29 results on '"Nobuyo Higashi-Kuwata"'
Search Results
2. Identification of SARS-CoV-2 Mpro inhibitors containing P1’ 4-fluorobenzothiazole moiety highly active against SARS-CoV-2
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Nobuyo Higashi-Kuwata, Kohei Tsuji, Hironori Hayashi, Haydar Bulut, Maki Kiso, Masaki Imai, Hiromi Ogata-Aoki, Takahiro Ishii, Takuya Kobayakawa, Kenta Nakano, Nobutoki Takamune, Naoki Kishimoto, Shin-ichiro Hattori, Debananda Das, Yukari Uemura, Yosuke Shimizu, Manabu Aoki, Kazuya Hasegawa, Satoshi Suzuki, Akie Nishiyama, Junji Saruwatari, Yukiko Shimizu, Yoshikazu Sukenaga, Yuki Takamatsu, Kiyoto Tsuchiya, Kenji Maeda, Kazuhisa Yoshimura, Shun Iida, Seiya Ozono, Tadaki Suzuki, Tadashi Okamura, Shogo Misumi, Yoshihiro Kawaoka, Hirokazu Tamamura, and Hiroaki Mitsuya
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Multidisciplinary ,General Physics and Astronomy ,General Chemistry ,General Biochemistry, Genetics and Molecular Biology - Abstract
Abstract COVID-19 caused by SARS-CoV-2 has continually been serious threat to public health worldwide. While a few anti-SARS-CoV-2 therapeutics are currently available, their antiviral potency is not sufficient. Here, we identify two orally available 4-fluoro-benzothiazole-containing small molecules, TKB245 and TKB248, which specifically inhibit the enzymatic activity of main protease (Mpro) of SARS-CoV-2 and significantly more potently block the infectivity and replication of various SARS-CoV-2 strains than nirmatrelvir, molnupiravir, and ensitrelvir in cell-based assays employing various target cells. Both compounds also block the replication of Delta and Omicron variants in human-ACE2-knocked-in mice. Native mass spectrometric analysis reveals that both compounds bind to dimer Mpro, apparently promoting Mpro dimerization. X-ray crystallographic analysis shows that both compounds bind to Mpro’s active-site cavity, forming a covalent bond with the catalytic amino acid Cys-145 with the 4-fluorine of the benzothiazole moiety pointed to solvent. The data suggest that TKB245 and TKB248 might serve as potential therapeutics for COVID-19 and shed light upon further optimization to develop more potent and safer anti-SARS-CoV-2 therapeutics.
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- 2023
3. Design, Synthesis and Evaluation of Anti-Hepatitis B Virus (Hbv) Activity of Novel Entecavir Analogues Having 4′-Cyano-6″-Fluoromethylenecyclopentene Skeletons as an Aglycone Moiety
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Hiroki Kumamoto, Nobuyo Higashi-Kuwata, Sanae Hayashi, Debananda Das, Haydar Bulut, Ryoh Tokuda, Shuhei Imoto, Kengo Onitsuka, Yuka Honda, Yuki Odanaka, Satoko Shinbara-Matsubayashi, Kazuhiro Haraguchi, Yasuhito Tanaka, and Hiroaki Mitsuya
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- 2023
4. Generation of Angiotensin-Converting Enzyme 2/Transmembrane Protease Serine 2-Double-Positive Human Induced Pluripotent Stem Cell-Derived Spheroids for Anti-Severe Acute Respiratory Syndrome Coronavirus 2 Drug Evaluation
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Nobuyo Higashi-Kuwata, Shigeharu G. Yabe, Satsuki Fukuda, Junko Nishida, Miwa Tamura-Nakano, Shin-ichiro Hattori, Hitoshi Okochi, and Hiroaki Mitsuya
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Microbiology (medical) ,General Immunology and Microbiology ,Ecology ,Physiology ,SARS-CoV-2 ,Induced Pluripotent Stem Cells ,COVID-19 ,Cell Biology ,Infectious Diseases ,Genetics ,Serine ,Humans ,Drug Evaluation ,Angiotensin-Converting Enzyme 2 - Abstract
The hiPSC-derived spheroids we generated are more expensive to obtain than the human cell lines currently available for anti-SARS-CoV-2 drug evaluation, such as Calu-3 cells; however, the spheroids have better infection susceptibility than the existing human cell lines. Although we are cognizant that there are human lung (and colonic) organoid models for the study of SARS-CoV-2, the production of those organoids is greatly more costly and time consuming than the generation of human iPSC-derived spheroid cells.
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- 2022
5. Restoration of Neutralization Activity Against Omicron BA.2 and BA.5 in Older Adults and Individuals With Risk Factors Following the Fourth Dose of Severe Acute Respiratory Syndrome Coronavirus 2 BNT162b2 Vaccine
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Masayuki Amano, Sachiko Otsu, Yasuko Ichikawa, Nobuyo Higashi-Kuwata, Shuzo Matsushita, Shinya Shimada, and Hiroaki Mitsuya
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Infectious Diseases ,SARS-CoV-2 ,Risk Factors ,Immunology and Allergy ,Humans ,COVID-19 ,Antibodies, Viral ,Antibodies, Neutralizing ,BNT162 Vaccine ,Aged - Published
- 2022
6. Potent and biostable inhibitors of the main protease of SARS-CoV-2
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Kohei Tsuji, Takahiro Ishii, Takuya Kobayakawa, Nobuyo Higashi-Kuwata, Chika Azuma, Miyuki Nakayama, Takato Onishi, Hiroki Nakano, Naoya Wada, Miki Hori, Kouki Shinohara, Yutaro Miura, Takuma Kawada, Hironori Hayashi, Shin-ichiro Hattori, Haydar Bulut, Debananda Das, Nobutoki Takamune, Naoki Kishimoto, Junji Saruwatari, Tadashi Okamura, Kenta Nakano, Shogo Misumi, Hiroaki Mitsuya, and Hirokazu Tamamura
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Multidisciplinary - Abstract
Potent and biostable inhibitors of the main protease (M
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- 2022
7. A novel anti-HBV agent, E-CFCP, restores Hepatitis B virus (HBV)-induced senescence-associated cellular marker perturbation in human hepatocytes
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Yuki Takamatsu, Sanae Hayashi, Hiroki Kumamoto, Shuhei Imoto, Yasuhito Tanaka, Hiroaki Mitsuya, and Nobuyo Higashi-Kuwata
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Cancer Research ,Infectious Diseases ,Virology - Published
- 2023
8. Identification of a novel long-acting 4’-modified nucleoside reverse transcriptase inhibitor against HBV
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Masakazu Kakuni, Naoki Kishimoto, Hiroki Kumamoto, Yasuhito Tanaka, Debananda Das, Nobutoki Takamune, Shogo Misumi, David Venzon, Hiromi Ogata-Aoki, Haydar Bulut, David A. Davis, Nobuyo Higashi-Kuwata, Hiroaki Mitsuya, Sanae Hayashi, Masaki Otagiri, Mai Hashimoto, and Shin-ichiro Hattori
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0301 basic medicine ,Hepatitis B virus ,Cell ,Pharmacology ,Tenofovir alafenamide ,Article ,Drug Administration Schedule ,Time ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Drug Development ,Drug Resistance, Viral ,medicine ,Animals ,Humans ,Potency ,Hepatitis B e Antigens ,Dosing ,Hepatology ,Reverse-transcriptase inhibitor ,business.industry ,Drug Administration Routes ,virus diseases ,RNA-Directed DNA Polymerase ,Entecavir ,Hepatitis B ,Reverse transcriptase ,Disease Models, Animal ,030104 developmental biology ,medicine.anatomical_structure ,Delayed-Action Preparations ,Toxicity ,Reverse Transcriptase Inhibitors ,030211 gastroenterology & hepatology ,business ,medicine.drug - Abstract
