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The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

Objective Intestinal inflammation resulting from manipulation-induced mast cell activation is a crucial mechanism in the pathophysiology of postoperative ileus (POI). Recently it has been shown that spleen tyrosine kinase (Syk) is involved in mast cell degranulation. Therefore, we have evaluated the effect of the Syk-inhibitor GSK compound 143 (GSK143) as potential treatment to shorten POI. Design In vivo: in a mouse model of POI, the effect of the Syk inhibitor (GSK143) was evaluated on gastrointestinal transit, muscular inflammation and cytokine production. In vitro: the effect of GSK143 and doxantrazole were evaluated on cultured peritoneal mast cells (PMCs) and bone marrow derived macrophages. Results In vivo: intestinal manipulation resulted in a delay in gastrointestinal transit at t=24 h (Geometric Center (GC): 4.4±0.3). Doxantrazole and GSK143 significantly increased gastrointestinal transit (GC doxantrazole (10 mg/kg): 7.2±0.7; GSK143 (1 mg/kg): 7.6±0.6), reduced inflammation and prevented recruitment of immune cells in the intestinal muscularis. In vitro: in PMCs, substance P (0–90 μM) and trinitrophenyl (0–4 μg/ml) induced a concentration-dependent release of β-hexosaminidase. Pretreatment with doxantrazole and GSK143 (0.03–10 μM) concentration dependently blocked substance P and trinitrophenyl induced β-hexosaminidase release. In addition, GSK143 was able to reduce cytokine expression in endotoxin-treated bone marrow derived macrophages in a concentration-dependent manner. Conclusions The Syk inhibitor GSK143 reduces macrophage activation and mast cell degranulation in vitro. In addition, it inhibits manipulation-induced intestinal muscular inflammation and restores intestinal transit in mice. These findings suggest that Syk inhibition may be a new tool to shorten POI.

A structure-guided small-molecule and chemoproteomics approach uncovers a catalytic site inhibitor selective for the jumonji subfamily of H3K27me3 demethylases; the inhibitor decreases lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages. JMJ demethylase inhibitors and inflammation Jumonji (JMJ) family histone demethylases have become recognized as key regulators of transcription, although the importance of their enzymatic activity - as opposed to their structural role - has been unclear. JMJD3 demethylases are specific for histone H3 trimethylated at Lys27 (H3K27me3) and are involved in the inflammatory response, as well as other physiological functions. Here, a structure-guided small-molecule and chemoproteomics approach is used to discover a catalytic-site inhibitor selective for the H3K27me3-specific JMJ subfamily. The inhibitor reduces lipopolysaccharide-induced pro-inflammatory cytokine production in human primary macrophages. This study demonstrates the importance of JMJ demethylase activity and suggests that small-molecule inhibitors of JMJ enzymes could have therapeutic applications. The jumonji (JMJ) family of histone demethylases are Fe 2+ - and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes 1 , 2 , 3 , 4 . These enzymes demethylate lysine residues in histones in a methylation-state and sequence-specific context 5 . Considerable effort has been devoted to gaining a mechanistic understanding of the roles of histone lysine demethylases in eukaryotic transcription, genome integrity and epigenetic inheritance 2 , 4 , 6 , as well as in development, physiology and disease 3 , 7 . However, because of the absence of any selective inhibitors, the relevance of the demethylase activity of JMJ enzymes in regulating cellular responses remains poorly understood. Here we present a structure-guided small-molecule and chemoproteomics approach to elucidating the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX) 8 . The liganded structures of human and mouse JMJD3 provide novel insight into the specificity determinants for cofactor, substrate and inhibitor recognition by the KDM6 subfamily of demethylases. We exploited these structural features to generate the first small-molecule catalytic site inhibitor that is selective for the H3K27me3-specific JMJ subfamily. We demonstrate that this inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. Our results resolve the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provide encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family.

Replying to B. Heinemann et al. Nature514, 10.1038/nature13688 (2014) We welcome the accompanying Comment 1 by Heinemann et al. , in which the authors use an extensive panel of sensitive KDM assays to independently confirm our results 2 that GSK-J1 is a potent KDM6 inhibitor. Additionally, Heinemann et al. 1 demonstrate that GSK-J1 has some, albeit weaker, activity towards KDM5B and KDM5C, for which we only had preliminary data available at the time of our original publication. As our jumonji assay portfolio expands, we have continued to update the GSK-J1 activity profile on the SGC website ( http://www.thesgc.org/chemical-probes/GSKJ1 ); this includes KDM5 inhibition activity by GSK-J1 similar to that reported by Heinemann. In conclusion, GSK-J1 remains the most selective KDM inhibitor yet disclosed and thus a valuable chemical tool.

