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Updated: May 19, 2026
In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing β-galactosidase
Published on: November 5, 2021
Diverse inhibitor chemotypes targeting Trypanosoma cruzi CYP51
Shamila S Gunatilleke1, Claudia M Calvet, Jonathan B Johnston
1Sandler Center for Drug Discovery, University of California San Francisco, San Francisco, California, United States of America.
Insights
New drug discovery efforts for Chagas Disease are a priority. Researchers screened over 100,000 compounds, identifying potent inhibitors of the Trypanosoma cruzi CYP51 enzyme, a promising target for new Chagas Disease treatments.
Area of Science:
- Medicinal Chemistry
- Parasitology
- Drug Discovery
Background:
- Chagas Disease is a neglected tropical disease causing significant morbidity and mortality.
- Limited pharmaceutical interest exists due to economic factors, necessitating new therapeutic strategies.
Purpose of the Study:
- To identify novel drug candidates for Chagas Disease by targeting the Trypanosoma cruzi CYP51 enzyme.
- To diversify the anti-Chagasic drug pipeline through direct probing of the T. cruzi CYP51 active site.
Main Methods:
- High-throughput screening of approximately 104,000 synthetic small molecules against T. cruzi CYP51.
- Target-based screening yielded 185 hits with nanomolar K(D) values.
- Cell-based assays on T. cruzi-infected cells identified 57 active compounds with EC(50) <10 µM.
Main Results:
- A top-performing compound inhibited T. cruzi with an EC(50) of 17 nM and was trypanocidal at 40 nM.
- The screening identified structurally diverse small molecules targeting T. cruzi CYP51.
- The study identified 57 active hits with potential therapeutic value.
Conclusions:
- The T. cruzi CYP51 enzyme is a permissive target for small molecule inhibitors.
- Cheminformatic analysis suggests similarities between CYP51 pharmacology and other cytochrome P450 targets.
- Existing lead compounds for other therapeutic targets may be explored for Chagas Disease treatment.
Background:
Chagas Disease, a WHO- and NIH-designated neglected tropical disease, is endemic in Latin America and an emerging infection in North America and Europe as a result of population moves. Although a major cause of morbidity and mortality due to heart failure, as well as inflicting a heavy economic burden in affected regions, Chagas Disease elicits scant notice from the pharmaceutical industry because of adverse economic incentives. The discovery and development of new routes to chemotherapy for Chagas Disease is a clear priority.
Methodology/Principal Findings:
The similarity between the membrane sterol requirements of pathogenic fungi and those of the parasitic protozoon Trypanosoma cruzi, the causative agent of Chagas human cardiopathy, has led to repurposing anti-fungal azole inhibitors of sterol 14α-demethylase (CYP51) for the treatment of Chagas Disease. To diversify the therapeutic pipeline of anti-Chagasic drug candidates we exploited an approach that included directly probing the T. cruzi CYP51 active site with a library of synthetic small molecules. Target-based high-throughput screening reduced the library of ∼104,000 small molecules to 185 hits with estimated nanomolar K(D) values, while cross-validation against T. cruzi-infected skeletal myoblast cells yielded 57 active hits with EC(50) <10 µM. Two pools of hits partially overlapped. The top hit inhibited T. cruzi with EC(50) of 17 nM and was trypanocidal at 40 nM.
Conclusions/Significance:
The hits are structurally diverse, demonstrating that CYP51 is a rather permissive enzyme target for small molecules. Cheminformatic analysis of the hits suggests that CYP51 pharmacology is similar to that of other cytochromes P450 therapeutic targets, including thromboxane synthase (CYP5), fatty acid ω-hydroxylases (CYP4), 17α-hydroxylase/17,20-lyase (CYP17) and aromatase (CYP19). Surprisingly, strong similarity is suggested to glutaminyl-peptide cyclotransferase, which is unrelated to CYP51 by sequence or structure. Lead compounds developed by pharmaceutical companies against these targets could also be explored for efficacy against T. cruzi.
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