Background & Aims While certain nucleos(t)ide reverse transcriptase inhibitors (NRTIs) are efficacious in treating HBV infection, their effects are yet to be optimized and the emergence of NRTI-resistant HBV variants is an issue because of the requirement for lifelong treatment. The development of agents that more profoundly suppress wild-type and drug-resistant HBVs, and that have a long-acting effect, are crucial to improve patient outcomes. Methods Herein, we synthesized a novel long-acting 4’-modified NRTI termed E-CFCP. We tested its anti-HBV activity in vitro, before evaluating its anti-HBV activity in HBV-infected human-liver-chimeric mice (PXB-mice). E-CFCP’s long-acting features and E-CFCP-triphosphate’s interactions with the HBV reverse transcriptase (HBV-RT) were examined. Results E-CFCP potently blocked HBVWTD1 production (IC50qPCR_cell=1.8 nM) in HepG2.2.15 cells and HBVWTC2 (IC50SB_cell=0.7 nM), entecavir (ETV)-resistant HBVETV-RL180M/S202G/M204V (IC50SB_cell=77.5 nM), and adefovir-resistant HBVADV-RA181T/N236T production (IC50SB_cell=14.1 nM) in Huh7 cells. E-CFCP profoundly inhibited intracellular HBV DNA production to below the detection limit, but ETV and tenofovir alafenamide (TAF) failed to do so. E-CFCP also showed less toxicity than ETV and TAF. E-CFCP better penetrated hepatocytes and was better tri-phosphorylated; E-CFCP-triphosphate persisted intracellularly for longer than ETV-triphosphate. Once-daily peroral E-CFCP administration over 2 weeks (0.02~0.2 mg/kg/day) reduced HBVWTC2-viremia by 2–3 logs in PXB-mice without significant toxicities and the reduction persisted over 1–3 weeks following treatment cessation, suggesting once-weekly dosing capabilities. E-CFCP also reduced HBVETV-RL180M/S202G/M204V-viremia by 2 logs over 2 weeks, while ETV completely failed to reduce HBVETV-RL180M/S202G/M204V-viremia. E-CFCP’s 4’-cyano and fluorine interact with both HBVWT-RT and HBVETV-RL180M/S202G-M204 -RT via Van der Waals and polar forces, being important for E-CFCP-triphosphate’s interactions and anti-HBV potency. Conclusion E-CFCP represents the first reported potential long-acting NRTI with potent activity against wild-type and treatment-resistant HBV. Lay summary Although there are currently effective treatment options for HBV, treatment-resistant variants and the need for lifelong therapy pose a significant challenge. Therefore, the development of new treatment options is crucial to improve outcomes and quality of life. Herein, we report preclinical evidence showing that the anti-HBV agent, E-CFCP, has potent activity against wild-type and treatment-resistant variants. In addition, once-weekly oral dosing may be possible, which is preferrable to the current daily dosing regimens.
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- 2021
9. Neutralization of SARS-CoV-2 with IgG from COVID-19-convalescent plasma
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Noriko Kinoshita, Kenji Maeda, Hiroaki Mitsuya, Kiyoto Tsuchiya, Kouki Matsuda, Haruhito Sugiyama, Shin-ichiro Hattori, Hiroyuki Gatanaga, Nobuyo Higashi-Kuwata, Shinichi Oka, Yuki Takamatsu, Norio Ohmagari, and Satoshi Kutsuna
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0301 basic medicine ,Convalescent plasma ,Coronavirus disease 2019 (COVID-19) ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,medicine.medical_treatment ,Science ,Antibodies, Viral ,Article ,Neutralization ,Microbiology ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,medicine ,Humans ,030212 general & internal medicine ,Receptor ,COVID-19 Serotherapy ,Multidisciplinary ,biology ,SARS-CoV-2 ,Chemistry ,Immunization, Passive ,COVID-19 ,Immunotherapy ,Antibodies, Neutralizing ,030104 developmental biology ,Viral infection ,biology.protein ,Medicine ,Antibody - Abstract
While there are various attempts to administer COVID-19-convalescent plasmas to SARS-CoV-2-infected patients, neither appropriate approach nor clinical utility has been established. We examined the presence and temporal changes of the neutralizing activity of IgG fractions from 43 COVID-19-convalescent plasmas using cell-based assays with multiple endpoints. IgG fractions from 27 cases (62.8%) had significant neutralizing activity and moderately to potently inhibited SARS-CoV-2 infection in cell-based assays; however, no detectable neutralizing activity was found in 16 cases (37.2%). Approximately half of the patients (~ 41%), who had significant neutralizing activity, lost the neutralization activity within ~ 1 month. Despite the rapid decline of neutralizing activity in plasmas, good amounts of SARS-CoV-2-S1-binding antibodies were persistently seen. The longer exposure of COVID-19 patients to greater amounts of SARS-CoV-2 elicits potent immune response to SARS-CoV-2, producing greater neutralization activity and SARS-CoV-2-S1-binding antibody amounts. The dilution of highly-neutralizing plasmas with poorly-neutralizing plasmas relatively readily reduced neutralizing activity. The presence of good amounts of SARS-CoV-2-S1-binding antibodies does not serve as a surrogate ensuring the presence of good neutralizing activity. In selecting good COVID-19-convalescent plasmas, quantification of neutralizing activity in each plasma sample before collection and use is required.
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- 2021
10. Design, Synthesis and X‐Ray Structural Studies of Potent HIV‐1 Protease Inhibitors Containing C‐4 Substituted Tricyclic Hexahydro‐Furofuran Derivatives as P2 Ligands
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Arun K. Ghosh, Satish Kovela, Ashish Sharma, Dana Shahabi, Ajay K. Ghosh, Denver R. Hopkins, Monika Yadav, Megan E. Johnson, Johnson Agniswamy, Yuan‐Fang Wang, Shin‐Ichiro Hattori, Nobuyo Higashi‐Kuwata, Manabu Aoki, Masayuki Amano, Irene T. Weber, and Hiroaki Mitsuya
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Pharmacology ,X-Rays ,Organic Chemistry ,HIV Protease Inhibitors ,Crystallography, X-Ray ,Ligands ,Biochemistry ,Article ,Structure-Activity Relationship ,HIV Protease ,Drug Design ,Drug Discovery ,HIV-1 ,Molecular Medicine ,General Pharmacology, Toxicology and Pharmaceutics - Abstract
The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporated novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2’-ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2’ ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.
