Date of Graduation

5-2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Adams, Paul D.

Committee Member

Thallapuranam, Suresh

Second Committee Member

Henry, Ralph L.

Third Committee Member

Koeppe, Roger E. II

Keywords

Cancer research; Cdc42; Drug candidate; Drug discovery; Inhibitors; Protein interactions; Protein purification; Ras proteins; Small molecules

Abstract

The Ras superfamily of GTPases has 167 proteins that are involved in various cellular processes such as proliferation, transformation, migration, and inhibition of cell death. Mutations, abnormal expression, and function of these proteins are observed in many diseases, including several forms of cancer. Even though these GTPases were among the first discovered oncogenes, no successful Ras drug candidate has successfully passed clinical trials. Drugs targeting these proteins have failed mainly because of the complexity of their regulation, their high affinity to GTP, and their structure’s dynamic nature. Recently, novel promising targeting approaches have renewed interest in the Ras drug discovery field. One such approach uses small molecules to bind to small pockets on the protein rendering it unable to operate and bind to effectors. In this study, inhibition of Cdc42, a Ras-related GTPase in the Rho subfamily, was investigated. Using various computational, biophysical, and biochemical assays, we characterized the binding of a novel inhibitor to Cdc42. The effects of this inhibitor to Cdc42 protein-protein interactions (PPIs) were also investigated. Results show that the small molecule binds and stabilizes Cdc42 making it more resistant to denaturation. It also affects the protein’s hydrolysis activity and binding to PAK1, a crucial Cdc42 effector. A method to efficiently isolate and purify a PAK1 derived peptide was also developed. The results from these experiments show that ZCL278 is an effective Cdc42 inhibitor. However, the low effects of this inhibitor and low solubility in water make it a less attractive drug candidate. Using the results from this study, more soluble, ZCL278-like small molecules with better drug parameters are presented here as potential Cdc42 inhibitors. These small molecules will serve as candidates for investigating and developing better inhibitors of Cdc42-induced abnormal cell signaling.

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