1,721,013 research outputs found
Label Transfer Reagents for the Investigation of Protein Kinase Complexes
Get PDFThesis (Ph.D.)--University of Washington, 2012Protein kinases are essential enzymes for cellular signaling, and are often regulated by participation in protein complexes. The mitogen-activated protein kinase (MAPK) p38 is involved in multiple pathways, and its regulation depends on its interactions with other signaling proteins. However, the weak and transient nature of these interactions makes the identification of p38 interacting proteins challenging. For this reason, we have developed label transfer reagents (LTRs) which allow labeling of p38 signaling complexes. These LTRs leverage the potency and selectivity of known p38 inhibitors to place a photo-crosslinker and tag in the vicinity of p38 and its binding partners. Upon UV irradiation, proteins that are in close proximity to p38 are covalently crosslinked, and labeled proteins are detected and/or purified through an orthogonal chemical handle. Here we demonstrate that p38-selective LTRs efficiently label a diversity of p38 binding partners, including substrates and activators. Furthermore, these LTRs can be used in immunoprecipitations for study of proteins not exogenously expressible. Several limitations of LTRs are also explored. Finally, copper-catalyzed click chemistry is optimized in a quantitative fashion for the labeling and purification of alkynylated proteins
Dynamic control of intracellular signaling and genome engineering in space and time
Get PDFThesis (Ph.D.)--University of Washington, 2017-06Cells continuously sense both their external and internal environments, integrate diverse and often conflicting information, and respond. The potential responses are remarkably varied: it could be to kill an invading pathogen, repair damaged DNA, proliferate to heal a wound, or undergo programmed cell death. The process of sensing, integrating, and responding to information is carried about by complex biochemical networks within the cell. These cellular networks dictate a cell’s behavior, function, and identity. Moreover, dysregulation of these networks is a hallmark of diseases as prevalent and damaging as cancer and diabetes. It is imperative that we advance our understanding of these cellular networks in order to improve treatments for when these networks go awry. Unfortunately, our ability to adequately investigate these networks is hampered by their sheer complexity. Inherent to cellular networks are multiple layers of complexity: they are complex in time, in space, and in their architecture (i.e. the arrangement of the interactions that comprise the network). Recently, high-throughput methods have greatly improved our ability to observe these networks, allowing the characterization of thousands of genes or proteins in a single experiment. Yet, our ability to interrogate cellular networks has not kept pace with our ability to observe them. As a result, studies often yield conclusions which are largely phenomenological with limited mechanistic insight. Mechanistic insights are critical to developing novel therapies or better utilizing existing ones. To gain such insights would require tools that confer tunable control of individual network components with spatial and temporal precision, allowing systematic dissection of a network. Up to this point, the development of such tools, generally engineered proteins that are controlled by small molecules or light, has been hampered by the reliance on empirical protein engineering strategies that are inefficient, arduous, and costly. To address this outstanding problem, we developed a computational framework for the systematic design of small-molecule controlled proteins. This framework utilizes protein design tools developed by David Baker’s lab, and we show it greatly expedites the development process. Using this framework, we engineered Chemically Inducible Activator of RAS (CIAR), which enables tunable, spatiotemporally precise control of RAS activation. RAS is frequently hyperactivated in human cancers, and using CIAR we characterized dynamic features or RAS biology which were inaccessible to previous methods. For instance, we found that RAS signaling kinetics differ between cell lines, which may reflect the divergent propensities of different cell types to develop RAS-driven cancers. We also show that RAS signaling can be rewired by small molecule inhibitors currently used to treat melanoma, which sheds light on a phenomenon where this drug actually promotes growth of secondary cancers. More recently, we have demonstrated that we can control RAS activation at different subcellular locations, such as the Golgi. It has been suggested that the subcellular location of RAS activation can dictate phenotypic outcomes, including whether a developing T-cell proliferates or undergoes apoptosis. CIAR will enable unprecedented examinations