Date of Graduation

5-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Shi, Wei

Committee Member

Lee, Sun-Ok

Second Committee Member

Du, Yuchun

Third Committee Member

Erf, Gisela F.

Fourth Committee Member

Thallapuranam, Suresh

Keywords

Anti-cancer; Cytotoxicity; High throughput screening; Ipomoeassin F; Sec61; Target protein identification

Abstract

Ipomoeassin F is a flagship congener of a resin glycoside family that inhibits growth of many tumor cell lines with only single-digital nanomolar IC50 values. However, biological and pharmacological mechanisms of ipomoeassin F have been undefined. To facilitate exploration of the biological and pharmacological properties, we performed sophisticate SAR (Structure–activity relationship) studies of ipomoeassin F to understand its pharmacophore and structure properties so that we can design favorable probes for further biological investigation. By applying appropriate deviates that possess fluorescent groups and similar bio-activity, the target protein was found to be localized in endoplasmic reticulum (ER). Through biotin affinity pull down and proteomics studies, the target protein Sec61α (protein transport protein Sec61 subunit alpha isoform 1) was successfully isolated and confirmed. The isolated protein validation by Western blot provides convincing evidence to support the conclusion that Sec61α is the primary molecular target. Subsequently, the binding mode between ipomoeassin F and Sec61α was proved to be noncovalent or reversible covalent binding with the help of reverse/delayed competition. Based on competition result between ipomoeassin F and the derivatives with different activities, future applications of those derivatives to search for detect Sec61α-specific bio-active compounds by high throughput screening (HTS) are proposed. As a novel plant-derived carbohydrate-based macrocyclic molecule, ipomoeassin F was proved to have an exclusive mode of action. The successfully identification of its target protein Sec61α provides a new molecular tool to further understand the Sec61α biological properties and its potential to be a new therapeutic target for drug discovery. Most importantly, the information about this distinct mode of action would lead to a new way to design and develop more effective anti-cancer drugs.

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