Development of carbonic anhydrase IX-targeting molecular-targeted photodynamic therapy.

The efficacy of molecular-targeted photodynamic therapy (MT-PDT) targeting carbonic anhydrase (CA) IX, a cancer-specific molecule, was demonstrated. CA ligand-directed photosensitizers 1-3 were evaluated for their ability to deactivate CAIX protein in cells. Compounds 2 and 3 selectively deactivated CAIX protein under 540 nm light without affecting internal standard proteins. Mechanistic studies revealed that compound 3 not only induced CAIX-selective light inactivation via singlet oxygen but also induced cell membrane damage, resulting in an anti-tumor effect. In vivo studies of CAIX-targeting MT-PDT revealed that treatment with compound 3 followed by light irradiation exhibited remarkable anti-tumor activity, leading to tumor degeneration and necrosis.

Synthesis of the diazatricycloundecane scaffold via gold(I)-catalysed Conia-ene-type 5-exo-dig cyclization and stepwise substituent assembly for the construction of an sp3-rich compound library.

The bridged diazatricycloundecane sp3-rich scaffold was synthesised via the gold(I)-catalysed Conia-ene reaction. The electron-donating property of the siloxymethyl group on alkyne 1 enabled 6-endo-dig cyclization, whereas the ethoxy carbonyl group on alkyne 4 led to 5-exo-dig cyclization with complete regioselectivity in the Conia-ene reaction. The resulting bridged diazatricycloundecane scaffold 5 allowed the construction of a library of sp3-rich compounds. Among the compounds synthesised, compounds 6e and 6f inhibited the hypoxia inducible factor 1 (HIF-1) downstream signaling pathway without affecting HIF-1α mRNA expression.

Discovery of three-dimensional bicyclo[3.3.1]nonanols as novel heat shock protein 90 inhibitors.

We developed an efficient synthetic method for constructing bicyclo[3.3.1]nonane, an sp3-carbon-rich three-dimensional scaffold consisting of two fused six-membered rings. Among the bicyclo[3.3.1]nonanols synthesized, several bicyclo[3.3.1]nonane derivatives were found to inhibit gene transcription by hypoxia-inducible factor-1 (HIF-1). The structure-activity relationship study revealed that the number of hydrophobic functional groups and a carboxylic acid moiety in the bicyclo[3.3.1]nonanols are important for inhibitory activities of both gene transcription by HIF-1 and cell growth. Bicyclo[3.3.1]nonanols fluctuated the amounts of client proteins of heat shock protein (HSP) 90 without inducing a heat shock response in cells and specifically inhibited the ATPase activity of HSP90. These results indicate that bicyclo[3.3.1]nonanols are novel HSP90 inhibitors with a different mechanism of action from conventional HSP90 inhibitors.

Iodophenyl-conjugated closo-dodecaborate as a promising small boron molecule that binds to serum albumin and accumulates in tumor.

The development of novel boron carriers applicable to various cancers is required for further expansion of boron neutron capture therapy (BNCT). In this study, we took advantage of the fact that serum albumin accumulates in tumors and developed a boron compound that interacts non-covalently with the serum albumin. 4-Iodophenylbutanamide was chosen as an albumin ligand and conjugated with closo-dodecaborate (boron-conjugated 4-iodophenylbutanamide: BC-IP). BC-IP was found to be water soluble with low cytotoxicity. The IC50 values of BC-IP were 475 µM for U87MG cells, 738 µM for HeLa cells, and > 1000 µM for A549 cells. The dissociation constant (Kd) value of BC-IP to HSA was 148 ± 8 µM, while that of disodium closo-dodecaborate (4) was > 1000 µM. Significant tumor accumulation was observed in the U87MG tumor mouse model 3 h after injection. The boron concentration in the tumor reached a maximum of 11 µgB/g at 3 h and gradually decreased to 2.4 and 2.3 µgB/g at 12 and 24 h, respectively.

Elucidation of structure-activity relationship of humulanolides and identification of humulanolide analog as a novel HSP90 inhibitor.

