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PDF file, 1764K, Supplementary Figure S1. Immunohistochemical staining for BRG1 and BRM in cancer cell lines. Supplementary Figure S2. E431* and E608* Supplementary Figure S3. Fluorescence in situ hybridization for the detection of FGFR1 amplification in a case of squamous lung cell carcinoma. Supplementary Figure S4. Immunoblot analysis of SWI/SNF and ACF chromatinremodeling proteins. Supplementary Figure S5. Survival of H1299 (BRG1-deficient) and HeLa (BRG1-proficient) cells after shRNA-mediated knockdown of BRM (shBRM). Supplementary Figure S6. The effects of BRM knockdown on the morphologies of BRG1-deficient and proficient cancer cells. Supplementary Figure S7. Effect of BRM knockdown on the induction of senescence in BRG1-deficient and -proficient non-small-cell lung carcinoma cells.

PDF files, 147K.

PDF file, 106K, Supplementary Table S1. BRG1-deficient and -proficient cell lines subjected to BRM knockdown Supplementary Table S2. Verification of gene alterations in cell lines subjected to BRM knockdown Supplementary Table S3. Analysis of missense BRG1 mutations using the Polyphen software Supplementary Table S4. Negative correlation between BRG1 deficiency and alterations in therapeutic target genes in 373 NSCLC cases from three studies (this study and Refs 32 and 33) Supplementary Table S5. Correlation between BRG1 deficiency and smoking in 369 NSCLC cases from three studies (this study and Ref 32 and 33).

Endogenous substrates are emerging biomarkers for drug transporters, which serve as surrogate probes in drug-drug interaction (DDI) studies. In this study, the results of metabolome analysis using wild-type and Oct1/2 double knockout mice suggested that N1-methyladenosine (m1A) was a novel organic cation transporter (OCT) 2 substrate. An in vitro transport study revealed that m1A is a substrate of mouse Oct1, Oct2, Mate1, human OCT1, OCT2, and multidrug and toxin exclusion protein (MATE) 2-K, but not human MATE1. Urinary excretion accounted for 77% of the systemic elimination of m1A in mice. The renal clearance (46.9 ± 4.9 ml/min per kilogram) of exogenously given m1A was decreased to near the glomerular filtration rates by Oct1/2 double knockout or Mate1 inhibition by pyrimethamine (16.6 ± 2.6 and 24.3 ± 0.6 ml/min per kilogram, respectively), accompanied by significantly higher plasma concentrations. In vivo inhibition of OCT2/MATE2-K by a single dose of 7-[(3R)-3-(1-aminocyclopropyl)pyrrolidin-1-yl]-1-[(1R,2S)-2-fluorocyclopropyl]-8-methoxy-4-oxoquinoline-3-carboxylic acid in cynomolgus monkeys resulted in the elevation of the area under the curve of m1A (1.72-fold) as well as metformin (2.18-fold). The plasma m1A concentration profile showed low diurnal and interindividual variation in healthy volunteers. The renal clearance of m1A in younger (21–45 year old) and older (65–79 year old) volunteers (244 ± 58 and 169 ± 22 ml/min per kilogram, respectively) was about 2-fold higher than the creatinine clearance. The renal clearances of m1A and creatinine were 31% and 17% smaller in older than in younger volunteers. Thus, m1A could be a surrogate probe for the evaluation of DDIs involving OCT2/MATE2-K. SIGNIFICANCE STATEMENT Endogenous substrates can serve as surrogate probes for clinical drug-drug interaction studies involving drug transporters or enzymes. In this study, m1A was found to be a novel substrate of renal cationic drug transporters OCT2 and MATE2-K. N1-methyladenosine was revealed to have some advantages compared to other OCT2/MATE substrates (creatinine and N1-methylnicotinamide). The genetic or chemical impairment of OCT2 or MATE2-K caused a significant increase in the plasma m1A concentration in mice and cynomolgus monkeys due to the high contribution of tubular secretion to the net elimination of m1A. The plasma m1A concentration profile showed low diurnal and interindividual variation in healthy volunteers. Thus, m1A could be a better biomarker of variations in OCT2/MATE2-K activity caused by inhibitory drugs.

