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Wastewater surveillance can offer a comprehensive grasp of infectious disease prevalence and human health because wastewater contains various human-derived microbial pathogens, including viruses, bacteria, and fungi. However, methods capable of simultaneous detection of multiple groups of targets in the automated systems and large-scale surveillance are still under development. Here, we demonstrated the modification, involving the addition of bead-beating, to the existing COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater) enabled enhanced detection of various microorganisms, including SARS-CoV-2. The modified method, termed bead-beating COPMAN (BB-COPMAN), was evaluated through spike-and-recovery experiments and comparative analysis against three previously reported methods for simultaneous DNA/RNA detection. Our study targeted a range of microorganisms, including enveloped and non-enveloped RNA viruses (SARS-CoV-2, PMMoV), a DNA virus (crAssphage), archaea, gram-negative and gram-positive bacteria (E. coli, Lachnospiraceae), antibiotic resistance gene (ampC), and fungi (Candida albicans). The recovery rates of BB-COPMAN for gram-negative and gram-positive bacteria were 17 and 2.1-fold higher, respectively, compared to the method for DNA/RNA detection. Additionally, BB-COPMAN exhibited the highest extraction efficiency among the tested methods, achieving 1.2-5.7 times more DNA and 1.1-69 times more RNA yield on average. BB-COPMAN allowed the detection of SARS-CoV-2 from all nine samples and PMMoV at concentrations 39-97 times higher than other methods. Moreover, BB-COPMAN detected larger amounts of DNA for four out of six DNA targets than the previously reported DNA/RNA detection method. These results demonstrated that BB-COPMAN enables enhanced detection of multiple targets in a single flow of nucleic acid extraction, making the method well-suited for automated systems. In conclusion, BB-COPMAN is a promising method in wastewater surveillance for assessing the prevalence of wide range of pathogenic microorganisms., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuka Adachi Katayama, Shin Hayase, Ryo Iwamoto, Tomohiro Kuroita, Tomohiko Okuda, and Yusaku Masago reports a relationship with Shionogi and Co Ltd. that includes: employment. Ryo Iwamoto and Tomohiro Kuroita reports a relationship with AdvanSentinel Inc. that includes: employment. Masaaki Kitajima reports a relationship with Shionogi and Co Ltd. and AdvanSentinel Inc. that includes: funding grants. Masaaki Kitajima, Ryo Iwamoto, Yusaku Masago, Shin Hayase, and Yuka Adachi Katayama has patent #METHOD FOR DETECTING AND QUANTIFYING NUCLEIC ACID FROM ENVIRONMENTAL SAMPLES (WO/2022/181739) pending to Shionogi and Co Ltd., and Hokkaido University., (Copyright © 2023. Published by Elsevier B.V.)

