Comprehensive analysis of binding sites in tubulin to develop small molecule ligands

Mühlethaler, Tobias. Comprehensive analysis of binding sites in tubulin to develop small molecule ligands. 2022, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/88830/

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In eukaryotic cells, microtubules are highly dynamic filaments of the cytoskeleton where they are involved in vital tasks like cell division, intracellular transport, and cell polarization. Microtubules are assembled from their building block tubulin, and both microtubules and tubulin are known to bind a multitude of regulators. In cells, protein partners bind to microtubules to either modulate their behavior or to use them to fulfill various cellular functions. In addition to proteins, several dozen small molecule ligands are known to influence microtubule dynamics and they target six distinct binding sites on tubulin. As tumor cells exhibit faster rates of cell division than healthy cells, it is not surprising that mitotic inhibition is one of the many strategies to treat cancer. Indeed, five of these ligands are currently used in the clinic as monotherapeutic antitubulin drugs. While their mechanism of action differ, all original ligands are derived from natural sources and have complex chemical structures. Based on these observations, we aimed in the present thesis to address the two outstanding open questions whether there are additional binding sites present in tubulin and if it is possible to develop a small molecule ligand targeting microtubules.
To tackle these two questions, we first performed an X-ray crystallography based fragment screen. Using this approach, we found 56 chemically diverse fragments that bound to 10 distinct binding sites on tubulin. Moreover, we combined a computational binding-site search with the results from our fragment screen to comprehensively map all binding sites in tubulin. Indeed, we found 18 novel sites that are not targeted by any of the known ligands. Out of these, 11 sites are also not targeted by any known tubulin protein partner. These findings demonstrate that our combined computational and crystallographic approach is a powerful tool to map binding sites in any protein for which a well diffracting crystal system is available.
With the structural information at hands on a plethora of new fragments bound to tubulin, we went on to develop novel small molecule ligands targeting tubulin. As a first step and proof of principle, we chose a fragment that binds to the well-known colchicine site and has structural resemblance to nocodazole, a microtubule-destabilizing agent that was proven to be too toxic to use in the clinic. This fragment establishes an intricate interaction network with tubulin, consisting of four hydrogen bonds. Indeed, when probing for the importance of these hydrogen bonds by removing them individually we learned that all of them are necessary to retain binding. While leaving the interaction network unchanged, we still had the option to grow our fragment by attaching continuously larger substitutions and in this way, we managed to improve our fragment from an IC50 of 54 μM to a final IC50 of 0.94 μM in our best compound. These results demonstrate that already a small fragment can exhibit optimal interactions to its target and that indeed our fragment hits can be used to obtain potent tubulin inhibitors.
As our pilot project showed that our fragments can be improved, we went on to apply our freshly gained insight to a novel tubulin-binding site. This time we chose a fragment linking strategy, in which several fragments that bind close to each other are combined. First, we took a fragment that contained a common binding motif as a starting point to gain specificity. Second, we looked for additional fragments binding in close proximity that could be chemically linked to our starting fragment. The two additional fragments that we selected suggested that expanding our starting fragment with a phenyl moiety should increase its affinity. Indeed, with our linking effort we obtained a first ligand with an IC50 of 48 μM, whereas none of the starting fragments showed any effect on cells. Additional rounds of modification of this first ligand brought us our most potent compound, which we named Todalam. Todalam has an IC50 value of 8.8 μM and showed microtubule depolymerizing effects in vitro as well as in cells. Furthermore, we proposed a novel, twofold mechanism of action for Todalam: First, Todalam acts like a “plug” locking tubulin in its curved conformation and second, it links free tubulin to ring like structures that are incompatible with microtubule assembly. This is to the best of our knowledge the first rationally designed tubulin-targeting agent with a novel binding site and a novel molecular mechanism of action.
Overall, we demonstrated that crystallographic fragment screening is a very powerful tool both for finding novel binding sites as well as for developing small molecule ligands. Our results set a basis for future ligand design efforts by providing a diverse set of novel tubulin-binding sites and ligands. They further offer two lead-like compounds that are ready to be developed into small lead molecules.
Advisors:Steinmetz, Michel and Maier, Timm and Glockshuber, Rudolf
Faculties and Departments:05 Faculty of Science > Departement Chemie > Former Organization Units Chemistry > Physikalische Chemie (Maier)
UniBasel Contributors:Mühlethaler, Tobias and Maier, Timm
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:14761
Thesis status:Complete
Number of Pages:VI, 240, VII-XCVIII
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss147615
edoc DOI:
Last Modified:26 Jul 2022 04:30
Deposited On:25 Jul 2022 12:20

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