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Bio-Layer Interferometry Technology Helps Identify mTORC1-Specific Inhibitors

A high-throughput screen of drugs revealed a new role for a class of antihistamine piperazine compounds as potential anti-cancer agents.


Jennifer Gray Tomilov, PhD


November 18, 2019 - Bio-layer interferometry (BLI) technology was utilized to screen 1600 repurposed compounds and identify a novel class of direct mTOR binders that specifically inhibits only mTORC1 complex activity and may comprise a new class of anti-cancer therapeutics.


Sonia Allen, PhD, with the University of California, Davis, in Davis, CA, and colleagues published their findings in the July 17, 2018 issue of Biochemical Pharmacology.


The authors stated, “the mechanistic target of rapamycin (mTOR) is a 289 kDa serine/threonine kinase protein that controls various energetic functions at both the cellular and organism level.” Further, “mTOR participates in two major complexes, mTORC1 and mTORC2.” Functional effects of drugs affecting mTORC1 or mTORC2 may be quantified by S6 kinase and Akt phosphorylation assays, respectively.


Binding kinetics data was generated by implementing BLI technology to initially screen a library of 1600 compounds for binding activity to native mTOR protein. From this initial screen, 56 compounds were selected for dose-dependent mTOR binding analysis, and 16 of these compounds illustrated a dose-dependent binding response to mTOR target protein.


The binding constants (Kd) for four dose-dependent inhibitors of mTOR were quantified with BLI technology and indicated as 10 μM for testosterone propionate, 55 μM for carvedilol, 5 μM for hydroxyprogesterone, and 400 nM for cinnarizine. Interestingly, follow-up assays for S6 kinase activity and Akt phosphorylation indicated that whereas testosterone propionate, carvedilol, and hydroxyprogesterone showed inhibition of both mTORC1 and mTORC2 signaling, cinnarizine was a specific inhibitor of only mTORC1 signaling.


As cinnarizine was shown to inhibit the function of mTORC1 but not mTORC2, the group found that the piperazine analogs of cinnarizine, hydroxyzine, meclizine, and flunarizine showed a similar mTORC1-specific functional inhibition. These drugs are known for their antihistaminic properties.


To better understand the mechanism for this action, the group found that the addition of a benzene ring to hydroxyzine to create the piperazine buclizine completely eliminated mTORC1 inhibitory activity, indicating the structural importance of this class of drugs for mTORC1.


Further, both flunarizine and cinnarizine have been shown to independently mediate dose-dependent killing against lymphoma and multiple myeloma in vitro. Flunarizine has been shown in combination therapy to potentiate the effect of vincristine toxicity in B16 melanoma cells and to potentiate the effect of melphalan toxicity against sarcoma.


Together, these data illustrate the effective approach of using direct binding kinetics screening to rapidly identify new mechanisms for repurposed drugs. In this case, Dr. Allen states, “we identified in cinnarizine the first member of a new class of mTORC1-inibitory drugs, all of which are antihistamine piperazines.” These drugs may also be great candidates compared with the traditional mTORC1-specific inhibitor rapamycin. “Cinnarizine and hydroxyzine have been prescribed for over 60 years, and…they both have extensive pharmacological and safety profiles, compared with rapamycin, an immunosuppressant that cannot be given for an extended period of time,” concluded Dr. Allen.


Full author disclosures provided in manuscript.


Biochemical Pharmacology. Published July 17, 2018

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