Cancer is the most common cause of death in Japan. Fundamental and clinical studies on cancer were conducted from the viewpoint of Western medicine so far. However, a sustained complete remission has not been achieved yet. In order to alleviate the side effects of anticancer drugs, some traditional herbal medicines (Kampo medicines) have been prescribed to cancer patients. We have been studying on antitumor substances in medicinal herbs and found an antitumor medicinal herb named Rhus verniciflua (lacquer, Urushi in Japanese). To investigate the antitumor effect in vitro, a plant extract mixture was prepared from six medicinal herbs containing lacquer. The plant extract mixture containing lacquer (Rv-PEM) inhibited the proliferation of several mouse and human tumor cell lines. Rv-PEM had more potent inhibitory effect on the proliferation of human leukemia cell lines (MOLT-3, KG-1) than on other tumor cell lines. The IC50 values of Rv-PEM on MOLT-3 and KG-1 cells were 0.208 and 0.293 mg/mL, respectively. After treating Rv-PEM to the tumor cells, DNA fragmentation and Caspase-3 and -9 activity increased in the treated cells. The mechanisms of the inhibitory proliferation activity of Rv-PEM would involve apoptosis of human leukemia cells (MOLT-3, KG-1, K-562) by the mitochondrial pathway.
Docosahexaenoic acid (DHA), a predominant of n-3 polyunsaturated fatty acids (n-3 PUFA), has numerous beneficial physiological effects, including neuroprotection and cardiovascular protection. Recently, a possible involvement of n-3 PUFA in pain control has gathered considerable attention because numerous studies have reported a regulatory role of n-3 PUFAs. However, the mechanisms underlying how DHA exerts antinociceptive effect remain unknown. Here, we performed elucidation of mechanisms underlying DHA-induced antinociception. DHA administration dose-dependently exerted an antinociceptive effect. This effect was abolished by pretreated with the β-funaltrexamine (β-FNA), a μ-opioid receptor antagonist, and the nartrindole (NTI), a δ-opioid receptor antagonist, but not by the nor-binaltorphimine (nor-BNI), a κ-opioid receptor antagonist. In the radioligand binding assay, DHA itself did not have the affinity for μ-, δ- and κ- opioid receptor. Furthermore, the pretreatment of anti β-endorphin antiserum inhibited DHA-induced antinociception. The plasma levels of β-endorphin increased 30 min after DHA administration. The β-endorphin immunoreactivity in the brain increased at 30 min after DHA treatment. Expression of GPR40 protein was widely observed in the brain as well as the spinal cord. The intracerebroventricular but not intrathecal injection of DHA and GW9508, a GPR40/GPR120 agonist, significantly reduced formalin-induced pain behavior. The β-endorphin immunoreactivity in the brain increased at 10 and 20 min after intracerebroventricular injection of DHA and GW9508. These findings suggest that DHA-induced antinociception via β-endorphin release may be mediated through GPR40 signaling in the supraspinal area.
Schizophrenia is characterized by various behavioral abnormalities including cognitive dysfunction. Neonatal ventral hippocampus (NVH)-lesioned rats had been known as neurodevelopmental animal model similar to schizophrenia. Previous observations indicate that postpubertal NVH-lesioned rats exhibit impairments in prepulse inhibition (PPI), spontaneous locomotion, social interaction behavior and working memory. Here, we document the neurochemical basis of those defects in NVH-lesioned rats. Since Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is NMDA receptor downstream kinase, is essential for memory and learning acquisition, we developed a protocol to monitor the spatial changes in CaMKII autophosphorylation using immunohistochemical imaging of whole brain slices with anti-autophosphorylated CaMKII antibody in order to address mechanisms underlying impaired cognitive function in NVH-lesioned rats. Immunohistochemical analyses using anti-autophosphorylated CaMKII antibody revealed that CaMKII autophosphorylation was significantly reduced in the medial prefrontal cortex (mPFC) of NVH-lesioned rats compared with control animals. This immunohistochemical technique is useful to investigate temporal and special changes in CaMKII activity in rodent brain and to evaluate drugs to improve the cognitive impairment.
