Targeting infectious species by Structure-guided Fragment-based Drug Discovery and in silico approach

Abstract

We are living in a post-antibiotic era in which several human pathogens have developed multidrug resistance and few new antibiotics are being discovered. In addition to the challenge of the emergence of antibiotic resistance, the bacterial population also harbours mechanisms that increase pathogenicity and virulence by tolerating antibiotics and avoiding the host immune system. The emergence of species with multidrug resistance (MDR) and extensively drug resistant species (XDR) has alerted us to the danger of infection threats. For instance, Mycobacterium abscessus (Mab) is a rapidly growing multidrug-resistant species of nontuberculous mycobacteria (NTM) in addition to the well-characterised highly virulent species of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter subspecies) reported by the Centers for Disease Control and Prevention (CDC) and the World Health Organisation (WHO). There is therefore an urgent need to develop novel classes of antibiotics against these pathogens. In addition to resistance being problematic, antibiotic tolerance also adds another level of complexity during the process of treatment by compromising non-essential activities or creating dormant ‘persister’ states. The mechanisms of triggering persister phenotypes seem rather unclear but Toxin-antitoxin (TA) complexes have been reported to play a significant role in triggering persister phenotype. In this thesis, novel and validated targets are exploited using fragment-based drug discovery (FBDD) and in silico approaches to design unconventional antibiotics that could extend the scope of current treatment options and reverse some of the effects of living in a post-antibiotic era. Firstly, three biological targets that work as enzymes (FtsZ, MurB and CoaD) were investigated by cloning, purification, and crystallisation with the aim of solving structures of apo states or complexes with molecules in addition to fragment screening using one or more biophysical methods to identify suitable fragment complexes for structural characterization. The Second part of the thesis concerns toxin-antitoxin (TA) complexes, however most experiments focused on antitoxin components to avoid any possible binding of fragments or compounds to the active site of the toxin that might hinder its activity. Lastly, peptidomimetics were designed and screened in silico to find how this could build on the knowledge gained from previous experiments and improve on current strategies for targeting toxin-antitoxin complexes.