Date of Graduation

5-2022

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

Hestekin, Christa N.

Second Committee Member

Rhoads, Douglas D.

Third Committee Member

Beitle, Robert R. Jr.

Fourth Committee Member

Stites, Wesley E.

Keywords

Ras-related proteins; Ras superfamily; G proteins; Cancer; Small molecule inhibitor; CDC42; Rho subfamily; Activated kinase (ACK)

Abstract

The Ras superfamily of small G proteins are involved in cell-signaling processes that, if not regulated, may lead to cell multiplication, apoptosis inhibition, and tumorigenesis. They function as molecular switches, which through GTP/GDP exchange cycle, switch on or off cellular activities. Overexpression and/or hyperactivity of these proteins have been linked to many diseases including various cancers. CDC42, a member of the Rho subfamily of the Ras superfamily of small G proteins, participates in the regulation of many cellular processes including cell adhesion, mitosis, and cytoskeletal rearrangements. CDC42 binds to and activates many effector proteins including CDC42-activated kinase (ACK). Abnormal activities of CDC42/ACK have been associated with tumor development. It is paramount that small molecule inhibitors be developed to inhibit CDC42 interactions with its effector proteins leading to abnormal signaling activities. A small molecule, ZCL278, has been shown to interact with CDC42 and alter CDC42-stimulated overactivity. In this current work, we investigated the influence of ZCL278 on CDC42 wild type and CDC42F28L mutant as well as its influence on CDC42/ACK protein-protein interactions. Our In vitro binding assay data suggest that ZCL278 decreased binding capacity of CDC42 to ACK. ZCL278 was shown to lower the affinity of CDC42 for ACK by a nanomolar-to-micromolar jump in an extrinsic fluorescence titration experiment. Isothermal titration calorimetry experiments data showed that in absence of ZCL278, CDC42 bound to ACK with an apparent Kd value of 0.6 μM while in the presence of ZCL278 the apparent Kd peaked to 4 μM. Overall, our binding experiments suggested that 1) ZCL278 decreased the binding affinity of CDC42 to ACK, 2) ZCL278 bound to both CDC42 and CDC42F28L with micromolar affinity, 3) ZCL278 induced structural modifications in CDC42 and CDC42F28L upon binding, and 4) ZCL278 rendered CDC42 and CDC42F28L more thermally stable.

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