Development of a Three-Hybrid Split-Luciferase System for Interrogating Protein Kinase Inhibition

Persistent Link:
http://hdl.handle.net/10150/205450
Title:
Development of a Three-Hybrid Split-Luciferase System for Interrogating Protein Kinase Inhibition
Author:
Jester, Benjamin
Issue Date:
2011
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Embargo:
Embargo: Release after 06/15/2012
Abstract:
Eukaryotic protein kinases are one of the most important classes of human proteins, and a great deal of research has focused on the development of small molecule inhibitors as biological probes for the determination of their cellular function or as therapeutics for the treatment of disease, such as cancer. The need for new selective inhibitors and a better understanding of the selectivities of existing small molecules is readily apparent. Towards the goal of better understanding protein kinases and the molecules that inhibit them, I have developed a split-protein-based approach for the investigation of these kinase-small molecule interactions. Employing split-firefly luciferase as a reporter domain, we engineered a three-hybrid system capable of determining kinase inhibition through competitive interactions between an active site-directed ligand and a small molecule of interest. This method measures luciferase activity as a function of ligand binding, as opposed to the more traditional assays which quantify kinase activity directly, and alleviates the laborious process of protein purification. The model kinase PKA and the promiscuous ligand staurosporine were used in an initial test case to successfully validate the general design principles of our assay. The modular nature inherent to the assay's design enabled us to adapt it to roughly 300 additional protein kinases and two different ligands. We were able to establish a protocol for rapidly ascertaining the inhibition of a kinase by a library of 80 commercially available kinase inhibitors in a 96-well, high-throughput format. This protocol was then systematically applied to the AGC group of kinases to observe patterns of inhibition across similarly related kinases. We have further shown how these results might be correlated with the sequence identity between kinases to better anticipate inhibitor promiscuity. Finally, we were able to illustrate how a kinase-centric approach could be applied to correlate alterations to the kinase domain with changes in luminescence. This has use for the interrogation of different modes of inhibition as well as in identifying the specific determinants of inhibitor binding. In total, these efforts represent the optimization of a new, general platform for determining kinase inhibitor selectivity across the kinome, and it could potentially be applied universally to the interrogation of protein-ligand interactions.
Type:
text; Electronic Dissertation
Keywords:
split-protein complementation; three-hybrid; Chemistry; inhibitor selectivity; protein kinase
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Ghosh, Indraneel

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleDevelopment of a Three-Hybrid Split-Luciferase System for Interrogating Protein Kinase Inhibitionen_US
dc.creatorJester, Benjaminen_US
dc.contributor.authorJester, Benjaminen_US
dc.date.issued2011-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.releaseEmbargo: Release after 06/15/2012en_US
dc.description.abstractEukaryotic protein kinases are one of the most important classes of human proteins, and a great deal of research has focused on the development of small molecule inhibitors as biological probes for the determination of their cellular function or as therapeutics for the treatment of disease, such as cancer. The need for new selective inhibitors and a better understanding of the selectivities of existing small molecules is readily apparent. Towards the goal of better understanding protein kinases and the molecules that inhibit them, I have developed a split-protein-based approach for the investigation of these kinase-small molecule interactions. Employing split-firefly luciferase as a reporter domain, we engineered a three-hybrid system capable of determining kinase inhibition through competitive interactions between an active site-directed ligand and a small molecule of interest. This method measures luciferase activity as a function of ligand binding, as opposed to the more traditional assays which quantify kinase activity directly, and alleviates the laborious process of protein purification. The model kinase PKA and the promiscuous ligand staurosporine were used in an initial test case to successfully validate the general design principles of our assay. The modular nature inherent to the assay's design enabled us to adapt it to roughly 300 additional protein kinases and two different ligands. We were able to establish a protocol for rapidly ascertaining the inhibition of a kinase by a library of 80 commercially available kinase inhibitors in a 96-well, high-throughput format. This protocol was then systematically applied to the AGC group of kinases to observe patterns of inhibition across similarly related kinases. We have further shown how these results might be correlated with the sequence identity between kinases to better anticipate inhibitor promiscuity. Finally, we were able to illustrate how a kinase-centric approach could be applied to correlate alterations to the kinase domain with changes in luminescence. This has use for the interrogation of different modes of inhibition as well as in identifying the specific determinants of inhibitor binding. In total, these efforts represent the optimization of a new, general platform for determining kinase inhibitor selectivity across the kinome, and it could potentially be applied universally to the interrogation of protein-ligand interactions.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectsplit-protein complementationen_US
dc.subjectthree-hybriden_US
dc.subjectChemistryen_US
dc.subjectinhibitor selectivityen_US
dc.subjectprotein kinaseen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorGhosh, Indraneelen_US
dc.contributor.committeememberAspinwall, Craig A.en_US
dc.contributor.committeememberBandarian, Vaheen_US
dc.contributor.committeememberMontfort, Williamen_US
dc.contributor.committeememberGhosh, Indraneelen_US
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