Structure-activity relationship study, target identification, and pharmacological characterization of a small molecular IL-12/23 inhibitor, APY0201.

Interleukin-12 (IL-12) and IL-23 are proinflammatory cytokines and therapeutic targets for inflammatory and autoimmune diseases, including inflammatory bowel diseases, psoriasis, rheumatoid arthritis, and multiple sclerosis. We describe the discovery of APY0201, a unique small molecular IL-12/23 production inhibitor, from activated macrophages and monocytes, and demonstrate ameliorated inflammation in an experimental model of colitis. Through a chemical proteomics approach using a highly sensitive direct nanoflow LC-MS/MS system and bait compounds equipped with the FLAG epitope associated regulator of PIKfyve (ArPIKfyve) was detected. Further study identified its associated protein phosphoinositide kinase, FYVE finger-containing (PIKfyve), as the target protein of APY0201, which was characterized as a potent, highly selective, ATP-competitive PIKfyve inhibitor that interrupts the conversion of phosphatidylinositol 3-phosphate (PtdIns3P) to PtdIns(3,5)P2. These results elucidate the function of PIKfyve kinase in the IL-12/23 production pathway and in IL-12/23-driven inflammatory disease pathologies to provide a compelling rationale for targeting PIKfyve kinase in inflammatory and autoimmune diseases.

Organic anion transporter OAT1 is involved in renal handling of citrulline.

Because citrulline plasma concentration is elevated in kidney failure, citrulline could be a biomarker of renal insufficiency, although the mechanism regulating its disposition in the kidney has not been clarified. In rat kidney slices, citrulline uptake was apparently Na(+) dependent, saturable with K(m) 556 microM, and significantly inhibited by anionic (PAH) and cationic (TEA) compounds, but not by probenecid at 1 mM. Preincubation of kidney slices with glutarate increased citrulline uptake, while such an increase was not observed after preincubation of the slices in Na(+)-free buffer. This result suggested that a sodium-dependent dicarboxylate cotransporter is involved in citrulline uptake by rat kidney slices. In studies using transporter-overexpressing cells, human organic anion transporter 1 (OAT1) and rat Oat1 exhibited citrulline transport activity with K(m) values of 238 and 373 microM, respectively, while other OATs and organic cation transporters (OCTs) did not transport citrulline. Based on the relative activity factor method, the contribution of rat Oat1 to the overall uptake of citrulline in rat kidney slices was approximately 70%. Moreover, the interaction among citrulline, PAH, and probenecid uptakes via rat Oat1 suggested that there are multiple functional sites on Oat1 and that the citrulline site may be distinct from the PAH and probenecid site. Thus OAT1/Oat1 appears to be one of the major contributors to renal basolateral uptake of citrulline, and impaired activities of these transporters may contribute substantially to the increase in plasma citrulline in renal failure. Accordingly, citrulline may be useful for diagnosis of kidney function as is creatinine.

Changes of differentiation and proliferation in k562 cells with various levels of knockdown of cationic amino acid transporter 1.

We recently showed that arginine transport via cationic amino acid transporter 1 (CAT1), which transports arginine, lysine, ornithine, and histidine, is essential for erythropoiesis. In the present study, to confirm the importance of both arginine and CAT1 in erythropoiesis, we investigated the relationship of arginine uptake activity and differentiation and proliferation of blood cells by knockdown of the CAT1 gene using shRNA. Five short hairpin RNA (shRNA)-transfected K562 cell clones, in which the CAT1 mRNA expression level is decreased, and a vector-transfected clone were obtained. The differentiation to erythrocytes and proliferation of K562 cells were decreased by knockdown of CAT1. In addition, the initial uptake rate of [(3)H]arginine was decreased in the shRNA-transfected cell clones. The ratio of differentiation of CAT1-knockdown K562 cells was well correlated with the uptake activity for arginine by the cells (R(2)=0.59). These findings indicate that CAT1 is directly involved in erythropoiesis through supplying arginine to the blood cells.

Qualitative and quantitative assessment of video transmitted by DVTS (digital video transport system) in surgical telemedicine.