The present study examined the significance of enterohepatic circulation and the effect of rifampicin [an inhibitor of organic anion-transporting polypeptide 1B (OATP1B)] on the plasma concentrations of bile acid-O-sulfates (glycochenodeoxycholate-O-sulfate, lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate) in monkeys and human liver-transplanted chimeric mice (PXB mouse). Rifampicin significantly increased the area under the curve of bile acid-O-sulfates in monkeys (13-69 times) and PXB mice (13-25 times) without bile flow diversion. Bile flow diversion reduced the concentration of plasma bile acid-O-sulfates under control conditions in monkeys and the concentration of plasma glycochenodeoxycholate-O-sulfate in PXB mice. It also diminished diurnal variation of plasma lithocholate-O-sulfate, glycolithocholate-O-sulfate, and taurolithocholate-O-sulfate in PXB mice under control conditions. Bile flow diversion did not affect the plasma concentration of bile acid-O-sulfates in monkeys and PXB mice treated with rifampicin. Plasma coproporphyrin I and III levels were constant in monkeys throughout the study, even with bile flow diversion. This study demonstrated that bile acid-O-sulfates are endogenous OATP1B biomarkers in monkeys and PXB mice. Enterohepatic circulation can affect the baseline levels of plasma bile acid-O-sulfates and modify the effect of OATP1B inhibition.

Esaxerenone (CS-3150) is a novel, nonsteroidal, selective mineralocorticoid receptor blocker. The absorption, metabolism, distribution, and excretion of esaxerenone were assessed in in vitro studies and in a clinical study, where [14C]esaxerenone (150 μCi/20 mg) was administered orally to six healthy male subjects. The plasma concentrations of esaxerenone and its metabolites (M4, M11, and M1) were measured using liquid chromatography-tandem mass spectrometry. The recovery of radioactivity was 92.5%, with 38.5% and 54.0% excreted in the urine and feces, respectively. The half-life of radioactivity in blood and plasma was approximately 30 hours, similar to that of the unchanged form in plasma. The blood-to-plasma ratio was 0.628, demonstrating low partitioning to blood components. In plasma, esaxerenone was the most abundant moiety (40.8%), followed by O-glucuronide (21.4%; M4), acyl-glucuronide of amide-bond hydrolysate (8.0%; M11), and the deshydroxyethyl form (1.7%; M1). In vitro studies showed that esaxerenone was a substrate of CYP3A and multiple UDP-glucuronosyltransferase isoforms. Oxidation contributed approximately 30% to its clearance, as indicated by the excretion ratio of oxidized metabolites into urine and feces. Caco-2 studies showed that esaxerenone was a substrate of P-glycoprotein and breast cancer resistance protein; however, the excretion ratios of the unchanged form in the feces and urine were 18.7% and 1.6%, respectively, indicating that these transporters were not important for the absorption and elimination of esaxerenone. Low urinary excretion of esaxerenone suggested that the plasma exposure of esaxerenone was not affected by renal dysfunction. Multiple elimination pathways including oxidation, glucuronidation, and hydrolysis, and the low contribution of transporters, indicated limited drug-drug interaction potential.

To estimate the clinical impact of pharmacokinetic modulation via breast cancer resistance protein (BCRP), in vivo approaches in nonclinical settings are desired in drug development. Clinical observation has identified curcumin as a promising candidate for in vivo selective BCRP inhibition, in addition to several well known inhibitors, such as lapatinib and pantoprazole. This study aimed to confirm the inhibitory efficacy of curcumin on gastrointestinal BCRP function in cynomolgus monkeys and to perform comparisons with lapatinib and pantoprazole. Oral area under the plasma concentration-time curve (AUC) and bioavailability of well known BCRP (sulfasalazine and rosuvastatin), P-glycoprotein (fexofenadine, aliskiren, and talinolol), and CYP3A (midazolam) substrates were investigated in the presence and absence of inhibitors. Oral exposures of sulfasalazine and rosuvastatin were markedly elevated by curcumin with minimal changes in systemic clearance, whereas pharmacokinetic alterations after fexofenadine, aliskiren, and talinolol oral exposure were limited. Curcumin increased oral midazolam exposure without affecting systemic clearance, presumably owing to partial inhibition of intestinal CYP3A. Lapatinib increased the oral AUC for sulfasalazine to a greater extent than curcumin did, whereas pantoprazole had a smaller effect. However, lapatinib also exerted significant effects on fexofenadine, failed to selectively discriminate between BCRP and P-glycoprotein inhibition, and had an effect on oral midazolam exposure comparable with that of curcumin. Thus, pharmacokinetic evaluation in monkeys demonstrated that pretreatment with curcumin as an in vivo selective BCRP inhibitor was more appropriate than pretreatment with lapatinib and pantoprazole for the assessment of the impact of BCRP on gastrointestinal absorption in nonrodent models.

