ST-Segment Elevation in a Patient With Nausea, Vomiting, and Intracerebral Hemorrhage.

A 60-year-old man who presented with nausea, vomiting, and intracerebral hemorrhage developed inferior ST-segment elevation and angina. Coronary angiography showed no coronary obstruction. The patient was found to have a small bowel obstruction causing superior translocation of the heart. Relief of obstruction caused immediate resolution of electrocardiographic changes and symptoms. (Level of Difficulty: Beginner.).

Ring expansions of acyloxy nitroso compounds.

Treatment of cyclopentanone and cyclobutanone-derived oximes with lead (IV) tetraacetate gives the bright blue acyloxy nitroso compounds, which upon basic hydrolysis yields the ring expansion product cyclic hydroxamic acids in 12-81% yield. Reactions of substituted cyclopentanones provide ring expanded products where the -NOH group regioselectively inserts to the more substituted position and gives a better yield compared to the treatment of the same ketone with a basic solution of Piloty's acid. Reaction of phosphines with acyloxy nitroso compounds generally generates a ring-expanded Beckmann rearrangement product that can be hydrolyzed to the corresponding lactam. Acyloxy nitroso compounds that undergo rapid hydrolysis to HNO do not show this ring expansion reactivity. These results further demonstrate the versatility of acyloxy nitroso compound to yield structurally complex materials.

Non-targeted metabolomics of Brg1/Brm double-mutant cardiomyocytes reveals a novel role for SWI/SNF complexes in metabolic homeostasis.

Mammalian SWI/SNF chromatin-remodeling complexes utilize either BRG1 or Brm as alternative catalytic subunits to alter the position of nucleosomes and regulate gene expression. Genetic studies have demonstrated that SWI/SNF complexes are required during cardiac development and also protect against cardiovascular disease and cancer. However, Brm constitutive null mutants do not exhibit a cardiomyocyte phenotype and inducible Brg1 conditional mutations in cardiomyocyte do not demonstrate differences until stressed with transverse aortic constriction, where they exhibit a reduction in cardiac hypertrophy. We recently demonstrated the overlapping functions of Brm and Brg1 in vascular endothelial cells and sought here to test if this overlapping function occurred in cardiomyocytes. Brg1/Brm double mutants died within 21 days of severe cardiac dysfunction associated with glycogen accumulation and mitochondrial defects based on histological and ultrastructural analyses. To determine the underlying defects, we performed nontargeted metabolomics analysis of cardiac tissue by GC/MS from a line of Brg1/Brm double-mutant mice, which lack both Brg1 and Brm in cardiomyocytes in an inducible manner, and two groups of controls. Metabolites contributing most significantly to the differences between Brg1/Brm double-mutant and control-group hearts were then determined using the variable importance in projection analysis. Increased cardiac linoleic acid and oleic acid suggest alterations in fatty acid utilization or intake are perturbed in Brg1/Brm double mutants. Conversely, decreased glucose-6-phosphate, fructose-6-phosphate, and myoinositol suggest that glycolysis and glycogen formation are impaired. These novel metabolomics findings provide insight into SWI/SNF-regulated metabolic pathways and will guide mechanistic studies evaluating the role of SWI/SNF complexes in homeostasis and cardiovascular disease prevention.

Non-targeted metabolomics analysis of cardiac Muscle Ring Finger-1 (MuRF1), MuRF2, and MuRF3 in vivo reveals novel and redundant metabolic changes.