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- 2022
11. Highly Neutralizing COVID-19 Convalescent Plasmas Potently Block SARS-CoV-2 Replication and Pneumonia in Syrian Hamsters
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Kazumi Omata, Mutsumi Ito, Hiroaki Mitsuya, Tadaki Suzuki, Yuichiro Takeda, Noriko Nakajima, Kiyoko Iwatsuki-Horimoto, Kenji Maeda, Yoshihiro Kawaoka, Nobuyo Higashi-Kuwata, Masaki Imai, Tadashi Maemura, Yuki Takamatsu, and Maki Kiso
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Male ,Immunology ,Hamster ,Virus Replication ,Microbiology ,Virology ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Pathogen ,Lung ,Vero Cells ,COVID-19 Serotherapy ,biology ,Mesocricetus ,SARS-CoV-2 ,Immunization, Passive ,COVID-19 ,medicine.disease ,Blot ,Titer ,Pneumonia ,Disease Models, Animal ,medicine.anatomical_structure ,Viral replication ,Insect Science ,biology.protein ,Antibody - Abstract
Despite various attempts to treat SARS-CoV-2-infected patients with COVID-19-convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly-neutralizing COVID-19-convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing-activity-determined convalescent plasma samples, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in significant reduction of viral titers in lungs by up to 32-fold as compared to the viral titers in hamsters receiving control non-neutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using micro-computerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19-convalescent-plasmas variably contained antibodies against SARS-CoV-2 components including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent-neutralizing-activity-confirmed COVID-19-convalescent plasmas would be efficacious in treating patients with COVID-19. Importance Convalescent plasmas obtained from patients, who recovered from a specific infection, have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized-controlled-clinical-trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94-95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly-neutralizing-effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally-induced lung lesions. The present data provide a proof-of-concept that the presence of highly-neutralizing antibody in COVID-19-convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly-neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.
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- 2021
12. Indole Chloropyridinyl Ester-Derived SARS-CoV-2 3CLpro Inhibitors: Enzyme Inhibition, Antiviral Efficacy, Structure-Activity Relationship, and X-ray Structural Studies
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Brandon J. Anson, Dana Shahabi, Emma K. Lendy, Andrew D. Mesecar, Monika Yadav, Hiroaki Mitsuya, Jakka Raghavaiah, Nobuyo Higashi-Kuwata, Arun K. Ghosh, and Shin-ichiro Hattori
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Indoles ,Stereochemistry ,Pyridines ,medicine.medical_treatment ,Molecular Dynamics Simulation ,Crystallography, X-Ray ,Article ,chemistry.chemical_compound ,Structure-Activity Relationship ,Drug Discovery ,Chlorocebus aethiops ,medicine ,Structure–activity relationship ,Animals ,Humans ,Protease Inhibitors ,Carboxylate ,Binding site ,IC50 ,Vero Cells ,Coronavirus 3C Proteases ,Indole test ,Protease ,Alanine ,Binding Sites ,Chemistry ,SARS-CoV-2 ,COVID-19 ,Adenosine Monophosphate ,RNA Polymerase Inhibitor ,Vero cell ,Molecular Medicine - Abstract
Here, we report the synthesis, structure-activity relationship studies, enzyme inhibition, antiviral activity, and X-ray crystallographic studies of 5-chloropyridinyl indole carboxylate derivatives as a potent class of SARS-CoV-2 chymotrypsin-like protease inhibitors. Compound 1 exhibited a SARS-CoV-2 3CLpro inhibitory IC50 value of 250 nM and an antiviral EC50 value of 2.8 µM in VeroE6 cells. Remdesivir, an RNA-dependent RNA polymerase inhibitor, showed an antiviral EC50 value of 1.2 µM in the same assay. Compound 1 showed comparable antiviral activity with remdesivir in immunocytochemistry assays. Compound 7d with an N-allyl derivative showed the most potent enzyme inhibitory IC50 value of 73 nM. To obtain molecular insight into the binding properties of these molecules, X-ray crystal structures of compounds 2, 7b, and 9d-bound to SARS-CoV 3CLpro were determined, and their binding properties were compared.
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- 2021
13. Highly-Neutralizing COVID-19-Convalescent-Plasmas Potently Block SARS-CoV-2 Replication and Pneumonia in Syrian Hamsters
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Yoshihiro Kawaoka, Kiyoko Iwatsuki-Horimoto, Kenji Maeda, Yuichiro Takeda, Kazumi Omata, Noriko Nakajima, Maki Kiso, Tadashi Maemura, Yuki Takamatsu, Tadaki Suzuki, Masaki Imai, Hiroaki Mitsuya, Nobuyo Higashi-Kuwata, and Mutsumi Ito
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Lung ,Coronavirus disease 2019 (COVID-19) ,biology ,business.industry ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Hamster ,medicine.disease ,Virology ,Blot ,Pneumonia ,Titer ,medicine.anatomical_structure ,medicine ,biology.protein ,Antibody ,business - Abstract
Despite various attempts to treat SARS-CoV-2-infected patients with COVID-19-convalescent plasmas, neither appropriate approach nor clinical utility has been established. We examined the efficacy of administration of highly-neutralizing COVID-19-convalescent plasma (hn-plasmas) and such plasma-derived IgG administration using the Syrian hamster COVID-19 model. Two hn-plasmas, which were in the best 1% of 340 neutralizing-activity-determined convalescent plasma samples, were intraperitoneally administered to SARS-CoV-2-infected hamsters, resulting in significant reduction of viral titers in lungs by up to 32-fold as compared to the viral titers in hamsters receiving control non-neutralizing plasma, while with two moderately neutralizing plasmas (mn-plasmas) administered, viral titer reduction was by up to 6-fold. IgG fractions purified from the two hn-plasmas also reduced viral titers in lungs than those from the two mn-plasmas. The severity of lung lesions seen in hamsters receiving hn-plasmas was minimal to moderate as assessed using micro-computerized tomography, which histological examination confirmed. Western blotting revealed that all four COVID-19-convalescent-plasmas variably contained antibodies against SARS-CoV-2 components including the receptor-binding domain and S1 domain. The present data strongly suggest that administering potent-neutralizing-activity-confirmed COVID-19-convalescent plasmas would be efficacious in treating patients with COVID-19.ImportanceConvalescent plasmas obtained from patients, who recovered from a specific infection, have been used as agents to treat other patients infected with the very pathogen. To treat using convalescent plasmas, despite that more than 10 randomized-controlled-clinical-trials have been conducted and more than 100 studies are currently ongoing, the effects of convalescent plasma against COVID-19 remained uncertain. On the other hand, certain COVID-19 vaccines have been shown to reduce the clinical COVID-19 onset by 94-95%, for which the elicited SARS-CoV-2-neutralizing antibodies are apparently directly responsible. Here, we demonstrate that highly-neutralizing-effect-confirmed convalescent plasmas significantly reduce the viral titers in the lung of SARS-CoV-2-infected Syrian hamsters and block the development of virally-induced lung lesions. The present data provide a proof-of-concept that the presence of highly-neutralizing antibody in COVID-19-convalescent plasmas is directly responsible for the reduction of viral replication and support the use of highly-neutralizing antibody-containing plasmas in COVID-19 therapy with convalescent plasmas.
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- 2021
14. Highly Accurate, Specific And Sensitive Quantitation By Droplet Digital PCR Reveals The Stability of Intrahepatic Hepatitis B Virus cccDNA
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Kyoko Ito, Hiroaki Mitsuya, Natthaya Chuaypen, Keigo Kawashima, Pisit Tangkijvanich, Masanori Isogawa, Yuji Morine, Yasuhito Tanaka, Sanae Hayashi, Nobuyo Higashi-Kuwata, and Mitsuo Shimada
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Hepatitis B virus ,Chemistry ,medicine ,Digital polymerase chain reaction ,cccDNA ,medicine.disease_cause ,Virology - Abstract
The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. ddPCR measured a less than two-fold difference in the intrahepatic cccDNA content more accurately than conventional real-time PCR (qPCR), (R2=0.9416 and R2=0.8963, respectively) and had also higher sensitivity and specificity than qPCR. The results of ddPCR correlated more closely with serum HB core-related antigen (R2=0.9843) than HB surface antigen (HBsAg) (R2=0.9742) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced in PXB-mice after entecavir treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p=0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.