of such phenomena. In parallel, we took a similar approach to study the processes involved in genome engineering. The development of CRISPR/Cas9 for genome editing has led to intense interest in both its potential research and therapeutic applications. Yet, little is known regarding the dynamics of Cas9-mediated DNA cleavage and subsequent DNA repair. Using an approach analogous to that used to develop CIAR, we engineered a rapidly inducible Cas9 variant, chemically-inducible Cas9. Additionally, we developed the first assay for quantitative, temporally-resolved monitoring of double-strand breaks (DSBs), DSB-ddPCR. Using these two technologies, we conducted a first-ever examination of Cas9-mediated DNA cleavage and repair dynamics. We found that Cas9 cleavage is rapid, and that both cleavage and repair kinetics differ between loci. We envision these technologies will enable in-depth examinations of this heretofore unexplored region of CRISPR/Cas9 biology, with potential implications for its applications in research and the clinic. Taken together, the technologies developed in the course of this dissertation demonstrate the utility of precision tools for the study of complex cellular networks. CIAR and ciCas9 have already begun to yield insights into intracellular signaling and genome engineering and are the foundation for several ongoing investigations. Beyond these specific tools, the design methods and engineering strategies we have devised may have greater impact. It is our hope that they will aid the development of future technologies to probe the inner workings of our cells, deepening our understanding of the networks which drive human health and disease
Chemical Tools for Profiling the Allosteric Regulation and Interactomes of Inhibitor-Bound Protein Kinases
No full textThesis (Ph.D.)--University of Washington, 2020This dissertation describes the development of a chemoproteomic method for profiling the interaction network of inhibitor-bound kinase complexes and chemical probes for understanding how ATP-competitive inhibitors can allosterically modulate tyrosine kinases that contain a Src-like regulatory architecture. Small molecule inhibitors often only block a subset of the cellular functions of their protein targets. In many cases, how inhibiting only a portion of a multifunctional protein’s functions affects the state of the cell is not well understood. Therefore, tools that allow the systematic characterization of the cellular interactions that inhibitor-bound proteins make would be of great utility, especially for multifunctional proteins. In the second chapter of this thesis, I describe a chemoproteomic strategy for interrogating the cellular localization and interactomes of inhibitor-bound kinases. By developing a set of orthogonal inhibitors that contain a trans-cyclooctene (TCO) click handle, we are able to enrich and characterize the proteins complexed to a drug-sensitized variant of the multidomain kinase Src. We show that Src’s cellular interactions are highly influenced by the intermolecular accessibility of its regulatory domains, which can be allosterically modulated through its ATP-binding site. Furthermore, we find that the signaling status of the cell also has a large effect on Src’s interactome. Finally, we demonstrate that our TCO-conjugated probes can be used as a part of a proximity ligation assay to study Src’s localization and interactions in situ. Together, our chemoproteomic strategy represents a comprehensive method for studying the localization and interactomes of inhibitor-bound kinases and, potentially, other druggable protein targets. Small molecule kinase inhibitors that stabilize distinct ATP-binding site conformations can differentially modulate the global conformation of Src-family kinases (SFKs). However, it is unclear which specific ATP-binding site contacts are responsible for modulating the global conformation of SFKs and whether these inhibitor-mediated allosteric effects are general to other tyrosine kinases. In the third chapter of this thesis, I describe the development of chemical probes that allow us to deconvolute which features in the ATP-binding site are responsible for the allosteric modulation of the global conformation of Src. We find that the ability of an inhibitor to modulate the global conformation of Src’s regulatory domain-catalytic domain module relies mainly on the influence it has on the conformation of a structural element called helix C. Furthermore, by developing a set of orthogonal probes that target a drug-sensitized Src variant, we show that stabilizing Src’s helix C in an active conformation is sufficient to promote a Src-mediated, phosphotransferase-independent alteration in cell morphology. Finally, we report that ATP-competitive, conformation-selective inhibitors can influence the global conformation of tyrosine kinases beyond the SFKs, suggesting that the allosteric networks we observe in Src are conserved in kinases that have a similar regulatory architecture. Taken together, our study highlights that an ATP-competitive inhibitor’s interactions with helix C can have a major influence on the global conformation of some tyrosine kinases in vitro and in cells
