Identification of 4-(6-((2-methoxyphenyl)amino)pyrazin-2-yl)benzoic acids as CSNK2A inhibitors with antiviral activity and improved selectivity over PIM3.

We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4'-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity.

The Formation of a Camalexin Biosynthetic Metabolon.

Arabidopsis (Arabidopsis thaliana) efficiently synthesizes the antifungal phytoalexin camalexin without the apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting that the biosynthetic pathway of this compound is channeled by the formation of an enzyme complex. To identify such protein interactions, we used two independent untargeted coimmunoprecipitation (co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits and determined that the camalexin biosynthetic P450 enzymes copurified with these enzymes. These interactions were confirmed by targeted co-IP and Förster resonance energy transfer measurements based on fluorescence lifetime microscopy (FRET-FLIM). Furthermore, the interaction of CYP71A13 and Arabidopsis P450 Reductase1 was observed. We detected increased substrate affinity of CYP79B2 in the presence of CYP71A13, indicating an allosteric interaction. Camalexin biosynthesis involves glutathionylation of the intermediary indole-3-cyanohydrin, which is synthesized by CYP71A12 and especially CYP71A13. FRET-FLIM and co-IP demonstrated that the glutathione transferase GSTU4, which is coexpressed with Trp- and camalexin-specific enzymes, is physically recruited to the complex. Surprisingly, camalexin concentrations were elevated in knockout and reduced in GSTU4-overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather plays a role in a competing mechanism.

Neuronavigation in minimally invasive spine surgery.

OBJECT: Parallel advancements in image guidance technology and minimal access techniques continue to push the frontiers of minimally invasive spine surgery (MISS). While traditional intraoperative imaging remains widely used, newer platforms, such as 3D-fluoroscopy, cone-beam CT, and intraoperative CT/MRI, have enabled safer, more accurate instrumentation placement with less radiation exposure to the surgeon. The goal of this work is to provide a review of the current uses of advanced image guidance in MISS. METHODS: The authors searched PubMed for relevant articles concerning MISS, with particular attention to the use of image-guidance platforms. Pertinent studies published in English were further compiled and characterized into relevant analyses of MISS of the cervical, thoracic, and lumbosacral regions. RESULTS: Fifty-two studies were included for review. These describe the use of the iso-C system for 3D navigation during C1-2 transarticular screw placement, the use of endoscopic techniques in the cervical spine, and the role of navigation guidance at the occipital-cervical junction. The authors discuss the evolving literature concerning neuronavigation during pedicle screw placement in the thoracic and lumbar spine in the setting of infection, trauma, and deformity surgery and review the use of image guidance in transsacral approaches. CONCLUSIONS: Refinements in image-guidance technologies and minimal access techniques have converged on spinal pathology, affording patients the ability to undergo safe, accurate operations without the associated morbidities of conventional approaches. While percutaneous transpedicular screw placement is among the most common procedures to benefit from navigation, other areas of spine surgery can benefit from advances in neuronavigation and further growth in the field of image-guided MISS is anticipated.

Machine learning reveals STAT motifs as predictors for GR-mediated gene repression.

Glucocorticoids are potent immunosuppressive drugs, but long-term treatment leads to severe side-effects. While there is a commonly accepted model for GR-mediated gene activation, the mechanism behind repression remains elusive. Understanding the molecular action of the glucocorticoid receptor (GR) mediated gene repression is the first step towards developing novel therapies. We devised an approach that combines multiple epigenetic assays with 3D chromatin data to find sequence patterns predicting gene expression change. We systematically tested> 100 models to evaluate the best way to integrate the data types and found that GR-bound regions hold most of the information needed to predict the polarity of Dex-induced transcriptional changes. We confirmed NF-κB motif family members as predictors for gene repression and identified STAT motifs as additional negative predictors.

Identification of 4-(6-((2-methoxyphenyl)amino)pyrazin-2-yl)benzoic acids as CSNK2A inhibitors with antiviral activity and improved selectivity over PIM3.

We report the synthesis of 2,6-disubstituted pyrazines as potent cell active CSNK2A inhibitors. 4'-Carboxyphenyl was found to be the optimal 2-pyrazine substituent for CSNK2A activity, with little tolerance for additional modification. At the 6-position, modifications of the 6-isopropylaminoindazole substituent were explored to improve selectivity over PIM3 while maintaining potent CSNK2A inhibition. The 6-isopropoxyindole analogue 6c was identified as a nanomolar CSNK2A inhibitor with 30-fold selectivity over PIM3 in cells. Replacement of the 6-isopropoxyindole by isosteric ortho-methoxy anilines, such as 7c, generated analogues with selectivity for CSNK2A over PIM3 and improved the kinome-wide selectivity. The optimized 2,6-disubstituted pyrazines showed inhibition of viral replication consistent with their CSNK2A activity.

