ASP5736, a novel 5-HT5A receptor antagonist, ameliorates positive symptoms and cognitive impairment in animal models of schizophrenia.

We recently identified ASP5736, (N-(diaminomethylene)-1-(3,5-difluoropyridin-4-yl)-4-fluoroisoquinoline-7-carboxamide (2E)-but-2-enedioate), a novel antagonist of 5-HT5A receptor, and here describe the in vitro and in vivo characterization of this compound. ASP5736 exhibited a high affinity for the human 5-HT5A receptor (Ki = 3.6 ± 0.66 nM) and antagonized 5-carboxamidotryptamine (5-CT)-induced Ca(2+) influx in human cells stably expressing the 5-HT5A receptor with approximately 200-fold selectivity over other receptors, including other 5-HT receptor subtypes, enzymes, and channels except human 5-HT2c receptor (Ki = 286.8 nM) and 5-HT7 receptor (Ki = 122.9 nM). Further, ASP5736 dose-dependently antagonized the 5-CT-induced decrease in cAMP levels in HEK293 cells stably expressing the 5-HT5A receptor. We then evaluated the effects of ASP5736 on cognitive impairments in several animal models of schizophrenia. Working memory deficit in MK-801-treated mice and visual learning deficit in neonatally phencyclidine (PCP)-treated mice were both ameliorated by ASP5736. In addition, ASP5736 also attenuated MK-801- and methamphetamine (MAP)-induced hyperactivity in mice without causing sedation, catalepsy, or plasma prolactin increase. The addition of olanzapine did not affect ASP5736-induced cognitive enhancement, and neither the sedative nor cataleptogenic effects of olanzapine were worsened by ASP5736. These results collectively suggest that ASP5736 is a novel and potent 5-HT5A receptor antagonist that not only ameliorates positive-like symptoms but also cognitive impairments in animal models of schizophrenia, without adverse effects. Present studies also indicate that ASP5736 holds potential to satisfy currently unmet medical needs for the treatment of schizophrenia by either mono-therapy or co-administered with commercially available antipsychotics.

Pharmacological characterization of the novel γ-secretase modulator AS2715348, a potential therapy for Alzheimer's disease, in rodents and nonhuman primates.

γ-Secretase is the enzyme responsible for the intramembranous proteolysis of various substrates, such as amyloid precursor protein (APP) and Notch. Amyloid-ß peptide 42 (Aß42) is produced through the sequential proteolytic cleavage of APP by ß- and γ-secretase and causes the synaptic dysfunction associated with memory impairment in Alzheimer's disease. Here, we identified a novel cyclohexylamine-derived γ-secretase modulator, {(1R*,2S*,3R*)-3-[(cyclohexylmethyl)(3,3-dimethylbutyl)amino]-2-[4-(trifluoromethyl)phenyl]cyclohexyl}acetic acid (AS2715348), that may inhibit this pathological response. AS2715348 was seen to reduce both cell-free and cellular production of Aß42 without increasing levels of APP ß-carboxyl terminal fragment or inhibiting Notch signaling. Additionally, the compound increased Aß38 production, suggesting a shift of the cleavage site in APP. The inhibitory potency of AS2715348 on endogenous Aß42 production was similar across human, mouse, and rat cells. Oral administration with AS2715348 at 1 mg/kg and greater significantly reduced brain Aß42 levels in rats, and no Notch-related toxicity was observed after 28-day treatment at 100 mg/kg. Further, AS2715348 significantly ameliorated cognitive deficits in APP-transgenic Tg2576 mice. Finally, AS2715348 significantly reduced brain Aß42 levels in cynomolgus monkeys. These findings collectively show the promise for AS2715348 as a potential disease-modifying drug for Alzheimer's disease.

Role of the C-terminal cytoplasmic domain of FlhA in bacterial flagellar type III protein export.

For construction of the bacterial flagellum, many of the flagellar proteins are exported into the central channel of the flagellar structure by the flagellar type III protein export apparatus. FlhA and FlhB, which are integral membrane proteins of the export apparatus, form a docking platform for the soluble components of the export apparatus, FliH, FliI, and FliJ. The C-terminal cytoplasmic domain of FlhA (FlhA(C)) is required for protein export, but it is not clear how it works. Here, we analyzed a temperature-sensitive Salmonella enterica mutant, the flhA(G368C) mutant, which has a mutation in the sequence encoding FlhA(C). The G368C mutation did not eliminate the interactions with FliH, FliI, FliJ, and the C-terminal cytoplasmic domain of FlhB, suggesting that the mutation blocks the export process after the FliH-FliI-FliJ-export substrate complex binds to the FlhA-FlhB platform. Limited proteolysis showed that FlhA(C) consists of at least three subdomains, a flexible linker, FlhA(CN), and FlhA(CC), and that FlhA(CN) becomes sensitive to proteolysis by the G368C mutation. Intragenic suppressor mutations were identified in these subdomains and restored flagellar protein export to a considerable degree. However, none of these suppressor mutations suppressed the protease sensitivity. We suggest that FlhA(C) not only forms part of the docking platform for the FliH-FliI-FliJ-export substrate complex but also is directly involved in the translocation of the export substrate into the central channel of the growing flagellar structure.

Mouse hepatoblasts at distinct developmental stages are characterized by expression of EpCAM and DLK1: drastic change of EpCAM expression during liver development.

