Targeting chalcone binding sites in living Leishmania using a reversible fluorogenic benzochalcone probe

Area of Science:

• Parasitology
• Medicinal Chemistry
• Fluorescence Microscopy

Background:

• Chalcones possess diverse biological activities, including antileishmanial properties, but their drug development is hindered by unknown mechanisms of action.
• Understanding how chalcones interact with Leishmania parasites is crucial for developing new antileishmanial drugs.

Purpose of the Study:

• To utilize a benzochalcone fluorogenic probe (HAB) to visualize and study the intracellular binding of chalcones in live Leishmania amazonensis promastigotes.
• To investigate the pharmacological interactions of various chalcones and analogues with Leishmania using competitive binding assays.

Main Methods:

• Employing fluorescence microscopy to image the localization of the HAB probe within live Leishmania promastigotes, targeting acidocalcisomes, lysosomes, and mitochondria.
• Utilizing competitive flow cytometry and cell microscopy to assess the binding of diverse chalcones and analogues by their capacity to inhibit HAB probe binding.
• Analyzing the formation of yellow-emitting fluorescent complexes by HAB upon association with biological targets.

Main Results:

• The HAB probe successfully imaged intracellular targets in Leishmania, including acidocalcisomes, lysosomes, and mitochondria, forming yellow fluorescent complexes.
• Competitive assays indicated that certain chalcones competitively inhibited HAB binding (20-30%), while non-chalconic compounds showed minimal inhibition (3-5%).
• The study identified limitations including the toxicity of competing chalcones at high concentrations and the limited sensitivity of the HAB fluorophore.

Conclusions:

• The HAB probe serves as a valuable tool for studying chalcone pharmacology in Leishmania, reflecting general chalcone binding patterns.
• Further optimization of reversible fluorescent probes is needed to overcome toxicity and sensitivity limitations for improved indirect target detection strategies.
• This research provides insights into chalcone-parasite interactions, paving the way for more effective antileishmanial drug development.