ANTIBACTERIAL DRUG DISCOVERY: TARGETING RESISTANCE AND PERSISTENCE IN BACTERIA USING NOVEL SMALL MOLECULES

Abstract

Antibiotic resistance is one of the major global health concerns. Inappropriate and excessive use of antibiotics has led to the emergence of multi-drug resistant (MDR) bacterial strains, thus exacerbating the problem. Despite generous efforts, no new class of antibiotics have been approved against Gram-negative pathogens in over 50 years and antibiotic research is under a dry spell. The ESKAPE group of pathogens namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species present a major threat worldwide due to their unequalled ability to escape the available antibiotic treatments. Only four new classes of antibiotics have been approved for clinical use in the last decade, all of which target Gram-positive pathogens and none of which exhibit a novel mechanism of action. Importantly, Gram-negative bacteria make up four of the six ESKAPE pathogens and present the most acute threat owing to their complex cell envelope structure that acts as a formidable barrier to the existing antibiotic arsenal. The increasing emergence of multidrug-resistant bacteria and the drying antibiotic pipeline calls for the development of new antibacterial agents and novel strategies that can help address the global threat of antibiotic resistance. In an effort to explore new leads in the field of antimicrobial drug discovery against Gram negative bacteria, a whole-cell screening assay was performed on a collection of 10,956 small molecules against Escherichia coli ATCC 25922 and 30 hit compounds demonstrating remarkable antibacterial activity were identified. Herein, I describe the antibacterial spectrum, in vitro pharmacodynamics and mechanism of action (MOA) of one such lead series represented by IITR06144. IITR06144 exhibits an impressive broad spectrum activity against most MDR bacteria and a remarkable antibiofilm and anti-persister activity. It displays no associated toxicity, lack of resistance development and in vivo efficacy in a mice model of infection. With the aid of antisense RNA technology, fluorescence microscopy studies and use of E. coli gene mutants etc., it was observed that IITR06144 inhibits the bacterial growth by causing DNA damage and consequently inhibiting the cell division machinery. IITR06144 is a promising antibacterial molecule displaying most features of a theoretically ―ideal‖ antibiotic and extreme potential to overcome the problem of antibiotic resistance prevailing in the clinics. The use of antibiotics in combination represents one of the most readily available strategies to combat infections caused by MDR pathogens. Synergistic antibiotic combinations are willingly accepted by clinicians as they aid the revival of old antibiotics and can overcome the problem of ii toxicity associated with certain antibiotic classes. Hence, I further studied the ability of small molecule, IITR06144 to act in combination with clinically relevant antibiotics against a few representative ESKAPE pathogens. IITR06144 was observed to exhibit favourable synergistic interaction with vancomycin against Vancomycin Intermediate Staphylococcus aureus (VISA). The combination of IITR06144 and Vancomycin displayed excellent bactericidal activity, anti-biofilm activity and in vivo efficacy in Caenorhabditis elegans model of S. aureus infection. Elucidating the mode of action for novel antibacterials identified in small molecule screens remains an arduous task that severely limits drug discovery efforts. In this respect, I exploited the previously established strategy of using chemical-chemical interactions to identify cellular targets of few aforementioned molecules from the phenotypic screen. Small molecules were combined with known antibacterials belonging to diverse chemical classes and mechanisms and screened for growth inhibition against E. coli MC1061, using a two-point dose matrix approach. This approach uncovered several synergistic combinations identifying a novel lead series antibacterial, IITR07865 which represents a new class of inhibitors targeting the bacterial cell wall synthesis. Apart from drug resistance, the phenomenon of ―drug tolerance‖ exhibited by a subpopulation of bacterial cells known as ―persisters‖ ensues another major threat. Eradication of the persister populations in chronic ailments holds extreme importance for an improved long term recovery from recurring infections. In this part of the study, I first investigated the characteristics and mechanisms responsible for meropenem persistence in the ESKAPE pathogen, A. baumannii. Meropenem induced A. baumannii persisters were observed to exhibit enhanced efflux activity thus rendering them to be multi-drug tolerant. Further, I devised a mechanistic screen for novel anti-persister compounds leading to the identification of a GRAS (Generally Regarded As Safe) status small molecule with promising activity against bacterial persisters of A. baumannii and two other ESKAPE pathogens. This study has vital implications for the treatment of recalcitrant bacterial infections. Overall, this work addresses the challenge of antibiotic resistance and endeavours to minimize or overcome the problem by the identification of new antibacterial agents, development of small molecule-combination regimens or targeting antibiotic tolerant organisms. Importantly, this study reports the identification of novel synthetic and natural scaffolds that demonstrate excellent potential and hold promise for use in clinics in the fight against antibiotic resistance.