The muscle-specific ubiquitin ligases MuRF1, MuRF2, MuRF3 have been reported to have overlapping substrate specificities, interacting with each other as well as proteins involved in metabolism and cardiac function. In the heart, all three MuRF family proteins have proven critical to cardiac responses to ischemia and heart failure. The non-targeted metabolomics analysis of MuRF1-/-, MuRF2-/-, and MuRF3-/- hearts was initiated to investigate the hypothesis that MuRF1, MuRF2, and MuRF3 have a similarly altered metabolome, representing alterations in overlapping metabolic processes. Ventricular tissue was flash frozen and quantitatively analyzed by GC/MS using a library built upon the Fiehn GC/MS Metabolomics RTL Library. Non-targeted metabolomic analysis identified significant differences (via VIP statistical analysis) in taurine, myoinositol, and stearic acid for the three MuRF-/- phenotypes relative to their matched controls. Moreover, pathway enrichment analysis demonstrated that MuRF1-/- had significant changes in metabolite(s) involved in taurine metabolism and primary acid biosynthesis while MuRF2-/- had changes associated with ascorbic acid/aldarate metabolism (via VIP and t-test analysis vs. sibling-matched wildtype controls). By identifying the functional metabolic consequences of MuRF1, MuRF2, and MuRF3 in the intact heart, non-targeted metabolomics analysis discovered common pathways functionally affected by cardiac MuRF family proteins in vivo. These novel metabolomics findings will aid in guiding the molecular studies delineating the mechanisms that MuRF family proteins regulate metabolic pathways. Understanding these mechanism is an important key to understanding MuRF family proteins' protective effects on the heart during cardiac disease.

Investigating the proteome reactivity and selectivity of aryl halides.

Protein-reactive electrophiles are critical to chemical proteomic applications including activity-based protein profiling, site-selective protein modification, and covalent inhibitor development. Here, we explore the protein reactivity of a panel of aryl halides that function through a nucleophilic aromatic substitution (S(N)Ar) mechanism. We show that the reactivity of these electrophiles can be finely tuned by varying the substituents on the aryl ring. We identify p-chloro- and fluoronitrobenzenes and dichlorotriazines as covalent protein modifiers at low micromolar concentrations. Interestingly, investigating the site of labeling of these electrophiles within complex proteomes identified p-chloronitrobenzene as highly cysteine selective, whereas the dichlorotriazine favored reactivity with lysines. These studies illustrate the diverse reactivity and amino-acid selectivity of aryl halides and enable the future application of this class of electrophiles in chemical proteomics.

1,3,5-Triazine as a modular scaffold for covalent inhibitors with streamlined target identification.

Small-molecule inhibitors can accelerate the functional annotation and validate the therapeutic potential of proteins implicated in disease. Phenotypic screens provide an effective platform to identify such pharmacological agents but are often hindered by challenges associated with target identification. For many protein targets, these bottlenecks can be overcome by incorporating electrophiles into small molecules to covalently trap interactions in vivo and by employing bioorthogonal handles to enrich the protein targets directly from a complex proteome. Here we present the trifunctionalized 1,3,5-triazine as an ideal modular scaffold for generating libraries of irreversible inhibitors with diverse target specificities. A divergent synthetic scheme was developed to derivatize the triazine with an electrophile for covalent modification of target proteins, an alkyne as a click-chemistry handle for target identification, and a diversity element to direct the compounds toward distinct subsets of the proteome. We specifically targeted our initial library toward cysteine-mediated protein activities through incorporation of thiol-specific electrophiles. From this initial screen we identified two compounds, RB-2-cb and RB-11-ca, which are cell permeable and highly selective covalent modifiers for Cys239 of ß-tubulin (TUBB) and Cys53 of protein disulfide isomerase (PDI) respectively. These compounds demonstrate in vitro and cellular potencies that are comparable to currently available modulators of tubulin polymerization and PDI activity. Our studies demonstrate the versatility of the triazine as a modular scaffold to generate potent and selective covalent modifiers of diverse protein families for chemical genetics applications.

Synthesis of cyclic hydroxamic acids through -NOH insertion of ketones.

Treatment of cyclobutanone or cyclopentanone with N-hydroxybenzenesulfonamide under basic conditions yields the ring-expanded cyclic hydroxamic acid in 18-69% yield. Reactions of substituted cyclobutanones give ring expanded products where the -NOH group regio- and stereoselectively inserts to the more substituted position. This expansion likely proceeds through a mechanism that includes addition of the N-anion of N-hydroxybenzenesulfonamide to the ketone and a C-nitroso intermediate that rearranges to the final product.