Biology subjects

Floyd, S. R.

Publications and source records attributed to Floyd, S. R..

9 recordsLinked to original sources

bioRxiv2026

Identification of 4,5,6,7-Tetrabromo-1H-benzotriazole (TBB) as a Small Molecule MESH1 Inhibitor that Suppresses Ferroptosis

Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation and contributes to diverse pathologies including ischemia-reperfusion injury and neurodegenerative disorders. Current ferroptosis inhibitors largely function as nonspecific radical-trapping antioxidants, limiting their clinical utility. We previously identified MESH1 as a key regulator of ferroptosis through its NADPH phosphatase activity. Here, we identify 4,5,6,7-tetrabromo-1H-benzotriazole (TBB) as a small molecule inhibitor of MESH1 with an IC50 value of 4.7 {+/-} 0.3 {micro}M. X-ray crystallography revealed the molecular determinants of TBB recognition which are corroborated through structure-activity relationships of TBB analogs. TBB protected multiple cell lines against ferroptosis in vitro, and this effect was mitigated by MESH1 knockdown, consistent with on-target activity. Furthermore, TBB reduced neuronal death in an ex vivo brain slice model of Alzheimers disease. Collectively, these findings establish TBB as a bona fide small-molecule MESH1 inhibitor that suppresses ferroptosis and establishes MESH1 as a promising therapeutic target. Graphical AbstractDepicting mechanism of TBB suppressing ferroptosis through the inhibition of MESH1. Figure Created with Biorender.com O_FIG O_LINKSMALLFIG WIDTH=131 HEIGHT=200 SRC="FIGDIR/small/706832v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1fd60e9org.highwire.dtl.DTLVardef@1e56518org.highwire.dtl.DTLVardef@15010c2org.highwire.dtl.DTLVardef@17c313a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Investigating Rat-Brain Normal Tissue and Tumor FLASH Effects with a Novel Very High Energy Electron Beam

PurposeUltra-high dose rate (FLASH) irradiation is reported to reduce normal tissue toxicity while maintaining tumor control, however mechanism(s) remain obscure. To study FLASH mechanisms in brain tissue, we developed a novel experimental platform featuring a unique high-energy electron linear accelerator (High Intensity Gamma Source, HIGS) paired with an organotypic ex vivo brain metastasis model. MethodsWe varied inter-pulse spacing to modulate the mean dose rate (MDR) of our unique 35 MeV electron beam, while maintaining extremely high instantaneous dose rate (IDR), and used film dosimetry to characterize dosimetry and targeting accuracy. We combined this HIGS-FLASH beam with an organotypic rat brain slice/breast carcinoma co-culture model of brain metastasis to assess effects on normal and neoplastic tissues. Live cell and bioluminescence imaging demonstrated cancer cell growth effects, while normal tissue responses and immune activation were assessed with live cell imaging, cytokine profiles, and confocal microscopy. We performed comparison experiments with 20 MeV electrons from a Varian clinical linear accelerator (VCLA) operating at conventional dose rate. ResultsThe highest IDR of the HIGS-FLASH beam to-date was 20.7 {+/-} 0.6 MGy/s, with maximum MDR of 20.7 MGy/s (1 s pulse of 20.7 Gy). Beam targeting was accurate to < 1 mm and reproducible. HIGS-FLASH and VCLA dose rates equivalently decreased cancer cell growth. HIGS-FLASH irradiation significantly increased TNF and fractalkine levels and confocal microscopy revealed distinct changes in microglial morphology in normal brain slices, suggesting microglia activation following HIGS-FLASH irradiation. ConclusionsOur novel experimental platform produces extremely high dose rates and rapid normal/neoplastic tissue readouts for mechanistic research into the effects of FLASH radiation on the brain. HIGS-FLASH irradiation induces comparable cancer cell growth inhibition but differential effects on cytokines and microglial morphology, suggesting that acute innate immune responses may be involved in FLASH normal tissue effects in the brain.

cancer biology↗

bioRxiv2025

Evidence for Functional Regulation of the KLHL3/WNK Pathway by O-GlcNAcylation

The 42-member Kelch-like (KLHL) protein family are adaptors for ubiquitin E3 ligase complexes, governing the stability of a wide range of substrates. KLHL proteins are critical for maintaining proteostasis in a variety of tissues and are mutated in human diseases, including cancer, neurodegeneration, and familial hyperkalemic hypertension. However, the regulation of KLHL proteins remains incompletely understood. Previously, we reported that two KLHL family members, KEAP1 and gigaxonin, are regulated by O-linked {beta}-N-acetylglucosamine (O-GlcNAc), an intracellular form of glycosylation. Interestingly, some ubiquitination targets of KEAP1 and gigaxonin are themselves also O-GlcNAcylated, suggesting that multi-level control by this post-translational modification may influence many KLHL pathways. To test this hypothesis, we examined KLHL3, which ubiquitinates with-no-lysine (WNK) kinases to modulate downstream ion channel activity. Our biochemical and glycoproteomic data demonstrate that human KLHL3 and all four WNK kinases (WNK1-4) are O-GlcNAcylated. Moreover, our results suggest that O-GlcNAcylation affects WNK4 function in both osmolarity control and ferroptosis, with potential implications ranging from blood pressure regulation to neuronal health and survival. This work demonstrates the functional regulation of the KLHL3/WNK axis by O-GlcNAcylation and supports a broader model of O-GlcNAc serving as a general regulator of KLHL signaling and proteostasis.

