A fluorescence anisotropy assay designed for the characterization of tubulin-specific ligands

An international team of researchers with participation of the group led by Dr. Fernando Díaz, at Centro de Investigaciones Biológicas, has recently published in Nature Comm. an assay based on fluorescence anisotropy specifically designed to detect ligands directed towards the tubulin maytansine site. By means of this technique, they have been able to characterize the dissociation constants of already known ligands, as well as to discover two new natural products that interact with tubulin.

Chemotherapy directed against tubulin has revealed itself as one of the most effective in both blood and solid tumors (breast, ovarian and lung). These molecules directed specifically to microtubules (MTAs) alter their dynamics during mitosis interfering with cell division, angiogenesis and other essential processes for tumor development. However, despite their effectiveness, they present problems of general toxicity and development of resistance. One of the strategies in vogue is to use these drugs linked to monoclonal antibodies that recognize tumor cells. Thus, they reach their targets, but at very low concentrations, so they need to have a very high affinity and be effective at those very small stoichiometries. The compounds directed to the site of maytansine (recently discovered by the authors of the work) present these two characteristics: a single molecule that binds to the microtubule is capable of poisoning it and its affinity, of nanomolar order, allows it to bind at very low concentrations.

In the work now published in Nature Communications the authors have deepened in the development of tools for the discovery and development of new compounds directed to the maytansine site. For this purpose, they have synthesized, evaluated and used a fluorescence probe specific to this target that has allowed them to identify new compounds with the ability to be used as warheads and to evaluate their potency with a robotic assay based on fluorescence anisotropy which provides results in a simple and fast way.

The team at CIB has been responsible for developing an effective fluorescent probe, tuning the assay and carrying out the biochemical characterization of the ligands directed to the maytansine site, as well as its use for the discovery and evaluation of the two new compounds discovered. In addition, the determination of the crystallographic structures of the tubulin complexes with these two ligands have allowed the authors to define an additional interaction site, adjacent to that shared by all ligands of the maytansine site, which will allow the development of a new generation of MTAs for the treatment of cancer.

Reference: A fluorescence anisotropy assay to discover and characterize ligands targeting the maytansinee site of tubulin. Grégory Menchon, Andrea E. Prota, Daniel Lucena-Agell, Pascal Bucher, Rolf Jansen, Herbert Irschik, Rolf Müller, Ian Paterson, J. Fernando Díaz, Karl-Heinz Altmann & Michel O. Steinmetz (2018) Nature Communications, 9, doi:10.1038/s41467-018-04535-8