Inhibition of cysteine biosynthesis for the development of enhancers of antibiotic therapy

Abstract

Emerging antibiotic resistance represents a major hazard for public health. The necessity for development of new antibiotics especially against Gram-negative bacteria is high, although many issues must be faced, such as bacterial intrinsic protection by a complex outer membrane, deficit of knowledge regarding its permeability, low number of identified targets and lack of molecule diversity in libraries used for screening. Unsuccessful outcomes of projects focused on identification of new antibiotics led several big pharmaceutical companies to completely abolish their antimicrobial research. A promising strategy to fight bacterial resistance is to use enhancers of antibiotic therapy. Enhancers either block the main bacterial resistance mechanism or potentiate the action of a chosen antibiotic. Cysteine is a multifunctional amino acid and its auxothrops are not able to grow on minimal medium. It is the organic source of sulfur, which is donated for biosynthesis of sulfur containing molecules, while cysteine itself stabilizes protein tertiary structure by formation of bisulfide bonds and maintains intracellular redox status. It has been shown that deletion mutants of cysteine biosynthetic pathway have attenuated virulence, elevated levels of intracellular oxidative stress and they have been linked to decreased antibiotic resistance. Oxidative stress is one of the common mechanisms by which antibiotics affect bacteria, and therefore inhibition of bacterial enzymes involved in biosynthesis of cysteine, that are absent in mammals, presents promising strategy for the development of enhancers of antibiotic therapy. The project was focused on the inhibition of the last two enzymes in this pathway, serine acetyltransferase (SAT) and O-acetylserine sulfhydrilase (OASS) that, in enteric bacteria, exists in two isoforms, A and B. The chosen proteins were from Salmonella enterica serovar Typhimurium, which presents a major health risk all over the world and has been listed as a high priority pathogen by World Health Organization for the development of novel inhibiting compounds. In the course of the project, we optimized the conditions for expression and purification of target enzymes as recombinant proteins in E. coli. Expression constructs for OASS were prepared during the secondments at the University of Cambridge (supervisor Prof. Martin Welch). The proteins were later used for in vitro testing of novel reversible and irreversible inhibitors. For OASS, potent reversible inhibitors were identified previously. A cocrystal between OASS-A and the most potent inhibitor UPAR-415 was prepared that allowed us to confirm binding interactions and understand the molecular basis of enzyme inhibition. Since existing inhibitors presented issues in the permeability through the Gram-negative membrane, several derivatives were prepared in Prof. Costantino’s group (University of Parma) in order to improve their drug-likeness and penetration in bacteria. We determined their binding affinity and mechanism of action via enzyme assays and fluorimetric titrations. We identified novel potent inhibitors and the most promising ones were later selected for microbiological testing. In a collaboration with Prof. Jirgensons’ group (Latvian Institute of Organic Synthesis), we investigated covalent modification of OASS by mechanism-based inactivators. Fluoroalanine derivatives were assayed in order to determine their inactivation potency, mechanism of action and structure-activity relationship. Inhibitors of SAT were tested in our laboratory for the first time. For this purpose, we optimized and validated an indirect activity assay that can be used for inhibitors testing on small and large scale. We screened novel SAT inhibitors, selected by Prof. Costantino’s group (University of Parma) based on the in silico screening of an in house compound library and high-throughput screening of ChemDiv libraries, evaluated their potency and determined their mechanism of action. With this approach we identified the most potent SAT inhibitors reported so far. Promising protein inhibitors were later tested in microbiological assays to evaluate their effect on bacterial viability. This part of the project was partially carried out during the secondments in Aptuit Verona (supervisor Dr. Antonio Felici). We found out that they have issues in the permeability through the bacterial membrane, although in the presence of the permeability enhancer they were able to interfere with bacterial growth. To investigate their mechanism of action when inside bacteria, we developed a method for the measurement of the bacteria intracellular reduced thiols as a marker of cysteine availability.