The success of new therapies hinges on our ability to understand their molecular and cellular mechanisms of action. We modified BET bromodomain inhibitors, an epigenetic-based therapy, to create functionally conserved compounds that are amenable to click chemistry and can be used as molecular probes in vitro and in vivo. We used click proteomics and click sequencing to explore the gene regulatory function of BRD4 (bromodomain containing protein 4) and the transcriptional changes induced by BET inhibitors. In our studies of mouse models of acute leukemia, we used high-resolution microscopy and flow cytometry to highlight the heterogeneity of drug activity within tumor cells located in different tissue compartments. We also demonstrate the differential distribution and effects of BET inhibitors in normal and malignant cells in vivo. This study provides a potential framework for the preclinical assessment of a wide range of drugs.

Here we show that the inhibitor is not specific for the H3K27me3/me2-demethylase subfamily in vitro and in tissue culture assays. Thus, the inhibitor cannot be used alone for drawing conclusions regarding the specific role of H3K27me3/me2-demethylase activity in biological processes or disease.

Nutrients, including bioactive natural compounds, have been demonstrated to affect key metabolic processes implicated in tumor growth and progression, both in preclinical and clinical trials. Although the application of precision nutrition as a complementary approach to improve cancer treatments is still incipient in clinical practice, the development of powerful \"omics\" techniques has opened new possibilities for delivering nutritional advice to cancer patients. Precision nutrition may contribute to improving the plasticity and function of antitumor immune responses. Herein, we present the results of a randomized, prospective, longitudinal, double-blind, and parallel clinical trial (NCT05080920) in cancer patients to explore the immune-metabolic effects of a bioactive formula based on diterpenic phenols from rosemary, formulated with bioactive alkylglycerols (Lipchronic WO/2017/187000). The trial involved cancer patients, including those with lung cancer (LC), colorectal cancer (CRC), and breast cancer (BC), undergoing chemotherapy, targeted biological therapy, and/or immunotherapy. The main readouts of the study were the analysis of Lip on systemic inflammation, hemogram profile, anthropometry, lipid and glucose profiles, and tolerability. Additionally, a deep immune phenotyping of peripheral blood mononuclear cells (PBMCs) was performed to identify the functional effects of Lip on key mediators of the immune system. Lip was well tolerated. The lung cancer subgroup of patients showed a reduction in biomarkers of systemic inflammation, including the neutrophil-to-lymphocyte ratio (NLR). Furthermore, modulation of key players in the immune system associated with the experimental treatment Lip compared to the control placebo (Pla) treatment was revealed, with particularities among the distinct subgroups of patients. Our results encourage further research to apply molecular nutrition-based strategies as a complementary tool in the clinical management of cancer patients, particularly in the current era of novel immunotherapies. ClinicalTrials.gov, identifier NCT05080920.

Miguel García-Baró ha identificado en el desarrollo de la Filosofía tres navegaciones generales, y el sentido que le da a la expresión navegación filosófica es muy semejante al significado de las salidas del Quijote. La primera navegación es como la primera salida de don Quijote a las aventuras: sin escudero, sin dinero, sin haberse armado caballero, o sea, sin prevención alguna. En 99d del Fedón, Platón dice por boca de Sócrates que los pensadores habían navegado la primera vez como si pudieran tratar las cosas directamente, como si pudieran conducirse entre y con las cosas directamente, a fin de conocerlas. Pero esta confianza excesiva no resultó en conocerlas, sino en una crisis de incertidumbre que con el tiempo vendría a ser llamada escepticismo. No podía ser otro el desenlace si de la multitud de doctrinas incompatibles se afirma de todas ellas estar en la verdad. La segunda navegación de la Filosofía se ve germinal en Anaxímenes de Mileto, pero en Parménides y en Heráclito es ya un hecho consagrado. Consiste en la búsqueda de algo donde las cosas mismas se reflejen para no perecer ante la multitud de opiniones o ante un dogmatismo, ese algo fue el espejo del discurso, el Logos, el medio de la verdad donde se deja captar. El ser es estable, eterno, y este Logos nuestro que es el pensamiento, si acaso quiere aproximarse a la verdad, debe ser estable como el ser. Dos milenios de la Filosofía se han invertido en hacer del Logos, del juicio, el lugar primordial del conocimiento de la verdad; dos milenios y siga contando. Todavía cuando apareció la idea de una tercera navegación que descubra las formas más primitivas, elementales o remotas del conocimiento —en la persona de René Descartes—, el Logos o el juicio no perdió su lugar, pues esas formas remotas del conocimiento, precisamente, fueron buscadas para que nuestros juicios llegaran a esencias ideales.La primera navegación, primera salida de la Filosofía, consistió en el estudio de la naturaleza, incluyendo la búsqueda de un principio rector, tal y como se deja ver, por ejemplo, en Anaximandro de Mileto quien fuera el primero en escribir un tratado en prosa sobre los temas que, andado el tiempo, serían los de la Filosofía y de la ciencia, además de ser el primero en diseñar un mapa del mundo. La segunda navegación, que se emprende con el aprendizaje doloroso de los malogros de la primera, consiste en la teoría de la verdad, y como don Quijote, esta segunda salida no se realiza estando solo. La tercera se podría nombrar como teoría del conocimiento.