Thesis advisor: Eranthie Weerapana / Over the past two decades there have been a variety of programmed cell death (PCD) pathways to emerge. Among these emerging PCD pathways ferroptosis is has been of especial interest as its iron-dependent reliance on the generation of reactive oxygen species (ROS) links this PCD pathway to some of the most pervasive pathologies including cancer and neurodegeneration. In order to broaden our understanding of ferroptosis we applied a number of proteomic based methods in effort to identify novel ferroptotic mediators. Before the application of proteomic methods, we developed complementary pharmacological and genetic ferroptosis models. With these models we identified ferroptosis-induced changes in protein abundance. Using these data, we generated CRISPR-Cas9-mediated knockouts of protein disulfide isomerase A1 (PDIA1) and cytosolic acetyl-CoA acetyltransferase (ACAT2) that were found to exhibit altered susceptibility to the induction of ferroptosis. With data on protein abundance changes we then profiled ferroptosis-induced changes in abundance corrected cysteine reactivity. Many proteins displayed significant changes in cysteine reactivity that will require further investigation in order to determine if they are drivers or a downstream consequence of ferroptosis. Finally, we used a cell surface biotinylation reagent together with proteomics in effort to identify potential cell surface markers of ferroptosis. This lead to the identification of multiple known cell membrane proteins with ferroptosis altered cell surface labeling. Future studies will seek to confirm the observed alterations with complementary methods. Together these studies illustrate the dynamic responses of the proteome to the induction of ferroptosis. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Nitroxides are intermediates in the accepted reaction mechanisms of the antioxidant activity of diarylamines and hindered alkyl amines. The parent amines are used as additives to preserve synthetic and natural hydrocarbon-based materials from oxidative degradation. New methodology which enables monitoring of hydrocarbon autoxidations at low rates of radical generation has revealed that diarylnitroxides and hindered nitroxides are far better inhibitors of unsaturated hydrocarbon autoxidation than their precursor amines, implying intervention of a previously overlooked mechanism. Experimental and computational investigations suggest that the nitroxides catalyze the cross-dismutation of alkylperoxyl and hydroperoxyl radicals to yield a hydroperoxide and O2, thereby halting the autoxidation chain reaction. The hydroperoxyl radicals – key players in hydrocarbon combustion, but essentially unknown in autoxidation – are proposed to derive from a tunneling-enhanced intramolecular (1,4)- hydrogen-atom transfer/elimination sequence from oxygenated radical addition intermediates. These insights suggest that nitroxides are preferred additives for the protection of unsaturated hydrocarbonbased materials from autoxidation since they exhibit catalytic activity under conditions where their precursor amines are less effective and/or inefficiently converted to nitroxides in situ. Polyunsaturated fatty acids (PUFAs) are highly autoxidizable lipids that are integral structural components of biological membranes as well as substrates for enzymes the produce inflammatory mediators implicated in a host of degenerative diseases. In particular, the interactions between these substrates and their respective native enzymes are hotly pursued since elucidation of the underlying mechanisms could lead to the discovery of better small molecule inhibitors for the ailments to which they contribute. In the past decade, an additional mode of cellular degeneration has been unveiled in the process of ferroptosis whose hallmark includes a sharp increase in the cellular pool of PUFA derived hydroperoxides. As a result, there is further incentive to uncover all mechanisms by which these inflammatory precursors are developed. Herein, progress toward the synthesis of fluorinated PUFAs is presented. These are proposed to be useful to probe the interactions of PUFAs with lipoxygenase enzymes, which metabolize polyunsaturated fatty acids to their hydroperoxide derivatives.

