Publications by authors named "Tohru Nagamitsu"

53 Publications

Impairment of endothelium-dependent vasodilator function of retinal blood vessels in adult rats with a history of retinopathy of prematurity.

J Pharmacol Sci 2021 Aug 7;146(4):233-243. Epub 2021 May 7.

Department of Molecular Pharmacology, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

Retinopathy of prematurity (ROP) is a proliferative retinal vascular disease, initiated by delayed retinal vascular growth after premature birth. In the majority of cases, ROP resolves spontaneously; however, a history of ROP may increase the risk of long-term visual problems. In this study, we evaluated the endothelial function of retinal blood vessels in adult rats with a history of ROP. ROP was induced in rats by subcutaneous injection of a vascular endothelial growth factor receptor tyrosine kinase inhibitor (KRN633) on postnatal day (P) 7 and P8. On P56, vasodilator responses to acetylcholine, GSK1016790A (an activator of transient receptor potential vanilloid 4 channels), NOR3 (a nitric oxide [NO] donor), and salbutamol (a β-adrenoceptor agonist) were assessed. Compared to age-matched controls, retinal vasodilator responses to acetylcholine and GSK1016790A were attenuated in P56 rats with a history of ROP. No attenuation of acetylcholine-induced retinal vasodilator response was observed under inhibition of NO synthase. Retinal vasodilator responses to NOR3 and salbutamol were unaffected. These results suggest that the production of and/or release of NO is impaired in retinal blood vessels in adult rats with a history of ROP. A history of ROP might increase the risk of impaired retinal circulation in adulthood.
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http://dx.doi.org/10.1016/j.jphs.2021.04.008DOI Listing
August 2021

Mixture of clopidogrel bisulfate and magnesium oxide tablets reduces clopidogrel dose administered through a feeding tube.

J Pharm Health Care Sci 2021 May 4;7(1):18. Epub 2021 May 4.

Department of Clinical Pharmacy, Research and Education Center for Clinical Pharmacy, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

Background: In clinical practice, a mixed suspension of clopidogrel bisulfate and magnesium oxide (MgO) tablets is administered frequently via a feeding tube. However, there is no report on the changes occurring when suspensions of these two drugs are combined, including the effects or potential decrease in dose following tube administration. Thus, the purpose of our study was to investigate the (i) changes caused by mixing clopidogrel bisulfate (ion form) and MgO tablets and (ii) effects on the administered clopidogrel dose after passing through a feeding tube.

Methods: The molecular structure of clopidogrel generated in a mixture of clopidogrel bisulfate and a basic compound, such as sodium bicarbonate or MgO tablet, was determined by H-NMR after extraction and purification. The suspension of clopidogrel bisulfate tablet alone and the mixed suspension of clopidogrel bisulfate tablet and MgO tablet were passed through a feeding tube. We compared the yield of the molecular form of clopidogrel from each passed fraction.

Results: The substance obtained from the mixture of clopidogrel bisulfate tablet and sodium bicarbonate or MgO tablet was identified as the molecular form of clopidogrel, and chemical degradation did not occur under these conditions. In the tube passage test, the yield of clopidogrel (molecular form) from the mixture of clopidogrel bisulfate and MgO tablets was lower than that from the suspension of clopidogrel bisulfate tablet alone.

Conclusions: The mixture of clopidogrel bisulfate and MgO tablets caused a considerable reduction in the administered dose passed through the feeding tube. Therefore, it is recommended to administer the suspensions of clopidogrel bisulfate and MgO tablets separately for safe and effective pharmacotherapy.
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http://dx.doi.org/10.1186/s40780-021-00202-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8094535PMC
May 2021

One-Pot γ-Lactonization of Homopropargyl Alcohols via Intramolecular Ketene Trapping.

Org Lett 2021 04 22;23(7):2831-2835. Epub 2021 Mar 22.

Laboratory of Synthetic Natural Products Chemistry and Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

A one-pot γ-lactonization of homopropargyl alcohols via an alkyne deprotonation/boronation/oxidation sequence has been developed. Oxidation of the generated alkynyl boronate affords the corresponding ketene intermediate, which is trapped by the adjacent hydroxy group to furnish the γ-lactone. We have optimized the conditions as well as examined the substrate scope and synthetic applications of this efficient one-pot lactonization.
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http://dx.doi.org/10.1021/acs.orglett.1c00840DOI Listing
April 2021

Pharmacological depletion of retinal neurons prevents vertical angiogenic sprouting without affecting the superficial vascular plexus.

Dev Dyn 2021 Apr 2;250(4):497-512. Epub 2020 Nov 2.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

Background: In mice, a tri-layered (superficial, intermediate, and deep) vascular structure is formed in the retina during the third postnatal week. Short-term treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor inhibitors delays the formation of superficial vascular plexus and this allows us to investigate the developmental process of superficial and deep vascular plexuses at the same time. Using this model, we examined the effect of pharmacological depletion of retinal neurons on the formation of superficial and deep vascular plexuses.