Humulanolides are natural products isolated from Asteriscus, and the isolation and total synthesis of many types of humulanolides have been reported. In this study, we evaluated anti-proliferative activity of twelve humulanolides against various human cancer cell lines and found that humulanolide analog E, which was newly designed and synthesized, exhibited the highest anti-proliferative activity. Structure-activity relationship analysis revealed that α,ß-unsaturated carbonyl moieties in humulanolides play an important role for anti-proliferative activity. To identify molecular targets of humulanolide analog E, we investigated various cell-based and in vitro assays. Treatment with humulanolide analog E against human fibrosarcoma HT1080 cells increased the expression level of HSP70 protein and decreased the levels of AKT and CDK4, which are HSP90 client proteins. Moreover, humulanolide analog E inhibited refolding of denatured luciferase protein via suppression of HSP90 activity in vitro. These results suggest that humulanolide analog E possesses the anti-proliferative activity against human cancer cells by inhibiting HSP90 functions.

Biological evaluation for anti-inflammatory effect of africane-type sesquiterpenoids.

Africane-type sesquiterpenoids are a unique tricyclic carbon architecture sesquiterpenoid isolated as natural products. Δ9(15) -africanene has been reported to exhibit anti-inflammatory activity for carrageenan-induced rat foot edema. In this study, we reported structure-activity relationship study of africane-type sesquiterpenoids and found that some africane-type sesquiterpenoid analogs and their synthetic intermediate showed potent anti-inflammatory activity. To identify the mode of action of africane-type sesquiterpenoids and their synthetic intermediate, we evaluated the anti-inflammatory activity using lipopolysaccharide (LPS)-stimulated mouse macrophage RAW264.7 cells. Treatment with the africane-type compounds and their synthetic intermediate suppressed LPS-induced expressions of Cox-2 protein and mRNAs of the inflammatory cytokines IL-1ß and IL-6 at the concentrations that did not affect cell viability. Interestingly, although these africane-type compounds and their synthetic intermediate suppressed the pro-inflammatory cytokines' expressions, the compounds did not modulate NF-κB activation. These results suggest that the africane-type compounds and their synthetic intermediate are anti-inflammatory compounds that suppress the expression of LPS-induced inflammatory mediators independently of NF-κB activation.

Scalable Syntheses and Biological Evaluation of Africane-Type Sesquiterpenoids.

Practical total syntheses of africane-type sesquiterpenoids were realized by reexamination of a divergent strategy employing optimized three-component coupling followed by ring-closing metathesis and substrate-controlled cyclopropanation. This sequential eight-step conversion provided Δ9(15) -africanene, a common bicyclo[5.3.0]decane intermediate for the syntheses of africane derivatives, in more than twice the yield as in the previous approach. The scalability and robustness of this improved synthetic route were confirmed by gram-scale preparation of Δ9(15) -africanene. In vitro cell-based assays of the synthesized africane-type sesquiterpenoids disclosed that ester-incorporating derivatives showed cytotoxic activity against HeLa cells. The effect of relative and absolute configuration of africane-9,15-diol monoacetates on the cytotoxicity against HeLa cells was also investigated.

Correction: Mori et al. Involvement of DPY19L3 in Myogenic Differentiation of C2C12 Myoblasts. Molecules 2021, 26, 5685.

In the original article [...].

Involvement of LH3 and GLT25D1 for glucosyl-galactosyl-hydroxylation on non-collagen-like domain of FGL1.

Glucosyl-galactosyl-hydroxylation (GGH) is one type of post-translational modification, which is mainly observed in collagen-like domain-containing proteins. Using LC-MS/MS analysis, we found a GGH-like modification at Lys65 of fibrinogen-like protein 1 (FGL1), although it does not contain a collagen-like domain. To identify the glycosyltransferases responsible for this modification, we established LH3/GLT25D1-knockout FGL1-overexpressing HT1080 cell lines. The result showed that knockout of LH3 or GLT25D1 significantly inhibited the glycosylation. Furthermore, deficiency of GGH by point mutation of the FGL1 protein or knockout of the GGH-related glycosyltransferase reduced FGL1 protein levels. Taken together, these data indicate that Lys65 of FGL1 is glucosyl-galactosyl-hydroxylated by LH3 and GLT25D1. Our results provide novel insights to regulate various FGL1 functions.

Geology-dependent impacts of forest conversion on stream fish diversity.

