Biology subjects

Keillor, J. W.

Publications and source records attributed to Keillor, J. W..

5 recordsLinked to original sources

bioRxiv2026

Chemoproteomics discovery of a CNS-penetrant covalent inhibitor of PIKfyve

PIKfyve is a lipid kinase involved in regulating protein clearance mechanisms and is a promising target for the treatment of neurodegenerative diseases. Here, we present the discovery and optimization of a CNS-penetrant covalent PIKfyve inhibitor, DUN058, which achieves sustained target occupancy in vivo. Covalent screening hits, identified from chemoproteomics experiments performed in live cells, were rapidly optimized to deliver a brain-penetrant covalent inhibitor of PIKfyve. This covalency centered approach employed a suite of mass spectrometry, biochemical and in vivo assays to optimize compound potency, selectivity, and CNS permeability. The target nucleophile, cysteine 1970, is on a flexible loop that appears distal from the kinase active site, highlighting the power of chemoproteomics screening to identify novel nucleophilic amino acids for covalent modification. DUN058 achieves efficient covalency at the target cysteine, as well as highly selective covalent and reversible selectivity profiles. Covalent PIKfyve inhibition results in modulation of downstream pathway activity, including activation of the transcription factor TFEB, upregulation of protein clearance pathways, and increased GPNMB transcription and secretion of exosome markers. When dosed in vivo, DUN058 achieves sustained target occupancy in the brains of mice long after systemic compound clearance, holding promise for achieving a sustained duration of action in the CNS at low doses, without prolonged effects in the periphery. Taken together, the development of DUN058 is a demonstration of chemoproteomics-based discovery for a high value CNS target, providing an orally bioavailable and covalent PIKfyve inhibitor.

biochemistry↗

bioRxiv2024

Transglutaminase 2 function in glioblastoma tumor efferocytosis

Glioblastoma is an aggressive and incurable type of brain cancer. Regions of tissue necrosis are a distinctive pathological feature of this cancer. These arise through thrombosis of tumor vasculature, driven by tumor-derived pro-coagulation factors. In studies of transglutaminase 2 (TGM2), we observed that TGM2 mRNA expression in glioblastoma was primarily in a subset of tumor-infiltrating myeloid cells with hypoxia gene expression signatures. Analysis of xenograft and human glioblastoma samples by immunohistochemistry showed that macrophages in the vicinity of necrotic regions expressed very high levels of TGM2. These macrophages were engaged in the phagocytosis of apoptotic cells, a process known as efferocytosis. In cell culture, incubation of macrophages with apoptotic cells induced TGM2 expression in macrophages, and TGM2 inhibitors blocked efferocytosis. In patient-derived glioblastoma organoids cultured in 5% O2, a basal level of apoptosis was observed, and endogenous macrophages were observed in the process of clearing apoptotic cells. Clearance of apoptotic cells was reduced in organoids treated with a TGM2 inhibitor. Apoptotic cells and efferocytosis were both markedly lower in organoids grown in 20% O2. These data, together with previous work, define a model in which necrotic regions in glioblastoma induce hypoxia-driven apoptosis, which in turn promotes efferocytosis by macrophages. TGM2 is both a marker of efferocytosis and a target for efferocytosis inhibition in this process. Efferocytosis is a potent immunosuppressive mechanism, so this process provides an additional mechanism by which large glioblastoma tumors can evade immune responses.

cancer biology↗

bioRxiv2024

Stabilizing transglutaminase 2 in the open conformation results in reactive astrocytes being more neurosupportive

Astrocytes play critical roles in supporting structural and metabolic homeostasis in the central nervous system (CNS). CNS injury leads to the development of a range of reactive phenotypes in astrocytes whose molecular determinants are poorly understood. Finding ways to modulate astrocytic injury responses and leverage a pro-recovery phenotype holds promise in treating CNS injury. Recently, it has been demonstrated that ablation of astrocytic transglutaminase 2 (TG2) modulates the phenotype of reactive astrocytes in a way that improves neuronal injury outcomes both in vitro and in vivo. In an in vivo mouse model, pharmacological inhibition of TG2 with the irreversible inhibitor VA4 phenocopies the neurosupportive effects of TG2 deletion in astrocytes. In this study, we provide insights into the mechanisms by which TG2 deletion or inhibition result in a more neurosupportive astrocytic phenotype. Using a neuron-astrocyte co-culture model, we show that VA4 treatment improves the ability of astrocytes to support neurite outgrowth on an injury-relevant matrix. To better understand how pharmacologically altering TG2 affects its ability to regulate reactive astrocyte phenotypes, we assessed how VA4 inhibition impacts TG2s interaction with Zbtb7a, a transcription factor we have previously identified as a functionally relevant TG2 nuclear interactor. The results of these studies demonstrate that VA4 significantly decreases the interaction of TG2 and Zbtb7a. TG2s interactions with Zbtb7a, as well as a wide range of other transcription factors and chromatin regulatory proteins, suggest that TG2 may act as an epigenetic regulator to modulate gene expression. To begin to understand if TG2-mediated epigenetic modification may impact astrocytic phenotypes in our models, we interrogated the effect of TG2 deletion and VA4 treatment on histone acetylation and found significantly greater acetylation in both experimental groups. Consistent with these findings, previous RNA-sequencing and our present proteomic analysis also supported a predominant transcriptionally suppressive role of TG2 in astrocytes. Our proteomic data additionally unveiled pronounced changes in lipid and antioxidant metabolism in astrocytes with TG2 deletion or inhibition, which likely contribute to the enhanced neurosupportive function of these astrocytes.

neuroscience↗

bioRxiv2021

Deletion or inhibition of astrocytic transglutaminase 2 promotes functional recovery after spinal cord injury

Following CNS injury astrocytes become "reactive" and exhibit pro-regenerative or harmful properties. However, the molecular mechanisms that cause astrocytes to adopt either phenotype are not well understood. Transglutaminase 2 (TG2) plays a key role in regulating the response of astrocytes to insults. Here we used mice in which TG2 was specifically deleted in astrocytes (Gfap-Cre+/-TG2fl/fl, referred to here as TG2-A-cKO) in a spinal cord contusion injury (SCI) model. Deletion of TG2 from astrocytes resulted in a significant improvement in motor function following SCI. GFAP and NG2 immunoreactivity, as well as number of SOX9 positive cells, were significantly reduced in TG2-A-cKO_mice. RNA-seq analysis of spinal cords from TG2-A-cKO and control mice 3 days postinjury identified thirty-seven differentially expressed genes, all of which were increased in TG2-A-cKO mice. Pathway analysis reveals a prevalence for fatty acid metabolism, lipid storage and energy pathways, which play essential roles in neuron-astrocyte metabolic coupling. Excitingly, treatment of wild type mice with the selective TG2 inhibitor VA4 significantly improved functional recovery after SCI, similar to what was observed using the genetic model. These findings indicate the use of TG2 inhibitors as a novel strategy for the treatment of SCI and other CNS injuries.

neuroscience↗

bioRxiv2019

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis

Transglutaminase 2 (TG2) is a ubiquitous enzyme with transamidating activity. We report here that the expression and activity of TG2 are enhanced in cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 activity impair bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e. hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis. As a consequence, TG2 activation results in increased protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

microbiology↗