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Ricq, Emily

Modulation of histone modifications in the brain may represent a new mechanism for brain disorder therapy. Post-translational modifications of histones regulate gene expression, affecting major cellular processes such as proliferation, differentiation, and function. An important enzyme involved in one of these histone modifications is lysine specific demethylase 1 (LSD1). This enzyme is flavin-dependent and exhibits homology to amine oxidases. Parnate (2-phenylcyclopropylamine (2-PCPA); tranylcypromine) is a potent inhibitor of monoamine oxidases, and derivatives of 2-PCPA have been used for development of selective LSD1 inhibitors based on the ability to form covalent adducts with flavin adenine dinucleotide (FAD). Here we report the synthesis and in vitro characterization of LSD1 inhibitors that bond covalently to FAD. The two most potent and selective inhibitors were used to demonstrate brain penetration when administered systemically to rodents. First, radiosynthesis of a positron-emitting analogue was used to obtain preliminary biodistribution data and whole brain time–activity curves. Second, we demonstrate that this series of LSD1 inhibitors is capable of producing a cognitive effect in a mouse model. By using a memory formation paradigm, novel object recognition, we show that LSD1 inhibition can abolish long-term memory formation without affecting short-term memory, providing further evidence for the importance of reversible histone methylation in the function of the nervous system.

Olfactory dysfunction is broadly associated with neurodevelopmental and neurodegenerative diseases and predicts increased mortality rates in healthy individuals. Conventional measurements of olfactory health assess odor processing pathways within the brain and provide a limited understanding of primary odor detection. Quantification of the olfactory sensory neurons (OSNs), which detect odors within the nasal cavity, would provide insight into the etiology of olfactory dysfunction associated with disease and mortality. Notably, OSNs are continually replenished by adult neurogenesis in mammals, including humans, so OSN measurements are primed to provide specialized insights into neurological disease. Here, we have evaluated a PET radiotracer, [11C]GV1-57, that specifically binds mature OSNs and quantifies the mature OSN population in vivo. [11C]GV1-57 monitored native OSN population dynamics in rodents, detecting OSN generation during postnatal development and aging-associated neurodegeneration. [11C]GV1-57 additionally measured rates of neuron regeneration after acute injury and early-stage OSN deficits in a rodent tauopathy model of neurodegenerative disease. Preliminary assessment in nonhuman primates suggested maintained uptake and saturable binding of [18F]GV1-57 in primate nasal epithelium, supporting its translational potential. Future applications for GV1-57 include monitoring additional diseases or conditions associated with olfactory dysregulation, including cognitive decline, as well as monitoring effects of neuroregenerative or neuroprotective therapeutics.

Epigenetic mechanisms regulate gene expression and mediate interactions between genetic factors and environmental exposures. The enzymes responsible for epigenetic regulation may thus be important therapeutic targets for multifactorial neurological syndromes. KDM1A, the first histone lysine demethylase to be discovered, regulates the maturation of neurons and is inactivated by non-selective monoamine oxidase inhibitors such as the antidepressant tranylcypromine. This thesis entails the development of small-molecule tools to study KDM1A in a neurobiological context, with application towards the development of new therapeutic agents.

We leveraged the chemical scaffold of tranylcypromine to generate novel KDM1A inhibitors. In chapter 2, we profile these analogs using biochemical, cellular, and in vivo assays. We show that RN1 potently inhibits KDM1A, exhibits high brain uptake, and affects the behavior of mice in a novel object recognition assay. Thermal shift assays reveal engagement of KDM1A by tranylcypromine in the brains of systemically-treated rats, suggesting that inhibition of KDM1A by non-selective antidepressants in a clinical setting warrants further examination.

We sought to discover new mechanisms of KDM1A inhibition in order to gain further selectivity versus the monoamine oxidases. In chapter 3, we present outcomes of a high-throughput screen and secondary assays which reveal a predominant mode of KDM1A inhibition based on thiol-reactivity, and widespread contamination of test compounds by elemental sulfur. We show that KDM1A is inhibited by the FDA-approved drug disulfiram, and disclose two novel scaffolds for medicinal chemistry development.