To 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. Uptake 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 (600 mg, po). GCDCA-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. We 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.

Endogenous substrates are emerging biomarkers for drug transporters, which serve as surrogate probes in drug-drug interaction (DDI) studies. In this study, the results of metabolome analysis using wild-type and Oct1/2 double knockout mice suggested that

There was a miscalculation of coproporphyrin I AUC

Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC) composed of a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2) conjugated to a topoisomerase I inhibitor (DXd) at a drug-to-antibody ratio (DAR) of 7-8. Here, we examined the pharmacokinetic (PK) profiles of DS-8201a and DXd in cynomolgus monkeys, a cross-reactive species.Following intravenous (iv) administration of DS-8201a, the linker was stable in plasma, and systemic DXd exposure was low. DXd was rapidly cleared following iv dosing. Biodistribution studies revealed that intact DS-8201a was present mostly in the blood without tissue-specific retention. The major pathway of excretion for DXd was the faecal route following iv administration of radiolabelled DS-8201a. The only detectable metabolite in the urine and faeces was unmetabolized DXd. DXd is a substrate of organic anion transporting polypeptides, P-gp, and breast cancer resistance protein.In conclusion, the stable linker in circulation and the high clearance of DXd upon release resulted in the low systemic exposure to DXd. Furthermore, the minimal tissue-specific retention and rapid excretion of DXd into faeces as its unmetabolized form with potentially limited impact on drug − drug interaction as a victim were also critical elements of the PK profile of DS-8201a. Trastuzumab deruxtecan (DS-8201a) is an antibody-drug conjugate (ADC) composed of a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2) conjugated to a topoisomerase I inhibitor (DXd) at a drug-to-antibody ratio (DAR) of 7-8. Here, we examined the pharmacokinetic (PK) profiles of DS-8201a and DXd in cynomolgus monkeys, a cross-reactive species. Following intravenous (iv) administration of DS-8201a, the linker was stable in plasma, and systemic DXd exposure was low. DXd was rapidly cleared following iv dosing. Biodistribution studies revealed that intact DS-8201a was present mostly in the blood without tissue-specific retention. The major pathway of excretion for DXd was the faecal route following iv administration of radiolabelled DS-8201a. The only detectable metabolite in the urine and faeces was unmetabolized DXd. DXd is a substrate of organic anion transporting polypeptides, P-gp, and breast cancer resistance protein. In conclusion, the stable linker in circulation and the high clearance of DXd upon release resulted in the low systemic exposure to DXd. Furthermore, the minimal tissue-specific retention and rapid excretion of DXd into faeces as its unmetabolized form with potentially limited impact on drug − drug interaction as a victim were also critical elements of the PK profile of DS-8201a.

A series of fluoroquinolone compounds (compounds 1-9), which contain a common quinolone scaffold, inactivated the metabolic activity of CYP3A. The purpose of this study was to identify mechanism-based inhibition (MBI) among these fluoroquinolone compounds by metabolite profiling to elucidate the association of the substructure and MBI potential. Reversibility of MBI after incubation with potassium ferricyanide differed among the test compounds. Representative quasi-irreversible inhibitors form a metabolite-intermediate (MI) complex with the heme of CYP3A4 according to absorption analysis. Metabolite profiling identified the cyclopropane ring-opened metabolites from representative irreversible inhibitors, suggesting irreversible binding of the carbon-centered radical species with CYP3A4. On the other hand, the oxime form of representative quasi-irreversible inhibitors was identified, suggesting generation of a nitroso intermediate that could form the MI complex. Metabolites of compound 10 with a methyl group at the carbon atom at the root of the amine moiety of compound 8 include the oxime form, but compound 10 did not show quasi-irreversible inhibition. The docking study with CYP3A4 suggested that a methyl moiety introduced at the carbon atom at the root of the primary amine disrupts formation of the MI complex between the heme and the nitroso intermediate because of steric hindrance. This study identified substructures of fluoroquinolone compounds associated with the MBI mechanism; introduction of substituted groups inducing steric hindrance with the heme of P450 can prevent formation of an MI complex. Our series of experiments may be broadly applicable to prevention of MBI at the drug discovery stage.

