Identification of new Cdc25 dual specificity phosphatase inhibitors in a targeted small molecule array.

Dual specificity protein phosphatases (DSPases) are key regulators of signal transduction, oncogenesis and the cell cycle. Few potent or specific inhibitors of DSPases, however, are readily available for these pharmacological targets. We have used a combinatorial/parallel synthetic approach to rigidify the variable core region and modify the side chains of 4-(benzyl-(2-[2,5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)- 2-decanoylamino butyric acid (or SC-alphaalphadelta9), which is the most active element in a previously described library of phosphatase inhibitors (Rice, R. L.; Rusnak, J. M.; Yokokawa, F.; Yokokawa, S.; Messner, D. J.; Boynton, A. L.; Wipf, P.; Lazo, J. S. Biochemistry 1997, 36, 15965). Several analogues were identified as effective inhibitors of the protein tyrosine phosphatase (PTPase) PTP1B and the DSPases VHR and Cdc25B2. Two compounds, FY3-alphaalpha09 and FY21-alphaalpha09, were partial competitive inhibitors of Cdc25B2 with Ki values of 7.6+/-0.5 and 1.6+/-0.2 microM, respectively. FY21-alphaalpha09 possessed only moderate activity against PTP1B. Consistent with its in vitro anti-phosphatase activity, FY21-alphaalpha09 inhibited growth in MDA-MB-231 and MCF-7 human breast cancer cell lines. FY21-alphaalpha09 also inhibited the G2/M transition in tsFT210 cells, consistent with Cdc25B inhibition. Several architectural requirements for DSPase inhibition were revealed through modification of the side chain moieties or variable core region of the pharmacophore, which resulted in decreased compound potency. The structure of FY21-alphaalpha09 provides a useful platform from which additional potent and more highly selective phosphatase inhibitors might be generated.

Disruption of insulin-like growth factor-1 signaling and down-regulation of cdc2 by SC-alphaalphadelta9, a novel small molecule antisignaling agent identified in a targeted array library.

We previously reported the generation of a library of hydrophobic oxazole-based small molecules designed as inhibitors of phosphatases involved in cellular signaling and cell cycle control. One member of the targeted array library, 4-(benzyl-(2-[(2, 5-diphenyl-oxazole-4-carbonyl)-amino]-ethyl)-carbamoyl)-2-decanoylami no butyric acid (SC-alphaalphadelta9), inhibited cell growth in the G0/G1 phase of the cell cycle. To investigate potential mechanisms for SC-alphaalphadelta9 antiproliferative activity, we have used mouse embryonic fibroblasts transformed with simian virus 40 large T antigen mouse embryonic fibroblasts as a model system for a malignant phenotype that depends on overexpression of cell cycle regulators and autocrine stimulation by insulin-like growth factor-1. Structure-activity relationship studies with SC-alphaalphadelta9 and four library congeners demonstrated that antiproliferative activity was not a result of overall hydrophobicity. Rather, SC-alphaalphadelta9 decreased insulin-like growth factor-1 receptor tyrosine phosphorylation, receptor expression, mitogen-activated protein kinase activation and levels of the cyclin-dependent kinase Cdc2. Less toxic congeners only partially affected receptor expression, receptor tyrosine phosphorylation and Cdc2 levels. Thus SC-alphaalphadelta9, which is structurally distinct from other known small molecules that decrease intracellular Cdc2 levels, has profound effects on intracellular signaling. Furthermore, SC-alphaalphadelta9, but not vanadate or okadaic acid, selectively inhibited the growth of simian virus 40 large T antigen mouse embryonic fibroblasts compared to the parental cells. These results suggest that overexpression of Cdc2 and increased dependence on insulin-like growth factor-1 autocrine stimulation are responsible for the increased sensitivity of simian virus 40 large T antigen mouse embryonic fibroblasts to SC-alphaalphadelta9. The SC-alphaalphadelta9 pharmacophore could be a useful platform for the development of novel antisignaling agents.

Isolation and characterization of a cDNA for human mouse, and rat full-length stem cell growth factor, a new member of C-type lectin superfamily.

cDNA encoding stem cell growth factor (SCGF; 245 aa), a novel human growth factor for primitive hematopoietic progenitor cells, has been previously reported (Hiraoka, A., Sugimura, A., Seki, T., Nagasawa, T., Ohta, N., Shimonishi, M., Hagiya, M. and Shimizu, S. Proc. Natl. Acad. Sci. USA 94, 7577-7582, 1997). Here we report the cloning and characterization of a full-length SCGF cDNA. This protein consists of 323, 328 and 328 aa in the human, murine and rat forms, the latter two of which share 85.1% and 83.3% aa identity, and 90.4% and 90.4% aa similarity to the human protein, respectively. Because the newly identified human clone encodes the protein longer by 78 aa than that previously identified, we term the longer clone as hSCGF-alpha and the shorter one as hSCGF-beta. The computer-assisted homology search reveals that SCGF is a new member of the C-type lectin superfamily, and that SCGF shows the greatest homology to tetranectin among the members of the family (27.2-33.7% aa identity and 46.0-53.6% aa similarity). SCGF transcripts are detected in spleen, thymus, appendix, bone marrow and fetal liver. Fluorescent in situ hybridization mapping indicates that the SCGF gene is located on chromosome 19 at position q13.3 for human form and on chromosome 7 at position B3-B5 for murine form, which are close to flk-2/flt3 ligand and interleukin-11 genes of both human and murine species.

Generation of a monoclonal antibody that induces apoptosis of hematopoietic cells.

