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Targeted covalent inhibitors have had a major impact on human health and disease. In the past decade, several drugs with a covalent mechanism of action have been approved for the treatment of various cancers. Unfortunately, due to the lack of chemical diversity found naturally in protein biologics, we have been limited to designing covalent inhibitors that are small molecules or peptides. In recent years, however, through the use of genetic code expansion, we have been able to genetically encode new functionalities into proteins. These new functionalities include incorporating bioreactive unnatural amino acids (Uaas) into proteins, which allow for the formation of covalent bonds between binding proteins and their targets. Developing covalent biologics has great implications for the development of novel therapeutics and diagnostic tools. Chapter 1 introduces the reader to the recent chemical advancement of encoding bioreactive Uaas into proteins as well as successful development of anticancer and antiviral covalent protein drugs. Here, we describe the development of a new electrophilic bioreactive Uaa as well as two applications of protein binders with covalent mechanisms. In chapter 2, we describe the development of genetically encoding a SuFEx reactive meta-fluorosulfate tyrosine (mFSY) into proteins to expand the toolbox of bioreactive Uaas. mFSY has the ability to covalently target different side chain orientations as compared to known fluorosulfate Uaas fluorosulfate-L-tyrosine (FSY) and fluorosulfonyloxy-benzoyl-L-lysine (FSK). Chapter 3 describes the application of covalent biologics for targeted radionuclide therapies. We develop a covalent nanobody that binds to its target irreversibly, achieving exceptional tumor uptake, fast blood clearance, and low background. This radiolabeled covalent nanobody markedly increases radioisotope levels in tumors and extends tumor residence time while maintaining systematic clearance. Finally, in chapter 4 we demonstrate the selectivity a covalent mechanism of action contributes to protein binding by developing a covalent neuregulin capable of specifically crosslinking with HER4; this covalent binding alters the signaling axis of HER4 in a novel and unexpected manner.

The Protein Kinase D (PRKD) family of serine/threonine kinases have been shown to contribute to a complex set of biological functions such as signal transduction, cell proliferation, differentiation, cell vesicle trafficking, secretion, polarity, and locomotion. Our lab has demonstrated that PRKD affects multiple aspects of osteoclast formation, such as cell-to-cell fusion, migration, and differentiation. Nevertheless, the specific targets of PRKD phosphorylation in osteoclasts remain poorly characterized. In this thesis, we aim to identify the targets of PRKD in osteoclast cell culture and to study the network effect of dephosphorylating those identified targets in osteoclasts. We utilized two complementary phospho-proteomic approaches: 1) Quantitative phospho-proteomics approach to establish a global view of changes in thousands of phospho-sites upon PRKD inhibition, followed by bioinformatic analysis of the functional enrichment clusters and phospho-motif enrichment analysis to identify candidate targets, 2) Targeted phospho-proteomics using PRKD recombinant kinase, followed by bioinformatic analysis of the phospho-motif enrichment and assessment of candidate targets in the context of known cellular signaling. Using the quantitative proteomics analysis of the PRKD inhibitor-treated osteoclasts cell culture, we showed that the main cellular pathways influenced by PRKD inhibition are mRNA processing, transcription, and regulation of transcription, which corresponded to the candidate targets: SRRM2, HDAC5, and JUND. Additionally, by using the targeted phospho-proteomics method, we identified two novel PRKD targets: SIPA1L3 and LSP1 which are associated with the actin cytoskeleton and cell membrane and contribute to cell motility. Subsequent validations of these findings would play an important role in better understanding the cellular regulators of osteoclast formation and bone resorption and establishing new therapeutic modalities to limit bone resorption in large numbers of patients afflicted with osteolytic conditions.

