[Partnership Activity for Neglected Tropical Diseases].

Delivering new medicines to patients suffering from Neglected Tropical Diseases (NTD) is a major challenge. There are various hurdles to be overcome, such as the large number of patients in a large number of different regions, the lack of marketability, and resistance to medicines. Takeda Pharmaceutical Company Limited (Takeda) is following a corporate mission of "striving towards better health for patients worldwide though leading innovation in medicine". These guiding principles lead to the values of Integrity, Fairness, Honesty and Perseverance that make up what we call "Takeda-ism". As part of its contribution to R&D for NTDs, Takeda collaborates with global Product Development Partnerships (PDPs). In this symposium, the "Drug Discovery Booster" project to accelerate and expand discovery of new drugs for Leishmaniasis and Chagas disease with Drugs for Neglected Diseases initiative (DNDi) and other pharmaceutical companies is introduced. Proprietary compound libraries and the drug discovery expertise of various partners was applied to this new drug discovery approach. An overview of our research projects in malaria, tuberculosis, and NTD is also presented. In addition to these, Takeda's Access to Medicines (ATM) strategy and activities are introduced. Lastly, we discuss a new open innovation model which is accelerated by partnership with a variety of organizations and how Takeda achieves its sustainable development goal (SDG) targets.

Cell factory for γ-aminobutyric acid (GABA) production using Bifidobacterium adolescentis.

BACKGROUND: Bifidobacteria are gram-positive, probiotic, and generally regarded as safe bacteria. Techniques such as transformation, gene knockout, and heterologous gene expression have been established for Bifidobacterium, indicating that this bacterium can be used as a cell factory platform. However, there are limited previous reports in this field, likely because of factors such as the highly anaerobic nature of this bacterium. Bifidobacterium adolescentis is among the most oxygen-sensitive Bifidobacterium species. It shows strain-specific gamma-aminobutyric acid (GABA) production. GABA is a potent bioactive compound with numerous physiological and psychological functions. In this study, we investigated whether B. adolesentis could be used for mass production of GABA. RESULTS: The B. adolescentis 4-2 strain isolated from a healthy adult human produced approximately 14 mM GABA. It carried gadB and gadC, which encode glutamate decarboxylase and glutamate GABA antiporter, respectively. We constructed pKKT427::Pori-gadBC and pKKT427::Pgap-gadBC plasmids carrying gadBC driven by the original gadB (ori) and gap promoters, respectively. Recombinants of Bifidobacterium were then constructed. Two recombinants with high production abilities, monitored by two different promoters, were investigated. GABA production was improved by adjusting the fermentation parameters, including the substrate concentration, initial culture pH, and co-factor supplementation, using response surface methodology. The optimum initial cultivation pH varied when the promoter region was changed. The ori promoter was induced under acidic conditions (pH 5.2:4.4), whereas the constitutive gap promoter showed enhanced GABA production at pH 6.0. Fed-batch fermentation was used to validate the optimum fermentation parameters, in which approximately 415 mM GABA was produced. The conversion ratio of glutamate to GABA was 92-100%. CONCLUSION: We report high GABA production in recombinant B. adolescentis. This study provides a foundation for using Bifidobacterium as a cell factory platform for industrial production of GABA.

Bifidobacterium-Escherichia coli Shuttle Vector Series pKO403, with Temperature-Sensitive Replication Origin for Gene Knockout in Bifidobacterium.

A series of Bifidobacterium-Escherichia coli shuttle vectors (pKO403-lacZ'-Cm, pKO403-lacZ'-Sp, pKO403-lacZ'-p15A) were constructed based on the pKO403 backbone, which carries a temperature-sensitive replication origin. These vectors carry the lacZ'α fragment, overhung by two facing type IIS restriction sites, for blue-white selection and seamless gene cloning. These vectors are useful for gene knockout or multigene integration into the chromosome of Bifidobacterium.

Construction of a reporter system for bifidobacteria using chloramphenicol acetyltransferase and its application for evaluation of promoters and terminators.