Results: Neuronal cell loss induced by an intravitreal injection of N-methyl-d-aspartic acid on postnatal day (P) 8 delayed vascular development in the deep layer but not in the superficial layer in mice treated with KRN633, a VEGF receptor inhibitor, on P0 and P1. In KRN633-treated mice, neuronal cell loss decreased the number of vertical sprouts originating from the superficial plexus without affecting the number of angiogenic sprouts growing in front. Neuronal cell loss did not impair networks of fibronectin and astrocytes in the superficial layer.

Conclusions: Our results suggest that inner retinal neurons play a crucial role in forming the deep vascular plexus by directing the sprouts from the superficial blood vessels to the deep layer.
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http://dx.doi.org/10.1002/dvdy.263DOI Listing
April 2021

The process of revascularization in the neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

Cell Tissue Res 2020 Dec 8;382(3):529-549. Epub 2020 Sep 8.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

Misdirected vascular growth frequently occurs in the neovascular diseases in the retina. However, the mechanisms are still not fully understood. In the present study, we created capillary-free zones in the central and peripheral retinas in neonatal mice by pharmacological blockade of vascular endothelial growth factor (VEGF) signaling. Using this model, we investigated the process and mechanisms of revascularization in the central and peripheral avascular areas. After the completion of a 2-day treatment with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5, revascularization started on P8 in the central avascular area where capillaries had been dropped out. The expression levels of VEGF were higher in the peripheral than in the central avascular area. However, the expansion of the vasculature in the peripheral avascular retina remained suppressed until revascularization had been completed in the central avascular area. Additionally, we found disorganized endothelial cell division, misdirected blood vessels with irregular diameters, and abnormal fibronectin networks at the border of the vascular front and the avascular retina. In the central avascular area, a slight amount of fibronectin as non-vascular component re-formed to provide a scaffold for revascularization. Mechanistic analysis revealed that higher levels of VEGF attenuated the migratory response of endothelial cells without decreasing the proliferative activity. These results suggest that the presence of concentration range of VEGF, which enhances both migration and proliferation of the endothelial cells, and the structurally normal fibronectin network contribute to determine the proper direction of angiogenesis.
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http://dx.doi.org/10.1007/s00441-020-03276-9DOI Listing
December 2020

Synthesis and Absolute Configuration of Habiterpenol.

Org Lett 2020 07 17;22(13):5131-5134. Epub 2020 Jun 17.

Laboratory of Synthetic Natural Products Chemistry and Medicinal Research Laboratories, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

The synthesis of habiterpenol, a G2 checkpoint inhibitor, was achieved through the stereoselective Ti(III)-mediated radical cyclization of a β-epoxide as the key reaction. Moreover, the absolute configuration of habiterpenol was determined.
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http://dx.doi.org/10.1021/acs.orglett.0c01736DOI Listing
July 2020

Abnormal Vascular Phenotypes Associated with the Timing of Interruption of Retinal Vascular Development in Rats.

Biol Pharm Bull 2020 ;43(5):859-863

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences.

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.
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http://dx.doi.org/10.1248/bpb.b19-01065DOI Listing
January 2021

Changes in components of the neurovascular unit in the retina in a rat model of retinopathy of prematurity.

Cell Tissue Res 2020 Mar 2;379(3):473-486. Epub 2019 Dec 2.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

An impairment of cellular interactions between the elements of the neurovascular unit contributes to the onset and/or progression of retinal diseases. The present study aims to examine how elements of the neurovascular unit are altered in a rat model of retinopathy of prematurity (ROP). Neonatal rats were treated subcutaneously with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8 to induce ROP. Morphological assessments were performed of blood vessels, astrocytes and neuronal cells in the retina. Aggressive angiogenesis, tortuous arteries and enlarged veins were observed in the retinal vasculature of KRN633-treated (ROP) rats from P14 to P28, compared to age-matched control (vehicle-treated) animals. Morphological abnormalities in the retinal vasculature showed a tendency toward spontaneous recovery from P28 to P35 in ROP rats. Immunofluorescence staining for glial fibrillary acidic protein and Pax2 (astrocyte markers) revealed that morphological changes to and a reduction in the number of astrocytes occurred in ROP rats. The developmental cell death was slightly accelerated in ROP rats; however, no visible changes in the morphology of retinal layers were observed on P35. The abnormalities in astrocytes might contribute, at least in part, to the formation of abnormal retinal blood vessels and the pathogenesis of ROP.
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http://dx.doi.org/10.1007/s00441-019-03112-9DOI Listing
March 2020

An Unusual Extender Unit Is Incorporated into the Modular Polyketide Synthase of Scopranones Biosynthesis.

