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The ability of Mycobacterium tuberculosis ( Mtb ) to persist in its host may enable an evolutionary advantage for drug resistant variants to emerge. A potential strategy to prevent persistence and gain drug efficacy is to directly target the activity of enzymes that are crucial for persistence. We present a method for expedited discovery and structure-based design of lead compounds by targeting the hypoxia-associated enzyme L-alanine dehydrogenase (AlaDH). Biochemical and structural analyses of AlaDH confirmed binding of nucleoside derivatives and showed a site adjacent to the nucleoside binding pocket that can confer specificity to putative inhibitors. Using a combination of dye-ligand affinity chromatography, enzyme kinetics and protein crystallographic studies, we show the development and validation of drug prototypes. Crystal structures of AlaDH-inhibitor complexes with variations at the N6 position of the adenyl-moiety of the inhibitor provide insight into the molecular basis for the specificity of these compounds. We describe a drug-designing pipeline that aims to block Mtb to proliferate upon re-oxygenation by specifically blocking NAD accessibility to AlaDH. The collective approach to drug discovery was further evaluated through in silico analyses providing additional insight into an efficient drug development strategy that can be further assessed with the incorporation of in vivo studies.

The identification of novel targets for antimicrobial agents is crucial for combating infectious diseases caused by evolving bacterial pathogens. Components of bacterial toxin–antitoxin (TA) systems have been recognized as promising therapeutic targets. These widespread genetic modules are usually composed of two genes that encode a toxic protein targeting an essential cellular process and an antitoxin that counteracts the activity of the toxin. Uncontrolled toxin expression may elicit a bactericidal effect, so they may be considered “intracellular molecular bombs” that can lead to elimination of their host cells. Based on the molecular nature of antitoxins and their mode of interaction with toxins, TA systems have been classified into six groups. The most prevalent are type II TA systems. Due to their ubiquity among clinical isolates of pathogenic bacteria and the essential processes targeted, they are promising candidates for the development of novel antimicrobial strategies. In this review, we describe the distribution of type II TA systems in clinically relevant human pathogens, examine how these systems could be developed as the targets for novel antibacterials, and discuss possible undesirable effects of such therapeutic intervention, such as the induction of persister cells, biofilm formation and toxicity to eukaryotic cells.

Antibiotic resistance is a growing threat for modern medicine, making treatment of infectious diseases increasingly tedious. However, even non-resistant bacteria can survive treatment and cause recurrent infections. This phenomenon is often due to non-proliferating bacteria able to survive the treatment and to resume infection afterwards, also called recalcitrant bacteria. Bacterial recalcitrance, which encompasses tolerance and persistence, is defined by increased survival of bacteria in the presence of antimicrobial agents. In contrast to resistance, the mechanisms underlying recalcitrance are only partially understood. In this review, we summarise the recent advances in the understanding of recalcitrance, its detection, as well as anti-recalcitrance therapies that have been developed. Recalcitrance is thought to be caused by a reduction of bacterial metabolism, mostly driven by stringent and SOS responses, leading to bacterial dormancy. These dormant bacteria escape the action of many antibiotics, preventing the complete resolution of infection. However, strategies have been proposed to tackle recalcitrance. Recalcitrant bacteria are susceptible to drugs whose action is independent of metabolic activity, such as membrane-targeting compounds. Inhibitors blocking the entry of bacteria into dormancy or locking bacteria in a permanent state of dormancy could help avoid recurrence of the infection. Dormant bacteria could also be forced to resume growth through supply of nutrients or signalling molecules. A phage specifically targeting dormant bacteria was recently described and may be an important tool to fight bacterial recalcitrance. Recalcitrance has been neglected for a long time, being in the shadow of resistance. However, both phenomena need to be further investigated in the future to develop a complete array of antibacterial agents that will allow to permanently eradicate all types of bacterial infections.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), poses a global health challenge and is responsible for over a million deaths each year. Current treatment is lengthy and complex, and new, abbreviated regimens are urgently needed. Mtb adapts to nutrient starvation, a condition experienced during host infection, by shifting its metabolism and becoming tolerant to the killing activity of bactericidal antibiotics. An improved understanding of the mechanisms mediating antibiotic tolerance in Mtb can serve as the basis for developing more effective therapies. We performed a forward genetic screen to identify candidate Mtb genes involved in tolerance to the two key first-line antibiotics, rifampin and isoniazid, under nutrient-rich and nutrient-starved conditions. In nutrient-rich conditions, we found 220 mutants with differential antibiotic susceptibility (218 in the rifampin screen and 2 in the isoniazid screen). Following Mtb adaptation to nutrient starvation, 82 mutants showed differential antibiotic susceptibility (80 in the rifampin screen and 2 in the isoniazid screen). Using targeted mutagenesis, we validated the rifampin-hypersusceptible phenotype under nutrient starvation in Mtb mutants lacking the following genes: ercc3, moeA1, rv0049, and rv2179c. These findings shed light on potential therapeutic targets, which could help shorten the duration and complexity of antitubercular regimens.