Wastewater-based epidemiology (WBE) is a practical approach for detecting the presence of SARS-CoV-2 infections and assessing the epidemic trend of the coronavirus disease 2019 (COVID-19). The purpose of this study was to evaluate the minimum sampling frequency required to properly identify the COVID-19 trend during the downward epidemic period when using a highly sensitive RNA detection method. WBE was conducted using the Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids (EPISENS-S), a highly sensitive SARS-CoV-2 RNA detection method, at nine neighboring wastewater treatment plants (WWTPs). These WWTPs were in the same prefecture in Japan, and they had different sewer types, sampling methods, and sampling frequencies. The overall detection rate of SARS-CoV-2 RNA was 97.8 % during the entire study period when the geometric means of new COVID-19 cases per 100,000 inhabitants were between 3.3 and 7.7 in each WWTP. The maximum SARS-CoV-2 RNA concentration in wastewater was 2.14 × 10 4 copies/L, which corresponded to pepper mild mottle virus (PMMoV)-normalized concentrations of 6.54 × 10 -3 . We evaluated the effect of sampling frequencies on the probability of a significant correlation with the number of newly reported COVID-19 cases by hypothetically reducing the sampling frequency in the same dataset. When the wastewater sampling frequency occurred 5, 3, 2, and 1 times per week, these results exhibited significant correlations of 100 % (5/5), 89 % (8/9), 85 % (23/27), and 48 % (13/27), respectively. To achieve significant correlation with a high probability of over 85 %, a minimum sampling frequency of twice per week is required, even if sampling methods and sewer types are different. WBE using the EPISENS-S method and a sampling frequency of more than twice a week can be used to properly monitor COVID-19 wave epidemic trends, even during downward periods., Competing Interests: Declaration of competing interest Tomohiro Kuroita, Akimasa Yoshimura, and Ryo Iwamoto are employees of Shionogi & Co., Ltd. Masaaki Kitajima received research funding and patent royalties from Shionogi & Co., Ltd. Satoshi Okabe received research funding from Shionogi & Co., Ltd. Hiroki Ando have no competing interests to declare., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Wastewater-based epidemiology (WBE) is a promising approach for monitoring the spread of SARS-CoV-2 within communities. Although qPCR-based WBE is powerful in that it allows quick and highly sensitive detection of this virus, it can provide limited information about which variants are responsible for the overall increase or decrease of this virus in sewage, and this hinders accurate risk assessments. To resolve this problem, we developed a next generation sequencing (NGS)-based method to determine the identity and composition of individual SARS-CoV-2 variants in wastewater samples. Combination and optimization of targeted amplicon-sequencing and nested PCR allowed detection of each variant with sensitivity comparable to that of qPCR. In addition, by targeting the receptor binding domain (RBD) of the S protein, which has mutations informative for variant classification, we could discriminate most variants of concern (VOC) and even sublineages of Omicron (BA.1, BA.2, BA.4/5, BA.2.75, BQ.1.1 and XBB.1). Focusing on a limited domain has a benefit of decreasing the sequencing reads. We applied this method to wastewater samples collected from a wastewater treatment plant in Kyoto city throughout 13 months (from January 2021 to February 2022) and successfully identified lineages of wild-type, alpha, delta, omicron BA.1 and BA.2 as well as their compositions in the samples. The transition of these variants was in good agreement with the epidemic situation reported in Kyoto city during that period based on clinical testing. These data indicate that our NGS-based method is useful for detecting and tracking emerging variants of SARS-CoV-2 in sewage samples. Coupled with the advantages of WBE, this method has the potential to serve as an efficient and low cost means for the community risk assessment of SARS-CoV-2 infection., Competing Interests: Declaration of competing interest All authors (Miho Kuroiwa, Yoshinari Gahara, Hirohito Kato, Yuji Morikawa, Yuki Matsui, Takumi Adachi, Shin Kurosawa, Tomohiro Kuroita, Yoshinori Ando, and Masatomo Rokushima) report a relationship with Shionogi & Co., Ltd. that includes: employment., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Rapid and accurate diagnosis of individuals with SARS-CoV-2 infection is an effective way to prevent and control the spread of COVID-19. Although the detection of SARS-CoV-2 viral RNA by RT-qPCR is the gold standard for COVID-19 testing, the use of antigen-detecting rapid diagnostic tests (Ag-RDTs) is emerging as a complementary surveillance tool as Omicron case numbers skyrocket worldwide. However, the results from Ag-RDTs are less accurate in individuals with low viral loads., Results: To develop a highly sensitive and accurate Ag-RDT, 90 monoclonal antibodies were raised from guinea pigs immunized with SARS CoV-2 nucleocapsid protein (CoV-2-NP). By applying a capture antibody recognizing the structural epitope of the N-terminal domain of CoV-2-NP and a detection antibody recognizing the C-terminal tail of CoV-2-NP to an automated chemiluminescence flow-through membrane immunoassay device, we developed a novel Ag-RDT, CoV-2-POCube. The CoV-2-POCube exclusively recognizes CoV-2-NP variants but not the nucleocapsid proteins of other human coronaviruses. The CoV-2-POCube achieved a limit of detection sensitivity of 0.20 ~ 0.66 pg/mL of CoV-2-NPs, demonstrating more than 100 times greater sensitivity than commercially available SARS-CoV-2 Ag-RDTs., Conclusions: CoV-2-POCube has high analytical sensitivity and can detect SARS-CoV-2 variants in 15 min without observing the high-dose hook effect, thus meeting the need for early SARS-CoV-2 diagnosis with lower viral load. CoV-2-POCube is a promising alternative to currently available diagnostic devices for faster clinical decision making in individuals with suspected COVID-19 in resource-limited settings., (© 2023. The Author(s).)

