An on-flow high throughput assay to screen Leishmania donovani Nucleoside hydrolase’ inhibitors

Acting in the formation of the parasite’s DNA and RNA, the enzyme Nucleoside hydrolase (NH) plays an essential role for the protozoa survival.1 Leishmania donovani is the causative agent of visceral leishmaniasis, a lethal form of this disease that remains with few options of treatment. Therefore, it is crucial to continually search for potential inhibitors. This work proposes a high throughput assay by the direct and automated on-flow quantification of the formed hypoxanthine using a HPLC system, as shown in Figure 1. Firstly, LdNH was covalently immobilized on magnetic particles (MPs). After that, using a syringe pump, the resulting LdNH-MPs were trapped in a PTFE tube (10 cm) coupled to a magnets sequence in a quadrupole arrangement (4 units of 1 × 1cm), producing the LdNH microreactor. The following step was to optimize chromatographic conditions to separate substrate (inosine)/product (hypoxanthine). Chromatography separation of inosine and hypoxanthine was achieved by using an octyl column (Eclipse XBD C18 Agilent 5 µm, 15 × 0.46 cm) and, as the mobile phase in pump B, a solution containing triethylamine (1% in water, v/v, acidified with AcOH pH 6.0):MeOH (95:5), at flow rate 0.8 mL.min-1. For the microreactor, PBS (20 mM, pH 7.4, 300 mM NaCl) was used as mobile at a flow rate of 0.02 mL.min-1.
The developed method was validated, yielding the analytical curve (y = 16061x – 3497.3, R2 = 1). Kinetic studies furnished the Michaelis constant (KM) for the inosine substrate (2079.2 ± 87.1 µM). To validate this on-flow assay for screening purposes, a novel oxoquinoline ribonucleoside, named 17a, was evaluated. LdNH microreactor showed a concentration dependent response to this compound, with an IC50 of 42.3 ± 3.1 µM. Therefore, the proposed on-flow assay can be applied in inhibitors screening.