Biochemistry 2019 12 6;58(50):5066-5073. Epub 2019 Dec 6.

Kitasato Institute for Life Sciences , Kitasato University , 1-15-1 Kitasato, Minami-ku , Sagamihara , Kanagawa 252-0373 , Japan.

Scopranones, produced by sp. BYK-11038, are the novel bone morphogenetic protein inhibitors characterized by atypical two scoop-like moieties and a 3-furanone moiety. Two scoop-like moieties connected to a 3-furanone have not previously been reported in natural products, and their biosynthesis must occur via a unique pathway. Feeding experiments using C-labeled precursors indicated that scopranones were synthesized from three acetates and three butyrates in polyketide-type biosynthesis. Genome mining of sp. BYK-11038 revealed that the candidate biosynthetic gene cluster contains 21 open reading frames (ORFs), including three modular polyketide synthases (PKSs; SprA, SprB, and SprC), which were composed of 4 modules with one loading module and 18 additional ORFs (SprD to SprU) spanning a distance of 55 kbp. The characterization of in-frame deletion mutants and feeding experiments with the predicted extender units indicated that two genes, and , encoding discrete 3-oxoacyl-ACP synthases, and a gene, , encoding crotonyl-CoA reductase, were involved in assembling an unusual C branched extender unit, 2-(2-ethylbutyl)malonyl-CoA. Additionally, three ORFs, , , and , encoding cytochrome P450s and a monooxygenase, are important tailoring enzymes in post-PKS modification. SprT is an essential enzyme for decarboxylative ring contraction via oxidation, which converts the 2-pyranone to a 3-furanone.
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http://dx.doi.org/10.1021/acs.biochem.9b00908DOI Listing
December 2019

Attenuation of Retinal Endothelial Vasodilator Function in a Rat Model of Retinopathy of Prematurity.

Curr Eye Res 2019 12 18;44(12):1360-1368. Epub 2019 Jul 18.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

: Retinopathy of prematurity (ROP) is characterized by morphological abnormalities in retinal blood vessels, but how an episode of ROP affects vascular function remains to be fully elucidated. The purpose of the present study was to assess the distribution of pericyte/smooth muscle in retinal blood vessels and retinal vasodilator responses in a rat model of ROP.: ROP was induced in rats by the subcutaneous injection of the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg) on postnatal day (P) 7 and P8. The distribution of pericyte/smooth muscle in retinal blood vessels was examined on P14 and P35 by immunohistochemistry. Retinal vasodilator responses were assessed on P35 by measuring the diameter of retinal arterioles in fundus images.: In retinas of KRN633-treated (ROP) rats, progressive angiogenesis, tortuous arteries, enlarged veins, and enhanced expression of α-smooth muscle actin in pericytes on capillaries and veins were observed on P14. These abnormalities in retinal vasculature showed a tendency to normalize by P35. Vasodilation of retinal arterioles induced by acetylcholine, an endothelium-dependent vasodilator, was smaller in P35 ROP rats than age-matched controls, whereas retinal vasodilator responses to the nitric oxide (NO) donor NOR3 were unaltered.: Phenotypic changes in pericytes occur in the ROP model rats and endothelium-dependent vasodilatory mechanisms in retinal blood vessels are impaired. The impaired vasodilator function may contribute to the progression and pathogenesis of ROP.
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http://dx.doi.org/10.1080/02713683.2019.1641825DOI Listing
December 2019

Total Synthesis and Absolute Configuration of Simpotentin, a Potentiator of Amphotericin B Activity.

Org Lett 2019 07 27;21(14):5596-5599. Epub 2019 Jun 27.

Laboratory of Synthetic Natural Products Chemistry and Medicinal Research Laboratories, School of Pharmacy , Kitasato University , 5-9-1 Shirokane , Minato-ku, Tokyo 108-8641 , Japan.

The total synthesis of simpotentin (), a new potentiator of amphotericin B activity against , was achieved. Our research results enabled the access of all stereoisomers of and the elucidation of the unknown absolute configuration of . Furthermore, one of the stereoisomers is a better amphotericin B potentiator than and is an excellent lead compound for the development of a novel amphotericin B potentiator.
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http://dx.doi.org/10.1021/acs.orglett.9b01945DOI Listing
July 2019

Establishment of an abnormal vascular patterning model in the mouse retina.

