Dinitroanilines Bind α-Tubulin to Disrupt Microtubules

Abstract

Protozoan parasites are remarkably sensitive to dinitroanilines such as oryzalin, which disrupt plant but not animal microtubules. To explore the basis of dinitroaniline action, we isolated 49 independent resistant Toxoplasma gondii lines after chemical mutagenesis. All 23 of the lines that we examined harbored single point mutations in ␣-tubulin. These point mutations were sufficient to confer resistance when transfected into wild-type parasites. Several mutations were in the M or N loops, which coordinate protofilament interactions in the microtubule, but most of the mutations were in the core of ␣-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma ␣-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus ␣-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate ␣-tubulin. We propose that the dinitroanilines act through a novel mechanism, by disrupting M-N loop contacts. These compounds also represent the first class of drugs that act on ␣-tubulin function. INTRODUCTIONMicrotubules are polymers constructed from ␣-␤-tubulin heterodimers (Downing and Nogales, 1998a,b). These structures are rapidly assembled and disassembled to create essential components of eukaryotic cells, such as spindles and flagella. The dynamic nature of microtubules makes them susceptible to pharmacological agents. Microtubule-disrupting and microtubule-stabilizing drugs have provided great insight into tubulin and microtubule function; they also have tremendous practical use. Compounds that perturb microtubule dynamics are currently some of the most effective drugs to treat medical conditions, including cancer, gout, and helminth infection (Jordan et al., 1998). Dinitroanilines (oryzalin, ethafluralin, and trifluralin) disrupt the microtubules of plants, ranging from the single-celled alga Chlamydomonas reinhardtii to higher plants such as the monocot Eleusine indica (James et al., 1993;Anthony et al., 1998;Zeng and Baird, 1999). Dinitroanilines also disrupt the microtubules of protozoa, including both free-living species such as Tetrahymena and protozoan parasites such as Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum, and Toxoplasma gondii (Chan and Fong, 1990;Chan et al., 1991;Gu et al., 1995;Edlind et al., 1996;Stokkermans et al., 1996;Armson et al., 1999; Makioka et al., 2000a,b;Traub-Cseko et al., 2001). Remarkably, the activity of dinitroanilines is restricted to plants and protozoa; these compounds are ineffective against vertebrate or fungal microtubules (Chan and Fong, 1990;Hugdahl and Morejohn, 1993;Murthy et al., 1994;Edlind et al., 1996).T. gondii is a member of the Apicomplexa, a phylum of parasites that includes several medically and agriculturally significant pathogens (Black and Boothroyd, 2000). Apicomplexans are obligate intracell...

Cryoelectron tomography reveals periodic material at the inner side of subpellicular microtubules in apicomplexan parasites

“…Intact (or completely folded) microtubules containing the luminal densities were present in tomograms of Toxoplasma tachyzoites that were extracted at room temperature with 5% Triton for 30 min, followed by phosphate-buff ered saline adjusted to 2 M NaCl for 60 min before freezing (Fig. 4, C, D, F, and G), indicating that the uncommon microtubule topology is preserved in Apicomplexa and that these microtubules are exceptionally stable (4,5,13). When additionally treated with the microtubule depolymerizing drug oryzalin, microtubules in Toxoplasma extracts appeared shorter, and were occasionally ruptured.…”

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

“…(Dow et al, 2002;Werbovetz et al, 2003)), while GB-II-5 is approximately 5-fold more active against L. donovani axenic amastigotes and about 60-fold more active against T. brucei bloodstream forms than against intraerythrocytic P. falciparum (K. Werbovetz, unpublished data). Experimental evidence indicates that the binding site for oryzalin is on α-tubulin (Anthony and Hussey, 1999;Morrissette et al, 2004). In the apicomplexan parasite Toxoplasma gondii, molecular simulations suggest that the oryzalin binding site consists of α-tubulin residues Arg2, Glu3, Val4, Trp21, Phe24, His28, Ile42, Asp47, Arg64, Cys65, Thr239, Arg243, and Phe244 (Morrissette et al, 2004).…”

“…This does not necessarily account for the susceptibility differences between kinetoplastid and apicomplexan tubulin to dinitroanilines, as the amino acid at position 42 differs between Plasmodium α-tubulin and all the other tubulins listed in Figure 3 and the amino acid at position 65 differs between the Leishmania and Trypanosoma α-tubulin. Morrissette et al have suggested that amino acid differences at positions other than the binding site causing allosteric effects are responsible for the selectivity of dinitroanilines for plant and protozoal tubulin (Morrissette et al, 2004). Further structure-activity studies and molecular modeling data are required to develop a binding site model for the dinitroaniline sulfanilamides on kinetoplastid tubulin that will facilitate the design of improved ligands for this important drug target.…”

Resistência de Eleusine indica aos inibidores de ACCase

“…Vale ressaltar que essa espécie possui o biótipo com o único caso conhecido de gene recessivo conferindo resistência aos herbicidas inibidores de mitose (Morrissette et al, 2004). Um biótipo coletado na Malásia possui resistência múl-tipla aos inibidores de ACCase e de EPSPS (Heap, 2005).…”