Novel Inhibitors of Toxin HipA Reduce Multidrug Tolerant Persisters

Area of Science:

• Microbiology
• Molecular Biology
• Drug Discovery

Background:

• Persister cells are a subpopulation of bacteria exhibiting drug tolerance without genetic mutations, posing a significant challenge to antibiotic treatments.
• Toxin-antitoxin (TA) systems, particularly the HipA (high persistence A) toxin in Escherichia coli, are strongly implicated in the formation of persister cells.
• Targeting TA modules represents a promising strategy to overcome antibiotic tolerance and eradicate persistent infections.

Purpose of the Study:

• To identify novel inhibitors of the HipA toxin using structure-based virtual screening.
• To evaluate the efficacy of identified HipA inhibitors in reducing Escherichia coli persister cell formation.
• To establish a potential therapeutic framework for combating bacterial persistence by targeting TA modules.

Main Methods:

• Structure-based virtual screening was employed to discover potential HipA inhibitors.
• In vitro assays were used to determine the binding affinity (K D) of the most potent inhibitor.
• Ex vivo screening assessed the efficacy of the inhibitor in reducing persister cell fractions in the presence of antibiotics (ampicillin and kanamycin).

Main Results:

• Several novel HipA inhibitors were identified through virtual screening.
• The most potent inhibitor demonstrated a significant reduction in the persister cell fraction (over five-fold).
• This inhibitor exhibited an in vitro K D of 270 ± 90 nM and ex vivo EC50 values of 46 ± 2 μM (ampicillin) and 28 ± 1 μM (kanamycin).

Conclusions:

• Inhibiting the HipA toxin effectively reduces bacterial persistence independently of conventional antibiotic action.
• Targeting toxin-antitoxin modules offers a viable strategy for developing new therapies against persistent bacterial infections.
• This study provides a foundation for developing novel anti-persistence treatments by interfering with TA systems.