J Pharmacol Sci 2018 Apr 14;136(4):177-188. Epub 2018 Mar 14.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

Abnormalities in retinal blood vessels and neuronal function persist in eyes undergoing retinopathy of prematurity. In this study, we examined morphological and functional changes in retinal blood vessels and neurons in mice that had undergone short-term interruption of retinal vascular development through inhibition of vascular endothelial growth factor (VEGF) signaling. In mice treated with the VEGF receptor tyrosine kinase inhibitor KRN633 on postnatal day (P) 0 and 1, the vascular density in the retinal surface increased by P12, but development of deep retinal vascular plexus and choroidal vasculature was delayed until P14. Overall retinal morphology was mostly normal in KRN633-treated mice during the observation period (∼P28), with the exception of P8 and P14. On P28, abnormalities in retinal vascular patterns were evident, but electroretinogram and retinal blood perfusion were within the normal range. Abnormal architecture of retinal vasculature disturbs retinal hemodynamics; therefore, mice treated postnatally with VEGF receptor inhibitors could serve as an animal model for studying the regulatory mechanism of local retinal blood flow and the effect of persistent abnormal retinal vascular patterns on the risk of onset of retinal ischemia.
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http://dx.doi.org/10.1016/j.jphs.2018.03.002DOI Listing
April 2018

Retinal neuronal cell loss prevents abnormal retinal vascular growth in a rat model of retinopathy of prematurity.

Exp Eye Res 2018 03 2;168:115-127. Epub 2018 Feb 2.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

A short-term blockade of the vascular endothelial growth factor (VEGF)-mediated pathway in neonatal rats results in formation of severe retinopathy of prematurity (ROP)-like retinal blood vessels. The present study aimed to examine the role of retinal neurons in the formation of abnormal retinal blood vessels. Newborn rats were treated subcutaneously with the VEGF receptor tyrosine kinase inhibitor, KRN633 (10 mg/kg), or its vehicle (0.5% methylcellulose in water) on postnatal day (P) 7 and P8. To induce excitotoxic loss of retinal neurons, N-methyl-D-aspartic acid (NMDA) was injected into the vitreous chamber of the eye on P9. Changes in retinal morphology, blood vessels, and proliferative status of vascular cells were evaluated on P11 and P14. The number of cells in the ganglion cell layer and the thickness of the inner plexiform layer and inner nuclear layer were significantly decreased 2 days (P11) after NMDA treatment. The pattern and degree of NMDA-induced changes in retinal morphology were similar between vehicle-treated (control) and KRN633-treated (ROP) rats. In ROP rats, increases in the density of capillaries, the tortuosity index of arteries, and the proliferating vascular cells were observed on P14. The expansion of the endothelial cell network was prevented, and the capillary density and the number of proliferating cells were reduced in NMDA-treated retinas of both control and ROP rats. Following NMDA-induced neuronal cell loss, no ROP-like blood vessels were observed in the retinas. These results suggest that retinal neurons play an important role in the formation of normal and ROP-like retinal blood vessels.
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http://dx.doi.org/10.1016/j.exer.2017.12.007DOI Listing
March 2018

Design and Synthesis of A-Ring Simplified Pyripyropene A Analogues as Potent and Selective Synthetic SOAT2 Inhibitors.

ChemMedChem 2018 03 30;13(5):411-421. Epub 2018 Jan 30.

Department of Synthetic Natural Products Chemistry, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

Currently, pyripyropene A, which is isolated from the culture broth of Aspergillus fumigatus FO-1289, is the only compound known to strongly and selectively inhibit the isozyme sterol O-acyltransferase 2 (SOAT2). To aid in the development of new cholesterol-lowering or anti-atherosclerotic agents, new A-ring simplified pyripyropene A analogues have been designed and synthesized based on total synthesis, and the results of structure-activity relationship studies of pyripyropene A. Among the analogues, two A-ring simplified pyripyropene A analogues exhibited equally efficient SOAT2 inhibitory activity to that of natural pyripyropene A. These new analogues are the most potent and selective SOAT2 inhibitors to be used as synthetic compounds and attractive seed compounds for the development of drug for dyslipidemia, including atherosclerotic disease and steatosis.
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http://dx.doi.org/10.1002/cmdc.201700645DOI Listing
March 2018

Transient phenotypic changes in endothelial cells and pericytes in neonatal mouse retina following short-term blockade of vascular endothelial growth factor receptors.

Dev Dyn 2018 05 5;247(5):699-711. Epub 2018 Jan 5.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

Background: A short-term interruption of vascular development causes structural abnormalities in retinal vasculature. However, the detailed changes in vascular components (endothelial cells, pericytes, and basement membranes) remain to be fully determined. The present study aimed to provide a detailed description of morphological changes in vascular components following a short-term interruption of retinal vascular development in mice.

Results: Two-day treatment of neonatal mice with the vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor KRN633 (10 mg/kg, subcutaneously) on postnatal day (P)0 and P1 (P0/1) and P4 and P5 (P4/5) induced different degrees and patterns of impairment of retinal vascular development. Three days after completion of the treatment, the delayed radial vascular growth occurred in P0/1 group mice, whereas in P4/5 group mice, revascularization preferentially occurred in the central avascular area, and radial vascular growth remained suppressed by P10. Differences in α-smooth muscle actin expression in pericytes were noted in the processes between normal vascular formation and vascular regrowth. The changes in vascular cells were associated with the hypoxia-induced enhancement of VEGF expression in the superficial retinal layer.

