Tubulin Polymerization Inhibitor Screenings

Introduction

Microtubules (MTs) are comprised of α/β tubulin heterodimers which have polymerized into cylinderical structures. MTs serve as an essential component of a cell’s cytoskeleton as they regulate and participate in a variety of cellular functions that include motility, morphology, intracellular transport, signal transduction, and cell division (Fig. 1). The cell cycle consists of the sequential G1, S, G2, and M phases with MT polymerization and depolymerization (i.e., MT dynamics) playing a key role in the normal progression of this cycle to insure proper cell division (Fig. 1). The disruption of MT dynamics, and thereby the cell cycle, leads to cell death. As such, MTs are a well-recognized and often-studied target for cancer drug discovery efforts^1-4.

Tubulin Polymerization Inhibitor ScreeningsTubulin polymerization inhibitors function in one of two ways: 1. stabilization of MTs which leads to inhibition of MT dynamics and 2. depolymerization of MTs^1-4. While the overwhelming target of anti-cancer compound screenings is MTs (motor kinesins are another target), different tubulin polymerization inhibitor screening methods are utilized^5-8. An early step in cancer drug discovery studies is usually an initial high throughput screen of a large number of compounds (hundreds of thousands) against a line (or lines) of cancer cells. Further screening is performed on a small number of compounds, selected based on the potency of their anti-cancer effect. One such screen is in vitro tubulin polymerization under cell-free conditions which not only confirms that the compound(s) inhibit tubulin polymerization, but also enables calculation of the compounds’ V_maxand IC₅₀/EC₅₀values⁷. These screens are often performed by either researchers in the laboratory or contract research organizations (CROs) as part of a custom screening service. CROs are cost and time-effective due to potential complications related to low yields and purity of tubulin along with any needed assay optimization. In either case, the screening uses tubulin protein and/or polymerization assay kits that are commerically available.

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Cytoskeleton, Inc. offers custom tubulin screening services that use tubulin and polymerization assay kits produced and validated by Cytoskeleton scientists. The tubulin proteins/kits have been used in multiple cancer drug discovery studies, specifically for follow-up screening efforts once a potent compound has been identified with the larger primary screens^5-8. Below we briefly discuss these papers, focusing on the key role that in vitro tubulin polymerization cell-free assays play in cancer drug discovery.

When screening novel compounds for their effect on tubulin polymerization, comparisons are made against well-characterized tubulin inhibitors, some of which are current anti-cancer treatments. In Cheng et al.⁵, the most promising drug was chosen based on large scale screenings of a variety of drugs for anti-proliferative effects against multiple cancer cell lines. Cytoskeleton’s tubulin polymerization assay/tubulin protein was used to demonstrate that the inhibitory effect of the compound had a mechanism of action similar to vincristine⁵. Similarly, Mu et al.⁶ used Cytoskeleton’s tubulin products to screen a novel synthetic compound that selectively targeted cancer cells, finding that the compound inhibited tubulin polymerization in a much more moderate manner than vincristine⁶. Besides vincristine, inhibitors of tubulin polymerization also can act at or near the colchicine binding site^7,8. Nagarajan et al.⁷ characterized a compound that inhibited tubulin polymerization via the colchicine binding site. Likewise, Bernard et al.⁸ described novel compounds that reversibly inhibit MT formation through binding near the colchicine site.

Fig. 1. Microtubules and Cell Division

Summary

Cancer drug discovery studies utilize a variety of screening tools, including cell free in vitro tubulin polymerization screens. Cytoskeleton Inc. offers custom tubulin screening services supported by over 20 years of experience. For those scientists who are comfortable with performing the screens themselves, Cytoskeleton offers tubulin in various purities as a stand-alone protein and as part of a polymerization kit that includes all necessary screening reagents For more information or a quote, please contact us at tservice@cytoskeleton.com.

References

Islam M.N. & Iskander M.N. 2004. Microtubule binding sites as target for developing anti-cancer agents. Mini Rev. Med. Chem. 4, 1077-1104.
Jordan A. et al. 1998. Tubulin as a target for anti-cancer drugs: Agents which interact with the mitotic spindle. Med. Res. Rev. 18, 259-296.
Desai A. & Mitchison T.J. 1997. Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13, 83-117.
Li Q. & Sham H.L. 2002. Discovery and development of antimitotic agents that inhibit tubulin polymerisation for the treatment of cancer. Expert Opin. Ther. Pat. 12, 1663-1702.
Cheng Y.-Y. et al. 2013. Design, synthesis, and mechanism of action of 2-(3-hydroxy-5-methoxyphenyl)-6-pyrrolidinylquinolin-4-one as a potent anticancer lead. Bioorg. Med. Chem. Lett. 23, 5223-5227.
Mu Y. et al. 2015. The novel tubulin polymerization inhibitor MHPT exhibits selective anti-tumor activity against Rhabdomyosarcoma in vitro and in vivo. PLoS ONE. 10, e0121806.
Nagarajan S. et al. 2015. Tubulin inhibitor identification by bioactive conformation alignment pharmacophore (BCAP)-guided virtual screening.Chem. Biol. Drug. Des. doi: 10.1111/cbdd.12568.
Bernard D. et al. 2015. Select microtubule inhibitors increase lysosome acidity and promote lysosomal disruption in acute myeloid leukemia (AML) cells. Apoptosis. 20, 948-959.

About Custom Services

Cytoskeleton, Inc. has been a reliable source of compound screening services in the areas of pre-clinical drug development programs and early compound screening in primary HTS projects, as well as secondary screening and compound target validation. We also have extensive experience in gene design and expression with an eye for producing highly purified biological active proteins. Our expertise in protein purification is the basis for the complementary skill of assay design. We have produced many functional assays for kinesins (e.g., Eg5, CenPE, MKLP2), dynein [cytoplasmic]), myosins (e.g., cardiac, smooth, skeletal, and non-muscle isoforms), small G-proteins (e.g., Rho, Arf, Ral families), tubulins (e.g., tumor, plant, and fungal origins), and actin binding proteins; many of them are multi-protein assays that might have protein complexes of 3 or more subunits, e.g., a soluble sarcomere format and the Arp2/3 complex based assay. Our experiences in antibody and ELISA technology complements the cytoskeletal and signal transduction focus. We support all of our services with a dedicated technical services department and years of laboratory experience in the fields of cell biology, cancer biology, and neuroscience.

For more details on these four main areas click on one of the following:

Compound Screening and Drug Development

Protein Purification

Assay Design and Develoment

ELISA and antibody development

If you have an immediate question please send an e-mail describing your requirements to tservice@cytoskeleton.com.

Have questions? Email tservice@cytoskeleton.com or call (303) 322-2254.

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