Structure-Guided Design of Halofuginone Derivatives as ATP-Aided Inhibitors Against Bacterial Prolyl-tRNA Synthetase

Area of Science:

• Biochemistry
• Microbiology
• Drug Discovery

Background:

• Aminoacyl-tRNA synthetases (aaRSs) are crucial for protein synthesis and represent promising antimicrobial targets.
• Expanding inhibitory mechanisms against aaRSs offers new avenues for antibiotic development.

Purpose of the Study:

• To investigate halofuginone (HF) as an inhibitor of bacterial prolyl-tRNA synthetases (ProRSs).
• To design and synthesize novel HF analogs with improved potency and antibacterial activity.
• To validate bacterial ProRS as a viable target for novel antibacterial agents.

Main Methods:

• Cocrystallization of Staphylococcus aureus ProRS (SaProRS) with HF and an ATP analog.
• Structure-guided design and synthesis of HF analogs.
• Enzyme inhibition assays (IC50, Kd) and determination of antibacterial activity (MIC).
• In vitro assessment of bacterial drug resistance development.

Main Results:

• Halofuginone demonstrated moderate inhibition of ProRS from various pathogenic bacteria.
• Cocrystal structure elucidated the binding mode of HF to SaProRS, guiding analog design.
• Compound 3 exhibited potent inhibition of SaProRS (IC50 = 0.18 μM, Kd = 30.3 nM) and significant in vitro antibacterial activity (MIC = 1-4 μg/mL).
• Compound 3 displayed resistance development rates comparable to or slower than existing antibiotics.

Conclusions:

• The HF scaffold and its ATP-competitive inhibition mechanism are effective against bacterial ProRS.
• Compound 3 represents a promising lead compound for developing new antibiotics targeting bacterial ProRS.
• This study provides chemical validation for bacterial ProRS as a druggable antimicrobial target.