Wastewater-based epidemiology (WBE) is a promising tool to efficiently monitor COVID-19 prevalence in a community. For WBE community surveillance, automation of the viral RNA detection process is ideal. In the present study, we achieved near full-automation of a previously established method, COPMAN (COagulation and Proteolysis method using MAgnetic beads for detection of Nucleic acids in wastewater), which was then applied to detect SARS-CoV-2 in wastewater for half a year. The automation line employed the Maholo LabDroid and an automated-pipetting device to achieve a high-throughput sample-processing capability of 576 samples per week. SARS-CoV-2 RNA was quantified with the automated COPMAN using samples collected from two wastewater treatment plants in the Sagami River basin in Japan between 1 November 2021 and 24 May 2022, when the numbers of daily reported COVID-19 cases ranged from 0 to 130.3 per 100,000 inhabitants. The automated COPMAN detected SARS-CoV-2 RNA from 81 out of 132 samples at concentrations of up to 2.8 × 10 5  copies/L. These concentrations showed direct correlations with subsequently reported clinical cases (5-13 days later), as determined by Pearson's and Spearman's cross-correlation analyses. To compare the results, we also conducted testing with the EPISENS-S (Efficient and Practical virus Identification System with ENhanced Sensitivity for Solids, Ando et al., 2022), a previously reported detection method. SARS-CoV-2 RNA detected with EPISENS-S correlated with clinical cases only when using Spearman's method. Our automated COPMAN was shown to be an efficient method for timely and large-scale monitoring of viral RNA, making WBE more feasible for community surveillance., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Masaaki Kitajima reports financial support was provided by Shionogi and Co Ltd. Masaaki Kitajima reports was provided by AdvanSentinel Inc. Shin Hayase reports a relationship with Shionogi and Co Ltd. that includes: employment. Ryo Iwamoto reports a relationship with AdvanSentinel Inc. that includes: employment. Tomohiro Kuroita reports a relationship with AdvanSentinel Inc. that includes: employment. Yuka Adachi Katayama, reports a relationship with Shionogi and Co Ltd. that includes: employment. Yoshinori Ando reports a relationship with Shionogi and Co Ltd. that includes: employment. Tomohiko Okuda reports a relationship with Shionogi and Co Ltd. that includes: employment. Yusaku Masago reports a relationship with Shionogi and Co Ltd. that includes: employment. Shin Hayase has patent pending to Nucleic acid detection and quantification methods from environmental samples pending to Shionogi and Co Ltd., Hokkaido University. Yuka Adachi Katayama has patent pending to Nucleic acid detection and quantification methods from environmental samples pending to Shionogi and Co Ltd., Hokkaido University. Yusaku Masago has patent pending to Nucleic acid detection and quantification methods from environmental samples pending to Shionogi and Co Ltd., Hokkaido University., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater-based epidemiology (WBE) attracted attention as an objective and comprehensive indicator of community infection that does not require individual inspection. Although several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection methods from wastewater have been developed, there are obstacles to their social implementation. In this study, we developed the COPMAN (Coagulation and Proteolysis method using Magnetic beads for detection of Nucleic acids in wastewater), an automatable method that can concentrate and detect multiple types of viruses from a limited volume (∼10 mL) of wastewater. The COPMAN consists of a high basicity polyaluminum chloride (PAC) coagulation process, magnetic bead-based RNA purification, and RT-preamplification, followed by qPCR. A series of enzymes exhibiting a high tolerance to PCR inhibitors derived from wastewater was identified and employed in the molecular detection steps in the COPMAN. We compared the detectability of viral RNA from 10-mL samples of virus-spiked (heat-inactivated SARS-CoV-2 and intact RSV) or unspiked wastewater by the COPMAN and other methods (PEG-qPCR, UF-qPCR, and EPISENS-S). The COPMAN was the most efficient for detecting spiked viruses from wastewater, detecting the highest level of pepper mild mottle virus (PMMoV), a typical intrinsic virus in human stool, from wastewater samples. The COPMAN also successfully detected indigenous SARS-CoV-2 RNA from 12 samples of wastewater at concentrations of 2.2 × 10 4 to 5.4 × 10 5 copies/L, during initial stages of an infection wave in the right and the left bank of the Sagami River in Japan (0.65 to 11.45 daily reported cases per 100,000 people). These results indicate that the COPMAN is suitable for detection of multiple pathogens from small volume of wastewater in automated stations., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Masaaki Kitajima reports financial support was provided by Shionogi & Co Ltd. Yuka Adachi Katayama reports a relationship with Shionogi & Co Ltd. that includes: employment. Shin Hayase reports a relationship with Shionogi & Co Ltd. that includes: employment. Yoshinori Ando reports a relationship with Shionogi & Co Ltd. that includes: employment. Tomohiro Kuroita reports a relationship with Shionogi & Co Ltd. that includes: employment. Kazuya Okada reports a relationship with Shionogi & Co Ltd. that includes: employment. Ryo Iwamoto reports a relationship with Shionogi & Co Ltd. that includes: employment. Toru Yanagimoto reports a relationship with Shionogi & Co Ltd. that includes: employment. Yusaku Masago reports a relationship with Shionogi & Co Ltd. that includes: employment. Yuka Adachi Katayama (Yuka Katayama) has patent Nucleic acid detection & quantification methods from environmental samples pending to Shionogi & Co Ltd., Hokkaido university. Shin Hayase has patent Nucleic acid detection & quantification methods from environmental samples pending to Shionogi & Co Ltd., Hokkaido university. Ryo Iwamoto has patent Nucleic acid detection and quantification methods from environmental samples pending to Shionogi & Co Ltd., Hokkaido University. Masaaki Kitajima has patent Nucleic acid detection and quantification methods from environmental samples pending to Shionogi & Co Ltd., Hokkaido University. Yusaku Masago has patent Nucleic acid detection and quantification methods from environmental samples pending to Shionogi & Co Ltd., Hokkaido university. Judy Noguchi had proofreaded this manuscript in English for a fee. Judy Noguchi works in Kobe Gakuin University., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Pellagra is caused by abnormal intake and/or use of nicotinic acid and is known in part to be induced by the use of medications such as isoniazid or pirfenidone. We previously investigated atypical phenotypes of pellagra, such as nausea, using a mouse model of pellagra and found that gut microbiota play an important role in the development of these phenotypes. Here, we investigated the effect of Bifidobacterium longum BB536 on pellagra-related nausea caused by pirfenidone in our mouse model. Our pharmacological data indicated that pirfenidone (PFD) causes modulation of the gut microbiota profile, which appeared to play an important role in the development of pellagra-related nausea. A gut microbiota-mediated protective effect of B. longum BB536 against nausea caused by PFD was also identified. Finally, the urinary ratio of nicotinamide/N-methylnicotinamide was shown to be a biomarker of pellagra-like adverse effects induced by PFD, and it may contribute to the prevention of these effects in patients with idiopathic pulmonary fibrosis., Competing Interests: Tomohiro Kuroita, Natsumi Susai, and Takeshi Yoshioka are employees of Shionogi & Co., Ltd. Other authors declare no competing interests., (©2023 BMFH Press.)

Background: Pirfenidone (PFD) is widely used in patients with idiopathic pulmonary fibrosis (IPF) and its adverse effects, such as nausea and photosensitivity, are well known. Many patients with IPF have reduced doses or even cessation of PFD because of its side-effects. No solutions have been found for these side-effects because the current mechanistic insights are insufficient., Methods: Using the results of real-world data analysis from the US Food and Drug Administration Adverse Events Reporting System, we hypothesised that PFD-related symptoms may be similar to pellagra. Reverse translational experiments using female BALB/c mice were performed to validate and estimate this hypothesis. Niacin and its metabolite responses were compared between patients with IPF treated with PFD and those treated without PFD., Results: The pellagra hypothesis was translated from real-world data analysis. Pharmacological and comprehensive genetic investigations showed that PFD caused pellagra-related nausea and photosensitivity in a mouse model, which may have been mediated by the actions of nicotinamide N- methyltransferase (NNMT). Higher NNMT substrate responses were observed in urine from patients and mice with PFD than in those without PFD., Conclusions: PFD may cause pellagra or pellagra-like symptoms such as photosensitivity. Further studies are required to investigate whether niacin prevents pellagra-like symptoms caused by PFD in patients with IPF., Competing Interests: Conflict of interest: T. Kuroita, N. Susai and T. Yoshioka are employees of Shionogi & Co., Ltd. The other authors declare no competing interests., (Copyright ©The authors 2022.)

