Plasmodium falciparum asexual blood-stage (ABS) assay

Plasmodium falciparum 3D7 cells were used as a target strain for the assay. Mefloquine (Sigma-Aldrich) was used as a standard inhibitor. At day 1, 250 ml of 10 mM mefloquine was added to columns 12 and 24 of the sterile 384-well black, clear-bottom, cell culture assay plates followed by compound curves which were added using Echo. The P. falciparum 3D7 cells were counted and 20 ml of culture was prepared at 5% haematocrit, 0.3% parasitaemia (as one batch, i.e., 12 plates, prepare 260 ml, to allow for WellMate prime volume) for each plate. From this, 50 µl of culture was added to all wells on all plates using WellMate with small bore tubing on full (S-1) speed. The plates were placed on the bottom shelf of the incubator, with a maximum stack height of 4 plates, preferring the front of the shelf and were incubated for 72 h at 37 °C in an atmosphere of special gas mix (1% O₂, 3% CO₂, balance N₂).

At day 4, SybrGreen/lysis buffer was prepared by diluting defrosted SybrGreen aliquots as required (20 μl of 10,000 × req. for 12 plates) to 3× with lysis buffer (70 ml for 12 plates). Ten μl of the prepared buffer was added to each well on each of the assay plates and incubated overnight in the dark at room temperature. The plates were read on Victor plate reader using ‘384sybrgreen’ protocol (excitation 485 nm, emission 528 nm) after which the plate contents were aspirated into 5% Virkon, and disposed of after autoclaving. Percentage inhibition for each test compound was calculated using the following equation:

EC₅₀ values for standard inhibitors were determined using non-linear regression curve-fitting within Activity Base data analysis template.

FNDR-20123 was assessed in male and female gametocyte functional viability assay, as reported by Ruecker et al. [22]. Briefly, gametocyte cultures were seeded at 1% rings and 4% haematocrit under 3% O₂, 5% CO₂, 92% N₂ gas by using an asexual culture with 3% ring stages at day 0. Gametocyte cultures were tested for functional viability and maturity after 14 days. Testing functional viability was done by quantifying male gametocyte formation, which was carried out by withdrawing 200 μl of culture. Following this, culture was centrifuged, and the pellet was resuspended in 5 μl ookinete medium (RPMI medium with 25 mM HEPES, 50 mg/l hypoxanthine, 2 g/l sodium bicarbonate, 100 μM xanthurenic acid, and 10% A⁺ human serum). The culture was observed under a microscope. After validating maturity and upon exflagellation centres being > 50 per field, the gametocyte culture was resuspended in 7.5 ml complete medium. From this, 50 μl was dispersed into previously prepared 96-well plate (containing complete culture medium and FNDR-20123 at a concentration of 1 μM and prewarmed at 37 °C for 20 min). The plates were then incubated at 37 °C for 24 h. Gamete formation was induced on day 15 and observed under the microscope following the method described by Ruecker et al. [23]. The assay was performed using four independent biological replicates.