Hepatoblasts are hepatic progenitor cells that expand and give rise to either hepatocyte or cholangiocytes during liver development. We previously reported that delta-like 1 homolog (DLK1) is expressed in the mouse liver primordium at embryonic day (E) 10.5 and that DLK1(+) cells in E14.5 liver contain high proliferative and bipotential hepatoblasts. While the expression of epithelial cell adhesion molecule (EpCAM) in hepatic stem/progenitor cells has been reported, its expression profile at an early stage of liver development remains unknown. In this study, we show that EpCAM is expressed in mouse liver bud at E9.5 and that EpCAM(+)DLK1(+) hepatoblasts form hepatic cords at the early stage of hepatogenesis. DLK1(+) cells of E11.5 liver were fractionated into EpCAM(+) and EpCAM(-) cells; one forth of EpCAM(+)DLK1(+) cells formed a colony in vitro whereas EpCAM(-)DLK1(+) cells rarely did it. Moreover, EpCAM(+)DLK1(+) cells contained cells capable of forming a large colony, indicating that EpCAM(+)DLK1(+) cells in E11.5 liver contain early hepatoblasts with high proliferation potential. Interestingly, EpCAM expression in hepatoblasts was dramatically reduced along with liver development and the colony-forming capacities of both EpCAM(+)DLK1(+) and EpCAM(-)DLK1(+) cells were comparable in E14.5 liver. It strongly suggested that most of mouse hepatoblasts are losing EpCAM expression at this stage. Moreover, we provide evidence that EpCAM(+)DLK1(+) cells in E11.5 liver contain extrahepatic bile duct cells as well as hepatoblasts, while EpCAM(-)DLK1(+) cells contain mesothelial cell precursors. Thus, the expression of EpCAM and DLK1 suggests the developmental pathways of mouse liver progenitors.

Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver.

Hepatic oval cells are considered to be facultative hepatic stem cells (HSCs) that differentiate into hepatocytes and cholangiocytes in severely injured liver. Hepatic oval cells have also been implicated in tumorigenesis. However, their nature and origin remain elusive. To isolate and characterize mouse oval cells, we searched for cell surface molecules expressed on oval cells and analyzed their nature at the single-cell level by flow cytometric analysis and in the in vitro colony formation assay. We demonstrate that epithelial cell adhesion molecule (EpCAM) is expressed in both mouse normal cholangiocytes and oval cells, whereas its related protein, TROP2, is expressed exclusively in oval cells, establishing TROP2 as a novel marker to distinguish oval cells from normal cholangiocytes. EpCAM(+) cells isolated from injured liver proliferate to form colonies in vitro, and the clonally expanded cells differentiate into hepatocytes and cholangiocytes, suggesting that the oval cell fraction contains potential HSCs. Interestingly, such cells with HSC characteristics exist among EpCAM(+) cells of normal liver. Intriguingly, comparison of the colony formation of EpCAM(+) cells in normal and injured liver reveals little difference in the number of potential HSCs, strongly suggesting that most proliferating mouse oval cells represent transit-amplifying cells rather than HSCs.

Inducible expression of Wnt genes during adult hepatic stem/progenitor cell response.

In injured livers where hepatocyte growth is severely limited, facultative hepatic stem/progenitor cells, termed oval cells in rodents, are known to emerge and contribute to the regeneration process. Here, we investigated a possible involvement of Wnt signaling during mouse oval cell response and found significant upregulation of several Wnt genes including Wnt7a, Wnt7b, and Wnt10a. Accordingly, increase of beta-catenin protein was observed in oval cell compartments. Pharmacological activation of the canonical Wnt/beta-catenin signaling induced proliferation of cultured hepatic stem/progenitor cell lines. These results together implicate the role of Wnt/beta-catenin signaling in adult hepatic stem/progenitor cell response.

Role of the N-terminal domain of FliI ATPase in bacterial flagellar protein export.

FliI, the ATPase involved in bacterial flagellar protein export, forms a complex with its regulator FliH in the cytoplasm and hexamerizes upon docking to the export gate composed of integral membrane proteins. The extreme N-terminal region of FliI is involved not only in its interaction with FliH but also in its oligomerization, but the regulatory mechanism of oligomerization remains unclear. Using in-frame 10-residue deletions within the 100 residues of the N-terminal domain, we demonstrate that the first 20 residues are required for FliH binding and that the conformation of the N-terminal domain is sensitive to the export function, even though the oligomerization and FliH-binding ability are retained and the ATPase activity is maintained in most of the deletion variants.

Roles of the intramolecular disulfide bridge in MotX and MotY, the specific proteins for sodium-driven motors in Vibrio spp.

The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na+-driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.

Divergent evolutions of trinucleotide polymerization revealed by an archaeal CCA-adding enzyme structure.

CCA-adding enzyme [ATP(CTP):tRNA nucleotidyltransferase], a template-independent RNA polymerase, adds the defined 'cytidine-cytidine-adenosine' sequence onto the 3' end of tRNA. The archaeal CCA-adding enzyme (class I) and eubacterial/eukaryotic CCA-adding enzyme (class II) show little amino acid sequence homology, but catalyze the same reaction in a defined fashion. Here, we present the crystal structures of the class I archaeal CCA-adding enzyme from Archaeoglobus fulgidus, and its complexes with CTP and ATP at 2.0, 2.0 and 2.7 A resolutions, respectively. The geometry of the catalytic carboxylates and the relative positions of CTP and ATP to a single catalytic site are well conserved in both classes of CCA-adding enzymes, whereas the overall architectures, except for the catalytic core, of the class I and class II CCA-adding enzymes are fundamentally different. Furthermore, the recognition mechanisms of substrate nucleotides and tRNA molecules are distinct between these two classes, suggesting that the catalytic domains of class I and class II enzymes share a common origin, and distinct substrate recognition domains have been appended to form the two presently divergent classes.