A simple, robust, and affordable bioluminescent assay for drug discovery against infective African trypanosomes

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.

Iboga Inspired N-Indolylethyl-Substituted Isoquinuclidines as a Bioactive Scaffold: Chemoenzymatic Synthesis and Characterization as GDNF Releasers and Antitrypanosoma Agents

“…As part of our search for candidate compounds to develop novel antiparasitic drugs, the biological activity against bloodstream T. brucei brucei was investigated for compounds 6 , 7 , 10 exo , 11 , 13 , 14 , and 15 [ 33 ]. In order to estimate the biological selectivity, the cytotoxicity towards murine macrophages (cell line J774) was tested for the most active compounds (i.e., EC50 vs. T. brucei <10 μM).…”

“…The anti-trypanosomal activity of compounds was evaluated against the bioluminescent cell line of the bloodstream stage of Trypanosoma brucei brucei (called LUC cell line) as previously described [ 33 ]. Briefly, to a 96-well culture plate containing 2.2 μL/well DMSO (negative control) or compounds dissolved in DMSO (1% final concentration), 220 μL/well of a suspension of 1 × 10 5 parasites/mL was added.…”

Glucose 6-Phosphate Dehydrogenase from Trypanosomes: Selectivity for Steroids and Chemical Validation in Bloodstream Trypanosoma brucei

“…Bloodstream T. b. brucei (strain 427- cell line 514-1313) constitutively expressing an ectopic copy of the luminescent red-shifted biosensor LUC [ 72 ] was grown in HMI-9 medium complemented with 10% ( v / v ) Fetal Bovine Serum Tetracycline-free (FBS; GIBCO ® , Thermo-Fisher Scientific, Waltham, MA, USA) in a humidified incubator with 5% CO 2 and at 37 °C. Parasite viability was performed as described in [ 72 ].…”

“…Bloodstream T. b. brucei (strain 427- cell line 514-1313) constitutively expressing an ectopic copy of the luminescent red-shifted biosensor LUC [ 72 ] was grown in HMI-9 medium complemented with 10% ( v / v ) Fetal Bovine Serum Tetracycline-free (FBS; GIBCO ® , Thermo-Fisher Scientific, Waltham, MA, USA) in a humidified incubator with 5% CO 2 and at 37 °C. Parasite viability was performed as described in [ 72 ]. Briefly, 1 × 10 5 parasites/mL in a mid-exponential growth phase were seeded in a 96 well-plate (200 µL of the cell suspension/well) containing 2 µL of steroid derivatives at different concentrations (range: 0.035–50 µM), and incubated over 24 h. Thereafter, 200 µL from each well were transferred to a 96-well black plate and incubated with 20 µL of a solution containing: 1.5 mg/mL luciferin in PBS glucose 1% ( w / v ) and Triton X-100.…”

Past and future of trypanosomatids high-throughput phenotypic screening

“… 35 , 36 , 37 The number of parasites has also been indirectly obtained by SYBR Green, 38 , 39 a fluorescent nuclear probe. Some assays use transgenic parasites carrying fluorescent reporter genes e.g., GFP 40 , 41 , 42 and mCherry 43 or reporter enzymes e.g., beta-galactosidase, 41 , 44 , 45 , 46 beta-lactamase, 47 , 48 luciferase 49 , 50 , 51 , 52 whose activities can be readily detected in the presence of their substrates.…”