cell biology↗

bioRxiv2025

Targeting the ferroptosis pathway: A novel compound, AZD1390, protects the brain after ischemic stroke

BackgroundFerroptosis is an iron-dependent form of regulated cell death driven by lipid peroxidation. This process has been implicated in various diseases, including ischemic stroke. Ischemic stroke leads to oxidative stress, iron overload, and reactive oxygen species (ROS) accumulation, which collectively may trigger ferroptotic neuronal cell death. However, the regulatory mechanisms of ferroptosis in stroke remain poorly understood. Previous studies have identified ataxia telangiectasia mutated (ATM), a DNA damage kinase, as a critical regulator of ferroptosis. However, the therapeutic potential of this discovery remains unknown. MethodsWe investigated the effect of ATM inhibitors, including the brain-penetrant AZD1390, on ferroptosis using in vitro, ex vivo, and in vivo models of ischemic stroke. Our analysis included assessments of cell viability, lipid peroxidation, ferroptosis marker expression, and infarct volume. ResultATM inhibitors significantly alleviated ferroptosis-induced cell death in cultured cells and ex vivo murine brain slice cultures. In the oxygen-glucose deprivation (OGD) stroke model, treatment with AZD1390 reduced the expression of ferroptosis markers (xCT and PTGS2) and diminished neuronal cell death in rat and mouse brain slices. Furthermore, in a mouse model of ischemic stroke, AZD1390 decreased infarct volume confirming its therapeutic efficacy in vivo. ConclusionsThis study identifies ferroptosis as a critical mechanism in ischemic stroke-induced neuronal cell death and highlights ATM inhibition, particularly with AZD1390, as a promising therapeutic candidate for mitigating stroke-associated damage. Targeting ferroptosis may provide a translationally relevant strategy to mitigate neuronal injury and improve clinical outcomes for stroke patients.

neuroscience↗

bioRxiv2024

Estrogens increase cancer cell efferocytosis to establish an immunosuppressive tumor microenvironment

Phagocytic clearance of apoptotic cancer cells (efferocytosis) by tumor-associated macrophages (TAMs) contributes in a substantial manner to the establishment of an immunosuppressive tumor microenvironment. This puts in context our observation that the female steroid hormone 17{beta}- estradiol (E2) facilitates tumor immune resistance through cancer cell extrinsic Estrogen Receptor (ER) signaling in TAMs. Notable was the finding that E2 induces the expression of CX3CR1 in TAMs to enable efferocytosis of apoptotic cancer cells which results in the suppression of type I interferon (IFN) signaling. Mechanistically, E2 facilitates calcium-dependent activation of the transcription factor NFATC1, which in turn induces CX3CR1 expression. This drives macrophage polarization towards an immune-suppressive state, increasing the ability of TAMs to engulf pro- inflammatory apoptotic cancer cells. Genetic or pharmacological inhibition of the E2/ER/CX3CR1 axis reversed the efferocytic activity of TAMs, rescued E2-dependent suppression of type I IFN signaling, and potentiated intratumoral adaptive immune cell function. Efferocytosis following radiation-induced cancer cell apoptosis limits the efficacy of radiation therapy. Importantly, we determined that preconditioning with either ER-directed endocrine therapies or CX3CR1 inhibition enhanced the antitumor efficacy of radiation therapy by reversing macrophage suppression and reviving intratumoral T cell activation. Our work defines the mechanisms by which E2 increases the efferocytotic activity of TAMs to establish an immunosuppressive tumor microenvironment and demonstrates how this process can be reversed with endocrine therapies which target ER.

cancer biology↗

bioRxiv2024

Protein CoAlation on TXNRD2 regulates mitochondrial thioredoxin system to protect against ferroptosis