Conclusions: These findings suggest that the phenotype of vascular cells is altered following a short-term interruption of vascular development in the retina. Developmental Dynamics 247:699-711, 2018. © 2017 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24614DOI Listing
May 2018

Scopranones with Two Atypical Scooplike Moieties Produced by Streptomyces sp. BYK-11038.

Org Lett 2017 11;19(21):5980-5983

Graduate School of Pharmaceutical Sciences, Kitasato University , 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

Three new compounds, designated scopranones A-C, were isolated from the culture broth of a soil isolate, Streptomyces sp. BYK-11038, and shown to be inhibitors of bone morphogenetic protein (BMP) induced alkaline phosphatase activity in a BMP receptor mutant cell line. The structures were elucidated using NMR and other spectral data. The scopranones have an unusual structure with two atypical scooplike moieties linked at the tails to form part of a unique 3-furanone ring.
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http://dx.doi.org/10.1021/acs.orglett.7b03003DOI Listing
November 2017

Development of a new air-stable structure-simplified nafuredin-γ analog as a potent and selective nematode complex I inhibitor.

J Antibiot (Tokyo) 2017 May 22;70(5):647-654. Epub 2017 Feb 22.

Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.

Nafuredin-γ, obtained from natural nafuredin, has demonstrated a potent and selective inhibitory activity against nematode complex I. However, nafuredin-γ is unstable in air since its conjugated dienes are oxygen-labile. The instability in air was naturally solved by the synthesis of structure-simplified nafuredin-γ analogs without conjugated dienes. However, these modified analogs showed lower complex I inhibitory activities. Therefore, new air-stable structure-simplified nafuredin-γ analogs were designed and synthesized herein. Among all analogs synthesized, the one bearing a unique 1-azabicyclo[3.1.0]hexane scaffold showed the highest inhibitory activity (IC=170 nM) while presenting high selectivity against nematode complex I.
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http://dx.doi.org/10.1038/ja.2017.16DOI Listing
May 2017

A delay in vascularization induces abnormal astrocyte proliferation and migration in the mouse retina.

Dev Dyn 2017 03 3;246(3):186-200. Epub 2017 Feb 3.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, Tokyo, Japan.

Background: Astrocytes migrate into the retina through the optic nerve head by means of the axons of retinal ganglion cells, and spread radially toward the peripheral retina. Endothelial cells migrate along the astrocyte cellular network to form the retinal surface vasculature. Here, we examined the effects of a delay in retinal vascularization on the migration and proliferation status of astrocytes in mice.

Results: A dose-dependent delay in retinal vascularization was observed in mice that had been treated with KRN633 (1-10 mg/kg), a VEGF receptor inhibitor, on the day of birth and on the following day. Delayed vascularization resulted in a delay in the astrocyte network formation, and an increase in astrocyte number in the optic nerve head and the vascular front. The increase in the number of astrocytes may be attributed to increased proliferation and delayed migration. These abnormalities in astrocyte behavior correlated with the degree of delay in retinal vascularization. The vascularization delay also led to retinal hypoxia, which subsequently stimulated VEGF leading to an increase in vascular density.

Conclusions: These findings suggest that a delay in normal vascularization leads to abnormal astrocyte behavior, which results in the formation of abnormal astrocyte and endothelial cell networks in the mouse retina. Developmental Dynamics 246:186-200, 2017. © 2016 Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/dvdy.24484DOI Listing
March 2017

Synthesis and Structural Revision of Cyslabdan.

Chem Pharm Bull (Tokyo) 2016 ;64(9):1370-7

Graduate School of Pharmaceutical Sciences, Kitasato University.

Cyslabdan was isolated from the culture broth of Streptomyces sp. K04-0144 as a new potentiator of imipenem activity against methicillin-resistant Staphylococcus aureus. We accomplished the synthesis of cyslabdan according to a previously reported structure. However, we subsequently found that this structure was incorrect; our analysis of natural cyslabdan showed that it possessed R stereochemistry at the C8 position, not S, as had previously been reported. Thus, we completed the protecting-group-free synthesis of the correct structure of cyslabdan, which is described herein.
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http://dx.doi.org/10.1248/cpb.c16-00382DOI Listing
February 2017

Simplifungin and Valsafungins, Antifungal Antibiotics of Fungal Origin.

J Org Chem 2016 09 19;81(17):7373-83. Epub 2016 Aug 19.