Divergent Allosteric Control of IRE1alpha endoribonuclease using small molecule kinase inhibitors
No full textThesis (Master's)--University of Washington, 2014When unfolded proteins get accumulated in the endoplasmic reticulum (ER), signaling pathways called the unfolded protein response (UPR) get turned on in the cell. However under conditions of chronic ER stress, the cell undergoes apoptosis. Key events of this "Terminal UPR" are controlled by IRE1α - an ER bifunctional kinase/endoribonuclease (RNase), which when hyperactivated, oligomerizes causing widespread endonucleolytic decay of ER-localized mRNAs and repressive micro-RNA precursors triggering cell death. Somatic mutations of IRE1α found in human cancers prevent oligomerization and inhibit apoptosis caused by the RNase. Using these results, our lab developed an array of ATP-competitive kinase inhibitors - called KIRAs (Kinase Inhibiting RNase Attenuators) - that inhibit oligomerization of the IRE1α kinase domain and allosterically inhibit the RNase. In this work we have made efforts to expand the existing panel of KIRAs, looking to achieve greater potency and selectivity towards IRE1α and understand IRE1α mechanism of action
Understanding Src Kinase Regulatory Mechanisms and Drug Resistance Using Deep Mutational Scanning and Chemical Biology
No full textThesis (Ph.D.)--University of Washington, 2020Protein phosphorylation controls a wide variety of cellular processes such as growth, differentiation, proliferation, and apoptosis in eukaryotes. Kinases are signaling enzymes that dictate cellular phosphorylation state by phosphorylating specific protein substrates. Over 530 protein kinases are encoded by the human genome and roughly half of these enzymes have at least one accessory domain in addition to the catalytic domain. The Src Family Kinases (SFKs) are a well-studied family of multi-domain, non-receptor tyrosine kinases. SFKs participate in numerous signal transduction pathways, and their misregulation is implicated in a variety of diseases, including cancer. Therefore, SFKs are of general interest as a model for understanding multi-domain kinase regulation and as potential drug targets. In this thesis, I describe my efforts to use the SFKs as models for understanding inhibitor selectivity and drug resistance, which are major challenges in the field. I show that it is possible to develop ATP-competitive inhibitors that are highly selective for the SFK Lyn over other members of the SFKs. Obtaining such selectivity is significant because the ATP-binding sites of SFKs are almost identical and obtaining selectivity amongst such closely related kinases has proven to be particularly challenging. I show that it is possible to achieve selectivity for Lyn by developing inhibitors that target a region called the helix C. With a series of sequence swap experiments, I demonstrate that sensitivity to these Lyn-selective inhibitors is due to the identity of the linker residues that control the conformational flexibility of helix C rather than any specific ATP-binding site interactions. Our strategy may hold promise for selectivity targeting other protein kinases. I also describe efforts to better understand how kinases develop resistance to ATP-competitive inhibitors. To do this, we used saturation mutagenesis and a yeast screening assay to comprehensively identify sites of ATP-competitive inhibitor resistance in Src. Using this methodology, resistance to the drug dasatinib and to a panel of conformation-selective, ATP-competitive inhibitors was profiled. Our efforts led to identification of mutations that provide general resistance to ATP-competitive inhibitors and mutations that uniquely affect specific modes of ATP-competitive inhibition. Interestingly, some of the strongest resistance mutations identified do not directly affect inhibitor binding but, instead, appear to confer resistance through other mechanisms. I describe comprehensive biochemical and biophysical analyses to better understand how a region on the N-terminal lobe of Src’s catalytic domain, which was previously identified as participating in the regulation of Src through an unknown mechanism, confers broad resistance. As a major challenge in the field of kinase drug discovery is the emergence of drug resistance, our efforts may help better inform the development of chemotherapeutic treatments that are less prone to resistance
A high-throughput screening assay based on TR-FRET to identify inhibitors for T. brucei kinases