A library of secondary metabolites from microorganisms was screened to identify novel inhibitors against lipopolysaccharide (LPS), a strong stimulant of innate immunity. Novel cyclic peptides, ogipeptin A, B, C and D, were identified in the culture broth of the marine bacterium Pseudoalteromonas sp. SANK 71903. These compounds blocked LPS binding to the cluster of differentiation 14 (CD14) in vitro with IC50 values of 4.8, 6.0, 4.1 and 5.6 nm, respectively, and attenuated tumor necrosis factor-α secretion from LPS-stimulated macrophage-like cells. These compounds also displayed antimicrobial activity against Escherichia coli with minimum inhibitory concentrations ranging from 0.25 μg ml-1 to 1 μg ml-1. Thus, novel antibiotics that inhibited LPS-induced innate immune reactions were identified in this study.

In the course of our screening program for inhibitors of lipopolysaccharide binding to cellular receptor CD14, a potent inhibitory activity was detected in the cultured broth of Pseudoalteromonas sp. SANK 71903. Four active compounds, ogipeptins A, B, C and D, were isolated from the cultured broth. The structures of these compounds were elucidated by physicochemical data and spectral analyses, and they were determined to be new cyclic lipopeptides.

In the course of our screening, we discovered a novel compound, A-503451A, as a potent hypoxia-inducible factor (HIF) activator. In human hepatocarcinoma HepG2 cells, A-503451A induced HIF-mediated luciferase reporter gene expression and stabilized HIF-1α protein. A-503451A increased the mRNA expression levels and the protein secretion of HIF-dependent genes, vascular endothelial growth factor and erythropoietin. Addition of excess ferric chloride to the culture medium suppressed the HIF-induction activity of A-503451A. A-503451A did not have iron-chelating activity in vitro, but decreased the intracellular labile iron pool concentration. These data indicate that A-503451A is a unique HIF activator.

Esaxerenone (CS-3150) is a novel, nonsteroidal, selective mineralocorticoid receptor blocker. The absorption, metabolism, distribution, and excretion of esaxerenone were assessed in in vitro studies and in a clinical study, where [

Human leukocyte antigen (HLA)-DRB1*01:01 has been shown to be involved in nevirapine-induced hepatic hypersensitivity reactions. In the present study, in silico docking simulations and molecular dynamics simulations were performed to predict the interaction mode of nevirapine with the peptide binding groove of HLA-DRB1*01:01 and its possible effect on the position and orientation of the ligand peptide derived from hemagglutinin (HA). In silico analyses suggested that nevirapine interacts with HLA-DRB1*01:01 around the P4 pocket within the peptide binding groove and the HA peptide stably binds on top of nevirapine at the groove. The analyses also showed that binding of nevirapine at the groove will significantly change the inter-helical distances of the groove. An in vitro competitive assay showed that nevirapine (1000 &mu, M) increases the binding of the HA peptide to HLA-DRB1*01:01 in an allele-specific manner. These results indicate that nevirapine might interact directly with the P4 pocket and modifies its structure, which could change the orientation of loaded peptides and the conformation of HLA-DRB1*01:01, these changes could be distinctively recognized by T-cell receptors. Through this molecular mechanism, nevirapine might stimulate the immune system, resulting in hepatic hypersensitivity reactions.

To evaluate association of the dose-dependent effect of rifampicin, an OATP1B inhibitor, on the plasma concentration-time profiles among OATP1B substrates drugs and endogenous substrates.Eight healthy volunteers received atorvastatin (1 mg), pitavastatin (0.2 mg), rosuvastatin (0.5 mg), and fluvastatin (2 mg) alone or with rifampicin (300 or 600 mg) in a crossover fashion. The plasma concentrations of these OATP1B probe drugs, total and direct bilirubin, glycochenodeoxycholate-3-sulfate (GCDCA-S), and coproporphyrin I, were determined.The most striking effect of 600 mg rifampicin was on atorvastatin (6.0-times increase) and GCDCA-S (10-times increase). The AUCThese results suggest that direct bilirubin, GCDCA-S, and coproporphyrin I are promising surrogate probes for the quantitative assessment of potential OATP1B-mediated DDI.

