Antileishmanial high-throughput drug screening reveals drug candidates with new scaffolds

Abstract

Drugs currently available for leishmaniasis treatment often show parasite resistance, highly toxic side effects and prohibitive costs commonly incompatible with patients from the tropical endemic countries. In this sense, there is an urgent need for new drugs as a treatment solution for this neglected disease. Here we show the development and implementation of an automated high-throughput viability screening assay for the discovery of new drugs against Leishmania. Assay validation was done with Leishmania promastigote forms, including the screening of 4,000 compounds with known pharmacological properties. In an attempt to find new compounds with leishmanicidal properties, 26,500 structurally diverse chemical compounds were screened. A cut-off of 70% growth inhibition in the primary screening led to the identification of 567 active compounds. Cellular toxicity and selectivity were responsible for the exclusion of 78% of the pre-selected compounds. The activity of the remaining 124 compounds was confirmed against the intramacrophagic amastigote form of the parasite. In vitro microsomal stability and cytochrome P450 (CYP) inhibition of the two most active compounds from this screening effort were assessed to obtain preliminary information on their metabolism in the host. The HTS approach employed here resulted in the discovery of two new antileishmanial compounds, bringing promising candidates to the leishmaniasis drug discovery pipeline.

Figure 1. HTS assay validation.

A) Linear correlation between relative fluorescence unit (RFU) readings from resazurin reduction (y-axis) and parasite number from microscopy counting (x-axis). B) Dose response curve and EC₅₀ (black line for L. major and grey dashed line for L. donovani) of the reference compounds used as controls: EtBr, amphotericin B, miltefosine and paromomycin. C) Distribution of 33 control microplates showing 1% DMSO as the negative control (blue dots), EtBr EC₅₀-30 nM (black dots) and EtBr EC₁₀₀-10 µM as positive controls (yellow dots) and the Z-factor of 0.62. D) Distribution plot of the duplicate assay screen of 4,000 compounds (red dots) and controls in three different concentrations, following the same color standards as in C.

Figure 2. Antileishmanial HTS with 26,500 compounds.

A) Distribution plot of the 26,500 compounds (red dots), 1% DMSO (blue dots), EC₁₀₀ (yellow dots) and Z-factor = 0.80. B) Frequency distribution of the 26,500 compounds based on binned RFUs, highlighting active compound selection with a black dashed box. Data for reference compound EC₁₀₀ in yellow, for reference compound EC₅₀ in black and for the 26,500 compounds in red. C) A funnel representing selection of antileishmanial activity from 26,500 compounds to 124 hit compounds after primary screening (antileishmanial activity against promastigotes), secondary screening (toxicity exclusion) and no redundancy against Mycobacterium tuberculosis or HIV screenings done in-house.

Figure 3. CH872 and CA272 antileishmanial activity against intracellular L. major amastigotes.

A) Infected THP-1 cells in the presence of 1% DMSO as a negative control (left) and EC₁₀₀ obtained with 10 µM of amphotericin B as a positive control (right). B) Structures of CH872 (left) and CA272 (right). C) Infected THP-1 cells in the presence of 1 nM of CH872 (left) and 1 nM of CA272 (right) as non-active concentrations of the compounds and D) in the presence of 0.7 µM of CH872 (left) and 10 µM of CA272 (right) as effective concentrations from the dose-response curves. E) Dose-response curves of the compounds CH872 and CA272 plotting the infection ratio (continuous lines) and relative number of parasites compared to the DMSO control (dashed lines).

Figure 4. CH872 and CA272 stability in the presence of liver microsomes.

Disappearance of CH872 and CA272 in the presence of human (lines) and rat (dashed lines) liver microsomes.

Figure 5. New quinoline with antileishmanial activity.

Unlike other quinoline derivatives reported as antileishmanial compounds, CH872 contains a 4-OH group that allows this compound to equilibrate with its tautomer (CH872A). 8-Cl can facilitate the tautomerization by making a H-bond with the NH in CH872A.