Dinitroanilines bind alpha-tubulin to disrupt microtubules.
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 alpha-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 alpha-tubulin. Docking studies predict that oryzalin binds with an average affinity of 23 nM to a site located beneath the N loop of Toxoplasma alpha-tubulin. This binding site included residues that were mutated in several resistant lines. Moreover, parallel analysis of Bos taurus alpha-tubulin indicated that oryzalin did not interact with this site and had a significantly decreased, nonspecific affinity for vertebrate alpha-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 alpha-tubulin function.
Morrissette
Naomi S
NS
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. naomi@borcim.wustl.edu
Mitra
Arpita
A
Sept
David
D
Sibley
L David
LD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003049:Coccidiostats / Q000494:pharmacology*; D004136:Dinitrobenzenes / Q000494:pharmacology*; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013424:Sulfanilamides; D014122:Toxoplasma / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003049:Coccidiostats; D004136:Dinitrobenzenes; D013424:Sulfanilamides; D014404:Tubulin; C012465:oryzalin