Biochemical characterization and molecular dynamic simulation of β-sitosterol as a tubulin-binding anticancer agent.
Βeta-sitosterol (β-SITO), a phytosterol present in pomegranate, peanut, corn oil, almond, and avocado, has been recognized to offer health benefits and potential clinical uses. β-SITO is orally bioavailable and, as a constituent of edible natural products, is considered to have no undesired side effects. It has also been considered as a potent anticancer agent. However, the molecular mechanism of action of β-SITO as a tubulin-binding anticancer agent and its binding site on tubulin are poorly understood. Using a combination of biochemical analyses and molecular dynamic simulation, we investigated the molecular details of the binding interactions of β-SITO with tubulin. A polymer mass assay comparing the effects of β-SITO and of taxol and vinblastine on tubulin assembly showed that this phytosterol stabilized microtubule assembly in a manner similar to taxol. An 8-anilino-1-naphthalenesulfonic acid assay confirmed the direct interaction of β-SITO with tubulin. Although β-SITO did not show direct binding to the colchicine site on tubulin, it stabilized the colchicine binding. Interestingly, no sulfhydryl groups of tubulin were involved in the binding interaction of β-SITO with tubulin. Based on the results from the biochemical assays, we computationally modeled the binding of β-SITO with tubulin. Using molecular docking followed by molecular dynamic simulations, we found that β-SITO binds tubulin at a novel site (which we call the 'SITO site') adjacent to the colchicine and noscapine sites. Our data suggest that β-SITO is a potent anticancer compound that interferes with microtubule assembly dynamics by binding to a novel site on tubulin.
Experimental Cancer Therapeutics and Chemical Biology, Department of Biology, UM-DAE Centre for Excellence in Basic Sciences, Kalina, Santacruz (E), Mumbai 400098, India.
Tejashree
T
Mahaddalkar
Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan 173234, Himachal Pradesh, India.
Charu
C
Suri
Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Waknaghat, Solan 173234, Himachal Pradesh, India.
Pradeep Kumar
PK
Naik
Experimental Cancer Therapeutics and Chemical Biology, Department of Biology, UM-DAE Centre for Excellence in Basic Sciences, Kalina, Santacruz (E), Mumbai 400098, India. Electronic address: manu.lopus@cbs.ac.in.
Manu
M
Lopus
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000502:physiology; D018360:Crystallography, X-Ray; D004305:Dose-Response Relationship, Drug; D006041:Goats; D017433:Protein Structure, Secondary; D012855:Sitosterols / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
D000970:Antineoplastic Agents; D012855:Sitosterols; D014404:Tubulin; C025473:gamma-sitosterol
5,5′-Dithiobis(2-nitrobenzoic acid) (PubChem CID: 6254); 8-anilinonaphthalene-1-sulfonic acid (PubChem CID: 1369); Beta-sitosterol (PubChem CID: 222284); Cancer; Colchicine (PubChem CID: 6167); Guanosine 5′-triphosphate (PubChem CID: 6830); Microtubule; Molecular dynamic simulation; Taxol (PubChem CID: 36314); Tubulin; Vinblastine sulfate (PubChem CID: 241902); Βeta-sitosterol