DNA-encoded library (DEL) technology is a powerful next-generation drug discovery engine. DELs permit the design and synthesis of large, customized molecular libraries, which can be mined for new ligands by affinity selection. More advanced screening technology has emerged that enables direct activity-based DEL screening, but to date, this is limited to biochemical activity assays. Cellular assays, which are vital for discovering physiologically relevant novel chemical matter, are still incompatible with DEL. Here, I describe a prospective workflow that renders DEL compatible with cellular activity assays. Cells are grown directly on DEL beads, which effectively serve as microcarriers. The cells are engineered to secrete a reporter upon stimulation with an agonist, which is liberated from the DEL bead via photocleavage. The microcarrier is functionalized with affinity tag ligands, which capture the affinity-tagged secreted reporter. Toward this goal, I have engineered cell lines that secrete HaloTag-labeled fluorescent protein reporters, shown that reporter cell lines can grow and signal on microcarriers, and explored photocleavage conditions that permit efficient photochemistry while preserving cell viability. Additional studies were conducted to identify appropriate positive control ligands of a model cellular target, STING (stimulator of interferon genes). This thesis lays the groundwork for a DEL-compatible cellular assay paradigm that can be adapted to an array of cell-based assay strategies.

The complex molecular machinery of the cell controls its response to various signals and environmental conditions. A natural approach to study these molecular mechanisms and cellular processes is with protein interaction networks. Due to the complexity of these networks, sophisticated computational techniques are required to extract biological insights from them. In this thesis, I develop and apply network-based algorithms for three different challenges. 1. I develop a novel, highly-scalable algorithm for network-based label prediction methods that enables the integration of functional annotations and interaction networks across many species in order to predict the functions of genes in newly-sequenced bacteria. 2. To overcome the limitations of experimental approaches to find human proteins and processes that are hijacked by SARS-CoV-2, I adapt network propagation approaches for predicting human interactors of the virus. 3. Large-scale experimental techniques to screen chemicals for toxicity have tested their effects on many individual proteins. I integrate human protein-protein interactions with this data to gain insights into the molecular networks those chemicals affect. For each of these research problems, I perform comprehensive evaluations and downstream analyses to demonstrate both the accuracy of our approaches and their utility in obtaining a broader understanding of the molecular systems in question.

Accumulating evidence over the past few decades has revealed that astrocytes, a type of glial cell in the brain, are more involved in active brain communication than previously thought. In addition to serving a multitude of homeostatic roles in the brain, astrocytes also participate in bidirectional communication with neurons, a phenomenon that has important consequences for brain function. One way in which astrocytes communicate with neurons is through calcium-induced release of their own signaling molecules, termed gliotransmitters. A well-studied mechanism of calcium signaling in astrocytes is G protein-coupled receptor (GPCR) signaling, particularly Gq GPCR signaling. A less-studied calcium signaling pathway in astrocytes is through Gi/o GPCR activation. In neurons, activation of Gq GPCRs and Gi/o GPCRs leads to cellular excitation and inhibition, respectively. Whether the same effects occur in astrocytes, and the consequences of such signaling, is the focus of experiments in Chapter Two. I used both an endogenous and chemogenetic approach (i.e. DREADDs) to characterize the functional consequences of Gq and Gi/o GPCR signaling pathways in neurons and astrocytes. I found that activation of Gq GPCRs led to excitation in both neurons and astrocytes. In contrast, Gi/o GPCR activation led to cellular inhibition in neurons, but activation in astrocytes. Both Gq and Gi/o GPCR activation in astrocytes stimulated the release of glutamate that increased neuronal excitability. These results add additional complexity to neural communication in the brain and suggest that inhibitory signaling in particular may be a particular property of neurons. Chapter Three delves further into the consequences of GPCR signaling in astrocytes and whether it is involved in mediating long-term plasticity. While Chapter Two shows the consequences of activating astrocyte GPCRs exogenously, Chapter Three investigates the consequences of activating astrocyte GPCRs synaptically. In particular, I investigate the role of astrocytes in mediating long-term depression (LTD) at a subset of synapses in the dorsolateral striatum. Despite extensive research into neuronal circuitry in the striatum, little work has been done in elucidating the role of astrocytes in striatal function. I show that astrocytes respond to high-frequency stimulation-induced LTD through mGluR5, a type of glutamate Gq-coupled GPCR. In return, astrocytes release the gliotransmitter ATP, which is necessary for inducing LTD at corticostriatal synapses. Through both a loss of function and gain of function approach, I demonstrate that astrocytes are an integral participant of striatal plasticity. Taken together, these studies add to the growing evidence that astrocytes play active roles in information processing in the brain, a dogmatic shift in the previously-held notion that astrocytes serve strictly support functions.

