Dinitroanilines compounds inhibit tubulin polymerization in diverse plants and in protozoa but do not disrupt vertebrate or fungal microtubules. These compounds disrupt microtubules in protozoan parasites including Trypanosoma spp., Leishmania spp., Entamoeba spp., Plasmodium falciparum, Cryptosporidium parvum and Toxoplasma gondii. Unfortunately, the poor water solubility of dinitroanilines prevents their development as anti-parasitic agents. Unlike all previously characterized microtubule-disrupting drugs, which bind beta-tubulin, the dinitroanilines act on alpha-tubulin. The experiments in this proposal seek to define the effect of dinitroanilines on alpha-tubulin and to identify soluble derivatives of the dinitroanilines. The yeast S. cerevisiae is normally insensitive to dinitroanilines but expression of altered tubulin is predicted to confer dinitroaniline sensitivity. Sensitive and resistant yeast strains will be used to develop an inexpensive high throughput screen for effective and soluble dinitroaniline derivatives. The screen will exploit growth competition between sensitive and insensitive S. cerevisiae strains to discriminate between compounds that are non-specifically toxic to both strains and those that specifically disrupt plant/protozoan-like tubulin. Dinitroaniline derivatives will be evaluated in the yeast growth competition assay to identify compounds with increased solubility, sub-micromolar activity and specific disruption of plant/protozoan-like microtubules. Active compounds identified by the yeast screen will be tested for specific disruption of parasite microtubules using intracellular Toxoplasma gondii. Dinitroaniline interaction with alpha-tubulin will be characterized by mapping the dinitroaniline binding site on tubulin, characterizing the kinetics of dinitroaniline binding to the tubulin dimer, identifying conformation changes associated with dinitroaniline binding and describing dinitroaniline effects upon the dynamic behavior of microtubules. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Career Transition Award (K22)
Project #
1K22AI055790-01
Application #
6672840
Study Section
Microbiology and Infectious Diseases B Subcommittee (MID)
Program Officer
Coyne, Philip Edward
Project Start
2004-07-01
Project End
2006-06-30
Budget Start
2004-07-01
Budget End
2005-06-30
Support Year
1
Fiscal Year
2004
Total Cost
$159,680
Indirect Cost
Name
University of California Irvine
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Ma, Christopher; Tran, Johnson; Li, Catherine et al. (2008) Secondary mutations correct fitness defects in Toxoplasma gondii with dinitroaniline resistance mutations. Genetics 180:845-56
Ma, Christopher; Li, Catherine; Ganesan, Lakshmi et al. (2007) Mutations in alpha-tubulin confer dinitroaniline resistance at a cost to microtubule function. Mol Biol Cell 18:4711-20