T-00017: Application of 2-AAPA as a Research Tool for Thiol Oxidative Stress Studies

Summary of Technology

The following is an analysis of the technology that uses 2-AAPA as a research tool in thiol oxidative stress studies. Research shows that 2-AAPA causes significant increase in thiol oxidative stress and induces protein thiol glutathionylation. Research also indicates that 2-AAPA can increase human cancer cells’ sensitivity to radiation and chemotherapy which will help improve treatment efficiency. Further, SDSU research is currently using 2-AAPA as a tool to develop microtubule thiol glutathionylation as a novel target for the development of antimitotic agents. Overall, 2-AAPA is a unique compound that possesses potential in many fields of research that are searching for a tool to be used for thiol oxidative stress related research and development of an antimitotic drug with a novel mechanism of action.

Inventors: Xiangming Guan and Teresa Seefeldt

Description of Technology

The invention disclosure describes a tool that creates intracellular thiol oxidative stress using 2-acetylamino-3-[4-(2-acetylamino-2-carboxy-ethylsulfanylthiocarbonylaminophenylthiocarbamoylsulfanyl]propionic acid (2-AAPA). 2-AAPA inhibits intracellular glutathione reductase (GR). Inhibition of GR is known to decrease reduced glutathione (GSH) and increase glutathione disulfide (GSSG). Both are important variables in the ability to increase the amount of oxidative stress within cells. Increasing intracellular thiol oxidative stress is demonstrated from a significant decrease in the ratio of GSH to GSSG. Tests indicate a moderate decrease in GSH with a five- to seven-fold increase in GSSG. When a cell experiences oxidative stress, the cell becomes weak and susceptible to otherwise ineffective methods of medication. As a result, treatments such as chemotherapy and radiation therapy become more effective due to the cell’s increased sensitivity. South Dakota State University (SDSU) research has worked with various human cancer cell lines such as A431, MCF7, NCI-H226, and OVCAR-3. The oxidative stress studies have shown that increasing GSSG significantly enhances cancer sensitivity to X-ray irradiation as well as cancer sensitivity to doxorubicin and paclitaxel.

Associated with thiol oxidative stress, 2-AAPA was found to glutathionylate protein thiols. Currently, there are not many reagents available to selectively glutathionylate protein thiols. Protein thiol glutathionylation, as a consequence or part of cellular thiol oxidative stress, is thought to contribute to various disease states such as chronic heart disease and atherosclerosis, rheumatoid arthritis, AIDS, Parkinsons disease and Alzheimers disease. Therefore, 2-AAPA will be useful as a research tool in both studying these thiol oxidative stress related diseases and searching for their treatments.

Recently, the research group also confirmed that as part of protein thiol glutathionylation by 2-AAPA, the thiols of microtubules were gluathionylated in cancer cells. Thiols are critical for microtubule function. Consistently, 2-AAPA was found in UACC-62 (human melanoma cells) to depolymerize microtubules, to arrest the cell cycle at the G2/M phase, and to lead to cell apoptosis. These findings reveal that 2-AAPA is an antimitotic agent similar to vinblastine. Although 2-AAPA is not considered as a potent antimitotic agent since its IC50 is in the M range, the antimitotic effect achieved through microtubule glutathionylation has never been reported and can be developed as a novel approach to identify a new class of antimitotic agents. These antimitotic agents could be complimentary to existing antimitotic drugs.

Overall, the compound can be utilized in many areas of research. The compound can be easily prepared using a one step synthesis from commercially available reagents. The compound also exhibits excellent solubility in both organic and aqueous solvents, a desired pharmaceutical property. These favorable properties aid in providing applications for the compound as a research tool.

Market

2-AAPA used as a research tool could prove beneficial toward understanding applications associated with intracellular thiol oxidative stress. Research on the topic is beneficial to the biomedical and pharmaceutical industry, South Dakota State University, and other life science research scientists.

