Structure-Based Screening and Optimization of PafA Inhibitors with Potent Anti-Tuberculosis Activity.

Area of Science:

• Microbiology
• Drug Discovery
• Biochemistry

Background:

• Tuberculosis (TB) is a significant global health issue, exacerbated by rising drug resistance in Mycobacterium tuberculosis (Mtb).
• Novel therapeutic strategies targeting Mtb are crucial, necessitating drugs with new mechanisms of action (MOAs).
• The prokaryotic ubiquitin-like protein (Pup) conjugation system, involving PafA, is essential for Mtb survival under stress.

Purpose of the Study:

• To discover and characterize novel inhibitors of the Mtb PafA enzyme.
• To investigate the structure-activity relationship for developing potent and selective PafA inhibitors.
• To evaluate the efficacy of these inhibitors against Mtb survival in a relevant in vitro model.

Main Methods:

• Computer-aided drug screening to identify initial hit compounds.
• Molecular docking and experimental validation to elucidate binding modes.
• Structure-guided drug design to optimize inhibitor potency.
• Biochemical and cell-based assays to assess PafA inhibition and Mtb viability.
• In vitro assays mimicking macrophage environments (nitric oxide stress).

Main Results:

• Two potent PafA inhibitors, Pi-1-58 and Pi-2-26, were identified and characterized.
• Pi-1-58 was discovered via virtual screening, and Pi-2-26 was developed through structure-guided design.
• Both inhibitors demonstrated selective and specific inhibition of Mtb PafA.
• Pi-1-58 and Pi-2-26 significantly inhibited Mtb survival under nitric oxide stress, simulating host conditions.

Conclusions:

• The study successfully identified and optimized novel PafA inhibitors with potential anti-TB activity.
• These compounds offer a promising starting point for developing new anti-tuberculosis drugs targeting a novel MOA.
• The findings contribute to understanding PafA inhibition and provide a foundation for future drug development against Mtb.