Cholesterol 24-hydroxylase (CH24H, CYP46A1) is a cytochrome P450 family enzyme that maintains the homeostasis of brain cholesterol. Soticlestat, a potent and selective CH24H inhibitor, is in development as a therapeutic agent for Dravet syndrome and Lennox-Gastaut syndrome. Herein, we report the discovery of aryl-piperidine derivatives as potent and selective CH24H positron emission tomography (PET) tracers which can be used for dose guidance of a clinical CH24H inhibitor and as a diagnostic tool for CH24H-related pathology. Starting from compound 1 (IC 50  = 16 nM, logD = 1.7), which was reported as a CH24H inhibitor with lower lipophilicity, a 18 F-labeling site (3-fluoroazetidine) was incorporated by structure-based drug design (SBDD) utilizing the co-crystal structure of a compound 1 analog. Subsequent optimization to adjust key parameters for PET tracers, such as potency, lipophilicity, brain penetration, and unbound plasma protein binding, enabled compounds 3f (IC 50  = 8.8 nM) and 3g (IC 50  = 8.7 nM) as PET imaging candidates. Selectivity of these compounds for CH24H was validated by a brain distribution study using CH24H-WT and KO mice. In non-human primate PET imaging, [ 18 F]3f and [ 18 F]3g showed similar regional uptake in the brain, indicating that these tracers were specific to the CH24H-expressed regions and validated the expression of CH24H in the living brain by different tracers., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Dysregulation of nicotinamide adenine dinucleotide (NAD +) metabolism contributes to the initiation and progression of age-associated diseases, including chronic kidney disease (CKD). Nicotinamide N-methyltransferase (NNMT), a nicotinamide (NAM) metabolizing enzyme, regulates both NAD + and methionine metabolism. Although NNMT is expressed abundantly in the kidney, its role in CKD and renal fibrosis remains unclear. We generated NNMT-deficient mice and a unilateral ureter obstruction (UUO) model and conducted two clinical studies on human CKD to investigate the role of NNMT in CKD and fibrosis. In UUO, renal NNMT expression and the degraded metabolites of NAM increased, while NAD + and NAD + precursors decreased. NNMT deficiency ameliorated renal fibrosis; mechanistically, it (1) increased the DNA methylation of connective tissue growth factor (CTGF), and (2) improved renal inflammation by increasing renal NAD + and Sirt1 and decreasing NF-κB acetylation. In humans, along with CKD progression, a trend toward a decrease in serum NAD + precursors was observed, while the final NAD + metabolites were accumulated, and the level of eGFR was an independent variable for serum NAM. In addition, NNMT was highly expressed in fibrotic areas of human kidney tissues. In conclusion, increased renal NNMT expression induces NAD + and methionine metabolism perturbation and contributes to renal fibrosis., (© 2022. The Author(s).)

Niacin deficiency causes pellagra, the symptoms of which include dermatitis, diarrhoea and dementia. Investigating the mechanism underlying these phenotypes has been challenging due to the lack of an appropriate animal model. Here, we report a mouse model of pellagra-related nausea induced by feeding mice a low-niacin diet and administering isoniazid (INH), which is thought to induce pellagra. Mice fed a normal or low-niacin diet received INH (0·3 or 1·0 mg/mg per animal, twice daily, 5 d), and nausea was evaluated based on pica behaviour, which considered the rodent equivalent of the emetic reflex. Furthermore, the effect of therapeutic niacin administration on nausea was evaluated in this model. Urinary and hepatic metabolite levels were analysed by LC coupled with MS. INH-induced pica was observed in mice fed a low-niacin diet but not in those fed a normal diet. Levels of urinary metabolites, such as 1-methyl-2-pyridone-5-carboxamide, kynurenic acid and xanthurenic acid, were significantly reduced in the mice treated with INH compared with those that did not receive INH. Furthermore, niacin supplementation prevented pica and restored the levels of some metabolites in this mouse model. Our findings suggest that INH-related nausea is pellagra-like. We also believe that our newly established method for quantifying pica is a useful tool for investigating the mechanisms of pellagra-related nausea.

Purpose: Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that plays a major role in brain cholesterol homeostasis by converting cholesterol into 24S-hydroxycholesterol. The selective CH24H inhibitor soticlestat (TAK-935) is being pursued as a drug for treatment of seizures in developmental and epileptic encephalopathies. Herein, we describe the successful discovery and the preclinical validation of the novel radiolabeled CH24H ligand (3-[ 18 F]fluoroazetidin-1-yl){1-[4-(4-fluorophenyl)pyrimidin-5-yl]piperidin-4-yl}methanone ([ 18 F]T-008) and its tritiated analog, [ 3 H]T-008., Methods: In vitro autoradiography (ARG) studies in the CH24H wild-type (WT) and knockout (KO) mouse brain sections were conducted using [ 3 H]T-008. PET imaging was conducted in two adult rhesus macaques using [ 18 F]T-008. Each macaque received two test-retest baseline scans and a series of two blocking doses of soticlestat administered prior to [ 18 F]T-008 to determine the CH24H enzyme occupancy. PET data were analyzed with Logan graphical analysis using plasma input. A Lassen plot was applied to estimate CH24H enzyme occupancy by soticlestat., Results: In ARG studies, binding of [ 3 H]T-008 was specific to CH24H in the mouse brain sections, which was not observed in CH24H KO or in wild-type mice after pretreatment with soticlestat. In rhesus PET studies, the rank order of [ 18 F]T-008 uptake was striatum > cortical regions > cerebellum, which was consistent with CH24H distribution in the brain. Pre-blocking with soticlestat reduced the maximum uptake and increased the washout in all brain regions in a dose-dependent manner. Calculated global occupancy values for soticlestat at a dose of 0.89 mg/kg were 97-98%, indicating maximum occupancy., Conclusion: The preclinical in vitro and in vivo evaluation of labeled T-008 demonstrates that [ 18 F]T-008 is suitable for imaging CH24H in the brain and warrants further studies in humans., (© 2021. The Author(s).)