Tuberculosis (TB) is a critical infectious disease world-wide, and the increasing development of antibiotic resistance drives the search for effective host-directed therapies. One molecular target of potential host-directed therapy is Type 1 Interferon, (IFN-I or IFNβ), an excess of which correlates with TB progression. The mechanisms underlying IFNβ overproduction are still unclear. In this dissertation we review cellular mechanisms, including mitochondrial function and metabolism, oxidative stress, and the Integrated Stress Response, which are involved in IFNβ production and macrophage function. We also describe an experimental model of human-like TB, the B6J.C3-Sst1C3HeB/Fej Krmn (B6.Sst1S) mouse, which provides a unique and convenient system for studying mechanisms of necrosis in TB granulomas. We use primary macrophages from the B6.Sst1S mouse to establish a mechanism that links the B6.Sst1S genotype to a cascade of dysregulation that drives IFNβ superinduction and susceptibility to TB infection. TNF is necessary for granuloma formation in vivo, but in the context of transcriptional dysregulation and excess free iron, it drives oxidative stress, which amplifies IFNβ induction to pathologic levels. This induction is maintained by positive feedback through the double stranded RNA-dependent Protein Kinase (PKR). We demonstrate that interruption of this cascade by iron chelation or inhibition of lipid peroxidation attenuates IFNβ induction and improves subsequent infection outcomes. We conclude by comparing the in vitro model system to an in vivo necrotic TB granuloma, describing similarities between our system and human TB, and discussing the connections between IFN-I and autoimmune and degenerative disease and the broader application of the B6.Sst1S model system to studies of human immunity.

Autoxidation, a radical chain reaction, is largely responsible for the degradation of most man-made and biological materials. These include chemical products such as lubricants, plastics and rubber; as well as biological molecules and membranes within our bodies. The development of means to hinder this process has been a major focus of the petroleum, chemical, pharmaceutical and biotechnology industry over the past century. The two most common strategies to emerge from these efforts have been the use of compounds that either prevent the initiation of autoxidation or trap the propagating radicals, so-called radical-trapping antioxidants (RTAs). Herein, we describe our efforts towards the design and development of extremely potent heterocyclic diarylamine RTAs, and their activity in a variety of applications ranging from isotropic organic solution to mammalian cells. We have elucidated the important structural motifs and mechanistic considerations necessary for the development of next-generation arylamine RTAs. Some of the substituted heterocyclic diarylamines analogs we disclose are among the best inhibitors of high temperature autoxidations described to date. Alongside, we developed novel analytical tools to facilitate the studies and acquisition of results for characterizing RTA activity in organic solutions and lipid bilayers. These fluorescent probes are highly relevant and allow for the determination of hydroperoxide and acid concentrations rapidly, as well as screen (or counter-screen) RTAs in liposomal membranes. Our methodologies address numerous drawbacks from frequently used ‘plug-and-play’ assays and we anticipate they will fill a current unmet need in both industrial and academic laboratories worldwide. Moreover, the recent characterization of ferroptosis – a novel regulated necrotic-like cell-death pathway associated with the accumulation of lipid hydroperoxides – has paved a way forward for studying oxidation induced damage in a biological context. Utilizing our expertise in lipid peroxidation and inhibition, we elucidated the prominent role of autoxidation in the execution of ferroptotic cell death. Alongside, our analytical tools and RTAs have also enabled the identification and characterization of novel ferroptosis inhibitors. Furthermore, this has prompted the development of a correlation to predict anti-ferroptosis activity of small-molecules using simple spectrophotometric assays.

Ferroptosis is a recently characterized iron-dependent form of regulated cell death associated with the accumulation of (phospho)lipid hydroperoxides. Since its characterization, there has been a spirited debate in the literature over the origin of the lipid hydroperoxides in ferroptotic cells. Many investigators have implicated lipoxygenases (LOXs), enzymes known to catalyze the oxidation of polyunsaturated fatty acids (especially linoleate and arachidonate) to yield lipid hydroperoxides. Previous work by our group, investigated the induction and suppression of ferroptosis in human embryonic kidney (HEK-293) cells transfected to overexpress the three most widespread isoforms of LOX (5-LOX, 15-LOX-1 and p12-LOX). The results suggested that LOX catalysis is not required for ferroptosis. Our previous work did not include investigations into cells transfected to overexpress 15-LOX-2. However, a series of recent publications has since implicated the 15-LOX-2/PEPB1 complex as a key player in ferroptotic cell death. Therefore, in this work, HEK-293 cells were transfected to overexpress the 15-LOX-2 isoform, as confirmed by immunodetection, and were subject to induction and suppression of ferroptosis pharmacologically. A library of small molecules was assembled consisting of LOX inhibitors, radical-traping antioxidants (RTAs) and LOX inhibitors that display off-target RTA activity. Consistent with our previous investigations, only LOX inhibitors with radical trapping activity or iron chelators were effective at suppressing ferroptosis. Furthermore, the poor performance of 15-LOX-2 inhibitors at rescuing cells transfected to overexpress 15-LOX-2 from ferroptosis does not support the role of the 15-LOX-2/PEBP1 complex as a central mediator of ferroptotic lipid peroxidation. We also report the details of corresponding investigations in cell lines that are reported to express high levels of LOXs and that have been used to establish characteristics of ferroptosis, including HT-22 mouse hippocampal cells (15-LOX-1 and/or 15-LOX-2) and HT-1080 human fibrosarcoma cells (all LOXs). The two cellular models were also subject to cell-rescue studies with our small molecule library. Again, only LOX inhibitors that possess radical-trapping antioxidant activity or which are good iron chelators could rescue cells from ferroptosis. These results underscore our previous conclusion that although lipoxygenase activity may contribute to the pool of cellular lipid hydroperoxides, autoxidation drives ferroptotic cell death.