Graduate School of Pharmaceutical Sciences, Kitasato University , 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

The targets of antifungal antibiotics in clinical use are more limited than those of antibacterial antibiotics. Therefore, new antifungal antibiotics with different mechanisms of action are desired. In the course of our screening for antifungal antibiotics of microbial origins, new antifungal antibiotics, simplifungin (1) and valsafungins A (2) and B (3), were isolated from cultures of the fungal strains Simplicillium minatense FKI-4981 and Valsaceae sp. FKH-53, respectively. The structures of 1 to 3 including their absolute stereochemistries were elucidated using various spectral analyses including NMR and collision-induced dissociation (CID)-MS/MS as well as chemical approaches including modifications to the Mosher's method. They were structurally related to myriocin. They inhibited the growth of yeast-like and zygomycetous fungi with MICs ranging between 0.125 and 8.0 μg/mL. An examination of their mechanisms of action by the newly established assay using LC-MS revealed that 1 and 2 inhibited serine palmitoyltransferase activity, which is involved in sphingolipid biosynthesis, with IC50 values of 224 and 24 nM, respectively.
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http://dx.doi.org/10.1021/acs.joc.6b00952DOI Listing
September 2016

Biosynthesis of mercapturic acid derivative of the labdane-type diterpene, cyslabdan that potentiates imipenem activity against methicillin-resistant Staphylococcus aureus: cyslabdan is generated by mycothiol-mediated xenobiotic detoxification.

J Ind Microbiol Biotechnol 2016 Mar 27;43(2-3):325-42. Epub 2015 Oct 27.

School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.

Genome mining of cyslabdan-producing Streptomyces cyslabdanicus K04-0144 revealed that a set of four genes, cldA, cldB, cldC, and cldD (the cld cluster), which formed a single transcriptional unit, were involved in the biosynthesis of cyslabdan that potentiates imipenem activity against methicillin-resistant Staphylococcus aureus. Experimental studies supported the heterologous expression of the cld cluster of S. cyslabdanicus K04-0144 in S. avermitilis SUKA22, and transformants carrying the cld cluster produced not only cyslabdan A (1), but also its new derivatives, 17-hydroxyl-1 (2) and 2-hydroxyl-1 (3), in the culture broth. An analysis of diterpene metabolites in the mycelia showed that a large amount of a novel intermediate had accumulated and its structure was elucidated as (7S, 8S, 12E)-8,17-epoxy-7-hydroxylabda-12,14-diene (4). The cld-like cluster (rmn cluster) was also detected in the genome of S. anulatus GM95 by searching our in-house genome databases, and the heterologous expression of the rmn cluster in S. avermitilis SUAK22 demonstrated that the rmn cluster was involved in the biosynthesis of the labdane-type bicyclic diterpene, raimonol (7). CldA/RmnA catalyzed the generation of geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and isopentenyl diphosphate. CldB/RmnB converted GGPP to (+)-copalyl diphosphate, and CldD/RmnD generated labda-8(17),12(E),14-triene (5). CldC introduced two oxygen atoms at C-7 and C-8,17 to generate 4, while RmnC hydroxylated 5 at C-7 to generate 7. The heterologous expression of the cld cluster suggested that four gene products catalyzed to generate 4, but not 1. The deletion mutant of the gene encoding the mycothiol (MSH)-S-conjugate amidase (mca) of S. avermitilis SUKA22 carrying the cld cluster failed to produce 1, but accumulated 4 in the mycelia, whereas S. avermitilis SUKA22 and its mca-deletion mutant carrying the cld cluster both produced the MSH-S-conjugate of 4. The intermediate 4 was converted into the MSH-S-conjugate with MSH, which was achieved through a non-enzymatic nucleophilic reaction. The MSH-S-conjugate of 4 generated was further hydrolyzed to generate the mercapturic acid derivative, 1, by MSH-S-conjugate amidase and 1 was excreted from the mycelia.
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http://dx.doi.org/10.1007/s10295-015-1694-6DOI Listing
March 2016

New pyripyropene A derivatives, highly SOAT2-selective inhibitors, improve hypercholesterolemia and atherosclerosis in atherogenic mouse models.

J Pharmacol Exp Ther 2015 Nov 3;355(2):299-307. Epub 2015 Sep 3.

Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan (T.O., M.O., T.N., D.M., H.T.); Department of Medicine, Jichi Medical University, Tochigi, Japan (T.O., H.Y., S.I.); and Department of Internal Medicine, Section on Molecular Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina (T.O., M.A.D., L.L.R.)

Sterol O-acyltransferase 2 (SOAT2; also known as ACAT2) is considered as a new therapeutic target for the treatment or prevention of hypercholesterolemia and atherosclerosis. Fungal pyripyropene A (PPPA: 1,7,11-triacyl type), the first SOAT2-selective inhibitor, proved orally active in vivo using atherogenic mouse models. The purpose of the present study was to demonstrate that the PPPA derivatives (PRDs) prove more effective in the mouse models than PPPA. Among 196 semisynthetic PPPA derivatives, potent, SOAT2-selective, and stable PRDs were selected. In vivo antiatherosclerotic activity of selected PRDs was tested in apolipoprotein E knockout (Apoe(-/-)) mice or low-density lipoprotein receptor knockout (Ldlr(-/-)) mice fed a cholesterol-enriched diet (0.2% cholesterol and 21% fat) for 12 weeks. During the PRD treatments, no detrimental side effects were observed. Among three PRDs, Apoe(-/-) mice treated with PRD125 (1-,11-O-benzylidene type) at 1 mg/kg/day had significantly lower total plasma cholesterol concentration by 57.9 ± 9.3%; further, the ratio of cholesteryl oleate to cholesteryl linoleate in low-density lipoprotein was lower by 55.6 ± 7.5%, respectively. The hepatic cholesteryl ester levels and SOAT2 activity in the small intestines and livers of the PRD-treated mice were selectively lowered. The atherosclerotic lesion areas in the aortae of PRD125-treated mice were significantly lower at 62.2 ± 13.1%, respectively. Furthermore, both PRDs were also orally active in atherogenic Ldlr(-/-) mice. Among the PRDs tested, PRD125 was the most potent in both mouse models. These results suggest that SOAT2-selective inhibitors such as PRD125 have a high potential as poststatin agents for treatment and/or prevention in patients with atherosclerosis and hypercholesterolemia.
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http://dx.doi.org/10.1124/jpet.115.227348DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613958PMC
November 2015

Design, synthesis, and biological evaluation of air-stable nafuredin-γ analogs as complex I inhibitors.

Bioorg Med Chem 2015 Mar 22;23(5):932-43. Epub 2015 Jan 22.

Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

Nafuredin-γ (2), converted from nafuredin (1) under mild basic conditions, demonstrates potent and selective inhibitory activity against helminth complex I. However, 2 is unstable in air because the conjugated dienes are oxygen-labile. To address this, we designed and synthesized air-stable nafuredin-γ analogs. Although the complex I inhibitory activities of all the new nafuredin-γ analogs were lower than that of 2, all were in the high nM range (IC50: 300-820nM).
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http://dx.doi.org/10.1016/j.bmc.2015.01.030DOI Listing
March 2015

Novel terpenes generated by heterologous expression of bacterial terpene synthase genes in an engineered Streptomyces host.

J Antibiot (Tokyo) 2015 Jun 21;68(6):385-94. Epub 2015 Jan 21.

Kitasato Institute for Life Sciences, Kitasato University, Kitasato, Sagamihara, Kanagawa, Japan.

Mining of bacterial genome data has revealed numerous presumptive terpene synthases. Heterologous expression of several putative terpene synthase genes in an engineered Streptomyces host has revealed 13 newly discovered terpenes whose GC-MS and NMR data did not match with any known compounds in spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowed assignment of the structures of 13 new cyclic terpenes. Among these newly identified compounds, two were found to be linear triquinane sesquiterpenes that have never previously been isolated from bacteria or any other source. The remaining 11 new compounds were shown to be diterpene hydrocarbons and alcohol, including hydropyrene (1), hydropyrenol (2), tsukubadiene (11) and odyverdienes A (12) and B (13) each displaying a novel diterpene skeleton that had not previously been reported.
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http://dx.doi.org/10.1038/ja.2014.171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4727541PMC
June 2015

Synthesis and biological activity of 5-(4-methoxyphenyl)-oxazole derivatives.

Bioorg Med Chem Lett 2015 Jan 22;25(2):313-6. Epub 2014 Nov 22.

Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Electronic address:

5-(4'-Methoxyphenyl)-oxazole (MPO), originally reported as a synthetic compound, was isolated from fungal culture broth as an inhibitor of hatch and growth of Caenorhabditis elegans. Nineteen MPO derivatives were chemically synthesized, but showed no effect on C. elegans hatch and growth. These findings strongly suggested that the whole structure of MPO is essential for anti-C. elegans activity.
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http://dx.doi.org/10.1016/j.bmcl.2014.11.042DOI Listing
January 2015

In vitro metabolism of pyripyropene A and ACAT inhibitory activity of its metabolites.

J Antibiot (Tokyo) 2015 Jan 9;68(1):27-34. Epub 2014 Jul 9.

Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.

