Design, Development, and Evaluation of Tools to Study Cellular ADP-ribose Polymer Metabolism

Persistent Link:
http://hdl.handle.net/10150/202973
Title:
Design, Development, and Evaluation of Tools to Study Cellular ADP-ribose Polymer Metabolism
Author:
Steffen, Jamin D.
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.
Abstract:
The metabolism of ADP-ribose polymers (PAR) is involved in several cellular processes with a primary focus on maintaining genomic integrity. PAR metabolism following genotoxic stress is transient due to a close coordination between poly(ADP-ribose) polymerases (PARPs) which synthesize PAR and poly(ADP-ribose) glycohydrolase (PARG) which degrades PAR. PARP-1 inhibitors have emerged as promising anticancer therapeutics by increasing chemotherapy sensitivity and selectively target tumors harboring DNA repair defects. Several pharmaceutical companies have PARP-1 inhibitors in clinical trials for treatment of cancer. PARP-1 inhibitors are generally well tolerated, although they typically have poor selectivity among PARPs, and potentially other NAD binding enzymes. The promise of PARP-1 inhibitors as cancer therapeutics has led this dissertation research towards developing alternative tools and approaches to target PAR metabolism.One approach described is an evaluation of high-throughput PARP-1 screening assays as potential tools to discover new classes of PARP-1 inhibitors. These assays were compared to a widely used radiolabeling PARP-1 assay. They were found to offer several advantages that include simplicity, sensitivity, reproducibility, accuracy and eliminating the need for radioactive materials.The primary focus of this dissertation research was to develop PARG inhibitors as an alternative way of targeting PAR metabolism. Lack of viable genetically engineered animals, effective siRNA, and useful pharmacological 20 inhibitors has prevented PARG from being evaluated as a therapeutic target. This dissertation describes the first systematic approach, using Target related Affinity Profiling (TRAP) technology, for the discovery of PARG inhibitors. Identification of several hits led to the first detailed structural activity relationship (SAR) studies defining a pharmacophore for PARG inhibition. Interestingly, these molecules show varying degrees of PARP-1 inhibition, providing the first direct evidence for homology in the active sites of PARP-1 and PARG. Evaluation of a lead inhibitor has provided the first evidence for PARG inhibition in intact cells. Further optimization resulted in a cell permeable inhibitor with reduced toxicity and poor selectivity, providing evidence for a new class of inhibitors that disrupt PAR metabolism by inhibiting both enzymes. The use of dual PARG/PARP-1 inhibitors represents a new approach for therapeutic development of anticancer agents. Finally, directions aimed to overcome remaining challenges are discussed.
Type:
text; Electronic Dissertation
Keywords:
PARP-1; pthalamic acids; salicylanilides; structure-activity relationships; Pharmaceutical Sciences; ADP-ribose; PARG
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Pharmaceutical Sciences
Degree Grantor:
University of Arizona
Advisor:
Jacobson, Myron K.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleDesign, Development, and Evaluation of Tools to Study Cellular ADP-ribose Polymer Metabolismen_US
dc.creatorSteffen, Jamin D.en_US
dc.contributor.authorSteffen, Jamin D.en_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.abstractThe metabolism of ADP-ribose polymers (PAR) is involved in several cellular processes with a primary focus on maintaining genomic integrity. PAR metabolism following genotoxic stress is transient due to a close coordination between poly(ADP-ribose) polymerases (PARPs) which synthesize PAR and poly(ADP-ribose) glycohydrolase (PARG) which degrades PAR. PARP-1 inhibitors have emerged as promising anticancer therapeutics by increasing chemotherapy sensitivity and selectively target tumors harboring DNA repair defects. Several pharmaceutical companies have PARP-1 inhibitors in clinical trials for treatment of cancer. PARP-1 inhibitors are generally well tolerated, although they typically have poor selectivity among PARPs, and potentially other NAD binding enzymes. The promise of PARP-1 inhibitors as cancer therapeutics has led this dissertation research towards developing alternative tools and approaches to target PAR metabolism.One approach described is an evaluation of high-throughput PARP-1 screening assays as potential tools to discover new classes of PARP-1 inhibitors. These assays were compared to a widely used radiolabeling PARP-1 assay. They were found to offer several advantages that include simplicity, sensitivity, reproducibility, accuracy and eliminating the need for radioactive materials.The primary focus of this dissertation research was to develop PARG inhibitors as an alternative way of targeting PAR metabolism. Lack of viable genetically engineered animals, effective siRNA, and useful pharmacological 20 inhibitors has prevented PARG from being evaluated as a therapeutic target. This dissertation describes the first systematic approach, using Target related Affinity Profiling (TRAP) technology, for the discovery of PARG inhibitors. Identification of several hits led to the first detailed structural activity relationship (SAR) studies defining a pharmacophore for PARG inhibition. Interestingly, these molecules show varying degrees of PARP-1 inhibition, providing the first direct evidence for homology in the active sites of PARP-1 and PARG. Evaluation of a lead inhibitor has provided the first evidence for PARG inhibition in intact cells. Further optimization resulted in a cell permeable inhibitor with reduced toxicity and poor selectivity, providing evidence for a new class of inhibitors that disrupt PAR metabolism by inhibiting both enzymes. The use of dual PARG/PARP-1 inhibitors represents a new approach for therapeutic development of anticancer agents. Finally, directions aimed to overcome remaining challenges are discussed.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectPARP-1en_US
dc.subjectpthalamic acidsen_US
dc.subjectsalicylanilidesen_US
dc.subjectstructure-activity relationshipsen_US
dc.subjectPharmaceutical Sciencesen_US
dc.subjectADP-riboseen_US
dc.subjectPARGen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplinePharmaceutical Sciencesen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorJacobson, Myron K.en_US
dc.contributor.committeememberHulme, Christopheren_US
dc.contributor.committeememberWondrak, Georg T.en_US
dc.contributor.committeememberHorton, Nancy C.en_US
dc.contributor.committeememberPolt, Robin L.en_US
dc.contributor.committeememberJacobson, Myron K.en_US
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