A novel Golgi mannosidase inhibitor: Molecular design,synthesis, enzyme inhibition,and inhibition of spheroid formation

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell–cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.     

Identification and characterization of a novel Golgi protein, golgin-67

In the course of screening a lambdagt11 human leukemic T-cell cDNA expression library with an antibody specific to the mitotic target of Src, Sam68, we identified and cloned a cDNA encoding a novel protein with a predicted molecular mass of 51.4 kDa. Polyclonal antibodies raised to a His(6)-tagged construct of this protein, detected a approximately 67-kDa protein in immunoprecipitation experiments, and cytological studies showed that this protein localized to the Golgi complex, through colocalization experiments with specific Golgi markers. Therefore, we designated this protein golgin-67. Sequence analysis revealed that golgin-67 is a highly coiled-coil protein, with potential Cdc2 and Src kinase phosphorylation motifs. It has sequence homologies to other Golgi proteins, including the coatamer complex I vesicle docking protein, GM130. Structurally, golgin-67 resembles, golgin-84, an integral membrane Golgi protein with an N-terminal coiled-coil domain and a single C-terminal transmembrane domain. The C-terminal region of golgin-67, which contains a predicted transmembrane domain, was demonstrated to be essential for its Golgi localization.     

Cohen syndrome-associated protein, COH1, is a novel, giant Golgi matrix protein required for Golgi integrity

Loss-of-function mutations in the gene COH1, also known as VPS13B, lead to autosomal recessive Cohen syndrome. However, the cellular distribution and function of the encoded protein COH1 (3997 amino acids), which lacks functional homologies to other mammalian proteins, have remained enigmatic. We show here that COH1 is a peripheral Golgi membrane protein that strongly co-localizes with the cis-Golgi matrix protein GM130. Consistent with its subcellular localization, COH1 depletion using RNAi causes fragmentation of the Golgi ribbon into ministacks. Disruption of Golgi organization observed in fibroblasts from Cohen syndrome patients suggests that Golgi dysfunction contributes to Cohen syndrome pathology. In conclusion, our findings establish COH1 as a Golgi-associated matrix protein required for Golgi integrity.     

Theromin, a novel leech thrombin inhibitor

We purified the most potent thrombin inhibitor described to date from the rhynchobdellid leech Theromyzon tessulatum. Designated theromin, it was purified to apparent homogeneity by gel permeation and anion exchange chromatography followed by two reverse-phase steps of high performance liquid chromatography. The primary sequence of theromin (a homodimer of 67 amino acid residues including 16 cysteine residues) was determined by a combination of reduction and s-beta-pyridylethylation, Edman degradation, trypsin enzymatic digestion, and matrix-assisted laser desorption mass spectrometry measurement. Theromin exhibits no sequence homology with any other thrombin inhibitors. Furthermore, theromin significantly diminishes, in a dose-dependent manner, the level of human granulocyte and monocyte activation induced by lipopolysaccharides. In summary, this potent thrombin inhibitor promises to have high biomedical significance.     

CspD, a novel DNA replication inhibitor induced during the stationary phase in Escherichia coli

CspD is a stationary phase-induced, stress response protein in the CspA family of Escherichia coli. Here, we demonstrate that overproduction of CspD is lethal, with the cells displaying a morphology typical of cells with impaired DNA replication. CspD consists mainly of beta-strands, and the purified protein exists exclusively as a dimer and binds to single-stranded (ss)DNA and RNA in a dose-dependent manner without apparent sequence specificity. CsdD effectively inhibits both the initiation and the elongation steps of minichromosome replication in vitro. Electron microscopic studies revealed that CspD tightly packs ssDNA, resulting in structures distinctly different from those of SSB-coated DNA. We propose that CspD dimers, with two independent beta-sheets interacting with ssDNA, function as a novel inhibitor of DNA replication and play a regulatory role in chromosomal replication in nutrient-depleted cells.     

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.     

Inhibition of induced acute lung edema by a novel protein kinase C inhibitor

The effect of the protein kinase C enzyme inhibitor H-7 on the noncardiogenic lung edema induced by phorbol myristate acetate (PMA) in mice was examined. Lung edema was assessed by measurement of 125I-labeled albumin leak into the lung. The results showed that pretreatment of mice with H-7 nearly prevents the albumin leak induced by PMA, whereas post-PMA treatment with H-7 had less of an effect on the albumin leak, although it was still significant.     