GM1-gangliosidosis and Morquio B are rare lysosomal storage diseases associated with a neurodegenerative disorder or dwarfism and skeletal abnormalities, respectively. These diseases are caused by deficiencies in the lysosomal enzyme human β-D-galactosidase (h-β-GAL), which lead to accumulations of the h-β-GAL substrates, GM1 ganglioside and keratan sulfate due to mutations in the h-β-GAL gene. H-β-GAL is an exoglycosidase that catalyzes the hydrolysis of terminal β-linked galactose residues. Here, we present the crystal structures of h-β-GAL in complex with its catalytic product galactose or with its inhibitor 1-deoxygalactonojirimycin. H-β-GAL showed a novel homodimer structure; each monomer was comprised of a catalytic TIM barrel domain followed by β-domain 1 and β-domain 2. The long loop region connecting the TIM barrel domain with β-domain 1 was responsible for the dimerization. To gain structural insight into the molecular defects of h-β-GAL in the above diseases, the disease-causing mutations were mapped onto the three-dimensional structure. Finally, the possible causes of the diseases are discussed.
Polyglutamine tract-binding protein 1 (PQBP1) is a nuclear protein that regulates transcription and pre-mRNA splicing. In addition, the mutations in the PQBP1 gene are known to cause hereditary mental retardation. This review summarizes current knowledge about the solution structure of PQBP1. PQBP1 is an intrinsically disordered protein: its polar-rich domain and C-terminal domain are disordered under physiological conditions. PQBP1 binds to its target molecule U5-15kD via a continuous 23-residue segment of the C-terminal domain. The function of PQBP1 in the pre-mRNA splicing is also discussed.
The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of this malaria parasite resistant to conventional drug therapy has stimulated the search for antimalarial compounds with novel modes of action. Here the structure-function relationship studies for two Plasmodium proteins are presented. One example is the structural studies for S-adenosyl-L-homocysteine hydrolase from Plasmodium falciparum (PfSAHH) and the other example is those for 1-deoxy-D-xylulose reductoisomerase from Plasmodium falciparum (PfDXR). In the former study, the clue for design of species specific PfSAHH inhibitors was obtained by the structural comparison of the active site of PfSAHH with that of human SAHH (HsSAHH). Our study revealed that the inhibitor selectivity depends on the difference of only one amino acid residue in the active site; Cys59 in PfSAHH vs. Thr60 in HsSAHH. In the latter study, the inhibition of PfDXR enzyme by fosmidomycin has proved to be efficient in the treatment of uncomplicated malaria in recent clinical trials conducted in Gabon and Thailand. Our crystal structure analyses of PfDXR/inhibitor complexes revealed the molecular basis of fosmidomycin's action in P. falciparum. We expect that the structure-function relationship studies on Plasmodium proteins are useful for developing the more effective antimalarial compounds.
G protein-coupled receptors (GPCRs) are major therapeutic drug targets and represent more than 30% of the market share of all prescription drugs. The high-resolution three-dimensional structures of the target receptors provide good initial models for structure-based approaches to drug screening and drug design, which are considered to accelerate drug discovery. However, significant bottlenecks at the expression, purification and crystallization stages of structure determination of GPCRs have existed. Here, we review recent techniques for the determination of GPCR structures. In particular, we focus on the protein engineering techniques that have been used to overcome bottlenecks in expression/purification and crystallization, including our development of a platform using budding yeast for the rapid construction and evaluation of GPCR variants for structural studies. We also present our success in determining the crystal structure of the histamine H1 receptor (H1R) in complex with doxepin, an inverse agonist antihistamine. The H1R structure revealed the low selectivity of doxepin to aminergic receptors and provides key information that should aid the development of highly selective antihistamines.
The genus Morbillivirus includes measles virus, canine distemper virus and rinderpest virus. These are highly contagious and exhibit high mortality. These viruses have the attachment glycoprotein, hemagglutinin (H), at the virus surface, which bind to signaling lymphocyte activation molecule (SLAM) and Nectin 4 as receptors for the entry. However, the molecular mechanism for this entry has been limitedly understood. Here we summarize the current topics, (1) newly identified receptor, Nectin 4, (2) crystal structures of H-receptor complexes and (3) detail biochemical studies of the H-F communication for the entry. These provide insight on the mechanism of morbillivirus entry event and furthermore drug developments.