How to tame your genes: mechanisms of inflammatory gene repression by glucocorticoids.

Glucocorticoids (GCs) are widely used therapeutic agents to treat a broad range of inflammatory conditions. Their functional effects are elicited by binding to the glucocorticoid receptor (GR), which regulates transcription of distinct gene networks in response to ligand. However, the mechanisms governing various aspects of undesired side effects versus beneficial immunomodulation upon GR activation remain complex and incompletely understood. In this review, we discuss emerging models of inflammatory gene regulation by GR, highlighting GR's regulatory specificity conferred by context-dependent changes in chromatin architecture and transcription factor or co-regulator dynamics. GR controls both gene activation and repression, with the repression mechanism being central to favourable clinical outcomes. We describe current knowledge about 3D genome organisation and its role in spatiotemporal transcriptional control by GR. Looking beyond, we summarise the evidence for dynamics in gene regulation by GR through cooperative convergence of epigenetic modifications, transcription factor crosstalk, molecular condensate formation and chromatin looping. Further characterising these genomic events will reframe our understanding of mechanisms of transcriptional repression by GR.

Enhancer RNA Expression in Response to Glucocorticoid Treatment in Murine Macrophages.

Glucocorticoids are potent anti-inflammatory drugs; however, their molecular mode of action remains complex and elusive. They bind to the glucocorticoid receptor (GR), a nuclear receptor that controls gene expression in almost all tissues in a cell type-specific manner. While GR's transcriptional targets mediate beneficial reactions in immune cells, they also harbor the potential of adverse metabolic effects in other cell types such as hepatocytes. Here, we have profiled nascent transcription upon glucocorticoid stimulation in LPS-activated primary murine macrophages using 4sU-seq. We compared our results to publicly available nascent transcriptomics data from murine liver and bioinformatically identified non-coding RNAs transcribed from intergenic GR binding sites in a tissue-specific fashion. These tissue-specific enhancer RNAs (eRNAs) correlate with target gene expression, reflecting cell type-specific glucocorticoid responses. We further associate GR-mediated eRNA expression with changes in H3K27 acetylation and BRD4 recruitment in inflammatory macrophages upon glucocorticoid treatment. In summary, we propose a common mechanism by which GR-bound enhancers regulate target gene expression by changes in histone acetylation, BRD4 recruitment and eRNA expression. We argue that local eRNAs are potential therapeutic targets downstream of GR signaling which may modulate glucocorticoid response in a cell type-specific way.

Pure Endoscopic Supracerebellar Infratentorial Approach to the Pineal Region: A Case Series.

BACKGROUND: Surgical approaches to pineal lesions present a challenge because of limited visibility and maneuverability within the posterior fossa. The supracerebellar infratentorial (SCIT) technique has emerged as an approach to pineal lesions. We aim to demonstrate the efficacy of the endoscopic SCIT technique through a case series conducted at our institution and highlight the advantages of the endoscopic technique over the microscopic alternative. OBJECTIVE: To evaluate the effectiveness and safety of the endoscopic SCIT approach. METHODS: We conducted a retrospective review of pure endoscopic SCIT cases conducted at our institution. Demographic information, preoperative and postoperative imaging, neurological status, surgical data, and complications were recorded. RESULTS: Six patients who underwent pure endoscopic SCIT surgery were identified for analysis. The average lesion volume was 14.12 ± 7.24 cm3. The median postoperative length of stay was 5.0 days. The average surgical duration was 3.54 ± 0.71 hours. All operations were performed in prone position with zero- and 30-degree endoscopes. Pathology included one each of the following lesions: Pineoctyoma, metastatic melanoma, atypical teratoma rhabdoid tumor, ependymoma, epidermoid, abscess. Gross total resection (GTR) was achieved in 5/6 patients, and near-total resection was achieved in 1/6 patients. Surgical complications included one case of postoperative infection. CONCLUSION: The purely endoscopic SCIT approach is a safe and effective approach for deep-seated pineal lesions. This approach allows for visibility and maneuverability around the lesion and facilitates high rates of GTR.