Pyripyropene A (PPPA, 1) of fungal origin, a selective inhibitor of acyl-CoA:cholesterol acyltransferase 2 (ACAT2), proved orally active in atherogenic mouse models. The in vitro metabolites of 1 in liver microsomes and plasma of human, rabbit, rat and mouse were analyzed by ultra fast liquid chromatography and liquid chromatography/tandem mass spectrometry. In the liver microsomes from all species, successive hydrolysis occurred at the 1-O-acetyl residue, then at the 11-O-acetyl residue of 1, while the 7-O-acetyl residue was resistant to hydrolysis. Furthermore, dehydrogenation of the newly generated 11-alcoholic hydroxyl residue occurred in human and mouse-liver microsomes, while oxidation of the pyridine ring occurred in human and rabbit liver microsomes. On the other hand, hydrolysis of the 7-O-acetyl residue proceeded only in the mouse plasma. These data indicated that the in vitro metabolic profiles of 1 have subtle differences among animal species. All of the PPPA metabolites observed in liver microsomes and plasma markedly decreased ACAT2 inhibitory activity. These findings will help us to synthesize new PPPA derivatives more effective in in vivo study than 1.
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http://dx.doi.org/10.1038/ja.2014.91DOI Listing
January 2015

Treatment of mid-pregnant mice with KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, induces abnormal retinal vascular patterning in their newborn pups.

Birth Defects Res B Dev Reprod Toxicol 2014 Aug 15;101(4):293-9. Epub 2014 May 15.

Department of Molecular Pharmacology, Shirokane, Minato-ku, Tokyo, Japan.

We previously reported that treatment of mid-pregnant mice with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, caused fetal growth restriction resulting from diminished vascularization in the placenta and fetal organs. In this study, we examined how the treatment of mid-pregnant mice with KRN633 affects the development and morphology of vascular components (endothelial cells, pericytes, and basement membrane) in the retinas of their newborn pups. Pregnant mice were treated with KRN633 (5 mg/kg) once daily from embryonic day 13.5 until the day of delivery. Vascular components were examined using immunohistochemistry with specific markers for each component. Radial vascular growth in the retina was slightly delayed until postnatal day 4 (P4) in the newborn pups of KRN633-treated mothers. On P8, compared with the pups of control mothers, the pups of KRN633-treated mothers exhibited decreased numbers of central arteries and veins and abnormal branching of the central arteries. No apparent differences in pericytes or basement membrane were observed between the pups of control and KRN633-treated mothers. These results suggest that a critical period for determining retinal vascular patterning is present at the earliest stages of retinal vascular development, and that the impaired vascular endothelial growth factor signaling during this period induces abnormal architecture in the retinal vascular network.
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http://dx.doi.org/10.1002/bdrb.21112DOI Listing
August 2014

Synthesis and biological activity of Citridone A and its derivatives.

J Antibiot (Tokyo) 2014 Jun 19;67(6):445-50. Epub 2014 Mar 19.

Graduate School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan.

Citridone A (1), originally isolated as a potentiator of antifungal miconazole activity from a fungal culture broth, has a phenyl-R-furopyridone structure. Because of its unique ring structure, 11 derivatives were chemically synthesized and their biological activity was evaluated. Derivatives 17, 20 and 21 potentiated miconazole activity against Candida albicans. Furthermore, 1, 14, 20 and 21 were found to inhibit yellow pigment production in methicillin-resistant Staphylococcus aureus.
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http://dx.doi.org/10.1038/ja.2014.14DOI Listing
June 2014

Effects of pre- and post-natal treatment with KRN633, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, on retinal vascular development and patterning in mice.

Exp Eye Res 2014 Mar 23;120:127-37. Epub 2014 Jan 23.

Department of Molecular Pharmacology, Kitasato University School of Pharmaceutical Sciences, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.

The impaired function of angiogenic factors, including vascular endothelial growth factor (VEGF), during pregnancy is associated with preeclampsia and intrauterine growth restriction. To determine how the attenuation of VEGF signals during retinal vascular development affects retinal vascular growth and patterns, we examined the effects of pre- and post-natal treatment of mice with KRN633, a VEGF receptor tyrosine kinase inhibitor, on retinal vascular development and structure. Delays in retinal vascular development were observed in the pups of mother mice that were treated daily with KRN633 (5 mg/kg/day) from embryonic day 13.5 until the day of delivery. A more marked delay was seen in pups treated with the inhibitor (5 mg/kg/day) on the day of birth and on the following day. Pups treated postnatally with KRN633 showed abnormal retinal vascular patterns, such as highly dense capillary networks and decreased numbers of central arteries and veins. The high-density vascular networks in KRN633-treated pups showed a greater sensitivity to VEGF signaling inhibition than the normal vascular networks in vehicle-treated pups. Compared to vehicle-treated pups, more severe hypoxia and stronger VEGF mRNA expression were observed in avascular areas in KRN633-treated pups. These results suggest that a short-term loss of VEGF function at the earliest stages of vascular development suppresses vascular growth, leading to abnormal vascular patterning, at least in part via mechanisms involving VEGF in the mouse retina.
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http://dx.doi.org/10.1016/j.exer.2014.01.009DOI Listing
March 2014