libraries were performed. Concerning the virtual screening of the in house library, a low micromolar enzyme binder was identified but once again permeability emerged as the main cause for the lack of activity in bacteria. On the other hand, the virtual screening of the commercial focused libraries led to the identification of a hit compound endowed with good in vitro and in cell activity, that is currently object of study to validate its use as antibacterial adjuvant.