Pellagra is caused by an abnormal intake and/or use of niacin, but its phenotypes are diverse. The phenotypes of pellagra can also be atypical, such as nausea. We previously reported a mouse model of pellagra-related nausea. However, the mechanism of this model is unclear. In this study, we found that the gut microbiota, which is thought to be a source of niacin, played an important role in the development of pellagra-related nausea in germ-free mice. We also investigated the gut microbiome. We compared urinary niacin metabolite levels and the dermal response between mice fed a normal diet and those fed a low-niacin diet to investigate the putative trigger of pellagra. Epoxyeicosatrienoic and hydroxyeicosatetraenoic acid levels were higher in mice fed a low-niacin diet compared with those fed a normal diet. Furthermore, histological studies indicated a dermatological response to the low-niacin diet. Interestingly, higher levels of oxidised fatty acids in response to the germ-free state were also observed. These findings indicate successful establishment of our newly established mouse model of pellagra via the gut microbiota. We believe that this model could enable the discovery of the putative cause of pellagra and phenotypes of pellagra that have not been recognised yet., (©2022 BMFH Press.)

Cholesterol 24-hydroxylase (CH24H, CYP46A1), a brain-specific cytochrome P450 (CYP) family enzyme, plays a role in the homeostasis of brain cholesterol by converting cholesterol to 24 S -hydroxycholesterol (24HC). Despite a wide range of potential of CH24H as a drug target, no potent and selective inhibitors have been identified. Here, we report on the structure-based drug design (SBDD) of novel 4-arylpyridine derivatives based on the X-ray co-crystal structure of hit derivative 1b . Optimization of 4-arylpyridine derivatives led us to identify 3v ((4-benzyl-4-hydroxypiperidin-1-yl)(2,4'-bipyridin-3-yl)methanone, IC 50 = 7.4 nM) as a highly potent, selective, and brain-penetrant CH24H inhibitor. Following oral administration to mice, 3v resulted in a dose-dependent reduction of 24HC levels in the brain (1, 3, and 10 mg/kg). Compound 3v (soticlestat, also known as TAK-935) is currently under clinical investigation for the treatment of Dravet syndrome and Lennox-Gastaut syndrome as a novel drug class for epilepsies.

Tuberculosis remains a leading cause of death from a single bacterial infection worldwide. Efforts to develop new treatment options call for expansion into an unexplored target space to expand the drug pipeline and bypass resistance to current antibiotics. Lipoamide dehydrogenase is a metabolic and antioxidant enzyme critical for mycobacterial growth and survival in mice. Sulfonamide analogs were previously identified as potent and selective inhibitors of mycobacterial lipoamide dehydrogenase in vitro but lacked activity against whole mycobacteria. Here we present the development of analogs with improved permeability, potency, and selectivity, which inhibit the growth of Mycobacterium tuberculosis in axenic culture on carbohydrates and within mouse primary macrophages. They increase intrabacterial pyruvate levels, supporting their on-target activity within mycobacteria. Distinct modalities of binding between the mycobacterial and human enzymes contribute to improved potency and hence selectivity through induced-fit tight binding interactions within the mycobacterial but not human enzyme, as indicated by kinetic analysis and crystallography.

Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24S-hydroxycholesterol, the primary mechanism of cholesterol catabolism in the brain. The therapeutic potential of CH24H activation has been extensively investigated, whereas the effects of CH24H inhibition remain poorly characterized. In this study, the therapeutic potential of CH24H inhibition was investigated using a newly identified small molecule, soticlestat (TAK-935/OV935). The biodistribution and target engagement of soticlestat was assessed in mice. CH24H-knockout mice showed a substantially lower level of soticlestat distribution in the brain than wild-type controls. Furthermore, brain-slice autoradiography studies demonstrated the absence of [ 3 H]soticlestat staining in CH24H-knockout mice compared with wild-type mice, indicating a specificity of soticlestat binding to CH24H. The pharmacodynamic effects of soticlestat were characterized in a transgenic mouse model carrying mutated human amyloid precursor protein and presenilin 1 (APP/PS1-Tg). These mice, with excitatory/inhibitory imbalance and short life-span, yielded a remarkable survival benefit when bred with CH24H-knockout animals. Soticlestat lowered brain 24S-hydroxycholesterol in a dose-dependent manner and substantially reduced premature deaths of APP/PS1-Tg mice at a dose lowering brain 24S-hydroxycholesterol by approximately 50%. Furthermore, microdialysis experiments showed that soticlestat can suppress potassium-evoked extracellular glutamate elevations in the hippocampus. Taken together, these data suggest that soticlestat-mediated inhibition of CH24H may have therapeutic potential for diseases associated with neural hyperexcitation.