Lipid peroxidation is believed to play a key role in the onset and progression of degenerative disease. Interestingly, although cholesterol is the most abundant lipid in the human body, our understanding of its autoxidation and subsequent decomposition is relatively limited. In fact, until recently, cholesterol-7-hydroperoxide was accepted as the only primary product of cholesterol autoxidation in organic solution, however, our group exhibited that the 4-, 5-, and 6-hydroperoxides are also formed. Although this work facilitated thorough investigation of the complexities of both H-atom abstraction and addition in cholesterol autoxidation in organic solution, it did not account for the dynamic environment of a cell membrane. Herein, we report on the product distribution of these primary autoxidation products in lipid bilayers and how antioxidant supplementation, H-bonding interactions, and concentration of polyunsaturated fatty acid (PUFA) substrate influence both the product distribution and efficiency of autoxidation. Indeed, not only does H-bonding of the 3β-OH of cholesterol appear to shut-down C4 H-atom abstraction, the absence of kinetic chol-5α-OOH product is likely due to the poor potency of α- tocopherol (α-TOH), also as a result of H-bonding with phosphate head group of lipid membrane phospholipids. Therefore, within a lipid membrane the 7-hydroperoxide products predominate, consistent with literature precedent, however the factors involved are more complex than previously understood. Moreover, with the authentic cholesterol hydroperoxides in hand, we sought to determine if the different regioisomers exhibit different cytotoxicity. Glutathione peroxidases (GPXs) are cytoprotective enzymes that reduce harmful hydroperoxides to benign alcohols in vivo. Using RSL3, a small-molecule inhibitor for GPX4, we were able to sensitize mammalian cells to ferroptotic cell death via administration of our exogenously prepared chol-OOHs. Surprisingly, we found that the toxicities of each of 7α-OOH, 6β-OOH and 5α-OOH were only marginally augmented by RSL3 treatment, suggesting that they do not substantially sensitize cells to ferroptosis, perhaps because their decomposition to lipid peroxidation chain-initiating species (i.e. alkoxyl radicals) is not particularly efficient. Instead their cytotoxicities may derive from other mechanisms, such as the induction of apoptosis. This inspired our investigation of the fate of lipid hydroperoxides in vivo, namely the secondary products of the predominant 7-hydroperoxide species. Acid-catalyzed Hock fragmentation, known for the industrial synthesis of phenol and acetone from cumene or implication in the generation of 4-hydroxynonenal (4-HNE), of 5α- and 6β-OOH has been shown by our group to produce highly electrophilic secosterol species; we sought to investigate the same decomposition mechanism for 7α-OOH in light of our investigations in the lipid membrane. Interestingly, we found that Hock fragmentation of 7α-OOH does not exhibit products resulting from the anticipated O-vinyl oxocarbenium intermediate, rather, the mechanism appears to funnel through an α-epoxy carbenium to produce unprecedented A-ring cleavage and epoxide products. Herein, we describe our thorough analysis of this chol-7α-OOH Hock fragmentation and attempts to investigate the presence of these products in biological samples, similar to previous analyses of similar products in atherosclerotic plaque extracts. The products isolated and characterized through this work have provided new mechanistic insight with regards to the primary and secondary oxidation products of cholesterol in vivo; through further development of these findings, we hope to provide a better understanding of the implications of cholesterol oxidation in the pathogenesis of atherosclerosis.