Forest conversion is one of the greatest global threats to biodiversity, and land-use change and subsequent biodiversity declines sometimes occur over a variety of underlying geologies. However, how forest conversion and underlying geology interact to alter biodiversity is underappreciated, although spatial variability in geology is considered an integral part of sustaining ecosystems. We aimed to examine the effects of forest conversion to farmland, the underlying geology, and their interaction on the stream fishes' diversity, evenness, and abundance in northeastern Japan. We disentangled complex pathways between abiotic and biotic factors with structural equation modeling. Species diversity of stream fishes was indirectly shaped by the interaction of land use and underlying geology. Diversity declined due to nutrient enrichment associated with farmlands, which was mainly the result of changes in evenness rather than by changes in species richness. This impact was strongest in streams with volcanic geology with coarse substrates probably because of the differential responses of abundant stream fishes to nutrient enrichment (i.e., dominance) and the high dependency of these fishes on large streambed materials during their life cycles. Our findings suggest that remediation of deforested or degraded forest landscapes would be more efficient if the interaction between land use and underlying geology was considered. For example, the negative impacts of farmland on evenness were larger in streams with volcanic geology than in other stream types, suggesting that riparian forest restoration along such streams would efficiently provide restoration benefits to stream fishes. Our results also suggest that land clearing around such streams should be avoided to conserve species evenness of stream fishes.

Impactos Geológicamente Dependientes de la Conversión de Bosques sobre la Diversidad de Peces de Arroyo Resumen La conversión de los bosques es una de las mayores amenazas para la biodiversidad mundial y el cambio en el uso de suelo y las declinaciones subsecuentes de la biodiversidad a veces ocurren a lo largo de una variedad de geologías subyacentes. Sin embargo, la manera en que interactúan la conversión del bosque y la geología subyacente está subestimada a pesar de que la variabilidad espacial en la geología es considerada una parte integral del mantenimiento de un ecosistema. Fijamos como objetivo examinar los efectos de la conversión del bosque a tierras de cultivo, la geología subyacente y sus interacciones sobre la diversidad, uniformidad y abundancia de peces de arroyo en el noreste de Japón. Para esto, desentrañamos las vías complejas entre los factores bióticos y abióticos con modelados de ecuación estructural. La diversidad de especies de los peces de arroyo estuvo formada indirectamente por la interacción del uso de suelo y la geología subyacente. La diversidad declinó debido al enriquecimiento de nutrientes asociado con las tierras de cultivo, lo cual fue principalmente resultado de los cambios en la uniformidad de especies en lugar de cambios en la riqueza de especies. Este impacto fue más fuerte en los arroyos con geología volcánica y sustratos ásperos, probablemente debido a las respuestas diferenciales de los peces abundantes en el arroyo al enriquecimiento de nutrientes (es decir, dominancia) y la alta dependencia de estos peces por los grandes materiales del lecho durante su ciclo de vida. Nuestros hallazgos sugieren que la reparación de los paisajes de bosque deforestados o degradados sería más eficiente si se considera la interacción entre el uso de suelo y la geología subyacente. Por ejemplo, los impactos negativos de las tierras de cultivo sobre la uniformidad fueron mayores en los arroyos con geología volcánica que en otros tipos de arroyo, lo que sugiere que la restauración de los bosques ribereños a lo largo de dichos arroyos proporcionaría eficientemente los beneficios de restauración a los peces del arroyo. Nuestros resultados sugieren que el desmonte de tierras alrededor de dichos arroyos debería evitarse para conservar la uniformidad de especies de los peces de arroyo.

Identification of madangamine A as a novel lysosomotropic agent to inhibit autophagy.

Madangamines are marine natural products isolated from Xestospongia ingens, and madangamine A-E with a different D-ring structure have been reported. We have reported that madangamine A has strong anti-proliferative activity against various human cancer cell lines. In this study, to clarify the anti-proliferative activity of madangamine A, we searched for molecular target of the madangamine A in human cells. Treatment with madangamine A increased the levels of LC3-II and p62, autophagy-related proteins, concomitant with growth inhibition. Moreover, madangamine A resulted in lysosome enlargement and increase in lysosomal pH, which are same phenomena observed in chloroquine-treated cells. These results suggest that madangamine A is a novel lysosome inhibitor, and the anti-proliferative activity of madangamine A is due to the inhibition of lysosome function.

Involvement of DPY19L3 in Myogenic Differentiation of C2C12 Myoblasts.

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.

Seven-Step Synthesis of All-Nitrogenated Sugar Derivatives Using Sequential Overman Rearrangements.

