Live-imaging rate-of-kill compound profiling for Chagas disease drug discovery with a new automated high-content assay

Abstract

Chagas disease, caused by the protozoan intracellular parasite Trypanosoma cruzi, is a highly neglected tropical disease, causing significant morbidity and mortality in central and south America. Current treatments are inadequate, and recent clinical trials of drugs inhibiting CYP51 have failed, exposing a lack of understanding of how to translate laboratory findings to the clinic. Following these failures many new model systems have been developed, both in vitro and in vivo, that provide improved understanding of the causes for clinical trial failures. Amongst these are in vitro rate-of-kill (RoK) assays that reveal how fast compounds kill intracellular parasites. Such assays have shown clear distinctions between the compounds that failed in clinical trials and the standard of care. However, the published RoK assays have some key drawbacks, including low time-resolution and inability to track the same cell population over time. Here, we present a new, live-imaging RoK assay for intracellular T. cruzi that overcomes these issues. We show that the assay is highly reproducible and report high time-resolution RoK data for key clinical compounds as well as new chemical entities. The data generated by this assay allow fast acting compounds to be prioritised for progression, the fate of individual parasites to be tracked, shifts of mode-of-action within series to be monitored, better PKPD modelling and selection of suitable partners for combination therapy.

Temporal and Wash-Out Studies Identify Medicines for Malaria Venture Pathogen Box Compounds with Fast-Acting Activity against Both Trypanosoma cruzi and Trypanosoma brucei

“…gambiense [ 42 ] and has shown 100% cure of mice infected with T. cruzi when treated for 40 days [ 4 ]. It has recently been shown, using live-cell imaging, that SCYX-6759 demonstrated a 20-h lag phase of inhibition following exposure of T. cruzi infected H9C2 rat heart cells to 16 µM of compound, suggesting a slow MOA [ 46 ]. However, the study did not state the IC 50 value, nor the EC 100 .…”

“…However, a fast-acting and long-lasting compound is recommended by DND i as optimal for Chagas disease, as it is questionable whether a slow-acting compound is desirable due to the clinical failure of slow-acting azoles [ 25 ]. Thus, fast-acting compounds are often prioritised in drug discovery campaigns [ 46 ]. Since the fast- and slow-activity compounds identified herein showed activity against T. cruzi and T.b.…”

State-of-the-Art in the Drug Discovery Pathway for Chagas Disease: A Framework for Drug Development and Target Validation

“…20,56 HCS assays designed to determine whether the parasites simply prevent parasite replication (static effect) or induce a reduction in the number of amastigotes (cidal effect), as well as the time required to produce such effect (rate-of-kill), have also been described and represent increasingly important components of drug discovery cascades. [56][57][58] In addition to addressing the compounds toxicity on the host cells supporting parasite replication, other cell toxicity assays are usually included in the drug discovery cascade. A common model uses the human hepatocarcinoma cell line HepG2, since damage to this line suggests liver toxicity.…”

Paving the Way: Contributions of Big Data to Apicomplexan and Kinetoplastid Research

“…In the context of Chagas disease, high-content imaging has been used to study host-cell infection rates and drug screening ( Alonso-Padilla and Rodríguez, 2014 ; Alonso-Padilla et al., 2015 ; Sykes and Avery, 2015 ; Franco et al., 2019 ; Fesser et al., 2020 ; Portella et al., 2021 ; Svensen et al., 2021 ). For T. brucei , high-content imaging has been used for the measurement of transcriptional activity ( Hiraiwa et al., 2018 ).…”