Adult neurogenesis, the process by which new neurons develop in the adult mammalian central nervous system, was thought to be nonexistent by accepted scientific dogma until the discovery of adult neural stem cells (NSCs) in the 1990s. NSCs have been found in two regions of the adult brain: the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus, and have the capacity to differentiate into neurons, astrocytes, and oligodendrocytes. Importantly, hippocampal NSCs play key roles in learning and memory, and have been implicated in a number of pathologies, including Alzheimer’s disease. NSCs reside in complex niches that provide the physical, chemical, and biological signals regulating stem cell maintenance and differentiation. A thorough understanding of NSC biology and niche signals can provide both insight into the mechanisms of adult neurogenesis, and inform stem cell based therapeutics to treat neurological injury and disease. Since the discovery of NSCs, a body of work has emerged characterizing the wide array of signals and intracellular pathways that mediate NSC behavior. Some of these findings, however, point to complex signaling mechanisms, the further study of which requires techniques outside the standard biological “toolbox”. The goal of this dissertation, therefore, was to engineer novel tools to enable the study and control of complex signaling systems in NSCs, and their application towards novel biological discoveries.The work presented here investigates two aspects of NSC biology: heterogeneity and cell-cell signaling. Stem cells are inherently heterogeneous; NSCs give rise to diverse progeny, including neurons and astrocytes, and in vitro, NSCs can differentially respond to the same set of cues. To probe this heterogeneity, a single cell Western blotting (scWestern) platform was developed. scWesterns enabled the interrogation of the proteome of thousands of single cells in about four hours, and multiplexing allowed for up to eleven targets to be detected from a single cell. We utilized the scWestern to probe heterogeneity in NSC signaling upon mitogen stimulation and differentiation. These studies provided unprecedented insight into differential single NSC responses to homogenous presentation of proliferation and differentiation factors. Cell-cell signaling in the NSC niche is comprised of paracrine and juxtacrine signals presented by supportive niche cells. Ephrin-B2 on hippocampal astrocytes was recently discovered to induce NSC neuronal differentiation through the receptor EphB4. Ephs and ephrins are both cell-surface bound and require oligomerization for downstream signal activation, so further investigation of ephrin-B2-mediated neurogenesis can benefit from novel tool development. This system was the focus of three studies, comprising the remainder of the dissertation. First, to better recapitulate the physical interactions between membrane-bound receptors and ligands, a supported lipid bilayer system was developed to present laterally mobile, monomeric ephrin-B2 to NSCs. We observed EphB4/ephrin-B2 co-clustering, and for the first time showed membrane-bound monomeric ephrin-B2 activation of EphB4 signaling and NSC differentiation in a synthetic system. By employing spatial mutation strategies to control ephrin-B2 diffusion and receptor-ligand complex size, we demonstrated that EphB4 signaling and NSC differentiation are sensitive to spatial properties of apposing cell membranes. This finding reveals novel regulatory mechanisms of both EphB4 signaling and NSC niche dynamics.Established Eph:ephrin signaling targets do not overlap with known neurogenic factors, so we next investigated ephrin-B2-induced downstream signaling in NSCs. Utilizing multivalent ephrin-B2 conjugates to enhance stimulation and mass spectrometry to identify signaling effectors, a number of novel kinases were identified, including activated Cdc42 kinase 1 (Ack1), Fyn proto-oncogene (Fyn), and Src proto-oncogene (Src). CRISPR/Cas9 genome engineering was then employed to knock down these proteins, which prevented ephrin-B2-induced NSC neurogenesis, demonstrating a role for these kinases in signaling downstream of EphB4 stimulation. Ack1 is a novel effector of Eph:ephrin signaling, and Ack1 and Fyn are novel regulators of NSC differentiation; therefore, this work reveals a number of previously unknown biological mechanisms.Finally, to examine dynamics of EphB4 signaling in NSCs, optogenetic methods for ephrin-independent Eph clustering and activation in response to blue light were created. We first developed generalized tools to apply a recently published method of optically targeting, clustering, and stimulating transmembrane receptors towards any signaling system of interest. We then harnessed these tools to study EphB4 signaling in NSCs by testing a number of EphB4-targeting optogenetic constructs. While none of the tested vectors were able to cluster and activate EphB4, a number of interesting observations were made that point to future areas for optimization. Moreover, we were able to perform a proof-of-concept study of the application of the generalized optogenetic tools developed.In summary, this dissertation presents work on both the development of novel tools for probing stem cell complexities, and the implementation of these tools towards the discovery of a number of novel biological mechanisms.

