A simple, robust, and affordable bioluminescent assay for drug discovery against infective African trypanosomes. Issue 2 (20th January 2020)

• Record Type: Journal Article
• Authors: Benítez, Diego; Dibello, Estefania; Bonilla, Mariana; Comini, Marcelo A.
• Other Names: Monte‐Neto Rubens L. guestEditor.; Fernandez‐Prada Christopher guestEditor.; Moretti Nilmar S. guestEditor.
• Is Part Of: Drug development research. Volume 83:Issue 2(2022)
• Page Start: 253
• Page End: 263
• Publication Date: 2020-01-20
• Subjects: African trypanosomiasis -- high‐throughput screening -- luciferase -- Trypanosoma brucei, genetically encoded biosensor; Drug development -- Periodicals; Drugs -- Research -- Periodicals; 615.19
• DOI: 10.1002/ddr.21634
• Languages: English
• ISSNs: 0272-4391

Abstract

Abstract: African trypanosomiasis is a major problem for human and animal health in endemic countries, where it threatens millions of people and affects economic development. New drugs are needed to overcome the toxicity, administration, low efficacy, and resistance issues of the current chemotherapy. Robust, simple, and economical high‐throughput, whole‐cell‐based assays are required to accelerate the identification of novel chemical entities. With this aim, we generated a bioluminescent cell line of the bloodstream stage of Trypanosoma brucei brucei and established a screening assay. Trypanosomes were stably transfected to constitutively express a thermostable red‐shifted luciferase. The growth phenotype and drug sensitivity of the reporter cell line were essentially identical to that of the parental cell line. The endogenous luciferase activity, measured by a simple bioluminescence assay, proved to be proportional to parasite number and metabolic status. The assay, optimized to detect highly potent compounds in a 96‐well‐plate format, was validated by screening a small compound library (inter‐assay values for Z' factor and coefficient variation were 0.77 and 5.8%, respectively). With a hit‐confirmation ratio of ~97%, the assay was potent enough to identify several hits with EC50 ≤ 10 μM. Preliminary tests indicated that the assay can be scaled up to a 384‐well‐plate format without compromising its robustness. In summary, we have generated reporter trypanosomes and a simple, robust, and affordable bioluminescence screening assay with great potential to speed up the early‐phase drug discovery against African trypanosomes.