A target-based high throughput screen yields Trypanosoma brucei hexokinase small molecule inhibitors with antiparasitic activity

Abstract

Background: The parasitic protozoan Trypanosoma brucei utilizes glycolysis exclusively for ATP production during infection of the mammalian host. The first step in this metabolic pathway is mediated by hexokinase (TbHK), an enzyme essential to the parasite that transfers the gamma-phospho of ATP to a hexose. Here we describe the identification and confirmation of novel small molecule inhibitors of bacterially expressed TbHK1, one of two TbHKs expressed by T. brucei, using a high throughput screening assay.

Methodology/principal findings: Exploiting optimized high throughput screening assay procedures, we interrogated 220,233 unique compounds and identified 239 active compounds from which ten small molecules were further characterized. Computation chemical cluster analyses indicated that six compounds were structurally related while the remaining four compounds were classified as unrelated or singletons. All ten compounds were approximately 20-17,000-fold more potent than lonidamine, a previously identified TbHK1 inhibitor. Seven compounds inhibited T. brucei blood stage form parasite growth (0.03<or=EC(50)<3 microM) with parasite specificity of the compounds being demonstrated using insect stage T. brucei parasites, Leishmania promastigotes, and mammalian cell lines. Analysis of two structurally related compounds, ebselen and SID 17387000, revealed that both were mixed inhibitors of TbHK1 with respect to ATP. Additionally, both compounds inhibited parasite lysate-derived HK activity. None of the compounds displayed structural similarity to known hexokinase inhibitors or human African trypanosomiasis therapeutics.

Conclusions/significance: The novel chemotypes identified here could represent leads for future therapeutic development against the African trypanosome.

Figure 1. Validation of the HTS by LOPAC screening.

Plot of percent inhibition for duplicate screen of the 1280 LOPAC compounds. LOPAC compounds (blue), minimum control, which should equal ∼100% inhibition of signal readout (red), IC₅₀ control compounds (yellow), and maximum control compounds, which should equal ∼0% inhibition of signal readout (green), are indicated.

Figure 2. Scheme depicting HTS interrogation of a 220,233 small molecule library for TbHK1 inhibitors.

Figure 3. Structures of the cluster 1 and singleton HTS hits.

Figure 4. Ebselen and SID 17387000 are mixed inhibitors of TbHK1 with respect to ATP.

Lineweaver-Burk plots of inhibition with ebselen (A.) or SID 17387000 (B.). Assays were performed as described for cell lystates (see Materials and Methods) with ATP concentrations varied.

Figure 5. Ebselen and SID 17387000 inhibit TbHK activity from parasite cell lysate and cause a reduction in cellular G6P levels in BSF parasites.

Increasing amounts of (A.) ebselen or (B.) SID 17387000 were incubated with 2×10⁵ BSF cell equivalents for 15 min at RT and HK assays were performed as described in the Materials and Methods. (C.) Growth in the presence of Ebselen or SID 17387000 causes a reduction in cellular G6P levels. BSF parasites (1×10⁷) were cultured for 1 or 4 hours in the presence of 30 µM or 1 µM (10-fold the EC₅₀) ebselen or SID 17387000 followed by lysate preparation and comparison of G6P levels to an equivalent number of untreated parasites .