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- 2021
15. Droplet digital PCR assay provides intrahepatic HBV cccDNA quantification tool for clinical application
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Sanae Hayashi, Masanori Isogawa, Keigo Kawashima, Kyoko Ito, Natthaya Chuaypen, Yuji Morine, Mitsuo Shimada, Nobuyo Higashi-Kuwata, Takehisa Watanabe, Pisit Tangkijvanich, Hiroaki Mitsuya, and Yasuhito Tanaka
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Hepatitis B virus ,Carcinoma, Hepatocellular ,Multidisciplinary ,Science ,Interferon-alpha ,Mice, Transgenic ,Hepatitis B ,Polymerase Chain Reaction ,Recombinant Proteins ,Polyethylene Glycols ,Liver ,DNA, Viral ,Medicine ,Animals ,Humans ,DNA, Circular - Abstract
The persistence of covalently closed circular DNA (cccDNA) poses a major obstacle to curing chronic hepatitis B (CHB). Here, we used droplet digital PCR (ddPCR) for cccDNA quantitation. The cccDNA-specific ddPCR showed high accuracy with the dynamic range of cccDNA detection from 101 to 105 copies/assay. The ddPCR had higher sensitivity, specificity and precisely than qPCR. The results of ddPCR correlated closely with serum HB core-related antigen and HB surface antigen (HBsAg) in 24 HBV-infected human-liver-chimeric mice (PXB-mice). We demonstrated that in 2 PXB-mice after entecavir treatment, the total cccDNA content did not change during liver repopulation, although the cccDNA content per hepatocyte was reduced after the treatment. In the 6 patients with HBV-related hepatocellular carcinoma, ddPCR detected cccDNA in both tumor and non-tumor tissues. In 13 HBeAg-negative CHB patients with pegylated interferon alpha-2a, cccDNA contents from paired biopsies were more significantly reduced in virological response (VR) than in non-VR at week 48 (p = 0.0051). Interestingly, cccDNA levels were the lowest in VR with HBsAg clearance but remained detectable after the treatment. Collectively, ddPCR revealed that cccDNA content is stable during hepatocyte proliferation and persists at quantifiable levels, even after serum HBsAg clearance.
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- 2022
16. A small molecule compound with an indole moiety inhibits the main protease of SARS-CoV-2 and blocks virus replication
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Srinivasa Rao Allu, Emma K. Lendy, Jakka Raghavaiah, Alexander Wlodawer, Arun K. Ghosh, Debananda Das, David A. Davis, Kazutaka Murayama, Hiroaki Mitsuya, Shogo Misumi, Yuki Takamatsu, Nobuyo Higashi-Kuwata, Mi Li, Brandon J. Anson, Hironori Hayashi, Kazuya Hasegawa, Naoki Kishimoto, Nobutoki Takamune, Shin ichiro Hattori, Eiichi Kodama, Robert Yarchoan, Haydar Bulut, and Andrew D. Mesecar
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0301 basic medicine ,Indoles ,Pyridines ,Science ,medicine.medical_treatment ,viruses ,General Physics and Astronomy ,Antiviral Agents ,General Biochemistry, Genetics and Molecular Biology ,Article ,Cell Line ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Chlorocebus aethiops ,medicine ,Moiety ,Animals ,Humans ,Vero Cells ,Indole test ,Multidisciplinary ,Protease ,Alanine ,biology ,Chemistry ,SARS-CoV-2 ,Viral Proteases ,Antimicrobials ,Active site ,virus diseases ,General Chemistry ,Small molecule ,In vitro ,Adenosine Monophosphate ,COVID-19 Drug Treatment ,030104 developmental biology ,Coronavirus Protease Inhibitors ,Viral replication ,Biochemistry ,030220 oncology & carcinogenesis ,Indoline ,biology.protein - Abstract
Except remdesivir, no specific antivirals for SARS-CoV-2 infection are currently available. Here, we characterize two small-molecule-compounds, named GRL-1720 and 5h, containing an indoline and indole moiety, respectively, which target the SARS-CoV-2 main protease (Mpro). We use VeroE6 cell-based assays with RNA-qPCR, cytopathic assays, and immunocytochemistry and show both compounds to block the infectivity of SARS-CoV-2 with EC50 values of 15 ± 4 and 4.2 ± 0.7 μM for GRL-1720 and 5h, respectively. Remdesivir permitted viral breakthrough at high concentrations; however, compound 5h completely blocks SARS-CoV-2 infection in vitro without viral breakthrough or detectable cytotoxicity. Combination of 5h and remdesivir exhibits synergism against SARS-CoV-2. Additional X-ray structural analysis show that 5h forms a covalent bond with Mpro and makes polar interactions with multiple active site amino acid residues. The present data suggest that 5h might serve as a lead Mpro inhibitor for the development of therapeutics for SARS-CoV-2 infection., Here, using in vitro assays and structural analysis, the authors characterize the anti-SARS-CoV-2 properties of two small molcules, showing these to bind and target the virus main protease (Mpro), and to exhibit a synergistic antiviral effect when combined with remdesivir in vitro.
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- 2020
17. Design, Synthesis, and X-ray Studies of Potent HIV-1 Protease Inhibitors with P2-Carboxamide Functionalities
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Arun K. Ghosh, Yuan-Fang Wang, Nobuyo Higashi-Kuwata, Irene T. Weber, Shin-ichiro Hattori, Daniel W. Kneller, Hiroaki Mitsuya, Alessandro Grillo, Satish Kovela, Jakka Raghavaiah, and Megan E. Johnson
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Protease ,biology ,Bicyclic molecule ,Chemistry ,Ligand ,medicine.drug_class ,Stereochemistry ,medicine.medical_treatment ,Organic Chemistry ,Carboxamide ,Ether ,Biochemistry ,chemistry.chemical_compound ,HIV-1 protease ,Amide ,Drug Discovery ,medicine ,biology.protein ,Acetamide - Abstract
[Image: see text] The design, synthesis, biological evaluation, and X-ray structural studies are reported for a series of highly potent HIV-1 protease inhibitors. The inhibitors incorporated stereochemically defined amide-based bicyclic and tricyclic ether derivatives as the P2 ligands with (R)-hydroxyethylaminesulfonamide transition-state isosteres. A number of inhibitors showed excellent HIV-1 protease inhibitory and antiviral activity; however, ligand combination is critical for potency. Inhibitor 4h with a difluorophenylmethyl as the P1 ligand, crown-THF-derived acetamide as the P2 ligand, and a cyclopropylaminobenzothiazole P2′-ligand displayed very potent antiviral activity and maintained excellent antiviral activity against selected multidrug-resistant HIV-1 variants. A high resolution X-ray structure of inhibitor 4h-bound HIV-1 protease provided molecular insight into the binding properties of the new inhibitor.