No full textThesis (Master's)--University of Washington, 2018African Sleeping Sickness or Human African Trypanosomiasis (HAT) is one of the most under studied tropical diseases and continues to be a threat to more than 65 million people in sub-Saharan Africa. It is caused by the bite of a tsetse fly infected with a trypanosome known as Trypanosoma brucei. Therapeutic advances have been made in combating the disease, but a high mortality rate of 5%, and the unavailability of treatments for later stages, begets a need for more effective therapeutics. Because kinases are attractive drug targets, we explored the druggability of three Trypanosoma brucei kinases that have been demonstrated to be essential for the survival of the trypanosome, and thus potential drug targets for HAT. Here, we describe a TR-FRET-based assay for the three kinases to identify and confirm inhibitor hits from a kinase inhibitor library of 429 compounds. In our screen, we identified inhibitors belonging to the type II class of kinase inhibitors, with low nanomolar potency (IC50) against several of these three essential kinases. These inhibitors provide new hits for lead optimization with the aim of identifying new drug treatments for HAT
Development of Lysine-Targeted Probes for Protein Kinases
No full textThesis (Master's)--University of Washington, 2022Lysine is one of the most common amino acids in the proteome, but relatively few probes have been designed to label specific lysine. Properties of lysine such as its low intrinsic nucleophilicity and its ubiquity has made it a challenge to develop probes that label active site lysine in protein kinases. In this thesis, I describe the design, synthesis, and testing of three different lysine-targeted type I kinases probes based on different scaffolds. Two of these probes are fluorosulfates (Probe 1 and Probe 2) displayed from either a quinazoline or pyridine-pyrimidine scaffold. The third (Probe 3) is a sulfonyl fluoride probe with a trans- cyclooctyne (TCO) click handle, which is based on the previously developed probe XO44 probe. Based on lysate labeling experiments and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, neither alkyne-containing nor TCO-containing versions of Probe 1 and Probe 2 are able to specifically label protein kinases. Lysate labelingexperiments followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) demonstrate that Probe 3 can specifically label at least 7 protein kinases. Together, these results represent a comprehensive analysis of lysine-targeted type I kinases probes. The supplementary spreadsheets include the whole proteomic data for Probe 3 LC/MS/MS pull-down experiment (‘Proteomic data for Probe 3’); the protein kinases with non-significant label free intensities difference between the ‘Probe 3’ group and the ‘Probe 3 + Competitor 2’ group (‘Non-significant difference PKs’); the protein kinases only contain one label free intensity in the ‘Probe 3’ group (‘Non-representative PKs’); the protein kinases labeled by Probe 3 (‘Labeled PKs’)
Development of a Small Molecule Regulated Cre Recombinase and A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors
No full textThesis (Ph.D.)--University of Washington, 2016-12Chapter 1: Development of a Small Molecule Regulated Cre Recombinase Our lab has previously developed a chemical genetic method for controlling signaling enzymes with a small molecule. In this method, the endogenous regulatory domains of an enzyme of interest are replaced with a protein-protein interaction that acts as an artificial regulatory domain. This synthetic enzyme can be controlled by a potent, selective and cell permeable small molecule that disrupts the protein-protein interaction that serves as the artificial regulatory domain. Cre (Causes Recombination) Recombinase is a site-specific tyrosine recombinase from bacteriophage P1. Cre is commonly used to facilitate conditional knock-in, knock-out and inversion studies is mammals8. This chapter describes the development of a Cre Recombinase whose activity can be controlled in cells with the small molecule-regulated switch that we previously reported. Our small molecule-regulated Cre Recombinase allows rapid, reversible, and dose-dependent activation of Cre recombinase and has the potential to be used to conditionally activate or inactivate gene expression in vivo. Applications of our small molecule-regulated Cre Recombinase and efforts to determine the mechanism of small molecule regulation are described. Chapter 2: A Chemical-Genetic Strategy for the Investigation of Kinase Non-Catalytic Function using Covalent Conformation-Selective Inhibitors Protein kinases are a large family of >530 signaling proteins that allow a cell to respond appropriately to a variety of external stimuli (Manning et. al. 2002). Most current research on protein kinases focuses on studying their catalytic activity, but recent evidence has shown that the non-catalytic role of kinases–including scaffolding and DNA binding–is essential to cell survival (Rauch et. al 2011). A