Drug-like molecules with anti-trypanothione synthetase activity identified by high throughput screening

Area of Science:

• Biochemistry
• Drug Discovery
• Parasitology

Background:

• Trypanothione synthetase (TryS) is crucial for redox balance in trypanosomatids.
• TryS is a promising drug target for treating diseases caused by trypanosomatid parasites.
• Identifying novel TryS inhibitors can lead to new therapeutic strategies.

Purpose of the Study:

• To screen a large compound library for inhibitors of Trypanosoma brucei TryS (TbTryS).
• To characterize the activity and selectivity of identified inhibitors against TryS from various trypanosomatid species.
• To identify novel chemical scaffolds and understand the mechanism of inhibition.

Main Methods:

• High-throughput screening of a 51,624-compound library against TbTryS.
• Enzyme inhibition assays (IC50 determination) using TbTryS, Leishmania infantum TryS, and Trypanosoma cruzi TryS.
• Cell-based assays to evaluate anti-parasitic activity and host cell cytotoxicity (EC50 and selectivity index).
• Kinetic and mass spectrometry analysis to elucidate inhibition mechanisms.

Main Results:

• A true-hit rate of 0.056% was achieved, identifying several TbTryS inhibitors with IC50 values from 1.2 to 36 µM.
• Selected inhibitors demonstrated activity against homologous enzymes from L. infantum and T. cruzi (IC50 2.6–40 µM).
• Calmidazolium chloride and Ebselen showed broad-spectrum anti-TryS activity (IC50 2.6–13.8 µM).
• Novel inhibitor scaffolds (carboxy piperidine amide, amide methyl thiazole phenyl) were identified.
• Seven compounds exhibited high selectivity against T. b. brucei (selectivity index 11–182).
• Ebselen acts as a slow-binding inhibitor, irreversibly modifying a conserved cysteine residue.
• The most potent inhibitor displayed non-covalent, non-competitive inhibition.

Conclusions:

• The study identified novel chemical entities with potential as anti-trypanosomatid drugs.
• Ebselen and other multi-species inhibitors provide a foundation for developing broad-spectrum treatments.
• Understanding inhibition mechanisms aids in the rational design of future drug candidates.
• The identified scaffolds offer new starting points for medicinal chemistry optimization.