zampanolide and laulimalide

Zampanolide, first discovered in a sponge extract in 1996 and later identified as a microtubule-stabilizing agent in 2009, is a covalent binding secondary metabolite with potent, low nanomolar activity in mammalian cells. Zampanolide was not susceptible to single amino acid mutations at the taxoid site of β-tubulin in human ovarian cancer 1A9 cells, despite evidence that it selectively binds to the taxoid site. As expected, it did not synergize with other taxoid site microtubule-stabilizing agents (paclitaxel, ixabepilone, discodermolide), but surprisingly also did not synergize in 1A9 cells with laulimalide/peloruside binding site agents either. Efforts to generate a zampanolide-resistant cell line were unsuccessful. Using a standard wound scratch assay in cell culture, it was an effective inhibitor of migration of human umbilical vein endothelial cells (HUVEC) and fibroblast cells (D551). These properties of covalent binding, the ability to inhibit cell growth in paclitaxel and epothilone resistant cells, and the ability to inhibit cell migration suggest that it would be of interest to investigate zampanolide in preclinical animal models to determine if it is effective in vivo at preventing tumor growth and metastasis.

Topics: Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Female; Fibroblasts; Human Umbilical Vein Endothelial Cells; Humans; Lactones; Macrolides; Microtubules; Taxoids; Tubulin; Tubulin Modulators

In this chapter, we describe the methods used to determine the binding site and binding profile of zampanolide, a novel microtubule-stabilizing agent (MSA) that binds covalently to tubulin. These methods can be applied to other novel MSAs in which the binding site and mechanism of binding are unknown. Using the described methods, we have shown that zampanolide binds to the taxoid site on β-tubulin, but unlike most other MSAs is able to covalently modify this site. The purpose of this chapter is to provide a step-by-step protocol for determining the binding site of a novel MSA.

Topics: Animals; Binding Sites; Brain; Bridged Bicyclo Compounds, Heterocyclic; Cattle; Lactones; Macrolides; Microtubules; Paclitaxel; Polycyclic Compounds; Protein Binding; Tubulin Modulators