A 1,2,4-oxadiazole ring-containing compound DS-8500a was developed as a novel G protein-coupled receptor 119 agonist. In vivo metabolic fates of [14C]DS-8500a differently radiolabeled in the benzene ring or benzamide side carbon in rats were investigated.Differences in mass balances were observed, primarily because after the oxadiazole ring-opening and subsequent ring-cleavage small-molecule metabolites containing the benzene side were excreted in the urine, while those containing the benzamide side were excreted in the bile.DS-8500a was detected at trace levels in urine and bile, demonstrating extensive metabolism prior to urinary/biliary excretion. At least 16 metabolite structures were proposed in plasma, urine, and bile samples from rats treated with [14C]DS-8500a.Formation of a ring-opened metabolite (reduced DS-8500a) in hepatocytes of humans, monkeys, and rats was confirmed; however, it was not affected by typical inhibitors of cytochrome P450s, aldehyde oxidases, or carboxylesterases in human hepatocytes. Extensive formation of the ring-opened metabolite was observed in human liver microsomes fortified with an NADPH-generating system under anaerobic conditions.These results suggest an in vivo unique reductive metabolism of DS-8500a is mediated by human non-cytochrome P450 enzymes. A 1,2,4-oxadiazole ring-containing compound DS-8500a was developed as a novel G protein-coupled receptor 119 agonist. In vivo metabolic fates of [14C]DS-8500a differently radiolabeled in the benzene ring or benzamide side carbon in rats were investigated. Differences in mass balances were observed, primarily because after the oxadiazole ring-opening and subsequent ring-cleavage small-molecule metabolites containing the benzene side were excreted in the urine, while those containing the benzamide side were excreted in the bile. DS-8500a was detected at trace levels in urine and bile, demonstrating extensive metabolism prior to urinary/biliary excretion. At least 16 metabolite structures were proposed in plasma, urine, and bile samples from rats treated with [14C]DS-8500a. Formation of a ring-opened metabolite (reduced DS-8500a) in hepatocytes of humans, monkeys, and rats was confirmed; however, it was not affected by typical inhibitors of cytochrome P450s, aldehyde oxidases, or carboxylesterases in human hepatocytes. Extensive formation of the ring-opened metabolite was observed in human liver microsomes fortified with an NADPH-generating system under anaerobic conditions. These results suggest an in vivo unique reductive metabolism of DS-8500a is mediated by human non-cytochrome P450 enzymes.

In the course of our screening for inhibitors of lipopolysaccharide (LPS) binding to cellular receptor CD14, potent inhibitory activity was detected in the cultured broth of Pedobacter sp. SANK 72003. Three active compounds, pedopeptin A, B and C, were isolated from the broth and their structures were elucidated by physicochemical and spectral data to be new cyclic depsipeptides.

Lipopolysaccharide (LPS) is a strong endotoxin and is delivered to the cell surface signaling receptor, Toll-like receptor 4 and MD-2 complex, via soluble cluster of differentiation (CD) 14 or membranous CD14, resulting in the induction of the inflammatory response. To obtain new compounds that block LPS binding to CD14, we designed a high-throughput screening based on time-resolved intermolecular fluorescence resonance energy transfer. This cell-free screening system successfully led to the discovery of novel inhibitors of LPS-CD14 interaction from the library of the secondary metabolites of microorganisms. We identified the novel compounds pedopeptin A, B and C from a culture broth of Pedobacter sp. SANK 72003. Pedopeptins blocked LPS binding to CD14 in vitro with IC50 values of 20, 11 and 47 nM, respectively, and also inhibited LPS binding to the cells expressing CD14, leading to the suppression of cytokine production. Moreover, they showed antimicrobial activities against Escherichia coli with minimum inhibitory concentration ranging from 2 to 4 μg ml(-1).

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. Cancer Res; 73(17); 5508–18. ©2013 AACR.

