Current FDA-approved chemotherapeutic antimetabolites elicit severe side effects that warrant their improvement; therefore, we designed compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered that 5-(α-substituted-2-nitrobenzyloxy)methyluridine-5′-triphosphates were exquisite DNA synthesis terminators; therefore, we synthesized a library of 35 thymidine analogs and evaluated their activity using an MTT cell viability assay of MCF7 breast cancer cells chosen for their vulnerability to these nucleoside derivatives. Compound 3a, having an α-tert-butyl-2-nitro-4-(phenyl)alkynylbenzyloxy group, showed an IC50 of 9 ± 1 μM. The compound is more selective for cancer cells than for fibroblast cells compared with 5-fluorouracil. Treatment of MCF7 cells with 3a elicits the DNA damage response as indicated by phosphorylation of γ-H2A. A primer extension assay of the 5′-triphosphate of 3a revealed that 3aTP is more likely to inhibit DNA polymerase than to lead to termination events upon incorporation into the DNA replication fork.
Background: Current chemotherapeutic antimetabolites often exhibit severe side effects that limit their use as drugs; therefore, we designed nucleoside compounds with mechanisms of action focusing on inhibiting DNA replication rather than targeting multiple pathways. We previously discovered cytotoxic basemodified thymidine and thymine analogs that show higher selectivity against cancerous versus normal cells compared to the current antimetabolites used in cancer chemotherapy. We anticipated these antimetabolites have the potential to effectively inhibit viral DNA replication while showing low cytotoxicity. Methods: Base-modified thymidine and thymine analogs were synthesized and their anti-viral activity was evaluated in human cells infected with human pappiloma, John Cunningham, and BK viruses using quantitative DNA polymerase chain reaction assay. In addition, their toxicity toward host cells was determined using CellTiter-Glo assay, and compared to cytotoxicity toward human breast cancer cells. Results: Novel lead compounds with high activity against human papilloma (HPV) and John Cunningham (JCV) viruses have been identified. Their EC50 values lie in low micromolar range (1-2 µM), which is significantly less than that of cidofovir (9-10 µM), a current drug used against DNA viruses. Cytotoxicity of the leads toward the host cells was found to be in 200-300 µM range, which is generally higher than that observed toward MCF-7 human breast cancer cells. None of the tested compounds significantly inhibited BK viral DNA replication. Conclusion: The lead compounds affect the viruses substantially more selectively than the host cells, which makes them a novel class of bioactive compounds with the potential to become effective anti-viral drugs.
This study explored the role of apoE receptor-2 (apoER2), a unique member of the LDL receptor family proteins with a restricted tissue expression profile, in modulating diet-induced obesity and diabetes. Unlike wild type mice and humans in which chronic feeding of a high fat Western type diet leads to obesity and the pre-diabetic state of hyperinsulinemia prior to hyperglycemia onset, the Lrp8-/- mice with global apoER2 deficiency displayed lower body weight and adiposity, slower development of hyperinsulinemia, but accelerated onset of hyperglycemia. Despite their lower adiposity, adipose tissues in Western diet-fed Lrp8-/- mice were more inflamed compared to wild type mice. Additional experiments revealed that the hyperglycemia observed in Western diet-fed Lrp8-/- mice was due to impaired glucose-induced insulin secretion, ultimately leading to hyperglycemia, adipocyte dysfunction, and inflammation upon chronic feeding of the Western diet. Interestingly, bone marrow-specific apoER2 deficient mice were not defective in insulin secretion, exhibiting increased adiposity and hyperinsulinemia compared to wild type mice. Analysis of bone marrow-derived macrophages revealed that apoER2 deficiency impeded inflammation resolution with lower secretion of IFN-β and IL-10 in response to LPS stimulation of IL-4 primed cells. The apoER2-deficient macrophages also showed increased level of disabled-2 (Dab-2) as well as increased cell surface TLR4, suggesting that apoER2 participates in Dab-2 regulation of TLR4 signaling. Taken together, these results showed that apoER2 deficiency in macrophages sustains diet-induced tissue inflammation and accelerates obesity and diabetes onset while apoER2 deficiency in other cell types contributes to hyperglycemia and inflammation via defective insulin secretion.
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