This dissertation presents a multifaceted study exploring critical aspects of drug development, focusing on the transporters on Blood-Brain Barrier (BBB) and a novel approach for Non-Alcoholic Fatty Liver Disease/Steatohepatitis (NAFLD/NASH).

The first segment of this research offers an in-depth analysis of the BBB transporters, focusing on age-related changes in protein expression and the functional impact of polymorphisms. The Blood-Brain Barrier (BBB) serves as a selective barrier for a variety of small molecules, including chemical carcinogens, environmental toxins, and therapeutic drugs. This barrier is constructed from brain capillary endothelial cells, pericytes, and astrocytic end-feet, working in unison to protect neurons and maintain brain homeostasis throughout life. The selective nature of the BBB is attributed to the tight junctions within the capillaries, which prevent the free passage of small molecules, and also to an array of transporters that regulate the influx of essential nutrients and the exclusion of many xenobiotics. The goal of this part of the dissertation is to elucidate the effect of aging from neonates to elderly on the human BBB, with an emphasis on transporters. A secondary goal is to examine in detail the determinants of function of a key transporter in the human BBB, ATP-binding cassette transporter, ABCG2 (BCRP).

This part of the dissertation begins with an overview of the current understanding of elements that influence the operation of the BBB, particularly transporters. It starts with a review of the BBB's structural components and their joint function in sustaining barrier integrity. It then summarizes how the BBB controls the movement of nutrients and medicines into the brain through various transport methods. The discussion includes the development of the BBB, how it changes as we age, and how diseases may affect it, emphasizing the dynamics of BBB and the challenges it creates for creating brain-targeted drugs. The chapter then shifts to how genetic variants in transporters influence BBB function and drug disposition, focusing particularly on the prominently expressed ATP-binding cassette transporters ABCB1 and ABCG2. After the overview, this dissertation provides a rich set of comprehensive analyses how age and genetic variations in transporters impact the functionality of BBB to advance our understanding of the multifaceted regulatory framework that controls BBB in physiological and pathological contexts. Major gaps in our understanding of the BBB, which are addressed by this dissertation research, are highlighted.

Chapter 2 examines changes in the human BBB proteome throughout a human lifespan, noting the significant shifts in protein expression that influence barrier permeability and the transport of nutrients and drugs. It is acknowledged that the BBB matures after birth, adjusting its transport mechanisms to align with each stage of development, and later alters due to aging and neurodegenerative disorders. Yet, fully grasping these modifications in the human BBB is an ongoing challenge. This chapter introduces a comprehensive proteomic analysis of the evolution and senescence of proteins in brain microvessels (BMVs). Samples from healthy individuals across a wide age spectrum and Alzheimer’s disease patients were analyzed using LC-MS/MS. A plethora of proteins, including numerous SLC and ABC transporters, were identified. Network analysis of the BMV proteome suggested potential alterations in BBB permeability over time and pinpointed transporters crucial for nutrient supply and drug penetration that exhibit age-dependent expression patterns. This investigation sheds light on the dynamic regulation of BBB proteins, emphasizing how transporter variations with age can affect drug permeability. These findings are crucial for refining pharmacokinetic modeling and therapeutic approaches across different stages of life.

The dissertation (Part A) then pivots to explore how genetic factors may alter the functionality of transporters, potentially causing variances in drug distribution within the brain. It focuses particularly on ABCG2, a transporter highly expressed at the BBB, noting that genetic variations leading to functional changes can result in differing drug responses. Utilizing deep mutational scanning (DMS), an innovative technique that combines next-generation sequencing (NGS) with functional outcomes of numerous variants, this study evaluated 12,724 variants of the ABCG2 gene. Our experimental setup was crafted to assess over ten thousand of missense, synonymous, and deletion variants of ABCG2 in a high-throughput manner. The abundance of ABCG2 was quantified, its surface expression was measured, and the functional effects of each variant were examined using the anti-cancer drug, mitoxantrone. The resulting detailed functional map, visualized through heatmaps and integrated with the structural data of ABCG2, helped identify crucial residues essential for ABCG2's function and poly-specificity. This study enhances our understanding of ABCG2 and lays the groundwork for future investigations into other ABC transporters. It underscores the value of DMS in dissecting the intricacies of pharmacogenetics and the mechanisms underlying drug resistance.

In summary, this part of the dissertation presents a comprehensive examination of the factors important for the functionality of BBB, shedding new light on transporters. Importantly, we unveil the BBB's dynamic protein regulation across the human lifespan, demonstrating how age-related changes affect drug permeability through a detailed proteomic analysis. Furthermore, the dissertation explores how mutations influence transporter functionality, ABCG2 as an example for developing the platform. The innovative use of Deep Mutational Scanning (DMS) to assess thousands of ABCG2 variants provides a rich functional map, revealing key insights into the transporter's operation and offering a valuable resource for future pharmacogenetic and drug resistance research. Overall, these studies highlight the necessity of understanding the BBB's complex mechanisms to enhance drug delivery strategies and overcome barriers in treating neurological disorders including neurodegenerative diseases.