- Published
- 2019
18. Raltegravir blocks the infectivity of red-fluorescent-protein (mCherry)-labeled HIV-1JR-FL in the setting of post-exposure prophylaxis in NOD/SCID/Jak3−/− mice transplanted with human PBMCs
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Shin ichiro Hattori, Nobuyo Higashi-Kuwata, Seiji Okada, Manabu Aoki, Akinobu Hamada, Yumi Hashiguchi, Hiromi Ogata-Aoki, Matthew Danish, Hiroaki Mitsuya, Chiemi Shiotsu, Hisataka Kobayashi, Hironori Hayashi, and Hironobu Ihn
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0301 basic medicine ,Pharmacology ,Infectivity ,biology ,CD3 ,030106 microbiology ,Nod ,Swollen lymph nodes ,Raltegravir ,Peripheral blood mononuclear cell ,Virology ,03 medical and health sciences ,Peritoneal cavity ,medicine.anatomical_structure ,medicine ,biology.protein ,Lymph ,medicine.symptom ,medicine.drug - Abstract
Employing NOD/SCID/Jak3-/- mice transplanted with human PBMCs (hNOJ mice) and replication-competent, red-fluorescent-protein (mCherry; mC)-labeled HIV-1JR-FL (HIVmC), we examined whether early antiretroviral treatment blocked the establishment of HIV-1 infection. The use of hNOJ mice and HIVmC enabled us to visually locate infection foci and to examine the early dynamics of HIVmC infection without using a large amount of antiretroviral unlike in non-human primate models. Although when raltegravir (RAL) administration was begun 1 day after intraperitoneal (ip) inoculation of HIVmC, no plasma p24 or plasma HIV-1-RNA (pRNA) were detected in 10 of 12 hNOJ (hNOJmCRAL+) mice as assessed on the last day of the 14-day continuous twice-daily RAL administration, all 10 untreated hNOJmC (hNOJmCRAL-) mice became positive for p24 and pRNA and had significantly swollen lymph nodes in peritoneal cavity and abundant p24+/mC+/CD3+/CD4+ T cells and p24+/mC+/CD68+ monocytes/macrophages were identified in their omenta and mesenteric lymphoid tissues/lymph nodes upon necropsy of the mice on day 14. In 12 hNOJmCRAL+ mice, no significantly swollen lymph nodes were seen compared to hNOJmCRAL- mice; however, in the omentum of the 2 hNOJmCRAL+ mice that were positive for pRNA and in site RNA, mC+/p24+/CD3+/CD83+ cells were identified, suggesting that viral breakthrough occurred later in the observation period. The present data suggest that the use of hNOJ mouse model and HIVmC may shed light on the study of early-phase dynamics of HIV-1 infection and cellular events in post-exposure/pre-exposure prophylaxis.
- Published
- 2018
19. CMCdG, a Novel Nucleoside Analog with Favorable Safety Features, Exerts Potent Activity against Wild-Type and Entecavir-Resistant Hepatitis B Virus
- Author
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Shin-ichiro Hattori, Yasuhito Tanaka, Kamalendra Singh, Hiroaki Mitsuya, Debananda Das, Shuhei Imoto, Sanae Hayashi, Satoru Kohgo, Nobuyo Higashi-Kuwata, Shuko Murakami, Stefan G. Sarafianos, and David Venzon
- Subjects
DNA Replication ,Hepatitis B virus ,Guanine ,Viremia ,medicine.disease_cause ,Antiviral Agents ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,In vivo ,Cell Line, Tumor ,Drug Discovery ,Drug Resistance, Viral ,medicine ,Deoxyguanosine ,Animals ,Humans ,Pharmacology (medical) ,IC50 ,Serum Albumin ,030304 developmental biology ,Pharmacology ,0303 health sciences ,Chemistry ,virus diseases ,Nucleosides ,Entecavir ,Hep G2 Cells ,medicine.disease ,Hepatitis B ,Molecular biology ,Reverse transcriptase ,Infectious Diseases ,Purines ,Reverse Transcriptase Inhibitors ,030211 gastroenterology & hepatology ,Nucleoside ,medicine.drug - Abstract
We designed, synthesized, and characterized a novel nucleoside analog, (1S,3S,5S)-3-(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)-5-hydroxy-1-(hydroxymethyl)-2-methylene-cyclopentanecarbonitrile, or 4′-cyano-methylenecarbocyclic-2′-deoxyguanosine (CMCdG), and evaluated its anti-hepatitis B virus (anti-HBV) activity, safety, and related features. CMCdG’s in vitro activity was determined using quantitative PCR and Southern blotting assays, and its cytotoxicity was determined with a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, while its in vivo activity and safety were determined in human liver-chimeric mice infected with wild-type HBV genotype Ce (HBV(WT)(Ce)) and an entecavir (ETV)-resistant HBV variant containing the amino acid substitutions L180M, S202G, and M204V (HBV(ETV-R)(L180M/S202G/M204V)). CMCdG potently inhibited HBV production in HepG2.2.15 cells (50% inhibitory concentration [IC(50)], ∼30 nM) and HBV(WT)(Ce) plasmid-transfected Huh7 cells (IC(50), 206 nM) and efficiently suppressed ETV-resistant HBV(ETV-R)(L180M/S202G/M204V) (IC(50), 2,657 nM), while it showed no or little cytotoxicity (50% cytotoxic concentration, >500 μM in most hepatocytic cells examined). Two-week peroral administration of CMCdG (1 mg/kg of body weight/day once a day [q.d.]) to HBV(WT)(Ce)-infected human liver-chimeric mice reduced the level of viremia by ∼2 logs. CMCdG also reduced the level of HBV(ETV-R)(L180M/S202G/M204V) viremia by ∼1 log in HBV(ETV-R)(L180M/S202G/M204V)-infected human liver-chimeric mice, while ETV (1 mg/kg/day q.d.) completely failed to reduce the viremia. None of the CMCdG-treated mice had significant drug-related changes in body weights or serum human albumin levels. Structural analyses using homology modeling, semiempirical quantum methods, and molecular dynamics revealed that although ETV triphosphate (TP) forms good van der Waals contacts with L180 and M204 of HBV(WT)(Ce) reverse transcriptase (RT), its contacts with the M180 substitution are totally lost in the HBV(ETV-R)(L180M/S202G/M204V) RT complex. However, CMCdG-TP retains good contacts with both the HBV(WT)(Ce) RT and HBV(ETV-R)(L180M/S202G/M204V) RT complexes. The present data warrant further studies toward the development of CMCdG as a potential therapeutic for patients infected with drug-resistant HBV and shed light on the further development of more potent and safer anti-HBV agents.
- Published
- 2019
20. 7-Deaza-7-fluoro modification confers on 4′-cyano-nucleosides potent activity against entecavir/adefovir-resistant HBV variants and favorable safety
- Author
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Hiroaki Mitsuya, Yasuhito Tanaka, David Venzon, Shuko Murakami, Kohei Yamada, Debananda Das, Stefan G. Sarafianos, Kota Tomaya, Shin-ichiro Hattori, Sanae Hayashi, Masanori Isogawa, and Nobuyo Higashi-Kuwata
- Subjects
Models, Molecular ,0301 basic medicine ,Hepatitis B virus ,Guanine ,030106 microbiology ,Organophosphonates ,Mice, Transgenic ,Viremia ,Antiviral Agents ,Article ,Mice ,03 medical and health sciences ,Hepatitis B, Chronic ,Virology ,Drug Resistance, Viral ,medicine ,Adefovir ,Animals ,Humans ,IC50 ,Southern blot ,Pharmacology ,Nucleoside analogue ,Chemistry ,Adenine ,virus diseases ,Nucleosides ,Hep G2 Cells ,Entecavir ,Viral Load ,medicine.disease ,Molecular biology ,digestive system diseases ,Reverse transcriptase ,030104 developmental biology ,Nucleoside ,medicine.drug - Abstract
We designed, synthesized and identified a novel nucleoside derivative, 4’-C-cyano-7-deaza-7-fluoro-2’-deoxyadenosine (CdFA), which exerts potent anti-HBV activity (IC(50) ~26 nM) with favorable hepatocytotoxicity (CC(50)~56 μM). Southern blot analysis using wild-type HBV (HBV(WT))-encoding-plasmid-transfected HepG2 cells revealed that CdFA efficiently suppresses the production of HBV(WT) (IC(50)=153.7 nM), entecavir (ETV)-resistant HBV carrying L180M/S202G/M204V substitutions (HBV(ETV)(R); IC(50)=373.2 nM), and adefovir dipivoxil (ADV)-resistant HBV carrying A181T/N236T substitutions (HBV(ADV)(R); IC(50=)192.6 nM), whereas ETV and ADV were less potent against HBV(ETV)(R) and HBV(ADV)(R) (IC(50): >1,000 and 4,022.5 nM, respectively). Once-daily peroral administration of CdFA to human-liver-chimeric mice over 14 days (1 mg/kg/day) comparably blocked HBV(WT) and HBV(ETV)(R) viremia by 0.7 and 1.2 logs, respectively, without significant changes in body-weight or serum human-albumin levels, although ETV only slightly suppressed HBV(ETV)(R) viremia (CdFA vs ETV; p=0.032). Molecular modeling suggested that ETV-TP has good nonpolar interactions with HBV(WT) reverse transcriptase (RT(WT))’s Met204 and Asp205, while CdFA-TP fails to interact with Met204, in line with the relatively inferior activity against HBV(WT) of CdFA compared to ETV (IC(50): 0.026 versus 0.003 nM). In contrast, the 4’-cyano of CdFA-TP forms good nonpolar contacts with RT(WT)’s Leu180 and RT(ETV)(R)’s Met180, while ETV-TP loses interactions with RT(ETV)(R)’s Met180, explaining in part why ETV is less potent against HBV(ETV)(R) than CdFA. The present results show that CdFA exerts potent activity against HBV(WT), HBV(ETV)(R) and HBV(ADV)(R) with enhanced safety and that 7-deaza-7-fluoro modification confers potent activity against drug-resistant HBV variants and favorable safety, shedding light to further design more potent and safer anti-HBV nucleoside analogs.
- Published
- 2020
21. A novel central nervous system-penetrating protease inhibitor overcomes human immunodeficiency virus 1 resistance with unprecedented aM to pM potency
- Author
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Hideyuki Saito, Manabu Aoki, Hironori Hayashi, Nobuyo Higashi-Kuwata, Ravikiran S. Yedidi, Shin ichiro Hattori, Arun K. Ghosh, Robert Yarchoan, Noriko Nishida, Debananda Das, Hiroaki Mitsuya, Haydar Bulut, Nobutoki Takamune, Prasanth R. Nyalapatla, David A. Davis, Kanury V. S. Rao, Shogo Misumi, Yuki Takamatsu, Hirofumi Jono, Heather L. Osswald, Kazuya Hasegawa, and Hiromi Aoki-Ogata
- Subjects
Central Nervous System ,0301 basic medicine ,CNS penetration ,QH301-705.5 ,medicine.medical_treatment ,Science ,030106 microbiology ,Central nervous system ,Human immunodeficiency virus (HIV) ,Microbial Sensitivity Tests ,Biology ,medicine.disease_cause ,General Biochemistry, Genetics and Molecular Biology ,Virus ,protease inhibitor ,Inhibitory Concentration 50 ,03 medical and health sciences ,HIV Protease ,Drug Resistance, Viral ,drug-resistant HIV-1 ,medicine ,Animals ,Humans ,Potency ,Biology (General) ,Cells, Cultured ,Microbiology and Infectious Disease ,Protease ,General Immunology and Microbiology ,Cell growth ,GRL-142 ,General Neuroscience ,HIV Protease Inhibitors ,General Medicine ,Virology ,Rats ,3. Good health ,030104 developmental biology ,medicine.anatomical_structure ,HIV-1 ,Medicine ,Target protein ,Intracellular ,Research Article ,Protein Binding - Abstract
Antiretroviral therapy for HIV-1 infection/AIDS has significantly extended the life expectancy of HIV-1-infected individuals and reduced HIV-1 transmission at very high rates. However, certain individuals who initially achieve viral suppression to undetectable levels may eventually suffer treatment failure mainly due to adverse effects and the emergence of drug-resistant HIV-1 variants. Here, we report GRL-142, a novel HIV-1 protease inhibitor containing an unprecedented 6-5-5-ring-fused crown-like tetrahydropyranofuran, which has extremely potent activity against all HIV-1 strains examined with IC50 values of attomolar-to-picomolar concentrations, virtually no effects on cellular growth, extremely high genetic barrier against the emergence of drug-resistant variants, and favorable intracellular and central nervous system penetration. GRL-142 forms optimum polar, van der Waals, and halogen bond interactions with HIV-1 protease and strongly blocks protease dimerization, demonstrating that combined multiple optimizing elements significantly enhance molecular and atomic interactions with a target protein and generate unprecedentedly potent and practically favorable agents.
- Published
- 2017
22. 4′‐modified nucleoside analogs: Potent inhibitors active against entecavir‐resistant hepatitis B virus
- Author
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David Venzon, Hiroaki Mitsuya, Stefan G. Sarafianos, Shuko Murakami, Satoru Kohgo, Manabu Aoki, Kamalendra Singh, Kazuhiro Haraguchi, Masayuki Amano, Kenji Maeda, Satoru Takahashi, Debananda Das, Yoshikazu Sukenaga, Yuki Takamatsu, Nobuyo Higashi-Kuwata, Nicole S. Delino, Yasuhito Tanaka, and Sanae Hayashi
- Subjects
Hepatitis B virus ,Guanine ,Deoxyadenosines ,Hepatology ,virus diseases ,Viremia ,Entecavir ,Transfection ,Biology ,medicine.disease_cause ,medicine.disease ,Virology ,Article ,Reverse transcriptase ,Mice ,Drug Resistance, Viral ,HIV-1 ,medicine ,biology.protein ,Animals ,Nucleoside ,IC50 ,Polymerase ,medicine.drug - Abstract
Certain nucleoside/nucleotide reverse transcriptase (RT) inhibitors (NRTIs) are effective against human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV). However, both viruses often acquire NRTI resistance, making it crucial to develop more-potent agents that offer profound viral suppression. Here, we report that 4′-C-cyano-2-amino-2′-deoxyadenosine (CAdA) is a novel, highly potent inhibitor of both HBV (half maximal inhibitory concentration [IC50] = 0.4 nM) and HIV-1 (IC50 = 0.4 nM). In contrast, the approved anti-HBV NRTI, entecavir (ETV), potently inhibits HBV (IC50 = 0.7 nM), but is much less active against HIV-1 (IC50 = 1,000 nM). Similarly, the highly potent HIV-1 inhibitor, 4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA; IC50 = 0.3 nM) is less active against HBV (IC50 = 160 nM). Southern analysis using Huh-7 cells transfected with HBV-containing plasmids demonstrated that CAdA was potent against both wild-type (IC50 = 7.2 nM) and ETV-resistant HBV (IC50 = 69.6 nM for HBVETV‐RL180M/S202G/M204V), whereas ETV failed to reduce HBVETV‐RL180M/S202G/M204V DNA even at 1 μM. Once-daily peroral administration of CAdA reduced HBVETV‐RL180M/S202G/M204V viremia (P = 0.0005) in human-liver-chimeric/ HBVETV‐RL180M/S202G/M204V–infected mice, whereas ETV completely failed to reduce HBVETV‐RL180M/S202G/M204V viremia. None of the mice had significant drug-related body-weight or serum human-albumin concentration changes. Molecular modeling suggests that a shallower HBV-RT hydrophobic pocket at the polymerase active site can better accommodate the slightly shorter 4′-cyano of CAdA-triphosphate (TP), but not the longer 4′-ethynyl of EFdA-TP. In contrast, the deeper HIV-1-RT pocket can efficiently accommodate the 4′-substitutions of both NRTIs. The ETV-TP's cyclopentyl ring can bind more efficiently at the shallow HBV-RT binding pocket. Conclusion: These data provide insights on the structural and functional associations of HBV- and HIV-1-RTs and show that CAdA may offer new therapeutic options for HBV patients. (Hepatology 2015;62:1024-1036)
- Published
- 2015
23. Author response: A novel central nervous system-penetrating protease inhibitor overcomes human immunodeficiency virus 1 resistance with unprecedented aM to pM potency
- Author
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Manabu Aoki, Hironori Hayashi, Kalapala Venkateswara Rao, Debananda Das, Nobuyo Higashi-Kuwata, Haydar Bulut, Hiromi Aoki-Ogata, Yuki Takamatsu, Ravikiran S Yedidi, David A Davis, Shin-ichiro Hattori, Noriko Nishida, Kazuya Hasegawa, Nobutoki Takamune, Prasanth R Nyalapatla, Heather L Osswald, Hirofumi Jono, Hideyuki Saito, Robert Yarchoan, Shogo Misumi, Arun K Ghosh, and Hiroaki Mitsuya
- Published
- 2017
24. Raltegravir blocks the infectivity of red-fluorescent-protein (mCherry)-labeled HIV-1
- Author
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Hiromi, Ogata-Aoki, Nobuyo, Higashi-Kuwata, Shin-Ichiro, Hattori, Hironori, Hayashi, Matthew, Danish, Manabu, Aoki, Chiemi, Shiotsu, Yumi, Hashiguchi, Akinobu, Hamada, Hisataka, Kobayashi, Hironobu, Ihn, Seiji, Okada, and Hiroaki, Mitsuya
- Subjects
Mice, Knockout ,Anti-HIV Agents ,Genetic Vectors ,virus diseases ,Gene Expression ,Janus Kinase 3 ,HIV Infections ,Mice, SCID ,Viral Load ,Immunohistochemistry ,Article ,Disease Models, Animal ,Luminescent Proteins ,Mice ,Genes, Reporter ,Mice, Inbred NOD ,Raltegravir Potassium ,HIV-1 ,Leukocytes, Mononuclear ,Animals ,Humans ,Post-Exposure Prophylaxis - Abstract
Employing NOD/SCID/Jak3(−/−) mice transplanted with human PBMCs (hNOJ mice) and replication-competent, red-fluorescent-protein (mCherry; mC)-labeled HIV-1(JR-FL) (HIV(mC)), we examined whether early antiretroviral treatment blocked the establishment of HIV-1 infection. The use of hNOJ mice and HIV(mC) enabled us to visually locate infection foci and to examine the early dynamics of HIV(mC) infection without using a large amount of antiretroviral unlike in non-human primate models. Although when raltegravir (RAL) administration was begun 1 day after intraperitoneal (ip) inoculation of HIV(mC), no plasma p24 or plasma HIV-1-RNA (pRNA) were detected in 10 of 12 hNOJ [Formula: see text] mice as assessed on the last day of the 14-day continuous twice-daily RAL administration, all 10 untreated hNOJ(mC) [Formula: see text] mice became positive for p24 and pRNA and had significantly swollen lymph nodes in peritoneal cavity and abundant p24(+)/mC(+)/CD3(+)/CD4(+) T cells and p24(+)/mC(+)/CD68(+) monocytes/macrophages were identified in their omenta and mesenteric lymphoid tissues/lymph nodes upon necropsy of the mice on day 14. In 12 [Formula: see text] mice, no significantly swollen lymph nodes were seen compared to [Formula: see text] mice; however, in the omentum of the 2 [Formula: see text] mice that were positive for pRNA and in site RNA, mC(+)/p24(+)/CD3(+)/CD83(+) cells were identified, suggesting that viral breakthrough occurred later in the observation period. The present data suggest that the use of hNOJ mouse model and HIV(mC) may shed light on the study of early-phase dynamics of HIV-1 infection and cellular events in post-exposure/pre-exposure prophylaxis.
- Published
- 2017
25. Early phase dynamics of traceable mCherry fluorescent protein-carrying HIV-1 infection in human peripheral blood mononuclear cells-transplanted NOD/SCID/Jak3
- Author
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Hironobu Ihn, Tatsuyoshi Kawamura, Seiji Okada, Hiromi Ogata-Aoki, Shin ichiro Hattori, Hironori Hayashi, Matthew Danish, Nobuyo Higashi-Kuwata, Chiemi Shiotsu, Hiroaki Mitsuya, Hisataka Kobayashi, and Manabu Aoki
- Subjects
0301 basic medicine ,Pathology ,medicine.medical_specialty ,Viremia ,HIV Infections ,Nod ,Mice, SCID ,Biology ,Peripheral blood mononuclear cell ,Article ,03 medical and health sciences ,Mice ,In vivo ,Genes, Reporter ,Mice, Inbred NOD ,Virology ,medicine ,Mesenteric lymph nodes ,Animals ,Pharmacology ,Staining and Labeling ,virus diseases ,Janus Kinase 3 ,medicine.disease ,Disease Models, Animal ,Luminescent Proteins ,030104 developmental biology ,medicine.anatomical_structure ,Viral replication ,HIV-1 ,Leukocytes, Mononuclear ,Lymph ,Lymph Nodes ,mCherry - Abstract
We attempted to elucidate early-phase dynamics of HIV-1 infection using replication-competent, red-fluorescent-protein (mCherry)-labeled HIV-1(JR-FL) (HIV(JR-FL) (mC)) and NOD/SCID/Jak3(−/−) mice transplanted with Individual-A's human peripheral blood mononuclear cells (hPBMC)(hNOJ mice). On day 7 following HIV(JR-FL) (mC) inoculation, mCherry-signal-emitting infection foci were readily identified in the subserosa of 10 of 10 HIV(JR-FL) (mC)-inoculated hNOJ mice, although infection foci were not located without the mCherry signal in unlabeled HIV-1(JR-FL)-inoculated mice (n = 6). Even on day 14, infection foci were hardly located in the unlabeled HIV-1(JR-FL)-inoculated mice, while in all of 7 HIV(JR-FL) (mC)-inoculated hNOJ mice examined, mCherry-signal-emitting lymph nodes were easily identified, in which active viral replication was present. On day 14, a significantly larger number of mesenteric lymph nodes were seen in HIV(JR-FL) (mC)-exposed hNOJ mice than in HIV(JR-FL) (mC)-unexposed mice (P = 0.0025). The weights of mesenteric lymph nodes of those HIV(JR-FL) (mC) -exposed hNOJ mice were also greater than those of HIV(JR-FL) (mC)-unexposed mice (P = 0.0005). When hNOJ mice were inoculated with HIV(JR-FL) (mC)-exposed hPBMC from Individual-B, significantly greater viremia was seen than in cell-free HIV(JR-FL)(mC)-inoculated hNOJ mice as examined on day 7. In the lymph nodes of those mice inoculated with HIV(JR-FL)(mC)-exposed hPBMC from Individual-B, a substantial number of Individual-B's HIV(JR-FL)(mC)-infected cells were identified together with Individual-A's cells as examined on day 14. The present HIV(JR-FL) (mC)-infected mouse model represents the first system reported using traceable HIV(JR-FL)(mC) and human target cells, not using SIV or simian cells, which should be of utility in studies of early-phases of HIV-1 transmission and in evaluating the effects of potential agents for post-exposure and pre-exposure and pre-exposure prophylaxis.
