Repurposing of robenidine and characterization of novel analogues for treatment of infectious diseases

Abstract

Infectious diseases are one of the leading causes of morbidity and mortality worldwide. Diseases caused by single-celled organisms, such as bacteria and protista, cause billions of infections per year. One of the leading weapons in the fight against infectious diseases are antimicrobials. However, the efficacy of antimicrobials is decreasing as the development of antimicrobial resistance increases. At the same time as increasing levels of resistance are observed there is a lack of new antimicrobial agents entering the market and many big pharmaceutical companies have suspended antimicrobial drug discovery programs as financial return is small. Due to the lack of novel treatments for infectious diseases and increasing treatment failures it is essential that new chemical entities are explored to fill this gap. In this thesis a novel library of compounds based on the structure of robenidine, an approved antimicrobial used to prevent coccidiosis in chickens and rabbits, was investigated as potential antimicrobial agents. Initial experiments focussed on the antibacterial activity of the library against representative pathogenic bacteria. Activity was assessed according to CLSI guidelines. The spectrum of activity of the majority of analogues investigated was limited to Gram-positive bacteria, with promising MICs as low as 1.3 μM. However, through the use of outer-membrane permeabilising agents and spheroplast induction, it was discovered that the target site of robenidine and some of the related analogues was also present in Gram-negative organisms. This led to the development of a small subset of analogues which demonstrated intrinsic activity against the Gram-negative pathogens Escherichia coli and Pseudomonas aeruginosa with MICs as low as 52 μM. Furthermore, kill kinetic studies revealed that robenidine and related analogues had a bactericidal mechanism of action. The next series of experiments focussed on the characterisation of the antiparasitic activity of the library against the protists Trypanosoma brucei, Leishmania donovani and Giardia duodenalis. Several of the analogues demonstrated activity against these parasites with some promising results against Leishmania donovani including a small number of analogues with selectivity indices (SI) for the parasite above 20 (an SI of >10 is considered selective). In addition, activity against G. duodenalis was also promising (IC50 <1 μM). In total 121 analogues were tested against G. duodenalis with 13 being selective for Giardia with no antibacterial activity and limited, if any, toxicity towards mammalian cells. MICs for the most promising analogues were ≤ 2.8 μM. Electron microscopy studies to elucidate the mechanism or site of action of this class of antimicrobials against G. duodenalis demonstrated that the two most promising compounds both caused rapid disintegration of the cell membrane and the development of cyst-like structures, while one analogue also appeared to interfere with cell division. Finally, in order to test in vivo efficacy an animal model was effectively established in neonatal mice. In conclusion this thesis demonstrated for the first time the potential for this library of compounds to become therapeutic agents for a range of infectious diseases. In particular, the selective activity of several analogues for Giardia over other microorganisms and mammalian cells was demonstrated for the first time, highlighting the potential for this library of analogues. In addition, insight into the unique mechanism of action of a select group of compounds against G. duodenalis was demonstrated.