Identifying the molecular targets for the beneficial or detrimental effects of small-molecule drugs is an important and currently unmet challenge. We have developed a method, drug affinity responsive target stability (DARTS), which takes advantage of a reduction in the protease susceptibility of the target protein upon drug binding. DARTS is universally applicable because it requires no modification of the drug and is independent of the mechanism of drug action. We demonstrate use of DARTS to identify known small-molecule-protein interactions and to reveal the eukaryotic translation initiation machinery as a molecular target for the longevity-enhancing plant natural product resveratrol. We envisage that DARTS will also be useful in global mapping of protein-metabolite interaction networks and in label-free screening of unlimited varieties of compounds for development as molecular imaging agents.aging ͉ label-free ͉ proteomics ͉ small molecules

Triantennary N-acetyl galactosamine (GalNAc, GN3), a high-affinity ligand for the hepatocyte-specific asialoglycoprotein receptor (ASGPR), enhances the potency of second-generation gapmer antisense oligonucleotides (ASOs) 6–10-fold in mouse liver. When combined with next-generation ASO designs comprised of short S-cEt (S-2′-O-Et-2′,4′-bridged nucleic acid) gapmer ASOs, ∼60-fold enhancement in potency relative to the parent MOE (2′-O-methoxyethyl RNA) ASO was observed. GN3-conjugated ASOs showed high affinity for mouse ASGPR, which results in enhanced ASO delivery to hepatocytes versus non-parenchymal cells. After internalization into cells, the GN3-ASO conjugate is metabolized to liberate the parent ASO in the liver. No metabolism of the GN3-ASO conjugate was detected in plasma suggesting that GN3 acts as a hepatocyte targeting prodrug that is detached from the ASO by metabolism after internalization into the liver. GalNAc conjugation also enhanced potency and duration of the effect of two ASOs targeting human apolipoprotein C-III and human transthyretin (TTR) in transgenic mice. The unconjugated ASOs are currently in late stage clinical trials for the treatment of familial chylomicronemia and TTR-mediated polyneuropathy. The ability to translate these observations in humans offers the potential to improve therapeutic index, reduce cost of therapy and support a monthly dosing schedule for therapeutic suppression of gene expression in the liver using ASOs.

Summary Chronic activation of mammalian target of rapamycin complex 1 (mTORC1) and p70 S6 kinase (S6K) in response to hypernutrition contributes to obesity-associated metabolic pathologies including hepatosteatosis and insulin resistance. Sestrins are stress-inducible proteins that activate AMP-activated protein kinase (AMPK) and suppress mTORC1-S6K activity, but their role in mammalian physiology and metabolism has not been investigated. We show that Sestrin2, encoded by the Sesn2 locus whose expression is induced upon hypernutrition, maintains metabolic homeostasis in liver of obese mice. Sesn2 ablation exacerbates obesity-induced mTORC1-S6K activation, glucose intolerance, insulin resistance and hepatosteatosis, all of which are reversed by AMPK activation. Furthermore, concomitant ablation of Sesn2 and Sesn3 provokes hepatic mTORC1-S6K activation and insulin resistance even in the absence of nutritional overload and obesity. These results demonstrate an important homeostatic function for the stress-inducible Sestrin protein family in the control of mammalian lipid and glucose metabolism.

Sustained c-Jun N-terminal kinase (JNK) activation has been implicated in many models of cell death and tissue injury. Phosphorylated JNK (p-JNK) interacts with the mitochondrial outer membrane SH3 homology associated BTK binding protein (Sab, or SH3BP5). Using knockdown or liver-specific deletion of Sab, we aimed to elucidate the consequences of this interaction on mitochondrial function in isolated mitochondria and liver injury models in vivo. Respiration in isolated mitochondria was directly inhibited by p-JNK 1 adenosine triphosphate. Knockdown or liver-specific knockout of Sab abrogated this effect and markedly inhibited sustained JNK activation and liver injury from acetaminophen or tumor necrosis factor/galactosamine. We then elucidated an intramitochondrial pathway in which interaction of JNK and Sab on the outside of the mitochondria released protein tyrosine phosphatase, nonreceptor type 6 (SHP1, or PTPN6) from Sab in the inside of the mitochondrial outer membrane, leading to its activation and transfer to the inner membrane, where it dephosphorylates P-Y419Src (active), which required a platform protein, docking protein 4 (DOK4), on the inner membrane. Knockdown of mitochondrial DOK4 or SHP1 inhibited the inactivation of mitochondrial p-Src and the effect of p-JNK on mitochondria. Conclusions: The binding to and phosphorylation of Sab by p-JNK on the outer mitochondrial membrane leads to SHP1-dependent and DOK4-dependent inactivation of p-Src on the inner membrane; inactivation of mitochondrial Src inhibits electron transport and increases reactive oxygen species release, which sustains JNK activation and promotes cell death and organ injury.

.β-Thalassemia and HFE-related hemochromatosis are 2 of the most frequently inherited disorders worldwide. Both disorders are characterized by low levels of hepcidin (HAMP), the hormone that regulates iron absorption. As a consequence, patients affected by these disorders exhibit iron overload, which is the main cause of morbidity and mortality. HAMP expression is controlled by activation of the SMAD1,5,8/SMAD4 complex. TMPRSS6 is a serine protease that reduces SMAD activation and blocks HAMP expression. We identified second generation antisense oligonucleotides (ASOs) targeting mouse Tmprss6. ASO treatment in mice affected by hemochromatosis (Hfe -/-) significantly decreased serum iron, transferrin saturation and liver iron accumulation. Furthermore, ASO treatment of mice affected by β-thalassemia (HBB th3/+ mice, referred to here after as th3/+ mice) decreased the formation of insoluble membrane-bound globins, ROS, and apoptosis, and improved anemia. These animals also exhibited lower erythropoietin levels, a significant amelioration of ineffective erythropoiesis (IE) and splenomegaly, and an increase in total hemoglobin levels. These data suggest that ASOs targeting Tmprss6 could be beneficial in individuals with hemochromatosis, β-thalassemia, and related disorders.