The second part of the dissertation (Part B) shifts focus to the global health issue of NAFLD and its more severe form, NASH. It investigates Cis-Regulation Therapy (CRT) as a novel treatment approach, utilizing nuclease-deficient gene-editing technologies to modify gene regulatory elements for therapeutic ends. Approximately 30% of people worldwide are affected by NAFLD, and about 25% of these cases may advance to NASH. NASH represents a more serious stage of NAFLD, marked by liver inflammation and damage due to fat accumulation in the liver. About 25% of those with NAFLD progress to NASH, characterized by significant liver inflammation and damage due to fat accumulation. Currently, the pharmacological treatment options for NAFLD/NASH are severely limited. Our study investigates the potential of CRT as an innovative treatment strategy. In our research, we explored the effectiveness of CRT as a promising new treatment strategy. CRT employs nuclease-deficient gene-editing technologies, such as dead Cas9 (dCas9) combined with transcriptional modulators, to alter the activity of gene regulatory elements for therapeutic purposes. The goal of this part of the dissertation research specifically focuses on the nuclear receptor-like protein 1 (NURR1, NR4A2), a transcription factor critical in regulating inflammation which is a hallmark of NASH.

This part of the dissertation initiates with an overview of existing treatment options for NAFLD/NASH, pinpointing their limitations and the urgent need for more effective interventions. It further explores contemporary strategies in drug and therapeutic development targeting NAFLD/NASH, with a particular emphasis on animal models. After the overview, we present our findings that activating Nurr1 through CRISPR activation (CRISPRa) offers a promising therapeutic strategy for NAFLD/NASH within FATZO mouse models. This technique has shown efficacy in improving glucose metabolism abnormalities and reducing the CCL2-CCR2 axis, a critical inflammatory pathway, both before and after the onset of the disease. Our findings introduce a promising new therapeutic avenue for NAFLD/NASH, highlighting the capability of Nurr1 activation to control and possibly reverse the disease's progression.

In summary, this dissertation delivers a comprehensive analysis of the variables impacting the functionality of BBB transporters and presents a promising therapeutic approach for NAFLD/NASH. Through this research, we aim to pave new pathways for the advancement of treatments for neurological and hepatic disorders.

The study of protein protein-interactions (PPIs) can be approached with many lenses. Interaction networks can be mapped genetically or with proteomic methods. Biophysical characterization of specific partner interactions can clarify molecular mechanism of partner interaction. Screening methods can to identify novel interaction modulators, and site-directed chemical probes can used to demonstrate the importance of protein partner interaction in disease or biology. In this work I begin with an overview of the chemical biology toolbox for targeting non-catalytic cysteines residues. I apply one such tool, disulfide tethering, to two PPIs with the goal of broadening the PPIs which are targetable with covalent chemical probes. I then describe the use of cysteine-reactive molecules in understanding protein ligandability and its connection to protein dynamics. Finally, I study a promiscuous PPI receptor (Mac-1) to understand if inhibitors could be developed for specific ligands. Taken together, these data demonstrate the applicability of cysteine reactivity generally, and disulfide tethering specifically, to the study of PPIs.

Protein-protein interactions (PPIs) are central to biology and commonly dysregulated in disease. Despite their importance in cell homeostasis and their therapeutic potential, PPIs have rarely been a target for drug discovery. This has been due to three major challenges PPIs present to small molecule engagement: the flatness problem, the disorder problem, and the selectivity problem. PPIs which bind with large, flat interfaces typically do not have deep pockets accessible to small molecules, leading to low ligand efficiency. Intrinsically disordered proteins, likewise, are lacking in “hot spots” for small molecule binding. Finally, most proteins which participate in PPIs have more than one partner– 20% of so-called hub proteins with multiple interactions have over 20 binding partners– thus necessitating a selective small molecule to target a specific PPI edge as opposed to a general effector which inhibits the nodular hub protein. Disulfide tethering, a subset of fragment-base drug discovery (FBDD) offers a novel approach to the rapid discovery and development of potent, selective small molecule PPI modulators. This platform facilitates rapid and robust compound discovery and validation as well as simple, accessible scaffolds for hit-to-lead development, circumventing the difficulty of natural product total synthesis and mitigating costly late-stage failures in selectivity and safety. This work showcases multiple projects utilizing disulfide tethering to discover and optimize small molecule modulators for various “undruggable” targets within oncology, neurodegeneration, metabolic disease, and infectious disease.

Protein-protein interactions (PPIs) are vital for biology as they govern numerous essential biological processes, including signal transduction, enzymatic activity regulation, protein localization, and complex formation, collectively orchestrating cellular functions and maintaining homeostasis. The hub protein 14-3-3 interacts with hundreds of client proteins, including key signaling proteins CRAF and SOS1 and transcription factors Estrogen Receptor α (ERα), FOXO1, and TAZ. These 14-3-3/client interactions can be altered in disease, leading to undesirable activity and signaling. The Arkin Lab has pioneered a mass spectrometry-based site-directed disulfide tethering screen to identify PPI stabilizers and inhibitors. We have used this screen to discover stabilizers of various 14-3-3σ/client interactions including, CRAF, FOXO1, ERα, SOS1, and USP8. Disulfide fragment stabilizers lead to a 4 – 250-fold stabilization of 14-3-3σ/client complexes. To further characterize the effects of these stabilizers, we have developed Nanoluciferase bioluminescence resonance energy transfer (NanoBRET) assays to measure 14-3-3σ/CRAF, TAZ, and ERα interactions, where we have measured stabilizer EC50 values between 100 nM – 1 μM. We have optimized 14-3-3σ/CRAF stabilizers for selectivity and potency and measured increased PPI formation in cells, decreased CRAF kinase activity, and decreased signaling through the mitogen-activated protein kinase (MAPK) pathway. Finally, we have expanded the disulfide tethering technology to a new class of hub proteins to discover stabilizers of additional PPIs to modulate their interaction in disease.