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The action of the aspartyl protease renin is the rate-limiting initial step of the renin-angiotensin-aldosterone system. Therefore, renin is a particularly promising target for blood pressure as well as onset and progression of cardiovascular and renal diseases. New pyrimidine derivatives 5-14 were designed in an attempt to enhance the renin inhibitory activity of compound 3 identified by our previous fragment-based drug design approach. Introduction of a basic amine essential for interaction with the two aspartic acids in the catalytic site and optimization of the S1/S3 binding elements including an induced-fit structural change of Leu114 ('Leu-in' to 'Leu-out') by a rational structure-based drug design approach led to the discovery of N-(piperidin-3-yl)pyrimidine-5-carboxamide 14, a 65,000-fold more potent renin inhibitor than compound 3. Surprisingly, this remarkable enhancement in the inhibitory activity of compound 14 has been achieved by the overall addition of only seven heavy atoms to compound 3. Compound 14 demonstrated excellent selectivity over other aspartyl proteases and moderate oral bioavailability in rats., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A novel approach was conducted for fragment-based lead discovery and applied to renin inhibitors. The biochemical screening of a fragment library against renin provided the hit fragment which showed a characteristic interaction pattern with the target protein. The hit fragment bound only to the S1, S3, and S3 SP (S3 subpocket) sites without any interactions with the catalytic aspartate residues (Asp32 and Asp215 (pepsin numbering)). Prior to making chemical modifications to the hit fragment, we first identified its essential binding sites by utilizing the hit fragment's substructures. Second, we created a new and smaller scaffold, which better occupied the identified essential S3 and S3 SP sites, by utilizing library synthesis with high-throughput chemistry. We then revisited the S1 site and efficiently explored a good building block attaching to the scaffold with library synthesis. In the library syntheses, the binding modes of each pivotal compound were determined and confirmed by X-ray crystallography and the library was strategically designed by structure-based computational approach not only to obtain a more active compound but also to obtain informative Structure Activity Relationship (SAR). As a result, we obtained a lead compound offering synthetic accessibility as well as the improved in vitro ADMET profiles. The fragments and compounds possessing a characteristic interaction pattern provided new structural insights into renin's active site and the potential to create a new generation of renin inhibitors. In addition, we demonstrated our FBDD strategy integrating highly sensitive biochemical assay, X-ray crystallography, and high-throughput synthesis and in silico library design aimed at fragment morphing at the initial stage was effective to elucidate a pocket profile and a promising lead compound., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The aspartic proteinase renin is an attractive target for the treatment of hypertension and cardiovascular/renal disease such as chronic kidney disease and heart failure. We introduced an S1' site binder into the lead compound 1 guided by structure-based drug design (SBDD), and further optimization of physicochemical properties led to the discovery of benzimidazole derivative 10 (1-(4-methoxybutyl)- N -(2-methylpropyl)- N -[(3 S ,5 R )-5-(morpholin-4-yl)carbonylpiperidin-3-yl]-1 H- benzimidazole-2-carboxamide hydrochloride, TAK-272) as a highly potent and orally active renin inhibitor. Compound 10 demonstrated good oral bioavailability (BA) and long-lasting efficacy in rats. Compound 10 is currently in clinical trials.

Phosphodiesterase 10A (PDE10A) is a member of the PDE family of enzymes that degrades cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Our aim was to label a series of structurally related PDE10A inhibitors with carbon-11 and evaluate them as potential positron emission tomography (PET) radioligands for PDE10A using nonhuman primates. The series consisted of seven compounds based on the 3-(1H-pyrazol-5-yl)pyridazin-4(1H)-one backbone. These compounds were selected from the initial larger library based on a number of parameters such as affinity, selectivity for hPDE10A in in vitro tests, lipophilicity, and on the results of multidrug resistance protein 1 (MDR1)-LLCPK1 and the parallel artificial membrane permeability assays. Seven radioligands (KIT-1, 3, 5, 6, 7, 9, and 12) were radiolabeled with carbon-11 employing O-methylation on the hydroxyl moiety using [(11)C]methyl triflate. In vivo examination of each radioligand was performed using PET in rhesus monkeys; analysis of radiometabolites in plasma also was conducted using HPLC. All seven radioligands were labeled with high (>90%) incorporation of [(11)C]methyl triflate into their appropriate precursors and with high specific radioactivity. Carbon-11 labeled KIT-5 and KIT-6 showed high accumulation in the striatum, consistent with the known anatomical distribution of PDE10A in brain, accompanied by fast washout and high specific binding ratio. In particular [(11)C]KIT-6, named [(11)C]T-773, is a promising PET tool for further examination of PDE10A in human brain., (Copyright © 2015 John Wiley & Sons, Ltd.)

