Synthesis of,and Conformational Studies on, 2-Trifluoromethyl,Substituted Benzimidazole Ribofuranosides

Abstract

The 1-β-D-ribofuranosides of several 2-trifluoromethyl benzimidazoles were prepared by the fusion method, and their conformations, particularly about the glycosidic bond, determined by ¹H NMR spectroscopy.     

The spliceosome catalyzes debranching in competition with reverse of the first chemical reaction

Splicing of nuclear pre-mRNA occurs via two steps of the transesterification reaction, forming a lariat intermediate and product. The reactions are catalyzed by the spliceosome, a large ribonucleoprotein complex composed of five small nuclear RNAs and numerous protein factors. The spliceosome shares a similar catalytic core structure with that of fungal group II introns, which can self-splice using the same chemical mechanism. Like group II introns, both catalytic steps of pre-mRNA splicing can efficiently reverse on the affinity-purified spliceosome. The spliceosome also catalyzes a hydrolytic spliced-exon reopening reaction as observed in group II introns, indicating a strong link in their evolutionary relationship. We show here that, by arresting splicing after the first catalytic step, the purified spliceosome can catalyze debranching of lariat-intron-exon 2. The debranching reaction, although not observed in group II introns, has similar monovalent cation preferences as those for splicing catalysis of group II introns. The debranching reaction is in competition with the reverse Step 1 reaction influenced by the ionic environment and the structure of components binding near the catalytic center, suggesting that the catalytic center of the spliceosome can switch between different conformations to direct different chemical reactions.     

Oncogenes and inflammation rewire host energy metabolism in the tumor microenvironment

Here, we developed a model system to evaluate the metabolic effects of oncogene(s) on the host microenvironment. A matched set of “normal” and oncogenically transformed epithelial cell lines were co-cultured with human fibroblasts, to determine the “bystander” effects of oncogenes on stromal cells. ROS production and glucose uptake were measured by FACS analysis. In addition, expression of a panel of metabolic protein biomarkers (Caveolin-1, MCT1, and MCT4) was analyzed in parallel. Interestingly, oncogene activation in cancer cells was sufficient to induce the metabolic reprogramming of cancer-associated fibroblasts toward glycolysis, via oxidative stress. Evidence for “metabolic symbiosis” between oxidative cancer cells and glycolytic fibroblasts was provided by MCT1/4 immunostaining. As such, oncogenes drive the establishment of a stromal-epithelial “lactate-shuttle”, to fuel the anabolic growth of cancer cells. Similar results were obtained with two divergent oncogenes (RAS and NFκB), indicating that ROS production and inflammation metabolically converge on the tumor stroma, driving glycolysis and upregulation of MCT4. These findings make stromal MCT4 an attractive target for new drug discovery, as MCT4 is a shared endpoint for the metabolic effects of many oncogenic stimuli. Thus, diverse oncogenes stimulate a common metabolic response in the tumor stroma. Conversely, we also show that fibroblasts protect cancer cells against oncogenic stress and senescence by reducing ROS production in tumor cells. Ras-transformed cells were also able to metabolically reprogram normal adjacent epithelia, indicating that cancer cells can use either fibroblasts or epithelial cells as “partners” for metabolic symbiosis. The antioxidant N-acetyl-cysteine (NAC) selectively halted mitochondrial biogenesis in Ras-transformed cells, but not in normal epithelia. NAC also blocked stromal induction of MCT4, indicating that NAC effectively functions as an “MCT4 inhibitor”. Taken together, our data provide new strategies for achieving more effective anticancer therapy. We conclude that oncogenes enable cancer cells to behave as selfish “metabolic parasites”, like foreign organisms (bacteria, fungi, viruses). Thus, we should consider treating cancer like an infectious disease, with new classes of metabolically targeted “antibiotics” to selectively starve cancer cells. Our results provide new support for the “seed and soil” hypothesis, which was first proposed in 1889 by the English surgeon, Stephen Paget.     

Reverse genetics system for human rotaviruses

A reverse genetics technology is an incredibly useful technique both for a proper understanding of different aspects of virus biology and for the generation of complementary DNA (cDNA)-derived infectious viruses, which can act as safe and effective vaccines and viral vectors. Rotaviruses (RVAs), especially human RVAs (HuRVAs), had been very refractory to this technology until very recently. Here, we describe the historical background of the development of a long-awaited HuRVA reverse genetics system, culminating in the generation of replicative HuRVAs entirely from cloned cDNAs.