A series of fluoroquinolone antibacterial compounds were found to be irreversible (compounds 1-5) and quasi-irreversible (compounds 6-9) inhibitors of CYP3A4. The purpose of this study was to evaluate their mechanism-based inhibition (MBI) potency against CYP3A5. Compounds 1-5 were also irreversible inhibitors of CYP3A5, whereas compounds 6-9 showed neither irreversible nor quasi-irreversible inhibition of CYP3A5. Compounds 6 and 8 did not form a metabolite-intermediate complex with the heme of CYP3A5 during incubation. The structural analysis of the metabolites after incubation of compounds 1 and 6 with CYP3A5 revealed that their metabolites were identical to those produced by CYP3A4, including the precursors of which are speculated to account for the MBI of CYP3A4. The homology modeling of CYP3A5 suggests that four residues around the nitroso intermediate of compound 6 in the substrate-binding pocket of CYP3A4 correspond with the bulkier residues in CYP3A5-especially Phe210 in CYP3A5-which might contribute to the steric hindrance with the nitroso intermediate of compound 6. The substrate-binding pocket structure of CYP3A5 might prevent the nitroso intermediate from coordinate binding with the heme, thereby preventing quasi-irreversible inhibition. Our study may provide new insights into the observable differences between the inhibition of CYP3A4 and CYP3A5.

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.

In the course of our screening for activators of hypoxia-inducible factor (HIF), A-503451 A and virantmycin were isolated from the cultured broth of an actinomycete strain, Streptomyces sp. SANK 60101. From the same culture, the non-active homologs A-503451 B and D were also isolated. A-503451 A and virantmycin activated HIF-dependent reporter gene expression with EC

Synthesis and properties of an oligonucleotide uniformly modified with 2'-O,4-C-ethylene-bridged nucleic acid (ENA) units were compared with those of GRN163, which is modified with N3'-P5' thiophosphoramidates, with the sequence targeting human telomerase RNA subunit. Although an ENA phosphorothioate oligonucleotide, ENA-13, could be synthesized using ENA phosphoramidites on a 100-mg scale, synthesis of GRN163 was very hard even on a 1-micomol scale. In view of both stability of the duplex formation with complementary RNA and the efficiency of cellular uptake by endocytosis, ENA-13 was superior to GRN163. These findings suggest that ENA-13 has useful properties for antisense therapeutic application.

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–1and 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.

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.

An apoptotic cellular defense mechanism is triggered in response to viral dsRNA generated during the course of infection by many DNA and RNA viruses. We demonstrate that apoptosis induced by dsRNA or a paramyxovirus is independent of the action of interferon as it can proceed in a variety of cell lines and primary cells deficient in an interferon response. Initiation of apoptosis appears to be triggered by activation of a cellular transcription factor, the dsRNA-activated factor (DRAF1). DRAF1 is composed of interferon regulatory factor 3 (IRF-3) and the transcriptional coactivators CREB binding protein (CBP) or p300. We find that activation of IRF-3 in the absence of viral infection stimulates apoptosis. In addition, a negative interfering mutant blocks both target gene induction and apoptosis, demonstrating a requirement for gene expression by IRF-3/DRAF1 to promote apoptosis. IRF-3/DRAF1 target gene expression is also induced in response to a distinct apoptotic stimulus, the DNA damaging agent etoposide. The activity of the p53 tumor suppressor does not appear to be required for IRF-3/DRAF1-mediated apoptosis.

1-Deoxynojirimycin-trehalamine-fused and -linked compounds (10, 19a and 19b) were synthesized from 1-deoxy-2,3,4,6-tetra-O-benzylnojirimycin and trehazolamine, which was obtained from natural trehazolin as a degradation product. None of these synthetic compounds exceeded 1-deoxynojirimycin in the inhibitory activities towards rat intestinal maltase and yeast α- d -glucosidase.

BACKGROUND Tumor procoagulant is one of the factors responsible for disseminated intravascular coagulation and metastasis. The authors found procoagulant activity in LK52 human squamous cell carcinoma cells, which they designated cancer cell-derived blood coagulating activity 1 (CCA-1). A monoclonal antibody (MoAb) was generated to characterize this CCA-1 procoagulant activity. To date, antibodies that show an inhibitory effect on procoagulant activity as well as high reactivity in cancer cells are well known for their tissue factor specificity. METHODS Characterization of the procoagulant activity of CCA-1 was performed and an anti-CCA-1 MoAb, FS01, was generated. CCA-1 expression on the cancer cell surface was examined by flow cytometry. Procoagulant activity of various cancer cell lines and the inhibitory effect of the FS01 MoAb on this procoagulant activity was monitored by a clot timer. RESULTS The enzymologic character differed from that of cancer procoagulant (CP). The FS01 MoAb inhibited the procoagulant activity of CCA-1, but did not inhibit that of tissue factor. A positive correlation was observed between the expression intensity of CCA-1 and the inhibitory effect of the FS01 MoAb on the procoagulant activity of cancer cell lines. Expression of CCA-l was observed more frequently than that of tissue factor in human cancer cell lines. CONCLUSIONS The FS01 MoAb generated in the current study is a new antibody that reacts with various cancer cell lines, but not with normal cells. FS01 inhibits cancer cell-derived procoagulant activity and does not react with tissue factor and CP. CCA-1, which is recognized by the FS01 MoAb, appears to play a major role in cancer cell-derived procoagulant activity. Cancer 1998;82:1563-9. © 1998 American Cancer Society.