In chapter 4, we further profile the thiol-reactivity of KDM1A and show that catalytically-generated hydrogen peroxide negatively regulates demethylase activity. MALDI-TOF mass spectrometry indicates that hydrogen peroxide blocks labeling of cysteine 600, which we propose forms an intramolecular disulfide bond with cysteine 618. This activity-dependent regulation is unique among histone-modifying enzymes but consistent with redox sensitivity of epigenetic regulators. KDM1A may use this thiol/disulfide switch as a mechanism to sense other cellular oxidants, such as the monoamine neurotransmitter dopamine.

Conspectus Decades after its discovery, positron emission tomography (PET) remains the premier tool for imaging neurochemistry in living humans. Technological improvements in radiolabeling methods, camera design, and image analysis have kept PET in the forefront. In addition, the use of PET imaging has expanded because researchers have developed new radiotracers that visualize receptors, transporters, enzymes, and other molecular targets within the human brain. However, of the thousands of proteins in the central nervous system (CNS), researchers have successfully imaged fewer than 40 human proteins. To address the critical need for new radiotracers, this Account expounds on the decisions, strategies, and pitfalls of CNS radiotracer development based on our current experience in this area. We discuss the five key components of radiotracer development for human imaging: choosing a biomedical question, selection of a biological target, design of the radiotracer chemical structure, evaluation of candidate radiotracers, and analysis of preclinical imaging. It is particularly important to analyze the market of scientists or companies who might use a new radiotracer and carefully select a relevant biomedical question(s) for that audience. In the selection of a specific biological target, we emphasize how target localization and identity can constrain this process and discuss the optimal target density and affinity ratios needed for binding-based radiotracers. In addition, we discuss various PET test–retest variability requirements for monitoring changes in density, occupancy, or functionality for new radiotracers. In the synthesis of new radiotracer structures, high-throughput, modular syntheses have proved valuable, and these processes provide compounds with sites for late-stage radioisotope installation. As a result, researchers can manage the time constraints associated with the limited half-lives of isotopes. In order to evaluate brain uptake, a number of methods are available to predict bioavailability, blood–brain barrier (BBB) permeability, and the associated issues of nonspecific binding and metabolic stability. To evaluate the synthesized chemical library, researchers need to consider high-throughput affinity assays, the analysis of specific binding, and the importance of fast binding kinetics. Finally, we describe how we initially assess preclinical radiotracer imaging, using brain uptake, specific binding, and preliminary kinetic analysis to identify promising radiotracers that may be useful for human brain imaging. Although we discuss these five design components separately and linearly in this Account, in practice we develop new PET-based radiotracers using these design components nonlinearly and iteratively to develop new compounds in the most efficient way possible.

Ferroptosis, an iron-dependent, non-apoptotic cell death program, is involved in various degenerative diseases and represents a targetable vulnerability in certain cancers1. The ferroptosis-susceptible cell state can either preexist in cells arising from certain lineages or be acquired during cell-state transitions2–5. Precisely how ferroptosis susceptibility is dynamically regulated remains poorly understood. Using genome-wide CRISPR/Cas9 suppressor screens, we identify the peroxisome organelle as a critical contributor to ferroptosis sensitivity in human renal and ovarian carcinoma cells. By lipidomic profiling, we show that peroxisomes contribute to ferroptosis through the synthesis of polyunsaturated ether phospholipids (PUFA-ePLs), an understudied lipid class that provides substrates for lipid peroxidation, resulting in turn in induction of ferroptosis. Moreover, carcinoma cells that are initially sensitive to ferroptosis can switch to a ferroptosis-resistant state in vivo, a state associated with extensive PUFA-ePL downregulation. We further find that the pro-ferroptotic role of PUFA-ePLs can be extended beyond neoplastic cells to other cell types, including normal neurons and cardiomyocytes. Together, our work reveals important roles for the peroxisome–ether phospholipid axis in driving ferroptosis susceptibility and evasion, highlights PUFA-ePL as a distinct functional lipid group that is dynamically regulated during cell-state transitions, and suggests multiple regulatory nodes for therapeutic interventions in diseases involving ferroptosis.