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy

The relationshipbetween myogenin or MyoD expression and hypertrophy of the rat soleusproduced either by clenbuterol and 3,3',5-triiodo-L-thyronine(CT) treatment or by surgical overload was examined. Mature female ratswere subjected to surgical overload of the right soleus with the leftsoleus serving as a control. Another group received the same surgicaltreatment but were administered CT. Soleus muscles were harvested 4 wkafter surgical overload and weighed. Myosin heavy chain isoforms wereseparated by using polyacrylamide gel electrophoresis while myogeninand MyoD expression were evaluated by Northern analysis.CT and functional overload increased soleus muscle weight. CT treatmentinduced the appearance of the fast type IIX myosin heavy chain isoform,depressed myogenin expression, and induced MyoD expression. However,functional overload did not alter myogenin or MyoD expression inCT-treated or non-CT-treated rats. Thus pharmacologically andsurgically induced hypertrophy have differing effects on myogenin andMyoD expression, because their levels were associated with changes inmyosin heavy chain composition (especially type IIX) rather thanchanges in muscle mass.

     

Hygromycin A, a novel inhibitor of ribosomal peptidyltransferase

In cell-free systems from Escherichia coli, hygromycin A inhibits polypeptide synthesis directed by either poly(U) or phage R 17 RNA, and the reaction of puromycin with either natural peptidyl-tRNA, or AcPhe-tRNA, or the 3'-terminal fragment of AcLeu-tRNA (C-A-C-C-A-LeuAc). In contrast, the antibiotic does no inhibit the enzymatic binding of Phe-tRNA to ribosomes or the translocation of AcPhe-tRNA. It is concluded that hygromycin A is a specific inhibitor of the peptide bond formation step of protein synthesis. The action of hygromycin A on peptidyl transfer is similar to that of chloramphenicol, an antibiotic that shares some common structural features with hygromycin A. Both antibiotics inhibit the binding of C-A-C-C-A-Leu to the acceptor site of peptidyl transferase and stimulate that of C-A-C-C-A-LeuAc to the donor site of the enzyme. Moreover, hygromycin A blocks the binding of chloramphenicol to ribosomes, indicating that the binding sites of the antibiotics may be closely related. Hygromycin A is a more potent agent than chloramphenicol and binds quite strongly to ribosomes.     

Golgi endo-alpha-D-mannosidase from rat liver, a novel N-linked carbohydrate unit processing enzyme

An enzyme has been found in Triton-treated rat liver Golgi membranes which trims Glc1Man9GlcNAc to Man8GlcNAc with the release of Glc alpha 1-3Man. By removing a glucosylmannose disaccharide and yielding only one Man8GlcNAc isomer, this endo-alpha-D-mannosidase provides a processing route alternative to the sequential actions of alpha-glucosidase II and alpha-mannosidase I. The endomannosidase was fully active in the presence of 1-deoxynojirimycin and EDTA which inhibited exoglycosidase release of glucose and mannose, respectively, and these agents were, therefore, included in the standard assay. The specific activity of the endomannosidase was found to be 69-fold greater in Golgi than in rough endoplasmic reticulum (RER) membranes, and Golgi-RER mixing experiments excluded the possibility that the low activity in the RER was the result of some inhibitor present in this fraction. The neutral pH optimum (approximately 7.0) of the enzyme was consistent with a role in N-linked oligosaccharide processing. The existence of an endo-alpha-D-mannosidase pathway for glucose removal could provide an explanation for the incomplete block in oligosaccharide processing which is observed in cells with inhibited or deficient alpha-glucosidase.     

The protease inhibitor bikunin,a novel anti-metastatic agent

Bikunin is a Kunitz-type protease inhibitor predominantly found in human amniotic fluid. In cancers, administration of bikunin may block tumor cell invasion by a direct inhibition of tumor cell-associated plasmin activity as well as by inhibiting urokinase-type plasminogen activator (uPA) expression at the gene and protein levels, possibly through suppression of CD44 dimerization and/or the MAP kinase signaling cascade. Treatment of cancer patients with bikunin may be beneficial in the adjuvant setting to delay the onset of metastasis development and/or in combination with cytotoxic agents to improve treatment efficacy in patients with advanced ovarian cancer.     