Buthionine sulfoximine is a drug similar to 2-AAPA. According to the National Cancer Institute, buthionine sulfoximine “irreversibly inhibits gamma-glutamylcysteine synthetase, thereby depleting cells of glutathione, a metabolite that plays a critical role in protecting cells against oxidative stress. By depleting cells of glutathione, this agent may enhance the in vitro and in vivo cytotoxicities of various chemotherapeutic agents in drug-resistant tumors.” Although buthionine sulfoximine uses a similar approach toward increasing cancer cell sensitivity, tests performed at SDSU show 2-AAPA is different in creating thiol oxidative stress. 2-AAPA significantly increases the oxidized form of glutathione (GSSG) instead of depleting the reduced form glutathione (GSH). As a result, the ratio of GSH/GSSG is affected much more significantly by 2-AAPA than by BSO. Additionally, 2-AAPA produced protein thiol glutathionylation which has not been reported with BSO. As indicated earlier, protein thiol glutathionylation is associated with cells experiencing thiol oxidative stress. Thiol oxidative stress is associated with a number of disease states. Because many reagents are unable to produce protein thiol glutathionylation in using the method that 2-AAPA utilizes, 2-AAPA can be used as a research tool in studying protein thiol glutathionylation. .

Finally, 2-AAPA differs from existing taxanes (paclitaxel and docetaxel) and vinca alkaloids (vinblastine, vincristine, vindesine, and vinorelbine). Using microtubule (a protein) thiol glutathionylation as an approach to develop antimitotic drugs has never been reported and is a novel approach for the development of new classes of antimitotic drugs.

Given previous success on the topic, companies may be interested in sponsored research to generate new applications for 2-AAPA. Disclosed publications mentioned that researchers are experimenting to understand the impact of GR inhibition on intracellular thiol status as well on the antioxidant enzymatic defense systems. Studies within this scope or a different scope could generate commercial applications.

Publications and Meeting Presentations

• 2005: 230th National Meeting of the American Chemical Society, Washington, DC. 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid – a novel irreversible glutathione reductase inhibitor.
• 2005: 230th National Meeting of the American Chemical Society, Washington, DC. Investigation of 2-acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoyl-sulfanyl]propionic acid’s effect on intracellular glutathione reductase, glutathione, and cell growth with monkey kidney cells.
• April 2007: American Association for Cancer Research (AACR) Annual Meeting, Los Angeles, California. Increase in oxidative stress via glutathione reductase inhibition as a novel approach to enhance cancer sensitivity to chemotherapy.
• April, 2007: South Dakota Academy of Science 92nd Annual Meeting, Brookings, SD. Determination of the Ratio of Total Thiols and Disulfides – An Index of Intracellular Oxidative Stress.
• 2007: 234th National Meeting of the American Chemical Society, Boston, MA; American Chemical Society, Washington, DC. Increase in oxidative stress via glutathione modulation as a novel approach to enhance cancer sensitivity to doxorubicin.
• 2007: 234th National Meeting of the American Chemical Society, Boston, MA; American Society , Washington, DC. Carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoyl-sulfanyl]propionic acid (2-AAPA) on doxorubicin induced cardiotoxicity.
• April 7, 2008: J Biol Chem, Vol. 284(5): 2729-2737. Characterization of a novel dithiocarbamate glutathione reductase inhibitor and its use as a tool to modulate intracellular glutathione.
• March 9, 2009: Arch Biochem Biophys, Vol. 485(1): 56-62. Effects of glutathione reductase inhibition on cellular thiol redox state and related systems.
• April 24, 2009: Free Radical Biology & Medicine, Vol. 47(2): 176-83. Increase in Thiol Oxidative Stress Via Glutathione Reductase Inhibition as a Novel Appoach to Enhance Cancer Sensitivity to X-ray Irradiation.
• April 2010: AACR 101st Annual Meeting, Washington, DC. 2-AAPA: A novel anticancer agent that induces microtubule depolymerization and apoptosis.
• 2010 manuscript ready to submit: 2-Acetylamino-3-[4-(2-acetylamino-2-carboxyethylsulfanylcarbonylamino)phenylcarbamoylsulfanyl]propionic acid, an antimitotic agent with a novel mechanism of action.