major challenge in studying the non-catalytic roles of protein kinases is the lack of selective molecular tools for studying this aspect of kinase function. As a result, the non-catalytic functions of most proteins kinases are not well understood. A major focus of research in the field has been development of inhibitors that selectively bind distinct ATP-binding site conformations of kinases. Specifically, Type I inhibitors (Taylor et al, 2011), which bind catalytically active kinases, and type II inhibitors, which bind to catalytically inactive forms of the ATP-binding site (Ranjitkar et al, 2010; Ranjitkar et al, 2014; Seeliger et al, 2009; Okram et al, 2006). In many kinases, the conformation of the ATP-binding site and regulatory domains are allosterically coupled. Stabilizing different ATP-binding site forms with conformation-selective inhibitors has the potential to divergently modulate the non-catalytic roles of protein kinases in the cell (Rauch et al, 2011). The second half of this thesis describes work analyzing the divergent impacts of conformationally-selective inhibitors on the non-catalytic functions of Src Family Kinases (SFKs) in cells
Dissection of spatiotemporal intracellular signaling using engineered chemical and genetic tools
No full textThesis (Ph.D.)--University of Washington, 2020Cellular signaling proteins are responsible for transmitting intracellular signals and enacting the appropriate response to received stimuli. These messages are propagated through networks that rely on allosteric regulation, post-translational modifications, or other mechanisms for modulating signaling protein activity. Scientists seeking to engineer systems that mimic endogenous pathways must be able to control and precisely tune the spatiotemporal activity of the proteins involved in these complex networks. This thesis describes the development of chemical genetic tools that specifically control individual signaling nodes in a cell using three distinct approaches. The first approach utilizes conformation-selective inhibitors that modulate the allosteric regulation of Src Family Kinases (SFKs). SFKs are multi-domain kinases that transmit cellular signals through their catalytic and non-catalytic activities. Although dysregulation of kinases is often implicated in human diseases, studying individual kinases is difficult due to their conserved catalytic domain. By combining genetics with small molecule probe development, we created potent and specific kinase inhibitors that are capable of allosterically controlling the intramolecular regulation of an individual kinase. We applied this strategy to the SFKs and studied the phenotypic effects that resulted from conformation-selective inhibition. The second approach employed a small molecule-controlled, genetically-encoded rheostatic switch for studying signaling mediated by localized pools of RAS. In the final approach, a second-generation chemically-disrupted proximity (CDP) system was developed. This optimized CDP system can be incorporated into numerous chemical genetic systems that rely on intramolecular and intermolecular regulation. Together, these three approaches represent a broad platform for interrogating and engineering diverse cellular processes
Chemical Proteomic Tools for Studying Protein Kinase Active Sites
Get PDFThesis (Ph.D.)--University of Washington, 2012Protein kinases constitute one of the largest protein families in humans. These enzymes catalyze phosphorylation of serine, threonine or tyrosine residues in their protein substrates. As protein kinases regulate most signal transduction pathways in cells and play important roles in many cellular functions, deregulation of their activity can lead to a number of diseases including cancer, diabetes and inflammation. Targeted inhibition of protein kinases has therefore become an attractive therapeutic strategy for the treatment of a number of diseases. Small molecule inhibitors that target the active site of protein kinases can be used to study their catalytic function and regulation. We have designed and generated a set of small molecule ligands that bind to protein kinase active sites in a conformation–specific manner. These ligands have been used as proteomic tools to study the active sites of a wide range of protein kinases. These efforts have been particularly focused on a class of ligands, type II inhibitors, which stabilize an inactive conformation of the ATP–binding site, called the DFG–out. We have shown that type II inhibitors can be extensively modified and that these reagents can be used in a range of proteomic applications. These proteomic efforts have provided insight into the roles of specific kinases during signaling events. Furthermore, these chemical tools have significantly contributed towards our understanding of the structure and regulation of protein kinases
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