Identification of Novel Flavonoids and Ansa-Macrolides with Activities against Leishmania donovani through Natural Product Library Screening

Abstract

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis (VL), a potentially fatal disease if left untreated. Given the limitations of current therapies, there is an urgent need for new, safe, and effective drugs. To discover novel antileishmanial compounds from previously unexplored chemical spaces, we conducted a high-throughput screening (HTS) of 2562 natural compounds, assessing their activity against L. donovani promastigotes and intracellular amastigotes. Utilizing the criteria of ≥70% parasite growth inhibition and ≥70% host cell (THP-1) viability, we selected 100 inhibitors for half-maximal inhibitory concentration (IC₅₀) value determination. Twenty-six compounds showed activities in both forms of Leishmania with a selectivity index of over 3. Clustering analysis resulted in four chemical clusters with scaffolds of lycorine (cluster 1), 5-hydroxy-9,10-dihydro-4H,8H-pyrano[2,3-f]chromene-4,8-dione (cluster 2), and semi-synthetic derivatives of ansamycin macrolide (cluster 4). The enantiomer of lycorine, BMD-NP-00820, showed the highest anti-amastigote activity with an IC₅₀ value of 1.74 ± 0.27 μM and a selectivity index (SI) > 29. In cluster 3, the most potent compound had an IC₅₀ value of 2.20 ± 0.29 μM with an SI > 23, whereas in cluster 4, with compounds structurally similar to the tuberculosis drug rifapentine, BMD-NP-02085 had an IC₅₀ value of 1.76 ± 0.28 μM, but the SI value was 7.5. Taken together, the natural products identified from this study are a potential source for the discovery of antileishmanial chemotypes for further development.

Keywords: Leishmania; ansa-macrolide; flavonoid; high-throughput screening; natural product.

Figure 1

High−throughput screening (HTS) of a natural-product-based library against L. donovani and profiling of activities by clusters. (A) HTS of a 2562-natural-compound library against L. donovani promastigotes with Z′ factor. (B) Anti−amastigote versus host cell (THP−1) viability bi−plot of HTS, resulting in the intracellular Leishmania model. (C) Plotting of anti-amastigote activities (pIC₅₀) with the selectivity index. (D) Plotting of pIC₅₀ values of compounds from intracellular L. donovani versus L. donovani promastigote assays.

Figure 2

Chemical structure of compounds from clusters 1 and 2 and in vitro antileishmanial activity. (A) Chemical structure of lycorine and its enantiomer, cluster 1. (B) Chemical structure of cluster 2. (C) Dose–response curve (DRC) of BMD−NP−00820, BMD−NP−01562, and miltefosine in the promastigote assay and in the intracellular Leishmania assay (D). (E) Representative fluorescence image of DAPI−stained intracellular amastigotes treated with DMSO, miltefosine, or BMD−NP−00820.

Figure 3

Chemical structure of compounds from cluster 3 and in vitro antileishmanial activity. (A) The structure of the chemical scaffold, 5−hydroxy−9,10−dihydro−4H,8H−pyrano[2,3−f]chromene−4,8−dione, of cluster 3. (B) The structure of representative compounds from cluster 3. (C) Dose–response curve (DRC) of BMD−NP−01555, BMD−NP−01558, and BMD−NP−01559 in the promastigote assay and in the intracellular Leishmania assay (D).

Figure 4

Chemical structure of compounds from cluster 4, ansa−macrolides, and in vitro antileishmanial activity. (A) The structure of the chemical scaffold of identified ansa−macrolides. (B) The structure of representative compounds from cluster 4. (C) The chemical structure of rifampentine. (D) DRC of BMD−NP−02085, BMD−NP−02084, and BMD−NP−02070 in the promastigote assay and in the intracellular Leishmania assay (E).