Characterization and Exploitation of Bidirectional Allosteric Coupling in Multi-Domain Tyrosine Kinases using Conformation-Selective ATP-Competitive Inhibitors

Thesis (Ph.D.)--University of Washington, 2016-08Protein kinases are a large family of enzymes that play integral roles in cell signaling networks and are thus critical for effecting appropriate cellular responses to environmental stimuli. Much of kinase biological function has been studied in terms of catalytic activity: phosphorylation of substrate proteins as part of signaling cascades. However, recent evidence has shown that kinases play many important non-catalytic functions such as DNA-binding, scaffolding, and participating in a variety of physiologically relevant protein-protein interactions. While critical, these roles have not been thoroughly explored, in large part due to limited availability of selective ATP-competitive inhibitors. Selectivity for a specific kinase is difficult to achieve due to high structural homology between the ATP-binding sites of the 518 human kinases. Additionally, it has been shown over the past two decades that it is possible to stabilize structurally distinct ATP-binding site conformations using conformation-selective inhibitors, termed Type I and Type II inhibitors, in many kinases. In several cases, inhibition of kinase ATP-binding sites with Type I or Type II inhibitors has been shown to divergently affect cell signaling events as a result of allosteric coupling between important structural features in the ATP-binding site and distal protein-protein interaction sites on the inhibited kinase. Thus, it is important not only to build selective ATP-competitive inhibitors but to understand how their binding affects global kinase conformation through allosteric coupling. This thesis describes my work characterizing allosteric networks in multi-domain tyrosine kinases (Src-Family Kinases (SFKs) and Abl) using conformation-selective inhibitors as well as developing a method for using conformation-selective inhibitors in cells to better understand how non-catalytic function of a kinase of interest determines its role in cell signaling networks

Bioassay Development for Bispecific Antibodies—Challenges and Opportunities

Antibody therapeutics are expanding with promising clinical outcomes, and diverse formats of antibodies are further developed and available for patients of the most challenging disease areas. Bispecific antibodies (BsAbs) have several significant advantages over monospecific antibodies by engaging two antigen targets. Due to the complicated mechanism of action, diverse structural variations, and dual-target binding, developing bioassays and other types of assays to characterize BsAbs is challenging. Developing bioassays for BsAbs requires a good understanding of the mechanism of action of the molecule, principles and applications of different bioanalytical methods, and phase-appropriate considerations per regulatory guidelines. Here, we review recent advances and case studies to provide strategies and insights for bioassay development for different types of bispecific molecules

Targeting ABL-IRE1α Signaling Spares ER-Stressed Pancreatic β Cells to Reverse Autoimmune Diabetes.

(Cell Metabolism 25, 883–897; April 4, 2017) In the originally published version of this article, the immunoblot image of the HDAC1 nuclear extract protein control in Figure 3I was incorrectly cropped such that it included one extraneous lane. The corrected and original versions of Figure 3I are shown here. Furthermore, in the Discussion, after the sentence “Such compensatory, dysregulated UPR effects may be general as Perk deletion, likewise, hyperactivates IRE1α in β cells, which suffer early apoptosis, leading to postnatal diabetes,” we incorrectly cited Harding, H.P., and Ron, D. (2002). Endoplasmic reticulum stress and the development of diabetes: a review. Diabetes 51, S455–S461. The correct citation is: Harding, H.P., Zeng, H., Zhang, Y., Jungries, R., Chung, P., Plesken, H., Sabatini, D.D., and Ron, D. (2001). Diabetes mellitus and exocrine pancreatic dysfunction in perk−/− mice reveals a role for translational control in secretory cell survival. Molecular Cell 7, 1153–1163. The authors apologize for any confusion these errors may have caused. [Figure presented

Targeting ABL-IRE1α Signaling Spares ER-Stressed Pancreatic β Cells to Reverse Autoimmune Diabetes.

In cells experiencing unrelieved endoplasmic reticulum (ER) stress, the ER transmembrane kinase/endoribonuclease (RNase)-IRE1α-endonucleolytically degrades ER-localized mRNAs to promote apoptosis. Here we find that the ABL family of tyrosine kinases rheostatically enhances IRE1α's enzymatic activities, thereby potentiating ER stress-induced apoptosis. During ER stress, cytosolic ABL kinases localize to the ER membrane, where they bind, scaffold, and hyperactivate IRE1α's RNase. Imatinib-an anti-cancer tyrosine kinase inhibitor-antagonizes the ABL-IRE1α interaction, blunts IRE1α RNase hyperactivity, reduces pancreatic β cell apoptosis, and reverses type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. A mono-selective kinase inhibitor that allosterically attenuates IRE1α's RNase-KIRA8-also efficaciously reverses established diabetes in NOD mice by sparing β cells and preserving their physiological function. Our data support a model wherein ER-stressed β cells contribute to their own demise during T1D pathogenesis and implicate the ABL-IRE1α axis as a drug target for the treatment of an autoimmune disease

Mendelian randomization: can genetic epidemiology help redress the failures of observational epidemiology?

Establishing causal relationships between environmental exposures and common diseases is beset with problems of unresolved confounding, reverse causation and selection bias that may result in spurious inferences. Mendelian randomization, in which a functional genetic variant acts as a proxy for an environmental exposure, provides a means of overcoming these problems as the inheritance of genetic variants is independent of-that is randomized with respect to-the inheritance of other traits, according to Mendel's law of independent assortment. Examples drawn from exposures and outcomes as diverse as milk and osteoporosis, alcohol and coronary heart disease, sheep dip and farm workers' compensation neurosis, folate and neural tube defects are used to illustrate the applications of Mendelian randomization approaches in assessing potential environmental causes of disease. As with all genetic epidemiology studies there are problems associated with the need for large sample sizes, the non-replication of findings, and the lack of relevant functional genetic variants. In addition to these problems, Mendelian randomization findings may be confounded by other genetic variants in linkage disequilibrium with the variant under study, or by population stratification. Furthermore, pleiotropy of effect of a genetic variant may result in null associations, as may canalisation of genetic effects. If correctly conducted and carefully interpreted, Mendelian randomization studies can provide useful evidence to support or reject causal hypotheses linking environmental exposures to common diseases