Introduction: Phosphodiesterase 10A (PDE10A) is a dual-substrate PDE that hydrolyzes both cAMP and cGMP and is selectively expressed in striatal medium spiny neurons. Recent studies have suggested that PDE10A inhibition is a novel approach for the treatment of disorders such as schizophrenia and Huntington's disease. A positron emission tomography (PET) occupancy study can provide useful information for the development of PDE10A inhibitors. We discovered T-773 as a candidate PET radioligand for PDE10A and investigated its properties by in vitro autoradiography and a PET study in a monkey., Methods: Profiling of T-773 as a PET radioligand for PDE10A was conducted by in vitro enzyme inhibitory assay, in vitro autoradiography, and PET study in a monkey., Results: T-773 showed a high binding affinity and selectivity for human recombinant PDE10A2 in vitro; the IC50 value in an enzyme inhibitory assay was 0.77nmol/L, and selectivity over other PDEs was more than 2500-fold. In autoradiography studies using mouse, rat, monkey, or human brain sections, radiolabeled T-773 selectively accumulated in the striatum. This selective accumulation was not observed in the brain sections of Pde10a-KO mice. The binding of [(3)H]T-773 to PDE10A in rat brain sections was competitively inhibited by MP-10, a selective PDE10A inhibitor. In rat brain sections, [(3)H]T-773 bound to a single high affinity site of PDE10A with Kd values of 12.2±2.2 and 4.7±1.2nmol/L in the caudate-putamen and nucleus accumbens, respectively. In a monkey PET study, [(11)C]T-773 showed good brain penetration and striatum-selective accumulation., Conclusion: These results suggest that [(11)C]T-773 is a potential PET radioligand for PDE10A., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The pharmacological profile of a novel angiotensin II type 1 receptor blocker, azilsartan medoxomil, was compared with that of the potent angiotensin II receptor blocker olmesartan medoxomil. Azilsartan, the active metabolite of azilsartan medoxomil, inhibited the binding of [(125)I]-Sar(1)-I1e(8)-angiotensin II to angiotensin II type 1 receptors. Azilsartan medoxomil inhibited angiotensin II-induced pressor responses in rats, and its inhibitory effects lasted 24h after oral administration. The inhibitory effects of olmesartan medoxomil disappeared within 24h. ID(50) values were 0.12 and 0.55 mg/kg for azilsartan medoxomil and olmesartan medoxomil, respectively. In conscious spontaneously hypertensive rats (SHRs), oral administration of 0.1-1mg/kg azilsartan medoxomil significantly reduced blood pressure at all doses even 24h after dosing. Oral administration of 0.1-3mg/kg olmesartan medoxomil also reduced blood pressure; however, only the two highest doses significantly reduced blood pressure 24h after dosing. ED(25) values were 0.41 and 1.3mg/kg for azilsartan medoxomil and olmesartan medoxomil, respectively. In renal hypertensive dogs, oral administration of 0.1-1mg/kg azilsartan medoxomil reduced blood pressure more potently and persistently than that of 0.3-3mg/kg olmesartan medoxomil. In a 2-week study in SHRs, azilsartan medoxomil showed more stable antihypertensive effects than olmesartan medoxomil and improved the glucose infusion rate, an indicator of insulin sensitivity, more potently (≥ 10 times) than olmesartan medoxomil. Azilsartan medoxomil also exerted more potent antiproteinuric effects than olmesartan medoxomil in Wistar fatty rats. These results suggest that azilsartan medoxomil is a potent angiotensin II receptor blocker that has an attractive pharmacological profile as an antihypertensive agent., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Recombinant protein technology is an important tool in many industrial and pharmacological applications. Although the success rate of obtaining soluble proteins is relatively low, knowledge of protein expression/solubility under 'standard' conditions may increase the efficiency and reduce the cost of proteomics studies. In this study, we conducted a genome-scale experiment to assess the overexpression and the solubility of human full-length cDNA in an in vivo Escherichia coli expression system and a wheat germ cell-free expression system. We evaluated the influences of sequence and structural features on protein expression/solubility in each system and estimated a minimal set of features associated with them. A comparison of the feature sets related to protein expression/solubility in the in vivo Escherichia coli expression system revealed that the structural information was strongly associated with protein expression, rather than protein solubility. Moreover, a significant difference was found in the number of features associated with protein solubility in the two expression systems.

The angiotensin II (AII) antagonistic action of azilsartan (AZL) [2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}-1H-benzimidazole-7-carboxylic acid] was investigated in radioligand binding and function studies. AZL inhibited the specific binding of ¹²⁵I-Sar¹-Ile⁸-AII to human angiotensin type 1 receptors with an IC₅₀ of 2.6 nM. The inhibitory effect of AZL persisted after washout of the free compound (IC(50) value of 7.4 nM). Olmesartan, telmisartan, valsartan, and irbesartan also inhibited the specific binding with IC₅₀ values of 6.7, 5.1, 44.9, and 15.8 nM, respectively. However, their inhibitory effects were markedly attenuated with washout (IC₅₀ values of 242.5, 191.6, >10,000, and >10,000 nM). AZL also inhibited the accumulation of AII-induced inositol 1-phosphate (IP1) in the cell-based assay with an IC₅₀ value of 9.2 nmol; this effect was resistant to washout (IC₅₀ value of 81.3 nM). Olmesartan and valsartan inhibited IP1 accumulation with IC₅₀ values of 12.2 and 59.8 nM, respectively. The activities of these compounds were markedly reduced after washout (IC₅₀ value of 908.5 and 22,664.4 nM). AZL was defined as an inverse agonist in an experiment by using a constitutively active mutant of human angiotensin type 1 receptors. In isolated rabbit aortic strips, AZL reduced the maximal contractile response to AII with a pD'₂ value of 9.9. The inhibitory effects of AZL on contractile responses induced by AII persisted after the strips were washed; these inhibitory effects were more potent than those of olmesartan. These results suggest that AZL is a highly potent and slowly dissociating AII receptor blocker. Its tight receptor binding might be expected to produce potent and long-lasting antihypertensive effects in preclinical and clinical settings.

Appropriate resources and expression technology necessary for human proteomics on a whole-proteome scale are being developed. We prepared a foundation for simple and efficient production of human proteins using the versatile Gateway vector system. We generated 33,275 human Gateway entry clones for protein synthesis, developed mRNA expression protocols for them and improved the wheat germ cell-free protein synthesis system. We applied this protein expression system to the in vitro expression of 13,364 human proteins and assessed their biological activity in two functional categories. Of the 75 tested phosphatases, 58 (77%) showed biological activity. Several cytokines containing disulfide bonds were produced in an active form in a nonreducing wheat germ cell-free expression system. We also manufactured protein microarrays by direct printing of unpurified in vitro-synthesized proteins and demonstrated their utility. Our 'human protein factory' infrastructure includes the resources and expression technology for in vitro proteome research.