Cardiolipin (CL) is a phospholipid, which is exclusively located in mitochondria, and has a unique structure that consists of 2 phosphate residues and 4 kinds of fatty acyl chains. Cardiolipin plays an important role in regulating various kinds of mitochondrial proteins such as electron transport complexes, carrier proteins and phosphate kinases, and is also essential for the organization of particular mitochondrial structures such as cristae and contact sites. Mitochondrial phospholipase D hydrolyzes CL to produce phosphatidic acid, which is required for mitochondrial fusion. Oxidative stress-induced peroxidation of CL occurs because CL is rich in polyunsaturated fatty acids, especially linoleic acid. Accumulation of CL hydroperoxide (CLOOH) triggers the initiation of apoptosis. Formation of CLOOH causes the release of proapoptotic factors such as cytochrome c from the inner mitochondrial membrane and triggers opening of the permeability transition pore. Levels of CL decrease in the heart following ischemia or disease. Apoptosis is enhanced in temperature-dependent mutant cells whose amounts of CL reduce to half when compared to that of wild type cells. Low levels of CL cause the accumulation of CLOOH and enhance sensitivity to apoptosis. Accumulation of CLOOH in mitochondria causes instability of the membrane, because swelling of mitochondria is induced by the presence of CLOOH in the membrane and is significantly enhanced in CLOOH-loaded mitochondria by the addition of inducer of swelling.
We are studying the medicinal synthetic chemistry of biomolecular component mimics such as carbohydrates, nucleosides, amino acids, and peptides. In this review, the synthesis and biological activities of iminosugars as carbohydrate mimics are discussed. Glycosidases and glycosyltransferases are involved in a wide range of anabolic and catabolic process, including digestion, the lysosomal catabolism of glycoconjugates, glycoprotein biosynthesis. Hence, modifying or blocking these processes in vivo using inhibitors is a topic of great interest from the therapeutic point of view. Iminosugars are sugars in which the endocyclic oxygen is replaced by a basic nitrogen atom. They are regarded as transition state mimics in certain types of enzyme reactions. This makes the field of iminosugars as carbohydrate mimics an exciting area of research. We synthesized all of the stereoisomers of polyhydroxypiperidines such as fagomine, 1-deoxynojirimycine, and isofagomine. In addition, their both enantiomers, as substrates for a variety of glycosidases were evaluated. Secondly, the asymmetric synthesis of α-1-C-alkyl-arabinoiminofuranoses was achieved by asymmetric allylic alkylation, RCM, and Negishi cross coupling as key reactions. Surprisingly, the L-forms showed a quite potent inhibitory activity toward rat intestinal maltase, while the activities of the D-forms were much weaker. Some of the prepared L-forms showed potent inhibitory activities towards intestinal maltase, with IC50 values comparable to those of commercial drugs such as acarbose, voglibose, and miglitol, which are used in the treatment of type 2 diabetes. Among them, the inhibitory activity towards intestinal sucrase of α-1-C-L-butylarabinoiminofuranose was quite strong towards intestinal sucrase compared to the above commercial drugs.
Japanese patients with normal renal function were retrospectively analyzed to characterize increases in serum creatinine (SCr) observed following the use of a sulfamethoxazole-trimethoprim (SMX-TMP) combination product and identify factors affecting these increases. In the patients studied (n=49), an individual comparison was conducted for the three factors of age group [≤74 years (n=21) vs. ≥75 years (n=28)], sex [male (n=24) vs. female (n=25)], and total dose throughout the treatment period [≤7 g (n=24) vs. ≥8 g (n=25)] to determine the extent of SCr increase following SMX-TMP combination product use. SCr increased significantly following SMX-TMP combination product use in patients ≤74 years of age and ≥75 years of age, in both males and females, and in patients with a total dose of ≥8 g (8 to 96 g) (p<0.05). Multivariate logistic regression analysis was used to determine the independence of these factors. Total dose was identified as an independent factor and had an odds ratio of 6.571 [95% confidence interval=1.735-24.882, p=0.006]. Post-treatment percent increases in SCr were compared using pre-treatment levels as the baseline. The group with a total dose of ≥8 g (mean 29.8 g) had a significant SCr increase of 18.4% (p=0.002), while the increase in the ≤7 g (mean 5.3 g) group was only 4.5%. The data showed that SCr increased by about 20% when the total dose taken over the treatment period was around 30 g (about 2.4 g as TMP) and indicated that total dose contributes more than age and sex to the post-treatment increase in SCr.