Osamu Ando

A Synthetic Lethality–Based Strategy to Treat Cancers Harboring a Genetic Deficiency in the Chromatin Remodeling Factor BRG1

The occurrence of inactivating mutations in SWI/SNF chromatin-remodeling genes in common cancers has attracted a great deal of interest. However, mechanistic strategies to target tumor cells carrying such mutations are yet to be developed. This study proposes a synthetic-lethality therapy for treating cancers deficient in the SWI/SNF catalytic (ATPase) subunit, BRG1/SMARCA4. The strategy relies upon inhibition of BRM/SMARCA2, another catalytic SWI/SNF subunit with a BRG1-related activity. Immunohistochemical analysis of a cohort of non-small-cell lung carcinomas (NSCLC) indicated that 15.5% (16 of 103) of the cohort, corresponding to preferentially undifferentiated tumors, was deficient in BRG1 expression. All BRG1-deficient cases were negative for alterations in known therapeutic target genes, for example, EGFR and DDR2 gene mutations, ALK gene fusions, or FGFR1 gene amplifications. RNA interference (RNAi)-mediated silencing of BRM suppressed the growth of BRG1-deficient cancer cells relative to BRG1-proficient cancer cells, inducing senescence via activation of p21/CDKN1A. This growth suppression was reversed by transduction of wild-type but not ATPase-deficient BRG1. In support of these in vitro results, a conditional RNAi study conducted in vivo revealed that BRM depletion suppressed the growth of BRG1-deficient tumor xenografts. Our results offer a rationale to develop BRM-ATPase inhibitors as a strategy to treat BRG1/SMARCA4-deficient cancers, including NSCLCs that lack mutations in presently known therapeutic target genes.

Structural Basis of Digoxin That Antagonizes RORγt Receptor Activity and Suppresses Th17 Cell Differentiation and Interleukin (IL)-17 Production

The retinoic acid-related orphan nuclear receptor γt (RORγt)/RORγ2 is well known as a master regulator of interleukin 17 (IL-17)-producing helper T (Th17) cell development. To develop a therapeutic agent against Th17-mediated autoimmune diseases, we screened chemical compounds and successfully found that digoxin inhibited IL-17 production. Further studies revealed that digoxin bound to the ligand binding domain of RORγt and suppressed Th17 differentiation without affecting Th1 differentiation. To better understand the structural basis for the inhibitory activity of digoxin, we determined the crystal structure of the RORγt ligand-binding domain in complex with digoxin at 2.2 Å resolution. The structure reveals that digoxin binds to the ligand-binding pocket protruding between helices H3 and H11 from the pocket. In addition, digoxin disrupts the key interaction important for the agonistic activity, resulting in preventing the positioning of helix H12 in the active conformation, thus antagonizing coactivator interaction. Functional studies demonstrated that digoxin inhibited RORγt activity and decreased IL-17 production but not RORα activity. Digoxin inhibited IL-17 production in CD4+ T cells from experimental autoimmune encephalomyelitis mice. Our data indicates that RORγt is a promising therapeutic target for Th17-derived autoimmune diseases and our structural data will help to design novel RORγt antagonists.

The toxic and lethal effects of the trehalase inhibitor trehazolin in locusts are caused by hypoglycaemia

SUMMARY The main blood sugar of locusts is trehalose, which is hydrolysed to two glucose units by trehalase. Homogenates of locust flight muscles are rich in trehalase activity, which is bound to membranes. A minor fraction of trehalase is in an overt form while the remainder is latent, i.e. active only after impairing membrane integrity. Trehazolin, an antibiotic pseudosaccharide, inhibits locust flight muscle trehalase with apparent Ki- and EC50 values of 10–8 mol l–1 and 10–7 mol l–1, respectively. Trehazolin is insecticidal: 50 μg injected into locusts completely and selectively blocked the overt form of muscle trehalase (with little effect on latent activity) and killed 50% of the insects within 24 h. Here, it is demonstrated for the first time that trehazolin causes dramatic hypoglycaemia. Injection of 10 μg trehazolin caused glucose levels to fall by over 90% in 24 h, from 2.8 mmol l–1 to 0.23 mmol l–1, while trehalose increased from 61 mmol l–1 to 111 mmol l–1. Feeding glucose to the locusts fully neutralized the effects of a potentially lethal dose of trehazolin. This indicates that hypertrehalosaemia is not acutely toxic, whereas lack of glucose causes organ failure (presumably of the nervous system), and that sufficient haemolymph glucose can only be generated from trehalose by trehalase. The results also suggest that overt flight muscle trehalase is located in the plasma membrane with the active site accessible to the haemolymph. Trehalase inhibitors are valuable tools for studying the molecular physiology of trehalase function and sugar metabolism in insects.