A reporter assay system is an essential tool for investigating gene expression mechanisms. In the case of bifidobacteria, several convenient and sensitive reporter systems have been developed. Here, we developed a new reporter system for bifidobacteria using the chloramphenicol acetyltransferase gene (cat) from Staphylococcus aureus. This enzyme stoichiometrically produced free CoA-SH, which was analyzed quantitatively with Ellman's test using 2-nitrobenzoic acid (DTNB). The 2-nitro-5-thiobenzoate (TNB2-) produced showed a strong yellowish color with maximum absorbance at 412 nm. We also constructed a new pBCMAT plasmid series for CAT assays in bifidobacteria to evaluate promoters and terminators. Analyses using promoters from Bifidobacterium longum NCC2705 indicated that the CAT assay using these promoters is quantitative, has a wide measurement range, and is stable. In addition, this assay was useful for several bifidobacterial species, including B. longum, Bifidobacterium breve, and Bifidobacterium adolescentis. Compared with evoglow-Bs2, a fluorescent protein used under anaerobic conditions, the CAT assay showed about 0.25% background activity. In analyses using this CAT assay, we identified 11 promoters and 12 terminators of B. longum NCC2705. The genes encoding ribosomal proteins, elongation factors, and transfer RNAs possessed strong promoters, and terminators that include strong stem-loops and poly-U tails structures tended to show high activities. Although the abovementioned promoters made stronger contributions to expression activities than the terminators, the maximum fold difference in the activities among the tested terminators was approximately 17-fold. Modification of the -10 box and 5'-UTR in the promoters and the structure around the stem-loop in the terminators affected expression levels. These results suggest that the CAT assay is useful for various analyses of bifidobacterial gene expression.

Differences in the Concentration of the Fecal Neurotransmitters GABA and Glutamate Are Associated with Microbial Composition among Healthy Human Subjects.

Recent studies have shown that the gut microbiota modulates the physical and psychological functions of the host through several modes of action. One of them is mediating the production of active neurotransmitters, such as serotonin and gamma-aminobutyric acid (GABA). GABA is the major inhibitory neurotransmitter in the central nervous system. Here, we analyzed the relationship between fecal GABA concentration and microbial composition in more than 70 human participants. The gut microbiome composition was analyzed using next-generation sequencing based on 16S ribosomal RNA. High-performance liquid chromatography was used to evaluate the neurotransmitters GABA and glutamate. The GABA level was detected in a broad range (0-330 µg/g feces). The participants' samples were classified into high (>100 µg/g), medium (10-100 µg/g), and low (<10 µg/g) groups, based on fecal GABA concentration. The results reveal that the microbiome of the high-GABA samples had lower alpha diversity than the other samples. Beta diversity analysis showed significant (p < 0.05) separation between the high-GABA samples and others. Furthermore, we surveyed the abundance of specific GABA producer biomarkers among the microbiomes of tested samples. The family Bifidobacteriaceae exhibited high abundance in the microbiome of the high-GABA group. This study demonstrated that Bifidobacterium abundance was associated with high fecal GABA content in healthy human subjects. These results may aid the development of potential probiotics to improve microbial GABA production, which can support the maintenance of the physical and psychiatric health of the host.

Structure of a Core Promoter in Bifidobacterium longum NCC2705.

Bacterial promoters consist of core sequence motifs termed -35 and -10 boxes. The consensus motifs are TTGACA and TATAAT, respectively, which were identified from leading investigations on Escherichia coli However, the consensus sequences are not likely to fit genetically divergent bacteria. The sigma factor of the genus Bifidobacterium has a characteristic polar domain in the N terminus, suggesting the possibility of specific promoter recognition. We reevaluated the structure of Bifidobacterium longum NCC2705 promoters and compared them to other bacteria. Transcriptional start sites (TSSs) of the B. longum NCC2705 strain were identified using transcriptome sequencing (RNA-Seq) analysis to extract promoter regions. Conserved motifs of a bifidobacterial promoter were determined using regions upstream of TSSs and a hidden Markov model. As a result, consensus motifs of the -35 and -10 boxes were TTGTGC and TACAAT, respectively. To assess each base of both motifs, we constructed 37 plasmids based on pKO403-TPCTcon, including the hup promoter connected with a chloramphenicol acetyltransferase as a reporter gene. This reporter assay showed two optimal motifs of the -35 and -10 boxes, namely, TTGNNN and TANNNT, respectively. We further analyzed spacer lengths between the -35 and -10 boxes via a bioinformatics approach. The spacer lengths predominant in bacteria have been generally reported to be approximately 17 bp. In contrast, the predominant spacer lengths in the genus Bifidobacterium and related species were 11 bp, in addition to 17 bp. A reporter assay to assess the spacer lengths indicated that the 11-bp spacer length produced unusually high activity.IMPORTANCE The structures of sigma factors vary among bacterial strains, indicating that recognition rules may also vary. Therefore, we investigated the promoter structure of Bifidobacterium longum NCC2705 using a bioinformatics approach and wet analyses. The most frequent and optimal motifs were similar to other bacterial consensus motifs. The optimal spacer length between the two boxes was reported to be 17 bp. It is widely applied to a bioinformatics approach for other bacteria. Unexpectedly, conserved spacer lengths were 11 bp as well as 17 bp in the genus Bifidobacterium Moreover, the sigma factor of the genus Bifidobacterium has a characteristic domain in the N terminus which may contribute to the additional functions. Hence, it would be valuable to reevaluate the promoter in other organisms.

