Coumarin‐30 Enables Site‐Resolved Detection of Tubulin Ligands by Microscale Thermophoresis
Abstract
Abstract Tubulins are among the most successful targets for cancer chemotherapy. However, the emergence of drug resistance stimulates the continuous search for novel chemotherapeutics. We discovered that coumarin‐30, a widely available laser dye, binds to the colchicine site of tubulin, inhibiting microtubule dynamics and cancer cell division at submicromolar concentrations. Exploiting the excellent fluorescent properties of coumarin‐30, we developed a fast, accurate, and cost‐effective coumarin‐30‐based microscale thermophoresis (C‐MST) assay as an express method for detecting tubulin–ligand interactions and discriminating colchicine site binders from ligands targeting other protein pockets. Using this assay, we identified several potent tubulin polymerization inhibitors associating with the colchicine site and validated them through in vitro microtubule dynamics and cell cycle assays in cancer cells. Furthermore, the C‐MST assay was demonstrated to detect ligands targeting a novel binding site on tubulin, recently established through crystallographic fragment screening. We confirmed detection of a small‐molecule ligand targeting that site and further designed and characterized a series of its analogs. The ability of the C‐MST assay to detect tubulin binders regardless of their binding site or their effect on microtubule dynamics opens new avenues for developing unconventional modulators of tubulin–tubulin and tubulin–effector interactions thereby facilitating anticancer drug discovery.
Article Details
Authors (23)
Mikhail N. Anisimov
Department of Physics M. V. Lomonosov Moscow State University Moscow Russia
Maksim A. Boichenko
Department of Chemistry M. V. Lomonosov Moscow State University Moscow Russia
Anton A. Sivachev
Department of Chemistry M. V. Lomonosov Moscow State University Moscow Russia
Alexey N. Romanov
N. N. Semenov Institute of Chemical Physics Russian Academy of Sciences Moscow Russia
Ilya A. Lifshits
Department of Physics M. V. Lomonosov Moscow State University Moscow Russia
Julia N. Borzunova
Department of Physics M. V. Lomonosov Moscow State University Moscow Russia
Marina Janibekova
National Laboratory Astana Astana Kazakhstan
Vadim V. Mustyatsa
National Laboratory Astana Astana Kazakhstan
Timur P. Tikhonov
Department of Chemistry M. V. Lomonosov Moscow State University Moscow Russia
Andrey Yu. Plodukhin
Department of Chemistry M. V. Lomonosov Moscow State University Moscow Russia
Vitaly V. Shorokhov
Department of Chemistry M. V. Lomonosov Moscow State University Moscow Russia
Victor A. Tafeenko
Department of Chemistry M. V. Lomonosov Moscow State University Moscow Russia
Nina K. Ratmanova
N. D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences Moscow Russia
Sergey S. Zhokhov
Department of Chemistry M. V. Lomonosov Moscow State University Moscow Russia
Roman A. Novikov
Olga I. Kechko
Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia
Alexander A. Makarov
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
Vladimir A. Mitkevich
Engelhardt Institute of Molecular Biology Russian Academy of Sciences Moscow Russia
Ivan A. Andreev
N. D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences Moscow Russia
Ivan A. Vorobjev
National Laboratory Astana Astana Kazakhstan
Igor V. Trushkov
N. D. Zelinsky Institute of Organic Chemistry Russian Academy of Sciences Moscow Russia
Olga A. Ivanova
Department of Chemistry M. V. Lomonosov Moscow State University Moscow Russia
Nikita B. Gudimchuk
Department of Physics M. V. Lomonosov Moscow State University Moscow Russia