Four albumin-nitroxide conjugates were prepared and tested as metal-free organic radical contrast agents (ORCAs) for magnetic resonance imaging (MRI). Each human serum albumin (HSA) carrier bears multiple nitroxides conjugated via homocysteine thiolactones. These molecular conjugates retain important physical and biological properties of their HSA component, and the resistance of their nitroxide groups to bioreduction was retained or enhanced. The relaxivities are similar for these four conjugates and are much greater than those of their individual components: the HSA or the small nitroxide molecules. This new family of conjugates has excellent prospects for optimization as ORCAs.

Human Adenovirus type 6 (HAdV-C6) is a promising candidate for the development of oncolytic vectors as it has low seroprevalence and the intrinsic ability to evade tissue macrophages. However, its further development as a therapeutic agent is hampered by the lack of convenient cloning methods. We have developed a novel technology when a shuttle plasmid carrying the distal genome parts with modified E1A and E3 regions is recombined in vitro with the truncated HAdV-C6 genome. Using this approach, we have constructed a novel Ad6-hT-GM vector controlled by the hTERT promoter and expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) instead of 6.7K and gp19K E3 proteins. We have demonstrated that control by the hTERT promoter may result in delayed viral replication, which nevertheless does not significantly change the cytotoxic ability of recombinant viruses. The insertion of the transgene by displacing the E3-6.7K/gp19K region does not drastically change the expression patterns of E3 genes; however, mild changes in expression from major late promoter were observed. Finally, we have demonstrated that the treatment of human breast cancer xenografts in murine models with Ad6-hT-GM significantly decreased the tumor volume and improved survival time compared to mock-treated mice.

The NLRP3 inflammasome is a cytosolic protein complex important for the regulation and secretion of inflammatory cytokines including IL-1β and IL-18. Aberrant overactivation of NLRP3 is implicated in numerous inflammatory disorders. However, the activation and regulation of NLRP3 inflammasome signaling remains poorly understood, limiting our ability to develop pharmacologic approaches to target this important inflammatory complex. Here, we developed and implemented a high-throughput screen to identify compounds that inhibit inflammasome assembly and activity. From this screen we identify and profile inflammasome inhibition of 20 new covalent compounds across 9 different chemical scaffolds, as well as many known inflammasome covalent inhibitors. Intriguingly, our results indicate that NLRP3 possesses numerous reactive cysteines on multiple domains whose covalent targeting blocks activation of this inflammatory complex. Specifically, focusing on compound VLX1570, which possesses multiple electrophilic moieties, we demonstrate that this compound allows covalent, intermolecular crosslinking of NLRP3 cysteines to inhibit inflammasome assembly. Our results, along with the recent identification of numerous covalent molecules that inhibit NLRP3 inflammasome activation, suggests that NLRP3 serves as a cellular electrophile sensor important for coordinating inflammatory signaling in response to redox stress. Further, our results support the potential for covalent cysteine modification of NLRP3 for regulating inflammasome activation and activity.

SUMMARY: Pathogenic variants in the mitochondrial outer membrane GTPase MFN2 cause the peripheral neuropathy Charcot-Marie-Tooth Type 2A (CMT2A). These mutations disrupt MFN2-dependent regulation of diverse aspects of mitochondrial biology including organelle morphology, motility, mitochondrial-endoplasmic reticulum (ER) contacts (MERCs), and respiratory chain activity. However, no therapies currently exist to mitigate the mitochondrial dysfunction linked to genetic deficiencies in MFN2. Herein, we performed a drug repurposing screen to identify compounds that selectively activate the integrated stress response (ISR) – the predominant stress-responsive signaling pathway responsible for regulating mitochondrial morphology and function. This screen identified the compounds parogrelil and MBX-2982 as potent and selective activators of the ISR through the OMA1-DELE1-HRI signaling axis. We show that treatment with these compounds promotes adaptive, ISR-dependent remodeling of mitochondrial morphology and protects mitochondria against genetic and chemical insults. Moreover, we show that pharmacologic ISR activation afforded by parogrelil restores mitochondrial tubular morphology, promotes mitochondrial motility, rescues MERCs, and enhances mitochondrial respiration in MFN2 -deficient cells. These results demonstrate the potential for pharmacologic HRI activation as a viable strategy to mitigate mitochondrial dysfunction in CMT2A and other pathologies associated with MFN2 deficiency.

SUMMARY: The endoplasmic reticulum (ER) transporter solute carrier family 33 member 1 (SLC33A1) has emerged as an attractive therapeutic target in etiologically diverse diseases, ranging from lung cancer to neurodegenerative disorders. Yet, no pharmacologic SLC33A1 modulators have been described. Here, we show that the small molecule IXA4, a highly selective activator of the adaptive IRE1/XBP1s signaling arm of the unfolded protein response (UPR), binds to SLC33A1 and inhibits its activity. Genetic depletion of SLC33A1 phenocopies the selective induction of IRE1/XBP1s signaling brought about by IXA4 treatment. Chemoproteomic analyses and cryo-electron microscopy show that IXA4 binds SLC33A1 within the central channel to inhibit transport of its substrate metabolite(s). Binding of IXA4 to SLC33A1 leads to the accumulation of oxidized glutathione within the ER, hyperoxidizing the ER lumen and inducing activation of adaptive IRE1/XBP1s signaling. Consistent with this function, we find that pharmacologic inhibition of SLC33A1 with IXA4 selectively reduces viability of KEAP1-deficient lung adenocarcinoma cells that have elevated levels of glutathione, mimicking the sensitivity of these cells to genetic deletion of SLC33A1. Our work demonstrates a new physiologic role of SLC33A1 in regulation of ER redox homeostasis and designates IXA4 as a pharmacologic inhibitor of SLC33A1 that can be used to evaluate the biological impact and therapeutic utility of SLC33A1 inhibition in homeostasis and in disease.

SUMMARY: Pharmacological enhancement of endoplasmic reticulum (ER) proteostasis is an attractive strategy to mitigate pathology linked to etiologically-diverse protein misfolding diseases. However, despite this promise, few compounds have been identified that enhance ER proteostasis through defined mechanisms of action. We previously identified the phenylhydrazone-based compound AA263 as a compound that promotes adaptive ER proteostasis remodeling through mechanisms including preferential activation of the ATF6 signaling arm of the unfolded protein response (UPR). However, the protein target(s) of AA263 and the potential for further development of this class of ER proteostasis regulators had not been previously explored. Here, we employ chemical proteomics to demonstrate that AA263 covalently targets a subset of ER protein disulfide isomerases, revealing a potential molecular mechanism for the activation of ATF6 afforded by this compound. We then use medicinal chemistry to establish next-generation AA263 analogs showing improved potency and efficacy for ATF6 activation, as compared to the parent compound. Finally, we show that treatment with these AA263 analogs enhances secretory pathway proteostasis to correct the pathologic protein misfolding and trafficking of both a destabilized, disease-associated α1-antitrypsin (A1AT) variant and an epilepsy-associated GABA _A receptor variant. These results establish AA263 analogs with enhanced potential for correcting imbalanced ER proteostasis associated with etiologically-diverse protein misfolding disorders.