Discovery and functional characterization of a novel small molecule inhibitor of the intracellular phosphatase, SHIP2.

BACKGROUND AND PURPOSE: The lipid phosphatase known as SH2 domain-containing inositol 5'-phosphatase 2 (SHIP2) plays an important role in the regulation of the intracellular insulin signalling pathway. Recent studies have suggested that inhibition of SHIP2 could produce significant benefits in treatment of type 2 diabetes. However, there were no small molecule SHIP2 inhibitors and we, therefore, aimed to identify this type of compound. EXPERIMENTAL APPROACH: The phosphatase assay with malachite green was used for high-throughput screening. The pharmacological profiles of suitable compounds were further characterized in phosphatase assays, cellular assays and oral administration in normal and diabetic (db/db) mice. KEY RESULTS: During high-throughput screening, AS1949490 was identified as a potent SHIP2 inhibitor (IC(50)= 0.62 microM for SHIP2). This compound was also selective for SHIP2 relative to other intracellular phosphatases. In L6 myotubes, AS1949490 increased the phosphorylation of Akt, glucose consumption and glucose uptake. In FAO hepatocytes, AS1949490 suppressed gluconeogenesis. Acute administration of AS1949490 inhibited the expression of gluconeogenic genes in the livers of normal mice. Chronic treatment of diabetic db/db mice with AS1949490 significantly lowered the plasma glucose level and improved glucose intolerance. These in vivo effects were based in part on the activation of intracellular insulin signalling pathways in the liver. CONCLUSIONS AND IMPLICATIONS: This is the first report of a small molecule inhibitor of SHIP2. This compound will help to elucidate the physiological functions of SHIP2 and its involvement in various diseases, such as type 2 diabetes.

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.

Effect of zenarestat, an aldose reductase inhibitor, on endoneurial blood flow in experimental diabetic neuropathy of rat.

The effects of zenarestat, an aldose reductase inhibitor, on endoneurial blood flow (NBF) were explored in streptozotocin-induced diabetic rats. Rats were maintained on a diet of containing 0.09% zenarestat for 8 weeks, then NBF in the sciatic nerve was measured using microelectrode hydrogen polarography. NBF in the diabetic control rats was significantly lower than values in age-matched control rats, however, NBF was not significantly altered in diabetic rats treated with zenarestat. Direct application of nitric oxide synthase inhibitor, NG-nitro-L-arginine, did not affect NBF in diabetic control rats, whereas this application significantly reduced NBF both in age-matched control and zenarestat treated diabetic rats. Considerable levels of zenarestat were confirmed in the sciatic nerve in the drug treated rats. These data suggest that aldose reductase, such as zenarestat, might restore or prevent the alteration of endoneurial blood flow resulting from an impairment of nitric oxide function.

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.

Direct chiral resolution of lactic acid in food products by capillary electrophoresis.

Chiral resolution of native DL-lactic acid was performed by capillary electrophoresis using 2-hydroxypropyl-beta-cyclodextrin as a chiral selector. Various factors affecting chiral resolution, migration time, and peak area of lactic acid were studied. The running conditions for optimum separation of lactic acid were found to be 90 mM phosphate buffer (pH 6.0) containing 240 mM 2-hydroxypropyl-beta-cyclodextrin with an effective voltage of -30 kV at 16 degrees C, using direct detection at 200 nm. In order to enhance the sensitivity, sample injection was done under a pressure of 50 mbar for 200 s. On-line sample concentration was accomplished by sample stacking. With this system, D- and L-lactic acids in food products were analyzed successfully.

Cytotoxic substances produced by a fungal strain from a sponge: physico-chemical properties and structures.

Five novel metabolites, trichodenones A-C (1-3), harzialactone A (4) and B (5), have been isolated together with known R-mevalonolactone (6) from the culture broth of a strain of Trichoderma harzianum OUPS-N115 originally separated from the sponge Halichondria okadai. Their structures have been elucidated by spectral evidence. Among them, 1-3 exhibited significant cytotoxicity against cultured P388 cells.

Leptosins I and J, cytotoxic substances produced by a Leptosphaeria sp. Physico-chemical properties and structures.

Leptosins I and J, belonging to a series of epipolythiodioxopiperazines, have been isolated from the mycelium of a strain of Leptosphaeria sp. OUPS-4 attached to the marine alga Sargasssum tortile. Their relative stereostructures have been elucidated by chemical and spectral evidence. These compounds exhibited significant cytotoxic activity against cultured P388 cells.

Cloning and sequence analysis of the gene encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520.

The gene (chi40) encoding a thermostable chitinase from Streptomyces thermoviolaceus OPC-520 was cloned in Escherichia coli JM109 using pUC18. The nucleotide (nt) sequence of chi40 has been determined. A single open reading frame (ORF) encoded a protein consisting of 414 amino acids (aa) with a M(r) of 43,838. The deduced aa sequence of the cloned chitinase (Chi40) showed striking homology (74%) with Chi63 from Streptomyces plicatus. Comparison with other chitinases revealed that Chi40 contained the two conserved regions common to microbial and plant chitinases. Furthermore, the putative promoter region of chi40, which might be involved in the repression and induction of such catabolite-controlled genes, was detected by the DNA sequence alignments.

Purification and properties of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520.

A chitinase was purified from the culture filtrate of Streptomyces thermoviolaceus OPC-520. The enzyme showed a high optimum temperature (70 to 80 degrees C), a high optimum pH level (8.0 to 10.0), and heat stability. This enzyme showed high sequence homology with chitinases from Serratia marcescens QMB1466 and Bacillus circulans WL-12.

[FDP levels in the cerebrospinal fluid are elevated in patients with meningitis].

Since the level of fibrinogen degradation products (FDP) are elevated with severity of inflammation, we assumed that FDP in the cerebrospinal fluid (CSF) could be a marker of meningitis. We, therefore, measured FDP in the CSF of 6 patients with bacterial meningitis and 6 aseptic meningitis. The range of FDP levels in the CSF in patients without meningitis was 0.21 +/- 0.01 microgram/ml. While, the level of FDP in patients with aseptic meningitis (0.43 +/- 0.10 microgram/ml) and in bacterial meningitis (1.78 +/- 0.42 micrograms/ml) was significantly elevated (p less than 0.05). The value was significantly (p less than 0.01) higher in the group of septic meningitis than in aseptic meningitis. In one patient with septic meningitis, we could measure FDP in the CSF several times during the course of the disease, in which the level of FDP got into the high range earlier than the changes in levels of protein, glucose and cell counts in the CSF. FDP in the CSF well correlated to the clinical course of the meningitis. Eventually, we found that FDP in the CSF was definitely elevated in patients with bacterial meningitis, whereas it was slightly elevated in patients with aseptic meningitis. The measurement of FDP in the CSF, therefore, is concluded to be useful for the differential diagnosis of meningitis, and to assess the clinical course of meningitis.