Lipid peroxidation is well established to contribute to the etiology of many deteriorative conditions including neurodegeneration, cardiovascular disease, cancer, aging, and recently in ferroptosis—a regulated, necrotic modality of cell death that results from the accumulation of lipid hydroperoxides. Recent high-throughput screening efforts have uncovered ferrostatin-1 (Fer-1) and liproxstatin-1 (Lip-1) as two premiere inhibitors of ferroptosis. We propose that these compounds function as radical trapping antioxidants. We employ a systematic methodology of evaluating inherent radical trapping antioxidant (RTA) activity of Lip-1, Fer-1, and various aryl amine and aryl nitroxide analogues to put forward a biologically relevant mechanism of action based on our previous work in the field. Joining these observations with the efficacy of tetrahydronaphthyridinols (THNs), the results support a clear role of autoxidation in the execution of ferroptosis. Next, we expand the THN repertoire by targeting the payload towards the engine of our cells, the mitochondria. Decades of research have implicated mitochondrial dysfunction brought about by the peroxidation of mitochondrial membranes and the leaking of downstream oxidants, in the death of their symbiotic host cells. Isolated successes in the field have been demonstrated academically, though viable drugs remain to be developed, partially due to the lack of effective diagnostic tools. We endeavor to address some of these issues by investigating mitochondrially-targeted THNs (MitoTHNs) as a targeted chain-breaking antioxidant of unparalleled potency. Furthermore, we advance development of the THNs towards therapeutic applications by demonstrating their biodistribution in mice.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23553号 / 生博第464号 / 新制||生||62(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 垣塚 彰, 教授 井垣 達吏, 教授 豊島 文子 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Ferroptosis is a recently characterized cell death pathway associated with the iron-dependent accumulation of lipid hydroperoxides in phospholipid bilayers. The origin of these hydroperoxides has been an ongoing topic of debate and many researchers argue for a lipoxygenase (LOX) enzyme-controlled mechanism of initiation, given their known role as dioxygenases of polyunsaturated fatty acids (PUFAs). In response to this, our lab investigated the induction and inhibition of ferroptosis in human embryonic kidney (HEK-293) cells transfected to overexpress the three most prevalent LOX isoforms, 5-LOX, p12-LOX, and 15-LOX-1. These studies did not support a role for LOX in the execution of ferroptosis; LOX inhibition was not associated with ferroptosis suppression and in fact, anti-ferroptotic activity was directly tied to purported LOX inhibitors’ ability to act as radical-trapping antioxidants (RTAs). We have investigated the effects of LOX inhibitors on ferroptosis in human fibrosarcoma (HT-1080) cells, the cell line in which ferroptosis was initially characterized, and mouse hippocampal neuronal (HT-22) cells, the cell line in which the closely related cell death modality oxytosis was characterized. In sum, our findings mirror those obtained in HEK-293 cells, and the effectiveness of an inhibitor is tied to its off-target RTA activity, not inhibition of LOX. Moreover, we observed suppression of ferroptosis via necrostatin-1 (Nec-1), a known receptor-interacting serine/threonine-protein kinase 1 (RIPK1) (and necroptosis) inhibitor. Herein, we show that Nec-1 is not an RTA and exerts its effects by a yet unknown mechanism which we investigate in a series of exploratory experiments. Conjugated fatty acids – particularly α-ESA – have recently been reported to induce ferroptosis by an unclear mechanism. Theorizing this phenomenon was tied to the autoxidation of α-ESA’s conjugated trienic unit, we aimed to investigate the kinetic and biological properties of natural α-ESA alongside a deuterated isotopologue. Herein, we report preliminary work to derive biologically relevant rate constants for addition and hydrogen-atom transfer (HAT) of α-ESA. Moreover, we report our progress towards the synthesis of a deuterated α-ESA which will facilitate future study alongside its natural counterpart.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第25199号 / 医博第5085号 / 新制||医||1072(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 藤田 恭之, 教授 妹尾 浩 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM