Abstract
In recent years, the neglected diseases drug discovery community has elected phenotypic screening as the key approach for the identification of novel hit compounds. However, when this approach is applied, important questions related to the mode of action for these compounds remain unanswered. One of such questions is related to the rate of action, a useful piece of information when facing the challenge of prioritising the most promising hit compounds. In the present work, compounds of the “Leishmania donovani box” were evaluated using a rate of action assay adapted from a replicative intracellular high content assay recently developed. The potency of each compound was determined every 24 hours up to 96 hours, and standard drugs amphotericin B and miltefosine were used as references to group these compounds according to their rate of action. Independently of this biological assessment, compounds were also clustered according to their minimal chemical scaffold. Comparison of the results showed a complete correlation between the chemical scaffold and the biological group for the vast majority of compounds, demonstrating how the assay was able to bring information on the rate of action for each chemical series, a property directly linked to the mode of action. Overall, the assay here described permitted us to evaluate the rate of action of the “Leishmania donovani box” using two of the currently available drugs as references and, also, to propose a number of fast-acting chemical scaffolds present in the box as starting points for future drug discovery projects to the wider scientific community. The results here presented validate the use of this assay for the determination of the rate of action early in the discovery process, to assist in the prioritisation of hit compounds.
Highlights
Leishmania parasites are causative agents of different pathologies, ranging from self-curing but disfiguring cutaneous leishmaniasis (CL) to visceral leishmaniasis (VL), mainly caused by L. donovani species in humans and potentially lethal if untreated
The mean pEC50 values (pEC50 = -log EC50 (M)) at each time point are shown in the supplementary material (S1 Table for amastigotes per macrophage (AM/MAC) output and S2 Table for infected cells (INF) output)
Amphotericin B reached its maximum effect in both outputs between 24 and 48 hours, while miltefosine reached its maximum effect clearly later than 48 hours
Summary
Leishmania parasites are causative agents of different pathologies, ranging from self-curing but disfiguring cutaneous leishmaniasis (CL) to visceral leishmaniasis (VL), mainly caused by L. donovani species in humans and potentially lethal if untreated. Amphotericin B showed successful cure rates after a single-dose administration [2]. It requires intravenous administration, patient hospitalization and unbroken cold chain that, together with the high cost of the liposomal formulation, make it far from ideal for usage in endemic countries [3]. For the last 25 years, drug discovery focused primarily on target-based approaches [5], that rely on the ability of a drug to exert its effect through inhibition or interference with one or more essential targets or pathways These approaches always require an important initial effort for the identification of the right targets, as their success is dependent on target essentiality and druggability [6]. They are even more challenging for the kinetoplastids area due to the small number of drug targets fully validated in the field [7,8], and to the lack of knowledge about mode of action for currently available drugs, which potency seems to reflect the inhibition of multiple targets or pathways [9]
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