Rational Design of a Novel Tubulin Inhibitor with a Unique Mechanism of Action

Abstract

In this study, we capitalized on our previously performed crystallographic fragment screen and developed the antitubulin small molecule Todalam with only two rounds of straightforward chemical synthesis. Todalam binds to a novel tubulin site, disrupts microtubule networks in cells, arrests cells in G2/M, induces cell death, and synergizes with vinblastine. The compound destabilizes microtubules by acting as a molecular plug that sterically inhibits the curved-tostraight conformational switch in the α-tubulin subunit, and by sequestering tubulin dimers into assembly incompetent oligomers. Our results describe for the first time the generation of a fully rationally designed small molecule tubulin inhibitor from a fragment, which displays a unique molecular mechanism of action. They thus demonstrate the usefulness of tubulin-binding fragments as valuable starting points for innovative antitubulin drug and chemical probe discovery campaigns.

Frentizole, a Nontoxic Immunosuppressive Drug, and Its Analogs Display Antitumor Activity via Tubulin Inhibition

“…These results suggest that the replacement of the phenyl ring by the smaller ethyl groups requires a compensating increase in volume on the BZT moiety, and the observed differences might result from different binding modes. As anticipated, most of the synthesized compounds did not show antiproliferative activity, thus reflecting the strict structural requirements for eliciting antiproliferative activity, which is in good agreement with many previous studies on colchicine-site ligands showing that structural variations resulted in large potency changes [22,24,28,50]. Frentizole, although not the most potent antiproliferative agent of the series, has the broader antiproliferative profile and is, therefore, the optimal candidate for repurposing.…”

“…With the aim of testing the hypothesis that frentizole is a tubulin inhibitor, we synthesized frentizole and a series of analogs and evaluated them as antiproliferative agents against HeLa human cancer cells. Small structural changes can dramatically affect the potency of antiproliferative compounds, and colchicine-site binders are not an exception [22,24,28,50]. Therefore testing only a single compound (frentizole) might fail to uncover the desired activity.…”

“…There are more than seven structurally characterized drugbinding sites in tubulin: the colchicine site, the Vinca alkaloids site, the taxanes site, the laulimalide/peloruside site, the eribulin site, and the recently discovered gatorbulin-1 site [20] in the β subunit and the pironetin-binding site in the α subunit [21][22][23]. Additional potential drug-binding sites in tubulin have recently been uncovered [24,25]. The most clinically successful antimitotic agents, the taxanes and the Vinca minor alkaloid drugs, are natural product derivatives of very complex structures that render them susceptible to multidrug resistance (MDR) efflux proteins and result in unfavorable pharmacokinetic properties and high toxicity profiles.…”

The microtubule cytoskeleton: An old validated target for novel therapeutic drugs

“…Todalam has an inhibitory effect on in vitro tubulin assembly, disrupts microtubule networks in cells and arrests cells in G2/M. Thus, using a multidisciplinary approach combining structural biology, computational modelling, fragment screening, bio-guided and structure-guided chemical synthesis, the proof of concept that it is possible to synthesize a small molecule tubulin inhibitor that binds to a new, not yet described tubulin site has been achieved ( Mühlethaler et al, 2022 ). Although this approach gave promising results, it remains to be explored whether all the identified sites represent targets for drug development.…”

Alternative Approaches to Understand Microtubule Cap Morphology and Function

“…In addition, their implication in intracellular long-distance transport and scaffolding is behind the pharmacological regulation of MTs in inflammation and neurodegeneration. There is a plethora of MT-regulating compounds that bind to up to eight different pharmacological pockets in tubulin − and either activate or turn off the tubulin molecule, hence perturbing MT function through stabilization or disassembly. Often, MT stabilizing agents (MSAs) promote assembly and settle the structure of these filaments through the axial and/or the lateral contacts, whereas destabilizers (MDAs) prevent (curved-to-straight) conformational changes upon assembly or block the axial molecular interacting surfaces, thus preventing the formation of MTs.…”