Bioorthogonal Functionalization of Tubulin with Linear-Zampanolide

Medicinal chemists have directed their attention toward structure-activity relationships with the desire to determine the importance of various peripheral functional groups and ultimately improve biological and physiochemical properties of therapeutic lead compounds. When our program began almost three decades ago, little attention had been paid to the relationship between conformation and biological activity in complex natural products, even though minor substitution or stereochemical modifications affect their conformational preferences and in turn, their biological activity. This fact was almost certainly due to a misapprehension regarding their presumed flexibility and availability of analytical tools that could provide an accurate portrait of their conformational potential energy surface and solution conformational preferences. Our innovative program exploits the power of organic synthesis and conformational analysis to support medicinal chemistry that complements classic structure-activity profiles with insight into the spatial structural relationships critical to target engagement and biological activity. This project builds upon our previous studies of the potent, microtubule-stabilizing polyketide, zampanolide, which binds microtubules covalently. We recently identified linear-zampanolide, a unique analogue that maintains potent cytotoxicity through microtubule stabilization despite significant conformational flexibility. This surprising discovery provides an opportunity to systematically and independently modify two structural features of parent compound to provide new biological probes that covalently bind to tubulin. The current projects seeks to complement the previous studies by exploring modification of the solvent-exposed, C19-conjugated ester with trans-cyclooctene (TCO) functionality. Selective functionalization of tubulin will then be evaluated by in vivo generation of fluorescent probes for live- cell imaging through bioorthogonal tetrazine-TCO click chemistry. Despite their common b-tubulin binding site, taxol and zampanolide are known to produce distinct microtubule lattice structures and give rise to structural heterogeneity in the microtubule walls. Therefore, fluorescent derivatives of linear-zampanolide will provide a valuable new microtubule imaging agent as complement to ubiquitous use of fluorescent paclitaxel derivatives for this purpose. In addition, we will systematically modify the a,b-unsaturated ketone functionality of linear- zampanolide with the goal of enhancing covalent interactions within the tubulin-binding site. The goal of this endeavor is to favorably affect binding, off-rates and reversibility, and, in result, increase potency. The biological activity of all derivatives of new linear-zampanolide will be evaluated through standard cytotoxicity and tubulin-polymerization experiments prior to live cell imaging experiments.