We have generated a monoclonal antibody (MAb), FS01, which inhibits the procoagulant activity (CCA-1) produced by a human squamous cell carcinoma cell line, LK52. Expression of the antigen recognized by FS01 MAb in various cancer cell lines correlated well with the procoagulant activities of the expressing cell lines. Our objective was to characterize the molecule reacting with FS01 MAb and to analyze its involvement in the CCA-1 procoagulant activity. The molecule was identified as a glycolipid and found to be involved in the procoagulant activity because both procoagulant activity and reactivity to FS01 MAb were lost after endoglycoceramidase treatment of CCA-1. Furthermore, FS01 MAb recognized the Lewis Y (Le y ) antigen. To confirm the involvement of a glycolipid incorporating the Ley antigen in the procoagulant activity, we attempted to purify CCA-1 from LK52 culture supernatant. In one of the purification steps, a fraction containing low procoagulant activity (CCA-1p) separated from the Le y -positive fraction (CCA-1c). Although CCA-1c alone did not show procoagulant activity, the procoagulant activity of CCA-1p was augmented by CCA-1c and this augmentation was inhibited by FS01 MAb. Furthermore, CCA-1c enhanced the procoagulant activity of 33 cell lines tested as well as CCA-1p. In addition, purified Ley glycolipid from canine intestine augmented the procoagulant activity of CCA-1p, and this augmentation also could be inhibited by FS01 MAb. We conclude that Ley glycolipid is a co-factor for the procoagulant activity derived from cancer cells. Int. J. Cancer 73:903‐909, 1997. r 1997 Wiley-Liss, Inc.

The influence of polymerization conditions on the electrochemical properties and morphology of polyaniline are studied. It is found that the charge/discharge performance of chemically synthesized polyaniline is greatly influenced by the oxidizing reagents used for the polymerization. Cu(BF 4 ) 2 and Fe(ClO 4 ) 3 are particularly suitable reagents. Protons in the reaction media exert a strong influence on the morphology and electrochemical properties of polyaniline in both chemical and electrochemical polymerization processes. A novel process for chemically synthesizing polyaniline is developed to obtain polyaniline with superior electrochemical properties. In this process, polyaniline/ HBF 4 salt is oxidized with Cu(BF 4 ) 2 /acetonitrile and produces fibrous polyaniline with good doping/undoping reversibility.

Mechanisms of enzyme inhibition by trehazolin, a new inhibitor of trehalase (Ando et al. (1991) J. Antibiot. 44, 1165), were investigated using purified soluble silkworm trehalase and other glycosidases. Trehazolin inhibited trehalase with an IC50 value of 27 nM, whereas some other exo-alpha-glucosidases were inhibited only weakly, with IC50 values ranging from 7 to 370 microM. Other glycosidases tested were not inhibited by 500 microM trehazolin. The inhibition of trehalase by trehazolin was competitive with respect to trehalose. A notable feature of the inhibition was a slow progression of the association and dissociation of the enzyme-inhibitor complex. Preincubation of the enzyme and the inhibitor at 37 degrees C potentiated the inhibition by 10-times in a time-dependent manner up to 6 h. Dialysis of the inactivated enzyme recovered the enzymatic activity very slowly, and the rate constant for the dissociation at 37 degrees C was (7.3).10(-2) h-1. Trehalamine, a deglucosylated form of trehazolin, inhibited both silkworm trehalase and exo-alpha-glucosidases only weakly. The inhibition of trehalase by trehalamine was reversible. Rat isomaltase inhibition by trehazolin and sucrase inhibition by trehalamine were also reversible. Taken together, trehazolin is a specific slow, tight-binding inhibitor of trehalase, and the glucose moiety of the inhibitor is essential to the tight binding.