Real-time video pictures can be transmitted inexpensively via a broadband connection using the DVTS (digital video transport system). However, the degradation of video pictures transmitted by DVTS has not been sufficiently evaluated. We examined the application of DVTS to remote consultation by using images of laparoscopic and endoscopic surgeries. A subjective assessment by the double stimulus continuous quality scale (DSCQS) method of the transmitted video pictures was carried out by eight doctors. Three of the four video recordings were assessed as being transmitted with no degradation in quality. None of the doctors noticed any degradation in the images due to encryption by the VPN (virtual private network) system. We also used an automatic picture quality assessment system to make an objective assessment of the same images. The objective DSCQS values were similar to the subjective ones. We conclude that although the quality of video pictures transmitted by the DVTS was slightly reduced, they were useful for clinical purposes. Encryption with a VPN did not degrade image quality.

Role of polyamines derived from arginine in differentiation and proliferation of human blood cells.

L-Arginine is a precursor of polyamine, nitric oxide (NO), creatine, and agmatine and is essential for the differentiation and proliferation of blood cells, although the precise biological role of L-arginine is unclear. We have recently reported that the depletion of L-arginine in cultured medium prevented both proliferation and differentiation of blood cells (Shima et al., Blood First Edition Paper, October 6, 2005; DOI 10.1182). Since one of metabolic products of L-arginine in the cells is polyamine that associates with cell differentiation and proliferation, the effects of L-arginine on the human K562 cell line and human cord blood-derived CD34 positive cells were investigated by focusing on polyamines such as putrescine, spermidine, and spermine in the present study. When polyamines were added to the culture medium in the absence of L-arginine, the cells did not grow or differentiate well. However, when intracellular polyamines were depleted using ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), the proliferation and differentiation of K562 cells to erythrocytes were reduced even in the presence of L-arginine. Moreover, in the presence of DFMO, cell differentiation and proliferation were recovered by the addition of putrescine or spermidine in the presence of L-arginine. Accordingly, it was demonstrated that polyamines are essential for the proliferation and differentiation of the blood cells as the metabolites of L-arginine and the externally added polyamines are also effective by being taken up through polyamine transporter.

L-arginine import via cationic amino acid transporter CAT1 is essential for both differentiation and proliferation of erythrocytes.

In the present study, we examined the role in hematopoiesis of cationic amino acid transporter 1 (CAT1), which transports L-arginine, L-lysine, L-ornithine, and L-histidine. The expression level of human CAT1 (hCAT1) mRNA in mononuclear cells (MNCs) fractionated according to lineage-selective markers was examined by reverse transcriptase-polymerase chain reaction. The expression of CAT1 in glycophorin A-positive erythroid cells was 8 times higher than in nonfractionated MNC (control) cells. Characteristics of L-arginine uptake by K562 cells, an established leukemic cell line used as an erythroid model, were similar to those of CAT1 in regards to saturation kinetics, sodium independence, and substantial inhibition of L-arginine uptake by N-ethylmaleimide, which is a specific inhibitor of system y+ amino acid transporter. Removal of L-arginine from the culture medium prevented both proliferation and differentiation of K562 cells, while removal of L-lysine or L-histidine had little effect on differentiation, though proliferation was blocked. Hematopoietic stem cells obtained from human cord blood failed to develop into erythroid cells in the absence of L-arginine in the culture medium. These findings indicate that hCAT1 is involved in erythroid hematopoiesis through its role in importing L-arginine, which appears to be essential for the differentiation of red blood cells.

Rational design, synthesis, and structure-activity relationships of novel factor Xa inhibitors: (2-substituted-4-amidinophenyl)pyruvic and -propionic acids.

An inhibitor of factor Xa (fXa), the m-substituted benzamidine AXC1578 (1a), was structurally modified with the aim of increasing its potency. In particular, pyruvic acid and propionic acid substituents were incorporated into the P1 benzamidine moiety to introduce a favorable interaction with the oxy-anion hole in the catalytic triad region of fXa. This strategy was based on computational docking studies using the extracted active site of fXa. The validity of the computational model was supported by the acquisition of X-ray crystal structures of the 1a-trypsin and 3b-trypsin complexes (the homology around the active sites of fXa and trypsin is high). The above modifications significantly increased the inhibitory activity toward fXa, whereas the high selectivity for fXa versus thrombin was maintained or enhanced. Compounds 3b, 3c, 3e, and 4b are considered to be potential lead compounds for the development of orally active anticoagulant drugs because they demonstrated potent activity when administered orally to cynomolgus monkeys.