Abstract: In response to pathogens, immune cells induce protective effector functions, such as degranulation, phagocytosis and cytokine secretion, which are initiated by stimulatory or inhibitory immunoregulatory receptors expressed on leukocytes. Using a fish immunological model system, the focus of my thesis work was to examine immunoregulatory receptors in vertebrates by further characterizing members of the teleost Leukocyte Immune-type Receptor (LITR) family. IpLITRs share structural and phylogenetic features with certain mammalian receptors involved in activating and abrogating immune cell functions. As stimulatory IpLITR2.6b associates with signaling adaptor molecule IpFcRγ-L, I expressed a chimeric construct of IpLITR2.6b with IpFcRγ-L in mammalian immune cells to examine its functional abilities. Engagement of this receptor- adaptor induced intracellular signaling and activation of degranulation and phagocytosis. I also expressed IpLITR1.1b, a receptor previously reported to inhibit natural killer cell cytotoxicity to examine its functions further. This inhibitory receptor surprisingly facilitated phagocytosis. This represents dual functionality among select IpLITR-types, which, to our knowledge, has not been reported before in fish. Taken together, these results highlight conserved aspects of immunoregulatory receptor signaling and function while revealing new insights that may contribute to our overall understanding of immunity.

Reductionist biology has yielded tremendous insight into the basis of biochemistry and genetic disease. However, the remarkable failure of reductionist biology to explain complex problems, especially cancer, has led to the development of systems biology. The vast complexity of biological systems remains the most difficult problem in biology today. In order to understand this complexity, we need tools to massively multiplex measurements of a signaling network. Therefore, we developed lysate microarray technology to fill this need. In this work, we discuss three ways in which lysate microarrays were applied to human disease. In the first work, we discuss a key stage in malaria development. The liver-stage malaria parasite represents a promising target for intervention, and we present the first use of lysate microarray technology as a screening tool for host-parasite interactions in an infectious disease. We identified three cancer-related pathways that are modified in malaria infection, and studied the p53 pathway in depth. Our finding that the parasite downregulates p53 and that treatment with Nutlin-3 strongly decreases parasite load may lead to the development of a prophylactic malaria vaccine. In the second work, we began by screening drug combinations and varying dosing schedule in triple-negative breast cancers (TNBCs). We systematically explored stimulation space and collected a large lysate microarray dataset, which was used for statistical analysis. We identified a sensitization effect when a growth factor signaling inhibitor was presented before a genotoxic agent. This sensitization was generalizable among a subset of TNBCs and may generally be important for cancers driven by growth factor signaling, as we found the effect extends to nonTNBC cancers. We hope this data will be useful in guiding cancer treatment strategies in patients. In the third work, we study the changing role of the DNA Damage Response (DDR) as a cell line evolves towards cancer. We used the MCF10A progression series and studied how these cell lines respond to genotoxic agents. We identified differences in cell fates after treatment, and collected a large lysate microarray dataset for statistical analysis. Early analysis of the data indicates gross rewiring within the DDR between the MCF10A cell lines.