PANJARIAN, SHOGHAG
(2012)
INVESTIGATING ALLOSTERIC REGULATION OF ABL AND BCR-ABL KINASES: IMPLICATIONS FOR SMALL MOLECULE INHIBITORS.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
BCR-ABL is the oncogenic protein-tyrosine kinase responsible for the pathogenesis of chronic myelogenous leukemia (CML). Clinical management of CML has been revolutionized by imatinib, a selective ATP-competitive inhibitor of BCR-ABL kinase activity. Despite this clinical success, imatinib is less effective in advanced disease due to the emergence of drug resistant BCR-ABL mutants. Resistant mutations often arise in the drug binding site and include the most recalcitrant gatekeeper mutation, T315I. Other mutations arise outside the active site and allosterically reduce imatinib binding by promoting the active kinase conformation. Recently, a new class of allosteric BCR-ABL inhibitors, of which GNF-2 is the prototype, has been reported that targets the myristate-binding pocket of ABL. These compounds stabilize the inactive conformation of ABL and work in concert with ATP-competitive inhibitors to overcome imatinib resistance. Mounting evidence supports a regulatory influence of the non-catalytic SH3 and SH2 domains on BCR-ABL kinase domain. The major focus of this study was to exploit the intramolecular SH3:linker interaction, to stabilize the downregulated kinase domain conformation of BCR-ABL and sensitize the kinase to both imatinib and GNF-2. To achieve this goal, I engineered High Affinity Linker (HAL) variants of both ABL and BCR-ABL in which SH3:linker interaction was tightened through sequential addition of proline residues to the linker. Enhanced SH3:linker interaction induced long-range suppressive effects on the kinase activity in c-ABL, allosterically stabilized both the active site and the myristate-binding pocket, and sensitized BCR-ABL to small molecule inhibitors.
Src family kinases (SFKs) are important mediators of BCR-ABL signal transduction and oncogenesis in CML. SFKs also play important roles in clinical resistance to imatinib in the absence of BCR-ABL mutations. In the second part of my project, I explored the effect of SFK- selective inhibitor, pyrazolopyrimidine A-419259, on myeloid cells transformed with clinically relevant imatinib resistant BCR-ABL mutants. While proliferation of cells expressing BCR-ABL E255V and Y253H was inhibited by A-419259, BCR-ABL T315I cells were not. Surprisingly, cells expressing BCR-ABL-T315I maintained SFK activity in the presence of the inhibitor. This observation suggests that BCR-ABL-T315I induces cross-resistance to drugs that inhibit SFKs in CML through direct phosphorylation of the SFKs.
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Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
| Creators | Email | Pitt Username | ORCID  |
|---|
| PANJARIAN, SHOGHAG | sbp9@pitt.edu | SBP9 | |
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| ETD Committee: |
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| Date: |
14 December 2012 |
| Date Type: |
Publication |
| Defense Date: |
2 November 2012 |
| Approval Date: |
14 December 2012 |
| Submission Date: |
20 November 2012 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
181 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Pharmacology |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
allosteric regulation, kinases |
| Date Deposited: |
14 Dec 2012 16:33 |
| Last Modified: |
15 Nov 2016 14:07 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/16513 |
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