A New Escherichia coli Entry Vector Series (pIIS18) for Seamless Gene Cloning Using Type IIS Restriction Enzymes.

A series of new Escherichia coli entry vectors (pIIS18-SapI, pIIS18-BsmBI, pIIS18-BsaI, pIIS18-BfuAI-1, and pIIS18-BfuAI-2) was constructed based on a modified pUC18 backbone, which carried newly designed multiple cloning sites, consisting of two facing type IIS enzyme cleavage sites and one blunt-end enzyme cleavage site. These vectors are useful for seamless gene cloning.

O2-inducible H2O2-forming NADPH oxidase is responsible for the hyper O2 sensitivity of Bifidobacterium longum subsp. infantis.

Bifidobacteria are beneficial anaerobes, and their O2 sensitivity levels differ among species as a function of unknown molecular mechanisms. Bifidobacterium longum subspecies infantis (B. infantis), a predominant colonizer of the gastrointestinal tract of infants, showed a hyper O2-sensitive growth profile with accompanying a production of H2O2. In this study, we characterized an NADPH oxidase as a key enzyme responsible for this microbe's hyper O2 sensitivity. A dominant active elution peak of H2O2-forming NADPH oxidase activity was detected in the first step of column chromatography, and the purified NADPH oxidase (NPOX) was identified as a homolog of nitroreductase family proteins. The introduction of the gene encoding B. infantis NPOX (npoxA) into O2-tolerant Bifidobacterium minimum made the strain O2 sensitive and allowed it to produce H2O2. Knockout of the npoxA gene in B. infantis decreased the production of H2O2 and mitigated its B. infantis hyper O2 sensitivity. A transcript of B. infantis npoxA is induced by O2, suggesting that the aerobic production of toxic H2O2 is functionally conserved in B. infantis.

Discovery of a Novel Series of Pyrazolo[1,5-a]pyrimidine-Based Phosphodiesterase 2A Inhibitors Structurally Different from N-((1S)-1-(3-Fluoro-4-(trifluoromethoxy)phenyl)-2-methoxyethyl)-7-methoxy-2-oxo-2,3-dihydropyrido[2,3-b]pyrazine-4(1H)-carboxamide (TAK-915), for the Treatment of Cognitive Disorders.

It has been hypothesized that selective inhibition of phosphodiesterase (PDE) 2A could potentially be a novel approach to treat cognitive impairment in neuropsychiatric and neurodegenerative disorders through augmentation of cyclic nucleotide signaling pathways in brain regions associated with learning and memory. Following our earlier work, this article describes a drug design strategy for a new series of lead compounds structurally distinct from our clinical candidate 2 (TAK-915), and subsequent medicinal chemistry efforts to optimize potency, selectivity over other PDE families, and other preclinical properties including in vitro phototoxicity and in vivo rat plasma clearance. These efforts resulted in the discovery of N-((1S)-2-hydroxy-2-methyl-1-(4-(trifluoromethoxy)phenyl)propyl)-6-methyl-5-(3-methyl-1H-1,2,4-triazol-1-yl)pyrazolo[1,5-a]pyrimidine-3-carboxamide (20), which robustly increased 3',5'-cyclic guanosine monophosphate (cGMP) levels in the rat brain following an oral dose, and moreover, attenuated MK-801-induced episodic memory deficits in a passive avoidance task in rats. These data provide further support to the potential therapeutic utility of PDE2A inhibitors in enhancing cognitive performance.

Discovery of Clinical Candidate N-((1S)-1-(3-Fluoro-4-(trifluoromethoxy)phenyl)-2-methoxyethyl)-7-methoxy-2-oxo-2,3-dihydropyrido[2,3-b]pyrazine-4(1H)-carboxamide (TAK-915): A Highly Potent, Selective, and Brain-Penetrating Phosphodiesterase 2A Inhibitor for the Treatment of Cognitive Disorders.