We established interferon-gamma-resistant (IGR) cells from a human colon adenocarcinoma cell line, LoVo. Their resistance was extremely high, and the ED50 values of IFN-gamma were > 10(5) IU/ml. Interestingly, although IGR-5 cells were still sensitive to the antiproliferative effect of IFN-alpha, the cells lost responsiveness to the antiviral effects of both IFN-alpha and gamma. Another clone, IGR-53, was unresponsive to both the antiproliferative and antiviral effects of either IFN-alpha or gamma. Furthermore, the IFN-gamma-resistant phenotypes of IGR cells were apparently dominant to the parental LoVo cells based on complementation tests. Although IGR-53 cells lack IFN-gamma receptors, IGR-5 cells seemed to have functional IFN-gamma receptors and processing mechanisms of IFN-gamma bound to the receptors. Northern analysis showed that IGR-5 cells responded to IFN-gamma and alpha, but the enhancement of IRF-1 expression by IFN-gamma was markedly suppressed.

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.

The levels of tyrosinase mRNA and tyrosinase activity were analyzed in two amelanotic melanoma cell lines, D1178 (hamster) and G-361 (human). Neither tyrosinase mRNA nor tyrosinase activity were detected in D1178 cells. On the other hand, both tyrosinase mRNA and weak tyrosinase activity were detected in G-361 cells. Assuming that the different types of melanogenic inhibitors affected melanogenesis in these two amelanotic melanoma cells in different manners, we performed a screening of melanogenic inhibitors in these two cell lines. As an isolated tyrosinase suppressive melanogenic inhibitor, ascorbic acid and glutathione were identified from D1178 cells and G-361 cells, respectively. Furthermore, lactic acid was identified from D1178 cells as an isolated tyrosinase non-suppressive melanogenic inhibitor. B-16 mouse melanotic melanoma cells were depigmented by treatment with lactic acid. The melanogenesis suppression by lactic acid in B-16 cells was found to be due to inhibition of tyrosinase gene expression. J Invest Dermatol 100:150S– 155S, 1993

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.

1-Deoxynojirimycin-trehalamine-fused and -linked compounds (10, 19a and 19b) were synthesized from 1-deoxy-2,3,4,6-tetra-O-benzylnojirimycin and trehazolamine, which was obtained from natural trehazolin as a degradation product. None of these synthetic compounds exceeded 1-deoxynojirimycin in the inhibitory activities towards rat intestinal maltase and yeast α- d -glucosidase.

Natural azasugars have the ring oxygen substituted by nitrogen. They show potent inhibitory activity against glycosidases. The effect of substituting the ring carbon with nitrogen was examined with 1-azafagomine. 1-Azafagomine exhibited similar activity against processing glucosidase to that of fagomine.

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 μg, 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.

Several conductive polymers (polyacetylene, polypyrrole, polythiophene, polyiminodibenzyl, polycarbazole, polyfuran, polyphenothiazine) have been synthesized by a chemical polymerization method and examined for their suitability as positive electrode material for lithium secondary batteries. A test cell, using polypyrrole as a positive electrode, showed good charge/discharge characteristics. Oxidizing agents for the synthesis of polypyrrole have been investigated for further improvement, and polypyrrole prepared by a new method of synthesis using a Cu(BF4)2/nitrile system exhibited excellent performance.

Oligonucleotides uniformly modified with 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) units were synthesized using the phosphoramidite method on a hundred-milligram scale for the evaluation of thermodynamic and chemical properties. The properties of these ENA oligonucleotides with the sequences targeted to human telomerase RNA subunit (hTR) were compared with those of GRN163, which is an oligonucleotide modified with N3'-P5' thiophosphoramidates. The melting temperatures of the duplexes of ENA oligonucleotides with complementary RNA were higher than that of the duplex of GRN163. Moreover, ENA oligonucleotide ENA-13 was more highly stable than GRN163 under acidic conditions (pH 5.0). ENA-13, which contained contiguous guanine sequences, could not form a G-quadruplex, which formation is not feasible for binding to hTR as an antisense molecule. The above findings suggest that ENA oligonucleotides may be useful for antisense therapeutic applications.