Selzer, Ari
(2024)
Allosteric Modulation of Src-Family Kinases in Acute Myeloid Leukemia: Harnessing Natural Mechanisms of Kinase Regulation to develop Novel Therapeutics.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The Src-family of non-receptor tyrosine kinases (SFKs) contains eight mammalian members which play important roles in cell growth, survival, motility, and proliferation. Overactivation of these kinases are associated with the formation and progression of multiple forms of cancer including the blood cancer acute myeloid leukemia (AML).
Tyrosine kinase inhibitors (TKIs) are effective cancer therapeutics with dozens currently having FDA-approval. However, the therapeutic effectiveness of this drug class is limited by off-target effects and the rise of resistance mutations. The combination of allosteric and orthosteric TKIs for the same target, known as double-drugging, has shown potential in overcoming resistance mechanisms and increase on-target selectivity. ATP-site inhibitors for SFKs, particularly Hck and Fgr, have shown promising results in preclinical studies for AML. However, the development of an allosteric inhibitor to be used with these drugs could prove invaluable.
In this thesis I explored the conformational preference of the ATP-site SFK inhibitor A-419259 and characterized two novel allosteric modulators of Hck. Known as PDA1 and PDA2, these small molecules represent early leads for development as potential allosteric inhibitors. I solved a crystal structure of Hck with A-419259, which revealed that the activation loop of the protein is extended, in contrast to a structure of Hck with another ATP-site inhibitor (PP1) where the loop collapses into the active site. Using a kinase activity assay, I demonstrated that the extended A-loop conformation, which more closely resembles an active kinase, increases the ability of A-419259 to target the active form of Hck.
15N/1H HSQC NMR experiments identified a shared binding site for PDA1 and PDA2 on the regulatory SH3 domain of Hck. Hydrogen-deuterium exchange mass-spectrometry and kinase activity assays revealed that these compounds have opposite effects on kinase activity and conformation. Molecular dynamics simulations predicted that PDA1 forms contacts with the N-lobe of the catalytic kinase domain to maintain the protein in a closed inactive conformation. Finally, in-cell thermal stability assays revealed that PDA1 is able to bind Hck proteins in cells. Additionally, PDA1 and A-419259 have complementary resistance profiles, indicating the promise for their use in a double-drugging framework.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
14 October 2024 |
| Date Type: |
Publication |
| Defense Date: |
9 August 2024 |
| Approval Date: |
14 October 2024 |
| Submission Date: |
13 August 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
186 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Biophysics and Structural Biology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
Kinase, Src family kinases, Src, Hck, Drug Discovery, Structural Biology |
| Date Deposited: |
14 Oct 2024 16:04 |
| Last Modified: |
14 Oct 2024 16:04 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46920 |
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