The coagulation enzyme factor Xa (FXa) has been recognized as a promising target for the development of new antithrombotic agents. We previously found compound 1 to be an orally bioavailable FXa inhibitor in fasted monkeys; however, 1 showed poor bioavailability in rats and fed monkeys. To work out the pharmacokinetic problems, we focused our synthetic efforts on the chemical conversion of the 4-(imidazo[1,2- a]pyridin-5-yl)piperazine moiety of 1 to imidazolylpiperidine derivatives (fused and nonfused), which resulted in the discovery of the weakly basic imidazo[1,5- c]imidazol-3-one 3q as a potent and selective FXa inhibitor. Compound 3q showed favorable oral bioavailability in rats and monkeys under both fasted and fed conditions and antithrombotic efficacy in a rat model of venous thrombosis after oral administration, without a significant increase in bleeding time (unlike warfarin). On the basis of these promising properties, compound 3q was selected for further evaluation.

We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdsb encodes a protein of 170 amino acids with an NH(2)-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif, Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH(2)-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins of archaea and bacteria appear to be highly similar in terms of function.

Cypoviruses are insect viruses that produce a cytoplasmic crystalline particle called the polyhedron in which progeny virions are occluded. The virion structural protein, VP3, is implicated in the occlusion of viral particles into polyhedra. In this study, we determined the amino acid sequence of VP3 required for occlusion of viral particles into polyhedra and proposed that this sequence could be used as an immobilization signal to direct the stable incorporation of foreign proteins into polyhedra. A large-scale survey revealed that the immobilization signal could, in fact, direct the incorporation of a variety of human proteins into polyhedra. Immune reactivity and protein-protein interactions were detected on the surface of polyhedra containing immobilized foreign proteins, and these particles were shown to be highly stabilized against dehydration. We showed that these particles could be arrayed onto a glass slide by standard spotting and laser manipulation methods. Thus, this approach is well suited for protein expression, purification, and the development of protein microarrays.

A novel mechanism for controlling the proofreading and polymerase activities of archaeal DNA polymerases was studied. The 3'-5'exonuclease (proofreading) activity and PCR performance of the family B DNA polymerase from Thermococcus kodakaraensis KOD1 (previously Pyrococcus kodakaraensis KOD1) were altered efficiently by mutation of a "unique loop" in the exonuclease domain. Interestingly, eight different H147 mutants showed considerable variations in respect to their 3'-5'exonuclease activity, from 9% to 276%, as against that of the wild-type (WT) enzyme. We determined the 2.75A crystal structure of the H147E mutant of KOD DNA polymerase that shows 30% of the 3'-5'exonuclease activity, excellent PCR performance and WT-like fidelity. The structural data indicate that the properties of the H147E mutant were altered by a conformational change of the Editing-cleft caused by an interaction between the unique loop and the Thumb domain. Our data suggest that electrostatic and hydrophobic interactions between the unique loop of the exonuclease domain and the tip of the Thumb domain are essential for determining the properties of these DNA polymerases.

To search for restriction endonucleases, we used a novel plant-based cell-free translation procedure that bypasses the toxicity of these enzymes. To identify candidate genes, the related genomes of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii were compared. In line with the selfish mobile gene hypothesis for restriction-modification systems, apparent genome rearrangement around putative restriction genes served as a selecting criterion. Several candidate restriction genes were identified and then amplified in such a way that they were removed from their own translation signal. During their cloning into a plasmid, the genes became connected with a plant translation signal. After in vitro transcription by T7 RNA polymerase, the mRNAs were separated from the template DNA and translated in a wheat-germ-based cell-free protein synthesis system. The resulting solution could be directly assayed for restriction activity. We identified two deoxyribonucleases. The novel enzyme was denoted as PabI, purified and found to recognize 5'-GTAC and leave a 3'-TA overhang (5'-GTA/C), a novel restriction enzyme-generated terminus. PabI is active up to 90 degrees C and optimally active at a pH of around 6 and in NaCl concentrations ranging from 100 to 200 mM. We predict that it has a novel 3D structure.

Specific interactions between transcription factors and cis-acting DNA sequence motifs are primary events for the transcriptional regulation. Many regulatory elements appear to diverge from the most optimal recognition sequences. To evaluate affinities of a transcription factor to various suboptimal sequences, we have developed a new detection method based on the surface plasmon resonance (SPR) imaging technique. Transcription factor MafG and its recognition sequence MARE (Maf recognition elements) were adopted to evaluate the new method. We modified DNA immobilization procedure on to the gold chip, so that a double-stranded DNA array was successfully fabricated. We further found that a hydrophilic flexible spacer composed of the poly (ethylene glycol) moiety between DNA and alkanethiol self-assembled monolayers on the surface is effective for preventing nonspecific adsorption and facilitating specific binding of MafG. Multiple interaction profiles between MafG and six of MARE-related sequences were observed by the SPR imaging technique. The kinetic values obtained by SPR imaging showed very good correlation with those obtained from electrophoretic gel mobility shift assays, although absolute values were deviated from each other. These results demonstrate that the double-stranded DNA array fabricated with the modified multistep procedure can be applied for the comprehensive analysis of the transcription factor-DNA interaction.