Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) plays a key role in one-carbon (1C) metabolism in human mitochondria, and its high expression correlates with poor survival of patients with various types of cancer. An isozymeselective MTHFD2 inhibitor is highly attractive for potential use in cancer treatment. Herein, we disclose a novel isozymeselective MTHFD2 inhibitor DS44960156, with a tricyclic coumarin scaffold, which was initially discovered via high-throughput screening (HTS) and improved using structure-based drug design (SBDD). DS44960156 would offer a good starting point for further optimization based on the following features: (1) unprecedented selectivity (>18-fold) for MTHFD2 over MTHFD1, (2) a molecular weight of less than 400, and (3) good ligand efficiency (LE).

RNA interference (RNAi) induced by delivery of a small-interfering RNA (siRNA)-expressing vector was characterized in mice. siRNA-expressing plasmid DNA (pDNA) was injected by a hydrodynamics-based procedure along with pDNA encoding an exogenous target luciferase gene. A comparative study showed that stem-loop-type siRNA-expressing pDNA was superior, in terms of the transgene suppressive efficacy, to the tandem-type in the liver following systemic delivery of these pDNAs. Transgene suppression occurred in the liver, kidney, and lung as well as muscle. The degree of suppression was dependent on the dose of siRNA-expressing pDNA and the time at which transgene expression was determined following simultaneous injection of siRNA-expressing and target pDNAs. A reduction in transgene expression became apparent at 1 day after injection, whereas a lower degree of inhibition was obtained before this, as early as 6 h even in mice treated with an excess of siRNA-expressing pDNA. These results suggest that delivery of siRNA-expressing pDNA requires a period of time for induction of RNAi. A study of sequential injections revealed that prior injection of siRNA-expressing pDNA produced a significant suppression for at least 1 day, which disappeared within 4 days. Confocal microscopic studies indicated that the localization of the cells with successful delivery of transgene was different between primary and secondary hydrodynamicsbased injections, accounting for the less effective inhibition following the sequential injections. Taken together, these results demonstrate that vector-based in vivo RNAi is a dose-and time-dependent process and offers the possibility of suppressing endogenous targets in a variety of somatic cells.

While brown adipose tissue (BAT) is well‐recognized for its ability to dissipate energy in the form of heat, recent studies suggest multifaced roles of BAT in the regulation of glucose and lipid homeostasis beyond stimulating thermogenesis. One of the functions involves interorgan communication with metabolic organs, such as the liver, through BAT‐derived secretory factors, a.k.a., batokine. However, the identity and the roles of such mediators remain insufficiently understood. Here, we employed proteomics and transcriptomics in human thermogenic adipocytes and identified previously unappreciated batokines, including phospholipid transfer protein (PLTP). We found that increased circulating levels of PLTP, via systemic or BAT‐specific overexpression, significantly improve glucose tolerance and insulin sensitivity, increased energy expenditure, and decrease the circulating levels of cholesterol, phospholipids, and sphingolipids. Such changes were accompanied by increased bile acids in the circulation, which in turn enhances glucose uptake and thermogenesis in BAT. Our data suggest that PLTP is a batokine that contributes to the regulation of systemic glucose and lipid homeostasis as a mediator of BAT‐liver interorgan communication.

Our results demonstrate that sequence-specific suppression of mdr1 gene expression is possible at the mRNA level as well as the protein level in mice following intravenous delivery of siRNA effectors.