Phosphodiesterase (PDE) 2A inhibitors have emerged as a novel mechanism with potential therapeutic option to ameliorate cognitive dysfunction in schizophrenia or Alzheimer's disease through upregulation of cyclic nucleotides in the brain and thereby achieve potentiation of cyclic nucleotide signaling pathways. This article details the expedited optimization of our recently disclosed pyrazolo[1,5-a]pyrimidine lead compound 4b, leading to the discovery of clinical candidate 36 (TAK-915), which demonstrates an appropriate combination of potency, PDE selectivity, and favorable pharmacokinetic (PK) properties, including brain penetration. Successful identification of 36 was realized through application of structure-based drug design (SBDD) to further improve potency and PDE selectivity, coupled with prospective design focused on physicochemical properties to deliver brain penetration. Oral administration of 36 demonstrated significant elevation of 3',5'-cyclic guanosine monophosphate (cGMP) levels in mouse brains and improved cognitive performance in a novel object recognition task in rats. Consequently, compound 36 was advanced into human clinical trials.

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages.

BACKGROUND: Bifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial-host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant. RESULTS: A putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages. CONCLUSIONS: Our results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.

Design, synthesis, and pharmacological evaluation of 4-azolyl-benzamide derivatives as novel GPR52 agonists.

G protein-coupled receptor 52 (GPR52) agonists are expected to improve the symptoms of psychiatric disorders. During exploration for a novel class of GPR52 agonists with good pharmacokinetic profiles, we synthesized 4-(3-(3-fluoro-5-(trifluoromethyl)benzyl)-5-methyl-1H-1,2,4-triazol-1-yl)-2-methylbenzamide (4u; half maximal effective concentration (EC50)=75nM, maximal response (Emax)=122%) starting from a high-throughput screening hit 3 (EC50=470nM, Emax=56%). The structural features of a reported GPR52 agonist were applied to 3, led to design 4-azolylbenzamides as novel GPR52 agonists. A structure-activity relationship study of 4-azolylbenzamide resulted in the design of the 1,2,4-triazole derivative 4u, which demonstrated excellent bioavailability in rats (F=53.8%). Oral administration of 4u (10mg/kg) significantly suppressed methamphetamine-induced hyperlocomotion in mice. Thus, 4u is a promising lead compound for drug discovery research of GPR52 agonists.

Intraneuronally injected amyloid ß inhibits long-term potentiation in rat hippocampal slices.

Extracellular accumulation of amyloid beta (Aß) is a hallmark of Alzheimer's disease (AD). It has been reported that extracellular perfusion of Aß inhibits long-term potentiation (LTP), which is strongly related to memory in animal models. However, it has recently been proposed that intracellular Aß may be the first pathological change to occur in AD. Here, we have investigated the effect on LTP of intracellular injection of Aß (Aß(1-40), Aß(1-42)) into hippocampal pyramidal cells using patch-clamp technique. We found that injection of 1 nM Aß(1-42) completely blocked LTP, and extracellular perfusion of a p38 MAPK inhibitor or a metabotropic glutamate receptor blocker reversed these blocking effects on LTP. Furthermore, we have examined the effects of different concentrations of Aß(1-40) and Aß(1-42) on LTP and showed that Aß(1-40) required a 1,000-fold higher concentration to attenuate LTP than 1 nM Aß(1-42). These results indicate that LTP is impaired by Aß injected into genetically wild-type neurons in the sliced hippocampus, suggesting an acute action of intracellular Aß on the intracellular LTP-inducing machinery.

Complete genomic sequence of the O-desmethylangolensin-producing bacterium Clostridium rRNA cluster XIVa strain SY8519, isolated from adult human intestine.

The O-desmethylangolensin-producing Clostridium rRNA cluster XIVa strain SY8519 was isolated from the intestinal flora of a healthy human as a key isoflavonoid-metabolizing bacterium. Here, we report the finished and annotated genomic sequence of this organism.

Complete genomic sequence of the equol-producing bacterium Eggerthella sp. strain YY7918, isolated from adult human intestine.

Eggerthella sp. strain YY7918 was isolated from the intestinal flora of a healthy human. It metabolizes daidzein (a soybean isoflavonoid) and produces S-equol, which has stronger estrogenic activities than daidzein. Here, we report the finished and annotated genomic sequence of this organism.

