Inhibitors of Leishmania GDP-mannose pyrophosphorylase identified by high-throughput screening of small-molecule chemical library

Abstract

The current treatment for leishmaniasis is based on chemotherapy, which relies on a handful of drugs with serious limitations, such as high cost, toxicity, and a lack of efficacy in regions of endemicity. Therefore, the development of new, effective, and affordable antileishmanial drugs is a global health priority. Leishmania synthesizes a range of mannose-rich glycoconjugates that are essential for parasite virulence and survival. A prerequisite for glycoconjugate biosynthesis is the conversion of monosaccharides to the activated mannose donor, GDP-mannose, the product of a reaction catalyzed by GDP-mannose pyrophosphorylase (GDP-MP). The deletion of the gene encoding GDP-MP in Leishmania led to a total loss of virulence, indicating that the enzyme is an ideal drug target. We developed a phosphate sensor-based high-throughput screening assay to quantify the activity of GDP-MP and screened a library containing approximately 80,000 lead-like compounds for GDP-MP inhibitors. On the basis of their GDP-MP inhibitory properties and chemical structures, the activities of 20 compounds which were not toxic to mammalian cells were tested against ex vivo amastigotes and in macrophage amastigote assays. The most potent compound identified in the primary screen (compound 3), a quinoline derivative, demonstrated dose-dependent activity in both assays (50% inhibitory concentration = 21.9 microM in the macrophage assay) and was shown to be nontoxic to human fibroblasts. In order to elucidate signs of an early structure-activity relationship (SAR) for this class of compounds, we obtained and tested analogues of compound 3 and undertook limited medicinal chemistry optimization, which included the use of a number of SAR probes of the piperazinyl aryl substituent of compound 3. We have identified novel candidate compounds for the design and synthesis of antileishmanial therapeutics.

FIG. 1.

High-throughput screening enzymatic cascade. The assay ultimately detected the production of inorganic phosphate. Inorganic pyrophosphatase was added to the reaction mixture in excess, to minimize the chance of the identification of inhibitors of this enzyme.

FIG. 2.

Determination of the K_m values. The K_m for M-1-P (A) was determined to be 4.6 μM and the K_m for GTP (B) was determined to be 1.5 μM, as indicated by the lines.

FIG. 3.

Average control values for compound plates in chronological order of plates screened. (A) Average positive (squares) and negative (triangles) controls; (B) calculated Z′ values for individual library compound plates screened. A Z′ value of >0.4 was considered acceptable.

FIG. 4.

Structures of compound focus sets discussed herein. Three distinct classes can be discerned: 4-pyrizinylquinolines (A), thiadiazole-like (B), and pyrazolin-3,5-diones (C). Me, methyl; Et, ethyl.

FIG. 5.

Treatment of L. major-infected, mouse bone marrow-derived macrophages with GDP-MP inhibitors. (A and D) Infected macrophages were treated for 48 h with 30 μM test compounds. Data are plotted for seven compounds (two additional compounds tested had adverse effects on macrophages [see the Results section] and were not included); (B and E) infected macrophages were treated with various concentrations of compound 3 and its structural analogues; (C and F) infected macrophages were treated with different concentrations of compound 5; amphotericin B (AmpB) was used as a reference compound. Macrophages were infected with promastigotes (A to C) and amastigotes (D to F). The number of macrophages infected with at least one amastigote was determined by microscopy of fixed specimens, in duplicate. The assays were performed at least twice, in duplicate. Error bars indicate the standard errors of the means. *, statistically significant difference; +ve, positive.