Specific Targeting of Plant and Apicomplexa Parasite Tubulin through Differential Screening Using In Silico and Assay-Based Approaches

Abstract

Dinitroanilines are chemical compounds with high selectivity for plant cell α-tubulin in which they promote microtubule depolymerization. They target α-tubulin regions that have diverged over evolution and show no effect on non-photosynthetic eukaryotes. Hence, they have been used as herbicides over decades. Interestingly, dinitroanilines proved active on microtubules of eukaryotes deriving from photosynthetic ancestors such as Toxoplasma gondii and Plasmodium falciparum, which are responsible for toxoplasmosis and malaria, respectively. By combining differential in silico screening of virtual chemical libraries on Arabidopsis thaliana and mammal tubulin structural models together with cell-based screening of chemical libraries, we have identified dinitroaniline related and non-related compounds. They inhibit plant, but not mammalian tubulin assembly in vitro, and accordingly arrest A. thaliana development. In addition, these compounds exhibit a moderate cytotoxic activity towards T. gondii and P. falciparum. These results highlight the potential of novel herbicidal scaffolds in the design of urgently needed anti-parasitic drugs.

Keywords: Plasmodium falciparum; Toxoplasma gondii; Tubulin; cell-based assays; dinitroanilines; plant cells; small molecules; virtual screening.

Figure 1

Determination of the best P. falciparum. α-tubulin conformations for in-silico screening. (A) Conformations of the H1-S2 loop (residues 35–60) of P. falciparum α-tubulin, showing the overlap of the initial homology model based on homology predictions to mammalian tubulin (cyan) and lowest-energy S4MPLE (Sampler for Multiple Protein-Ligand Entities)-generated open-site geometry model, which takes into account the dinitroaniline binding ability (orange). The residues Ser6, Ile235 and Leu167 in the dinitroaniline binding site of the P. falciparum tubulin that were found involved in interactions for every conformation and used as criteria for selection are coloured in red, and their immediate neighbourhood (within 4Å) is in green. The loop in the homology model—in cyan—is seen to block access to the site. When displaced to allow dinitroaniline binding ability the predicted new position (in orange) opens access to the three key residues. (B) View of the “red” key residues in a protein surface model, showing that they (and their immediate neighborhood, in green) are buried in the homology model (upper panel) but accessible in the sampled conformer model (lower panel).

Figure 2

Effect of selected compounds on plant cell microtubule network organization. BY2 cells were incubated for 2 h with 0.25% DMSO (vehicle control); 25 µM dinitroaniline, 2 µM colchicine and 50 µM of the indicated compounds. Cells were then processed for tubulin immunofluorescence as described in Material and Methods. CM539 is an example of a compound that was found inactive. Bar = 20 µm.

Figure 3

Effect of selected compounds on mammalian cell microtubule network organization. HeLa cells were incubated for 2 h with 0.25% DMSO (vehicle control); 25 µM dinitroaniline, 2 µM colchicine and 50 µM of the indicated compounds. Cells were then processed for tubulin immunofluorescence as described in Material and Methods. Bar = 20 µm.

Figure 4

Comparison of the effects of CM094, CM571 and CM852 on mammalian and plant microtubule polymerization in vitro. (A) Pure bovine brain tubulin polymerization assay. Tubulin was allowed to polymerize at 37 °C. Fluorescence of DAPI bound to microtubules was measured to monitor microtubule polymerization, as described in the Material and Methods section. Experiments were performed in triplicate, in the presence of the indicated compounds. Results are presented as mean ± standard error of the mean (SEM). Effect of dinitroaniline (B), CM571 (C), CM852 (D), and CM094 (E) on soybean tubulin assembly. Recombinant soybean tubulin was polymerized as described in the Material and Methods section, in the presence of DMSO (control) or the indicated compounds.

Figure 5

Structure of the selected compounds.

Figure 6

Effect of selected compounds on T. gondii and P. falciparum. (A) The effect of the compounds on T. gondii replication within human fibroblasts was tested. The graph presents T. gondii growth inhibition curves for each treatment at concentrations ranging from 0.01 to 100 µM, in triplicates. The quantity of individual parasites within intracellular vacuoles exposed or not to the drug for 30 h post-invasion was automatically scored. The calculated IC₅₀ is indicated for each compound. (B) Plaque assays showing at 4 and 7 days the expansion of HFF cleared zones due to successive rounds of lytic cycle that were initiated by a single parasite. HFF monolayers plated on a 6-well plate were inoculated with 200 invasive tachyzoites per well for 1 h. The non-invading tachyzoites were washed away and cells were incubated with medium containing the vehicle (DMSO, 0) or different concentrations of CM094. After fixation and Crystal violet staining, cells were air dried and scanned before image processing and plaque area measurement. At day 7, post-inoculation, a highly significant reduction of parasite expansion was observed for the cultures exposed to 15 µM of CM094 and no plaque was detected at 30 µM while the HFF monolayer was well preserved. (C) Histograms showing the cumulated area of HFF cleared zones. p value = 0.0082. (D) The effect of selected compounds on P. falciparum intra-erythrocytic growth was tested. The growth of parasites within human red blood cells is determined using a classical SYBR Green assay followed by quantification of parasite DNA fluorescence. The graph presents growth curves for concentrations of each compounds ranging from 0 to 250 µM, in triplicates.