Long-term effects of the trehalase inhibitor trehazolin on trehalase activity in locust flight muscle.

SUMMARY Trehalase (EC 3.2.1.28) hydrolyzes the main haemolymph sugar of insects, trehalose, into the essential cellular substrate glucose. Trehalase in locust flight muscle is bound to membranes that appear in the microsomal fraction upon tissue fractionation, but the exact location in vivo has remained elusive. Trehalase has been proposed to be regulated by a novel type of activity control that is based on the reversible transformation of a latent (inactive) form into an overt (active) form. Most trehalase activity from saline-injected controls was membrane-bound (95%) and comprised an overt form (∼25%) and a latent form (75%). Latent trehalase could be assayed only after the integrity of membranes had been destroyed. Trehazolin, a potent tight-binding inhibitor of trehalase, is confined to the extracellular space and has been used as a tool to gather information on the relationship between latent and overt trehalase. Trehazolin was injected into the haemolymph of locusts, and the trehalase activity of the flight muscle was determined at different times over a 30-day period. Total trehalase activity in locust flight muscle was markedly inhibited during the first half of the interval, but reappeared during the second half. Inhibition of the overt form preceded inhibition of the latent form, and the time course suggested a reversible precursor–product relation (cycling) between the two forms. The results support the working hypothesis that trehalase functions as an ectoenzyme, the activity of which is regulated by reversible transformation of latent into overt trehalase.

Reduction of glycogen in eggs of the silkworm, Bombyx mori, by use of a trehalase inhibitor, trehazolin, and diapause induction in glycogen-reduced eggs.

A new trehalase inhibitor, trehazolin, caused a potent inhibition of ovary trehalase in the silkworm, Bombyx mori. A single injection of trehazolin into pupae (40&mgr;g/animal) did not interfere with the accumulation of proteins and lipids, but markedly reduced glycogen content in eggs accompanied by a remarkable increase in hemolymph trehalose levels. The most potent effect of trehazolin was expressed in eggs that developed at the mid-stage of pupal-adult development. In these eggs glycogen content was reduced to a trace level, less than 3% of that of the control. The reduced glycogen content was almost restored to the control level by injection of glucose but not by trehalose. Trehazolin treatment influenced oviposition and larval hatching, whereas embryogenesis went on normally in glycogen-reduced eggs. Injection of synthetic diapause hormone into non-diapause type hosts induced an incidence of 45% diapause in the eggs and increased their glycogen content. Surprisingly, injection of trehazolin never affected diapause induction by the hormone, despite considerably reduced glycogen content in these eggs. Thus, our findings provide a new method for production of eggs containing various amounts of glycogen, and a novel system for analyzing diapause-associated metabolism besides the well-known glycogen-sorbitol metabolism.

Investigation of Glycochenodeoxycholate Sulfate and Chenodeoxycholate Glucuronide as Surrogate Endogenous Probes for Drug Interaction Studies of OATP1B1 and OATP1B3 in Healthy Japanese Volunteers

PurposeTo assess the use of glycochenodeoxycholate-3-sulfate (GCDCA-S) and chenodeoxycholate 3- or 24-glucuronide (CDCA-3G or -24G) as surrogate endogenous substrates in the investigation of drug interactions involving OATP1B1 and OATP1B3.MethodsUptake of GCDCA-S and CDCA-24G was examined in HEK293 cells transfected with cDNA for OATP1B1, OATP1B3, and NTCP and in cryopreserved human hepatocytes. Plasma concentrations of bile acids and their metabolites (GCDCA-S, CDCA-3G, and CDCA-24G) were determined by LC–MS/MS in eight healthy volunteers with or without administration of rifampicin (600xa0mg, po).ResultsGCDCA-S and CDCA-24G were substrates for OATP1B1, OATP1B3, and NTCP. The uptake of [3H]atorvastatin, GCDCA-S, and CDCA-24G by human hepatocytes was significantly inhibited by both rifampicin and pioglitazone, whereas that of taurocholate was inhibited only by pioglitazone. Rifampicin elevated plasma concentrations of GCDCA-S more than those of other bile acids. The area under the plasma concentration–time curve for GCDCA-S was 20.3 times higher in rifampicin-treated samples. CDCA-24G could be detected only in plasma from the rifampicin-treatment phase, and CDCA-3G was undetectable in both phases.ConclusionsWe identified GCDCA-S and CDCA-24G as substrates of NTCP, OATP1B1, and OATP1B3. GCDCA-S is a surrogate endogenous probe for the assessment of drug interactions involving hepatic OATP1B1 and OATP1B3.