Here, we developed anti-murine myeloid cell (NFS-60 cell) monoclonal antibodies in order to characterize the further mechanism of cell-to-cell interaction between the stromal cells and the hematopoietic progenitor cells. The established antibody, designated mAb UNF, inhibited proliferation of NFS-60 cells and induced apoptosis. Incubation of NFS-60 cells with mAb UNF initiated the cell aggregation within 3 hours. This phenomena did not require newly synthesized proteins, since the pretreatment of 1 mM cycloheximide failed to prevent the agglutination. Fifty-eight percent of mouse hematopoietic stem cells, Lin-, c-Kit+, Sca-1+ bone marrow cells, were shown to express UNF antigen. The glycolipid prepared from NFS-60 cells was specifically immunostained by mAb UNF.

A targeted library of small-molecule, tyrosine, and dual-specificity phosphatase inhibitors derived from a rational core design and random side chain variation.

Tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosines while dual-specificity phosphatases (DSPases) dephosphorylate contiguous and semicontiguous phosphothreonine and phosphotyrosine on cyclin dependent kinases and mitogen-activated protein kinases. Consequently, PTPases and DSPases have a central role controlling signal transduction and cell cycle progression. Currently, there are few readily available potent inhibitors of PTPases or DSPases other than vanadate. Using a pharmacophore modeled on natural product inhibitors of phosphothreonine phosphatases, we generated a refined library of novel, phosphate-free, small-molecule compounds synthesized by a parallel, solid-phase combinatorial-based approach. Among the initial 18 members of this targeted diversity library, we identified several inhibitors of DSPases: Cdc25A, -B, and -C and the PTPase PTP1B. These compounds at 100 microM did not significantly inhibit the protein serine/threonine phosphatases PP1 and PP2A. Kinetic studies with two members of this library indicated competitive inhibition for Cdc25 DSPases and noncompetitive inhibition for PTP1B. Compound AC-alphaalpha69 had a Ki of approximately 10 microM for recombinant human Cdc25A, -B, and -C, and a Ki of 0.85 microM for the PTP1B. The marked differences in Cdc25 inhibition as compared to PTP1B inhibition seen with relatively modest chemical modifications in the modular side chains demonstrate the structurally demanding nature of the DSPase catalytic site distinct from the PTPase catalytic site. These results represent the first fundamental advance toward a readily modifiable pharmacophore for synthetic PTPase and DSPase inhibitors and illustrate the significant potential of a combinatorial-based strategy that supplements the rational design of a core structure by a randomized variation of peripheral substituents.

Laser printer optics with use of slant scanning of multiple beams.

Simultaneous scanning of multiple beams in an array is an effective method to realize high-speed and high-resolution printers. The arrayed multiple beams can be generated by devices such as grating, Wollaston prism, fiber array, and laser diode array. In any of these devices, the focused spots in an array have a period several tens of times larger than the spot diameter. We propose a simultaneous scanning method suitable for these devices in which the arrayed multiple beams are arranged in a slant angle to the scanning direction to produce consecutive scan lines. Laser print experiments with two or four beams were carried out, and high-performance printing of a 431.8-mm print width, 23.6 dot/mm (i.e., 600 dot/in.) resolution, and of 541-mm/s speed were realized.

[A statistical study on the orthodontic patients in Meikai University Hospital].

We analyzed statistically two thousand orthodontic patients in Meikai University Hospital (formerly Josai Dental University Hospital) who have undergone treatment since the establishment of the hospital (1970-1985). By classifying the data in various ways, we obtained the following statistics; 1) The number of new patients increased quickly after the first class graduated from our university; 2) The number of patients from six to fifteen years old was the largest, with the peak being nine years old; 3) For the time of the first visit, many new patients came during spring and summer vacations, and a few junior high school and high school students came just season before taking their entrance examination; 4) The female sex predominated in most age groups; however, among patients in their twenties, the male sex was predominant, and in recent years the male ratio tended to increase; 5) Most patients came from a radius of less than 20 km from the hospital (mostly from along Tohbu railway line), with few from beyond 30 km; 6) Anterior crossbite and anterior crowding cases were many, particularly, young patients comprised many of the anterior crossbite cases; and in adult patients large overjet cases increased with the advance of age.

Determination of malotilate and its metabolites in plasma and urine.

A method for the determination of malotilate (I), the corresponding monocarboxylic acid (II) and its decarboxylated product (III) in plasma is described. Plasma was extracted with chloroform spiked with internal standard. The residue, dissolved in methanol, was chromatographed on a reversed-phase column with a mobile phase of 60% acetonitrile and 1% acetic acid in water. The sensitivity limit for I, II and III was 50, 25 and 100 ng/ml of plasma, respectively. Compound I in the same plasma extract was also analysed by gas chromatography--electron-impact mass spectrometry. The base peaks m/z 160 for I and m/z 162 for internal standard (IV) were monitored; the sensitivity limit for I was 2.5 ng/ml of plasma. The determination of the metabolites of I, II and its conjugate (V), and isopropyl-hydrogen malonate (VI) in urine by high-performance liquid chromatography is also described. The limit of quantification for VI was 2.0 micrograms/ml, and the overall coefficient of variation of VI was 4.7%. The limit of quantification for II in urine was 0.5 micrograms/ml and that for V was 1.0 micrograms/ml as total II (II + V). The overall precision of the method was satisfactory. The method was used to determine plasma and urine concentrations in four dogs orally dosed with 100, 200 or 400 mg of malotilate.