Anti-Mitotic and Anti-Parasitic Drugs

Vinca Domain Peptides

Tubulin, the structural subunit of microtubules, was first identified as the colchicine-binding protein in 1967. Since then, numerous tubulin-binding drugs have been identified, which include the vinca domain inhibitors that cause cell death at pM and low nM range. Notable among these vinca domain agents are the naturally occurring peptides and depsipeptides such as the cryptophycins , hemiasterlin and dolastatin that block tubulin polymerization, disrupt spindle microtubules and induce the self-association of tubulin dimers into single-protofilament rings and spirals.

We have characterized the binding of these compounds to β-tubulin and identified a common binding site (hemiasterlin is shown above). Sequence alignment indicates that the residues forming the binding pocket are conserved in vertebrate and protozoan β-tubulins, but not in fungal β-tubulins , consistent with experimental results.

Dinitroanilines

Dinitroanilines , such as oryzalin, trifluralin and their analogs, represent another class of compounds that are widely used in herbicide formulations as they inhibit tubulin assembly and disrupt microtubules of plants. Interestingly, these compounds also disrupt microtubules of parasitic protozoa such as Trypanosoma spp ., Leishmania spp ., Plasmodium falciparum and Toxoplasma gondii, but are ineffective against vertebrate and fungal tubulins . Despite different resistance profiles, all the oryzalin-resistant Toxoplasma lines harbored single point mutations in α-tubulin and we were able to confirm that these were the first compounds shown to bind to α-tubulin. Using computational modeling, the binding site for oryzalin, trifluralin and a newly developed analog, GB-II-5, was identified on Leishmania, Plasmodium and Toxoplasma α-tubulins (as shown below).

We are currently working to define the mechanism of action of these drugs on the tubulin and microtubule structure. Based on our docking results, we are also performing in situ design of new drug analogs for use as therapeutics against malaria and other parasitic diseases. This work is an ongoing collaboration with Naomi Morrissette (UC Irvine), Karl Werbovetz (University of Ohio) and David Sibley (Washington University School of Medicine).

Taxol

Taxol represents an important drug in the fight against cancer, in particular ovarian, breast and lung cancer. It binds to β-tubulin and hyper-stabilizes microtubules preventing mitosis thereby stopping the growth of tumors. Using a microtubule model based on cryo-EM work from Ken Downing, we have performed the first super-computer simulations of a small piece of a microtubule, both with and without taxol bound. The comparison of these two simulations allows us to directly determine the molecular effect of taxol, and we see long-range allosteric changes within the tubulin dimer. This is very recent work that is not yet complete, however we feel that this will offer significant insight how these drugs function and should aid in the development of new paclitaxel or epothilone analogs.