Critical upstream signals of cytochrome C release induced by a novel Bcl-2 inhibitor

Cytochrome c release is a central step in the apoptosis induced by many death stimuli. Bcl-2 plays a critical role in controlling this step. In this study, we investigated the upstream mechanism of cytochrome c release induced by ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA14-1), a recently discovered small molecule inhibitor of Bcl-2. HA14-1 was found to induce cytochrome c release from the mitochondria of intact cells but not from isolated mitochondria. Cytochrome c release from isolated mitochondria requires the presence of both HA14-1 and exogenous Ca(2+). This suggests that both mitochondrial and extramitochondrial signals are important. In intact cells, treatment with HA14-1 caused Ca(2+) spike, change in mitochondrial membrane potential (Delta psi(m)) transition, Bax translocation, and reactive oxygen species (ROS) generation prior to cytochrome c release. Pretreatment with either EGTA acetoxymethyl ester or vitamin E resulted in a significant decrease in cytochrome c release and cell death induced by HA14-1. Furthermore pretreatment with RU-360, an inhibitor of the mitochondrial Ca(2+) uniporter, or with EGTA acetoxymethyl ester, but not with vitamin E, prevented the HA14-1-induced Delta psi(m) transition and Bax translocation. This suggests that ROS generation is an event that occurs after the Delta psi(m) transition and Bax translocation. Together these data demonstrate that the Ca(2+) spike, mitochondrial Bcl-2 presensitization, and subsequent Delta psi(m) transition, Bax translocation, and ROS generation are important upstream signals for cytochrome c release upon HA14-1 stimulation. The involvement of endoplasmic reticulum and mitochondrial signals suggests both organelles are crucial for HA14-1-induced apoptosis.     

cDNA array studies of natural and pharmacologically induced sleep

Sleep and Biological Rhythms -     

ZnT7, a novel mammalian zinc transporter,accumulates zinc in the Golgi apparatus

ZnT7, a novel member of the zinc transporter (ZnT) family, was identified by searching the expressed sequence tag (EST) databases with the amino acid sequence of ZnT1. Like the other ZnT proteins, the protein (387 amino acids) predicted from this gene contains six transmembrane domains and a histidine-rich loop between transmembrane domains IV and V. We show that Znt7 is widely transcribed in mouse tissues with abundant expression in the liver and small intestine and moderate expression in the kidney, spleen, brain, and lung. An affinity-purified antibody raised against the amino acids 299-315 of mouse ZnT7 specifically reacted with the proteins with apparent molecular masses of 85, 43, and 65 kDa in small intestine and lung tissues by Western blot analysis. Immunofluorescence microscope analysis reveals that ZnT7 is localized in the Golgi apparatus and cytoplasmic vesicles. Exposure of the ZnT7-expressing Chinese hamster ovary (CHO) cells to zinc causes an accumulation of zinc in the Golgi apparatus, suggesting that ZnT7 facilitates zinc transport from the cytoplasm into the Golgi apparatus.     

Cardioprotective effects of ingliforib, a novel glycogen phosphorylase inhibitor

Interventions such as glycogen depletion, which limit myocardial anaerobic glycolysis and the associated proton production, can reduce myocardial ischemic injury; thus it follows that inhibition of glycogenolysis should also be cardioprotective. Therefore, we examined whether the novel glycogen phosphorylase inhibitor 5-Chloro-N-[(1S,2R)-3-[(3R,4S)-3,4-dihydroxy-1-pyrrolidinyl)]-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide (ingliforib; CP-368,296) could reduce infarct size in both in vitro and in vivo rabbit models of ischemia-reperfusion injury (30 min of regional ischemia, followed by 120 min of reperfusion). In Langendorff-perfused hearts, constant perfusion of ingliforib started 30 min before regional ischemia and elicited a concentration-dependent reduction in infarct size; infarct size was reduced by 69% with 10 microM ingliforib. No significant drug-induced changes were observed in either cardiac function (heart rate, left ventricular developed pressure) or coronary flow. In open-chest anesthetized rabbits, a dose of ingliforib (15 mg/kg loading dose; 23 mg.kg(-1).h(-1) infusion) selected to achieve a free plasma concentration equivalent to an estimated EC(50) in the isolated hearts (1.2 microM, 0.55 microg/ml) significantly reduced infarct size by 52%, and reduced plasma glucose and lactate concentrations. Furthermore, myocardial glycogen phosphorylase a and total glycogen phosphorylase activity were reduced by 65% and 40%, respectively, and glycogen stores were preserved in ingliforib-treated hearts. No significant change was observed in mean arterial pressure or rate-pressure product in the ingliforib group, although heart rate was modestly decreased postischemia. In conclusion, glycogen phosphorylase inhibition with ingliforib markedly reduces myocardial ischemic injury in vitro and in vivo; this may represent a viable approach for both achieving clinical cardioprotection and treating diabetic patients at increased risk of cardiovascular disease.