IDH1 and IDH2 have critical roles in 2-hydroxyglutarate production in D-2-hydroxyglutarate dehydrogenase depleted cells.

D-2-hydroxyglutaric aciduria (D-2HGA) is a hereditary metabolic disorder characterized by the elevated levels of D-2-hydroxyglutaric acid (D-2HG) in urine, plasma and cerebrospinal fluid. About half of the patients have autosomal recessive mutations in D-2-hydroxyglutarate dehydrogenase (D2HGDH) gene. To analyze the origin of D-2HG in D2HGDH-depleted cells, we used small interfering RNA (siRNA) techniques. We found that knockdown of D2HGDH in MCF7 cells increased the levels of 2HG, mimicking D2HGDH mutant cells. Additional knockdown of isocitrate dehydrogenase 1 (IDH1) or isocitrate dehydrogenase 2 (IDH2) decreased the level of 2HG in D2HGDH knockdown MCF7 cells. Conversely, ectopic expression of IDH1 or IDH2 increased 2HG in MCF7 cells. These results suggest that IDH1 and IDH2 have roles in production of D-2HG in cells.

IQGAP1 selectively interacts with K-Ras but not with H-Ras and modulates K-Ras function

K-Ras is frequently mutated and activated especially in pancreatic cancers. To analyze K-Ras function, we have searched for K-Ras interacting proteins and found IQ motif containing GTPase activating protein 1 (IQGAP1) as a novel K-Ras binding protein. IQGAP1 has been known as a scaffold protein for B-Raf, MEK1/2 and ERK1/2. Here we showed that IQGAP1 selectively formed a complex with K-Ras but not with H-Ras, and recruited B-Raf to K-Ras. We found that IQ motif region of IQGAP1 interacted with K-Ras. Both active and inactive K-Ras interacted with IQGAP1, and effector domain mutants of K-Ras also associated with IQGAP1, indicating that IQGAP1 interacts with K-Ras irrespective of Ras-effectors like B-Raf. We also found that overexpression or knock-down of IQGAP1 affected the interaction between K-Ras and B-Raf, and IQGAP1 overexpression increased ERK1/2 phosphorylation in K-Ras dependent manner in PANC1 cells. Our data suggest that IQGAP1 has a novel mechanism to modulate K-Ras pathway.

In Silico and In Vitro Analysis of Interaction between Ximelagatran and Human Leukocyte Antigen (HLA)-DRB1*07:01

Idiosyncratic ximelagatran-induced hepatotoxicity has been reported to be associated with human leukocyte antigen (HLA)-DRB1*07:01 and ximelagatran has been reported to inhibit the binding of the ligand peptide to HLA-DRB1*07:01 in vitro. In order to predict the possible interaction modes of ximelagatran with HLA-DR molecules, in silico docking simulations were performed. Molecular dynamics (MD) simulations were also performed to predict the effect of ximelagatran on the binding mode of the ligand peptide to HLA-DRB1*07:01. A series of in silico simulations supported the inhibitory effect of ximelagatran on the binding of the ligand peptide to HLA-DRB1*07:01 in vitro. Furthermore, direct interactions of ximelagatran with HLA-DR molecules were evaluated in vitro, which supported the simulated interaction mode of ximelagatran with HLA-DRB1*07:01. These results indicated that ximelagatran directly interacts with the peptide binding groove of HLA-DRB1*07:01 and competes with the ligand peptide for the binding site, which could alter the immune response and lead to the idiosyncratic ximelagatran-induced hepatotoxicity.

Fate and effects of the trehalase inhibitor trehazolin in the migratory locust (Locusta migratoria).

Trehalose is the main haemolymph sugar in many insect species. To be utilized trehalose must be hydrolysed into its glucose units by trehalase (EC 3.2.1.28). Inhibitors of trehalase have attracted interest as possible pesticides and tools for studying the regulation of trehalose metabolism in insects. To make full use of these inhibitors requires knowledge of their fate and effects in vivo. To this end we have measured trehazolin in locusts using a method based on the specific inhibition of a trehalase preparation. After injection of 20 microg, trehazolin decreased in haemolymph with a half-life of 2.6 days and after 10 days almost 95% had disappeared. Trehazolin did not reach the intracellular water space of locust tissues, but appeared with full inhibitory potency in locust faeces, suggesting that it was not metabolized, but quantitatively eliminated via the gut. Haemolymph trehalose increased transiently upon trehazolin injection, it was maximal after 3 days, then decreased and reached control level after 10 days. Inhibition of flight muscle trehalase by trehazolin was prolonged and still conspicuous 21 days post injection, suggesting that trehazolin inhibits trehalase activity irreversibly in vivo and that recovery requires de novo enzyme synthesis.