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A radioligand binding assay for antitubulin activity in tumor cells
David H Young1, Fernando M Rubio, Paul O Danis
1Dow Agro Sciences LLC, Indianapolis, IN 46268, USA. dyoung@dow.com
Abstract:
The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.
Insights
The benzamide RH-5854 drug effectively targets tumor cells by inhibiting nuclear division through specific binding to beta-tubulin. This discovery enables a new cell-based assay for detecting antitubulin effects in cancer research.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- The benzamide RH-5854 exhibits high potency against tumor cells.
- It functions by arresting nuclear division through specific covalent binding to the beta-subunit of tubulin.
- This interaction occurs within the colchicine binding region of tubulin.
Purpose of the Study:
- To develop and validate a cell-based competitive binding assay for detecting antitubulin effects.
- To characterize the binding properties of 3H-RH-5854 to beta-tubulin in colon cancer cells.
- To demonstrate the specificity of the assay for antitubulin activity.
Main Methods:
- Utilized 3H-RH-5854 for binding studies in HCT-116 colon cancer cells.
- Developed a cell-based competitive binding assay.
- Tested inhibition and enhancement of 3H-RH-5854 binding with known antitubulin agents (colchicine site binders, paclitaxel, vinblastine) and non-tubulin targeting cytotoxic agents.
Main Results:
- Binding of 3H-RH-5854 to beta-tubulin in HCT-116 cells is saturable.
- Binding is inhibited by colchicine site binders and paclitaxel.
- Binding is enhanced by vinblastine, but not by other vinblastine site agents.
- Non-tubulin targeting agents did not affect 3H-RH-5854 binding, confirming assay specificity.
Conclusions:
- The cell-based 3H-RH-5854 binding assay accurately detects antitubulin effects in whole tumor cells.
- This assay offers an alternative to traditional methods using isolated tubulin.
- It accounts for cell-specific factors influencing drug sensitivity, such as tubulin variations and drug metabolism.