Mechanism of impairment of long-term potentiation by amyloid beta is independent of NMDA receptors or voltage-dependent calcium channels in hippocampal CA1 pyramidal neurons.

beta-Amyloid peptide (Abeta) is known to be involved in Alzheimer's disease (AD). Although the fibril form of Abeta is known to have neurotoxicity, it has been shown that not only the fibril form but also the oligomer form of Abeta may be related to the neuropathophysiology of AD, specifically to memory loss. Some studies have demonstrated that low concentrations of the Abeta oligomer impair long-term potentiation (LTP), a cellular model for learning and memory, after short exposure times in vivo and in vitro, although little is known about the mechanism involved in Abeta-mediated inhibition of LTP. In this study, we used the patch clamp whole-cell technique in rat hippocampal CA1 pyramidal neurons to study more precisely how the Abeta oligomer affects synaptic plasticity. The brief perfusion of slices with a low concentration (1microM) of Abeta(1-42) significantly impaired LTP induction of the excitatory input. The same concentration of Abeta did not affect basal transmission or paired-pulse facilitation. We also demonstrated that neither NMDAR-EPSCs nor the voltage-depended calcium channel (VDCC) currents were affected by the same concentration of Abeta(1-42) as used in the LTP experiments. These data suggest that Abeta mediated impairment of LTP induction is independent of NMDARs or VDCCs.

Studies on the total synthesis of streptazolin and its related natural products: first total synthesis of (+/-)-8alpha-hydroxystreptazolone.

The intramolecular Pauson-Khand reaction of 2-oxazolone derivatives with a suitable heptynyl appendage gave exclusively the corresponding 4-hydroxy-6-substituted-9-oxa-1-azatricyclo[6.2.1.0(5,11)]undec-5-ene-7,10-diones. On the basis of this newly developed Pauson-Khand reaction of 2-oxazolone-alkyne derivatives, the first total synthesis of (+/-)-8alpha-hydroxystreptazolone was accomplished in a highly stereoselective manner. In addition, (+/-)-7-epi-8alpha-hydroxystreptazolone was also synthesized.

Immunogenecity of synthetic peptides representing linear B-cell epitopes of VapA of Rhodococcus equi.

Amino acid 65-78 of membrane protein VapA of the facultative intracellular Rhodococcus equi contained an immunodominant N-terminal B-cell epitope (N15Y peptide). Safety and immunogenecity of a synthetic peptide consisting of the amino acid 65-78 of VapA (peptide N15Y) were evaluated first in mice and in healthy adult horses. A single dose of a peptide-VapA vaccine induced and only in presence of adjuvant, specific IgG antibodies in sera of mice. After challenge with virulent R. equi 3 weeks after immunization, tissue clearance was more delayed in immunized mice than in control mice. An antibody-mediated response (restricted to IgG1 and IgG2b subclasses) predominated in vaccinated mice sera and no specific lymphocytes proliferation was observed. Next, a total of 15 mares were given systemic inoculation of N15Y peptide with IMS3012 ( n = 4 ) or IMS2211 ( n = 4 ) or ISA35 ( n = 4 ) or placebo ( n = 2). Serological responses to the peptide vaccine were found in all but not in placebo group. A significant increase of IgGb subclass in sera of vaccinated mare with N15Y peptide in presence of IMS3012 was observed in comparison to IMS2211 or ISA35 or control group. Moreover, INF-gamma, IL-2 and IL-10 mRNA expression increased more significantly in peripheral blood lymphocytes of IMS3012 group than in IMS2211 or ISA35 group. Interestingly, a significant decrease of IL-4 mRNA expression (undetectable level) was observed with all adjuvants. These results support the use of peptide N15Y in presence of IMS3012 adjuvant in future studies of protection of foals against R. equi.

Efficient construction of the bicyclo[5.3.0]decenone skeleton based on the Rh(I)-catalyzed allenic Pauson-Khand reaction.

A reliable procedure for constructing a bicyclo[5.3.0]deca-1,7-dien-9-one ring system by rhodium-catalyzed Pauson-Khand reaction (PKR) of allenynes with a sulfonyl group has been developed. Investigation of the rhodium-catalyzed PKR on 19 examples of 1,2-nonadien-8-yne derivatives demonstrated that (i) acceptable yields could be consistently achieved through the proper choice of the rhodium catalyst ([RhCl(CO)(2)](2) or [RhCl(CO)dppp](2)) depending on the starting allenyne and that (ii) an ester functionality as well as hydroxy and siloxy groups could be tolerated in this rhodium-catalyzed PKR.