- Published
- 2016
26. Distribution of retinylester-storing stellate cells in the arrowtooth halibut, Atheresthes evermanni
- Author
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Haruki Senoo, Nobuyo Higashi-Kuwata, Mitsuharu Yuuda, Hiroshi Sone, Katsuyuki Imai, Mitsuru Sato, Takaharu Seki, Kazuo Koyasu, Kiwamu Yoshikawa, Toshiaki Irie, and Naosuke Kojima
- Subjects
Male ,medicine.medical_specialty ,Kupffer Cells ,Physiology ,medicine.drug_class ,Liver cytology ,Tretinoin ,Flounder ,Biology ,Kidney ,Halibut ,Biochemistry ,Retinoids ,Cecum ,Lipid droplet ,Internal medicine ,medicine ,Animals ,Tissue Distribution ,Retinoid ,Molecular Biology ,Chromatography, High Pressure Liquid ,Pylorus ,biology.organism_classification ,Molecular biology ,Intestines ,Microscopy, Electron ,Endocrinology ,medicine.anatomical_structure ,Liver ,Hepatic stellate cell ,Female ,Atheresthes - Abstract
Hepatic stellate cells play a major role in retinylester storage in mammals, but the retinoid-storing state in nonmammalian vertebrates remains to be elucidated. In this study, we examined retinoids and retinoid-storing cells in the arrowtooth halibut, Atheresthes evermanni. High-performance liquid chromatography analyses revealed the highest concentrations of stored retinoids (retinol and retinylester, 6199 nmol/g) in the pyloric cecum, a teleost-specific organ protruding from the intestine adjacent to the pylorus. Considerable amounts of retinoids were also stored in the intestine (3355 nmol/g) and liver (1891 nmol/g), and small amounts in the kidney (102 nmol/g). Very small amounts or no retinoids were detected in the heart, gill, skeletal muscle, and gonads (less than 2 nmol/g). Use of gold chloride staining and fluorescence microscopy to detect retinoid autofluorescence showed that, in the pyloric cecum and intestine, retinoid-storing cells were localized in the lamina propria mucosae. Under electron microscopy, cells containing well-developed lipid droplets, which are common morphological characteristics of the hepatic stellate cells of mammals, were observed in the lamina propria mucosae of the pyloric cecum. Thus, the distribution of stellate cells with retinoid-storing capacity differs between this halibut and mammals, suggesting that the retinoid-storing site has shifted during vertebrate evolution.
- Published
- 2006
27. Alternatively activated macrophages (M2 macrophages) in the skin of patient with localized scleroderma
- Author
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Hironobu Ihn, Takamitsu Makino, Motohiro Takeya, Nobuyo Higashi-Kuwata, and Yuji Inoue
- Subjects
Adult ,Pathology ,medicine.medical_specialty ,Biopsy ,Adipose tissue macrophages ,Antigens, Differentiation, Myelomonocytic ,Receptors, Cell Surface ,Inflammation ,Dermatology ,Biochemistry ,Scleroderma ,Scleroderma, Localized ,Antigens, CD ,Fibrosis ,medicine ,Humans ,Macrophage ,Localized Scleroderma ,Molecular Biology ,Skin ,Scleroderma, Systemic ,integumentary system ,medicine.diagnostic_test ,business.industry ,CD68 ,Macrophages ,Scavenger Receptors, Class A ,Middle Aged ,medicine.disease ,Immunohistochemistry ,Immunology ,Skin biopsy ,medicine.symptom ,business - Abstract
Localized scleroderma is a connective tissue disorder that is limited to the skin and subcutaneous tissue. Macrophages have been reported to be particularly activated in patients with skin disease including systemic sclerosis and are potentially important sources for fibrosis-inducing cytokines, such as transforming growth factor beta. To clarify the features of immunohistochemical characterization of the immune cell infiltrates in localized scleroderma focusing on macrophages, skin biopsy specimens were analysed by immunohistochemistry. The number of cells stained with monoclonal antibodies, CD68, CD163 and CD204, was calculated. An evident macrophage infiltrate and increased number of alternatively activated macrophages (M2 macrophages) in their fibrotic areas were observed along with their severity of inflammation. This study revealed that alternatively activated macrophages (M2 macrophages) may be a potential source of fibrosis-inducing cytokines in localized scleroderma, and may play a crucial role in the pathogenesis of localized scleroderma.
- Published
- 2009
28. Expression pattern of VEGFR-1, -2, -3 and D2-40 protein in the skin of patients with systemic sclerosis
- Author
-
Hironobu Ihn, Yuji Inoue, Takamitsu Makino, and Nobuyo Higashi-Kuwata
- Subjects
Adult ,Male ,Pathology ,medicine.medical_specialty ,Biopsy ,Lumen (anatomy) ,Dermatology ,Statistics, Nonparametric ,Microcirculation ,Pathogenesis ,Immunoenzyme Techniques ,Antibodies, Monoclonal, Murine-Derived ,Medicine ,Humans ,skin and connective tissue diseases ,Aged ,Sclerosis ,Vascular Endothelial Growth Factor Receptor-1 ,integumentary system ,medicine.diagnostic_test ,business.industry ,Middle Aged ,Vascular Endothelial Growth Factor Receptor-3 ,Vascular Endothelial Growth Factor Receptor-2 ,Endothelial stem cell ,Lymphatic system ,embryonic structures ,Skin biopsy ,cardiovascular system ,Immunohistochemistry ,Female ,business ,Biomarkers - Abstract
The earliest clinical symptoms of systemic sclerosis (SSc) relate to disturbances in the peripheral vascular system. However, detailed examination of the microcirculation including the lymphatics in the skin of patients with SSc has not been reported. The aim of our study was to examine the expression patterns of vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, VEGFR-3 and the lymphatic endothelial cell marker D2-40 in the skin of SSc patients. Skin biopsy specimens from nine patients with SSc were analysed by immunohistochemistry using antibodies against VEGFR-1, VEGFR-2, VEGFR-3 and D2-40 protein. The lumen area of lymphatic vessels was measured. The data were statistically analysed using the Mann-Whitney U test, Wilcoxon signed-ranks or Spearman's rank correlation coefficient method. The intensity of VEGFR-2 and VEGFR-3 staining in the skin of patients with SSc was significantly higher than that in healthy controls. The lumen area of lymphatic vessels in the skin of patients with SSc was significantly larger than that in healthy controls. This study details the expression of VEGFR and D2-40 in the skin of patients with SSc, and highlights a possible role of microcirculatory dysfunction in the pathogenesis of systemic sclerosis.
- Published
- 2011
29. Characterization of monocyte/macrophage subsets in the skin and peripheral blood derived from patients with systemic sclerosis
- Author
-
Yoshihiro Komohara, Hironobu Ihn, Satoshi Fukushima, Nobuyo Higashi-Kuwata, Faith C. Muchemwa, Yuji Inoue, Takamitsu Makino, Yuji Yonemura, Motohiro Takeya, Masatoshi Jinnin, and Hiroaki Mitsuya
- Subjects
education.field_of_study ,Pathology ,medicine.medical_specialty ,medicine.diagnostic_test ,integumentary system ,CD68 ,CD14 ,Monocyte ,Population ,Immunology ,Biology ,Peripheral blood mononuclear cell ,medicine.anatomical_structure ,Rheumatology ,Skin biopsy ,medicine ,Macrophage ,Immunology and Allergy ,education ,skin and connective tissue diseases ,CD163 ,Research Article - Abstract
Introduction Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc. Methods Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means. Results In the skin from SSc patients, the number of CD163+ cells or CD204+ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14+ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14+ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163+ cells belong to CD14brightCD204+ population. Conclusions This is the first report indicating CD163+ or CD204+ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14brightCD163+CD204+ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163+ or CD204+ macrophages in the skin.
- Published
- 2010
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