Development of Potent and Selective Bivalent Inhibitors for Protein Kinases Utilizing Phage Display

Persistent Link:
http://hdl.handle.net/10150/265559
Title:
Development of Potent and Selective Bivalent Inhibitors for Protein Kinases Utilizing Phage Display
Author:
Lamba, Vandana
Issue Date:
2012
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:
Release after 20-Nov-2013
Abstract:
Protein kinases function as key regulators in a variety of signaling pathways by executing the phosphorylation of a variety of protein substrates. Perturbation in the activity of numerous proteins kinases has been implicated in a large number of diseases including cancer, diabetes, inflammation and neurological disorders. Therefore, selective modulation of kinase activity is highly desirable for the dissection of complex signaling pathways and substantiating therapeutic targets. To develop potent and selective inhibitors for an array of kinases, our group has developed a fragment based bivalent methodology utilizing phage display. The strategy involves an ATP active site targeted small molecule which directs the selection of cyclic peptides, from a phage displayed library, on the target kinase surface through coiled coil interactions. The selected cyclic peptides can be conjugated to the ATP mimetic to generate bivalent inhibitors. In this thesis, I have expanded the scope of the bivalent phage-display selection approach. To interrogate the generality of this approach, we targeted several kinases from different groups within the human kinome using the staurosporine warhead. Fyn and PDGFRβ represented the tyrosine kinase group and CLK2 and Pim-1 kinases represented the CMGC and CaMK groups respectively. The selections against these four kinases did not result in potent inhibitors though they provided an avenue for the refinement of the bivalent phage-display approach as well as method development. Application of this methodology to AKT2 in the AGC family resulted in bivalent inhibitors which were interrogated for their selectivity and mode of action. The bivalent strategy was further explored for its utility to target inactive kinases, and success was achieved against AKT1. Finally, we demonstrated the modularity of ATP site targeted ligand by carrying out a selection against STK33 kinase using a new small molecule warhead, sunitinib. This resulted in potent and selective bivalent inhibitors for STK33. The use of different ATP site targeting molecules potentially increases the number of targetable kinases with our strategy. In all the selections, the identified cyclic peptides inhibited the kinase and showed a non-competitive mode of inhibition with respect to the kinase substrate. This suggests that the selected peptides do not target the substrate site and possibly bind to unidentified pockets on the kinase surface, which potentially provides new methods to target kinases outside the traditional ATP binding cleft. The strategy may prove to be a robust method to discover new allosteric sites on kinases as well as other proteins. The potent and selective bivalent inhibitors obtained by our strategy have the potential to provide insight towards the design of new non-ATP targeted approaches for inhibiting protein kinases and elucidating their specific functions.
Type:
text; Electronic Dissertation
Keywords:
Protein Kinases; Chemistry; Bivalent Inhibitors; Phage Display
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 Potent and Selective Bivalent Inhibitors for Protein Kinases Utilizing Phage Displayen_US
dc.creatorLamba, Vandanaen_US
dc.contributor.authorLamba, Vandanaen_US
dc.date.issued2012-
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.releaseRelease after 20-Nov-2013en_US
dc.description.abstractProtein kinases function as key regulators in a variety of signaling pathways by executing the phosphorylation of a variety of protein substrates. Perturbation in the activity of numerous proteins kinases has been implicated in a large number of diseases including cancer, diabetes, inflammation and neurological disorders. Therefore, selective modulation of kinase activity is highly desirable for the dissection of complex signaling pathways and substantiating therapeutic targets. To develop potent and selective inhibitors for an array of kinases, our group has developed a fragment based bivalent methodology utilizing phage display. The strategy involves an ATP active site targeted small molecule which directs the selection of cyclic peptides, from a phage displayed library, on the target kinase surface through coiled coil interactions. The selected cyclic peptides can be conjugated to the ATP mimetic to generate bivalent inhibitors. In this thesis, I have expanded the scope of the bivalent phage-display selection approach. To interrogate the generality of this approach, we targeted several kinases from different groups within the human kinome using the staurosporine warhead. Fyn and PDGFRβ represented the tyrosine kinase group and CLK2 and Pim-1 kinases represented the CMGC and CaMK groups respectively. The selections against these four kinases did not result in potent inhibitors though they provided an avenue for the refinement of the bivalent phage-display approach as well as method development. Application of this methodology to AKT2 in the AGC family resulted in bivalent inhibitors which were interrogated for their selectivity and mode of action. The bivalent strategy was further explored for its utility to target inactive kinases, and success was achieved against AKT1. Finally, we demonstrated the modularity of ATP site targeted ligand by carrying out a selection against STK33 kinase using a new small molecule warhead, sunitinib. This resulted in potent and selective bivalent inhibitors for STK33. The use of different ATP site targeting molecules potentially increases the number of targetable kinases with our strategy. In all the selections, the identified cyclic peptides inhibited the kinase and showed a non-competitive mode of inhibition with respect to the kinase substrate. This suggests that the selected peptides do not target the substrate site and possibly bind to unidentified pockets on the kinase surface, which potentially provides new methods to target kinases outside the traditional ATP binding cleft. The strategy may prove to be a robust method to discover new allosteric sites on kinases as well as other proteins. The potent and selective bivalent inhibitors obtained by our strategy have the potential to provide insight towards the design of new non-ATP targeted approaches for inhibiting protein kinases and elucidating their specific functions.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectProtein Kinasesen_US
dc.subjectChemistryen_US
dc.subjectBivalent Inhibitorsen_US
dc.subjectPhage Displayen_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.committeememberChristie, Hamishen_US
dc.contributor.committeememberMonti, Oliveren_US
dc.contributor.committeememberBandarian, Vaheen_US
dc.contributor.committeememberGhosh, Indraneelen_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.