The Cystine-xCT transporter-Glutathione (GSH)-GPX4 axis is the canonical pathway to protect against ferroptosis. While not required for ferroptosis-inducing compounds (FINs) targeting GPX4, FINs targeting the xCT transporter require mitochondria and its lipid peroxidation to trigger ferroptosis. However, the mechanism underlying the difference between these FINs is still unknown. Given that cysteine is also required for coenzyme A (CoA) biosynthesis, here we show that CoA supplementation specifically prevents ferroptosis induced by xCT inhibitors but not GPX4 inhibitors. We find that, auranofin, a thioredoxin reductase inhibitor, abolishes the protective effect of CoA. We also find that CoA availability determines the enzymatic activity of thioredoxin reductase, but not thioredoxin. Importantly, the mitochondrial thioredoxin system, but not the cytosolic thioredoxin system, determines CoA-mediated ferroptosis inhibition. Our data show that the CoA regulates the in vitro enzymatic activity of mitochondrial thioredoxin reductase (TXNRD2) by covalently modifying the thiol group of cysteine (CoAlation) on Cys-483. Replacing Cys-483 with alanine on TXNRD2 abolishes its in vitro enzymatic activity and ability to protect cells from ferroptosis. Targeting xCT to limit cysteine import and, therefore, CoA biosynthesis reduced CoAlation on TXNRD2, an effect that was rescued by CoA supplementation. Furthermore, the fibroblasts from patients with disrupted CoA metabolism demonstrate increased mitochondrial lipid peroxidation. In organotypic brain slice cultures, inhibition of CoA biosynthesis leads to an oxidized thioredoxin system, mitochondrial lipid peroxidation, and loss in cell viability, which were all rescued by ferrostatin-1. These findings identify CoA-mediated post-translation modification to regulate the thioredoxin system as an alternative ferroptosis protection pathway with potential clinical relevance for patients with disrupted CoA metabolism.

cell biology↗

bioRxiv2022

Ablative radiotherapy improves survival in autochthonous cancer mouse models

Genetically engineered mouse models (GEMMs) of cancer are powerful tools to study mechanisms of disease progression and therapy response, yet little is known about how these models respond to multimodality therapy used for curative intent in patients. Radiation therapy (RT) is frequently used to treat localized cancers with curative intent, delay progression of oligometastases, and palliate symptoms of metastatic disease. RT can be combined with surgery, systemic therapy, and/or immunotherapy for definitive treatment of solid tumors. Here we report the development, testing, and validation of a murine platform that faithfully emulates human stereotactic ablative radiotherapy (SABR). We demonstrate that SABR regimens used in clinical practice can be effectively delivered in mouse models and establish the intestinal tract as the dose limiting organ for abdominopelvic radiation with notable, but transient, lymphodepletion. SABR alters tumor stroma and immune environment, improves survival in GEMMs of primary prostate and colorectal cancer, and synergizes with androgen deprivation in prostate cancer. While SABR is capable of fully ablating xenografts it is unable to completely eradicate disease in GEMMs. These data show that some GEMMs are resistant to curative therapy suggesting the existence of treatment-resistant persister cells in these models.

cancer biology↗

bioRxiv2021

CometChip Enables Parallel Analysis of Multiple DNA Repair Activities

DNA damage can be cytotoxic and mutagenic and is directly linked to aging, cancer, and heritable diseases. To counteract the deleterious effects of DNA damage, cells have evolved highly conserved DNA repair pathways. Many commonly used DNA repair assays are relatively low throughput and are limited to analysis of one protein or one pathway. Here, we have explored the capacity of the CometChip platform for parallel analysis of multiple DNA repair activities. Taking advantage of the versatility of the traditional comet assay and leveraging micropatterning techniques, the CometChip platform offers increased throughput and sensitivity compared to the traditional comet assay. By exposing cells to DNA damaging agents that create substrates of Base Excision Repair, Nucleotide Excision Repair, and Non-Homologous End Joining, we show that the CometChip is an effective method for assessing repair deficiencies in all three pathways. With these advanced applications of the CometChip platform, we expand the efficacy of the comet assay for precise, high-throughput, parallel analysis of multiple DNA repair activities.

molecular biology↗

bioRxiv2019

BRD4 Prevents R-Loop Formation and Transcription-Replication Conflicts by Ensuring Efficient Transcription Elongation

Effective spatio-temporal control of transcription and replication during S-phase is paramount to maintaining genomic integrity and cell survival. Dysregulation of these systems can lead to conflicts between the transcription and replication machinery causing DNA damage and cell death. BRD4, a BET bromodomain protein and known transcriptional regulator, interacts with P-TEFb to ensure efficient transcriptional elongation by stimulating phosphorylation of RNA Polymerase II (RNAPII). Here we report that disruption of BET bromodomain protein function causes RNAPII pausing on the chromatin and DNA damage affecting cells in S-phase. We find that this persistent, RNAPII-dependent pausing leads to accumulation of RNA:DNA hybrids (R-loops), which are known to lead to transcription-replication conflicts (TRCs), DNA damage, and cell death. Furthermore, we show that resolution of R-loops abrogates BET bromodomain inhibitor-induced DNA damage, and that BET bromodomain inhibition induces both R-loops and DNA damage at sites of BRD4 occupancy. Finally, we see that the BRD4 C-terminal domain, which interacts with P-TEFb, is required to prevent R-loop formation and DNA damage caused by BET bromodomain inhibition and that oncogenes which promote transcription and replication exacerbate BET bromodomain inhibitor-induced DNA damage. Together, these findings demonstrate that BET bromodomain inhibitors can damage DNA via induction of R-loops and TRCs in highly replicative cells.

cancer biology↗