All-nitrogenated sugars (ANSs), in which all hydroxy groups in a carbohydrate are replaced with amino groups, are anticipated to be privileged structures with useful biological activities. However, ANS synthesis has been challenging due to the difficulty in the installation of multi-amino groups. We report herein the development of a concise synthetic route to peracetylated ANSs in seven steps from commercially available monosaccharides. The key to success is the use of the sequential Overman rearrangement, which enables formal simultaneous substitution of four or five hydroxy groups in monosaccharides with amino groups. A variety of ANSs are available through the same reaction sequence starting from different initial monosaccharides by chirality transfer of secondary alcohols. Transformations of the resulting peracetylated ANSs such as glycosylation and deacetylation are also demonstrated. Biological studies reveal that ANS-modified cholesterol show cytotoxicity against human cancer cell lines, whereas each ANS and cholesterol have no cytotoxicity.

Identification of vibsanin A analog as a novel HSP90 inhibitor.

Vibsanin A is the first natural product isolated from Viburnum awabuki and has several biological activities. We have reported that a vibsanin A analog, obtained from process of total synthesis of vibsanin A, has anti-proliferative activity against human cancer cell lines. In this study, we evaluated anti-proliferative effect of the vibsanin A analogs against various human cancer cell lines and examined molecular target of the analog in human cells. Among the vibsanin A analogs, vibsanin A analog C (VAC) showed anti-proliferative effect against various cancer cell lines, and the anti-proliferative activity was strongest among the vibsanin A analogs. Additionally, VAC fluctuated amounts of HSP90-related proteins in cells and inhibited HSP90-mediated protein refolding of luciferase in vitro. These results suggest that the anti-proliferative activity of VAC is due to HSP90 inhibition, and VAC has a potential as novel anti-cancer drug as HSP90 inhibitor.

A novel Golgi mannosidase inhibitor: Molecular design, synthesis, enzyme inhibition, and inhibition of spheroid formation.

Effective chemotherapy for solid cancers is challenging due to a limitation in permeation that prevents anticancer drugs from reaching the center of the tumor, therefore unable to limit cancer cell growth. To circumvent this issue, we planned to apply the drugs directly at the center by first collapsing the outer structure. For this, we focused on cell-cell communication (CCC) between N-glycans and proteins at the tumor cell surface. Mature N-glycans establish CCC; however, CCC is hindered when numerous immature N-glycans are present at the cell surface. Inhibition of Golgi mannosidases (GMs) results in the transport of immature N-glycans to the cell surface. This can be employed to disrupt CCC. Here, we describe the molecular design and synthesis of an improved GM inhibitor with a non-sugar mimic scaffold that was screened from a compound library. The synthesized compounds were tested for enzyme inhibition ability and inhibition of spheroid formation using cell-based methods. Most of the compounds designed and synthesized exhibited GM inhibition at the cellular level. Of those, AR524 had higher inhibitory activity than a known GM inhibitor, kifunensine. Moreover, AR524 inhibited spheroid formation of human malignant cells at low concentration (10 µM), based on the disruption of CCC by GM inhibition.

Development of Specific Fluorogenic Substrates for Human ß-N-Acetyl-D-hexosaminidase A for Cell-Based Assays.

Inhibitors of human ß-N-acetyl-D-hexosaminidase (hHEX) A and human O-GlcNAcase (hOGA) reportedly play roles in multiple diseases, suggesting their potential for pharmacological chaperone (PC) therapy of Sandhoff disease (SD) and Tay-Sachs disease (TSD), as lysosomal storage diseases, and Alzheimer's disease and progressive supranuclear palsy, respectively. In particular, hHEXA inhibitors as PCs have been shown to successfully enhance hHEXA levels, leading to the chronic form of SD and TSD. In the diagnosis of enzyme deficiencies in SD and TSD, artificial hHEXA substrates based on 4-methylumbelliferone as a fluorophore are available and generally used; however, they do not have sufficient performance to screen for potential inhibitors for a PC therapy from compound libraries. Further, there are currently few fluorogenic substrates for hHEXA suitable for such requirements and there are no substrates ideal for cell-based inhibitor screening. Here, we clarified the difference in enzyme active site structure between hHEXA and hOGA from their tertiary structures. To develop lysosome-localized hHEXA-specific fluorogenic substrates based on the difference in their active site structures, our developed quinone methide cleavage substrate design platform was applied for the molecular design of substrates. Thereafter, we synthesized via the shortest route and evaluated novel three-color fluorogenic substrates for hHEXA that exhibited excellent specificity and sensitivity in three human cell lines. The designed substrates represent the first-in-a class of new substrates that can be utilized to screen hHEXA inhibitors in adherent human cultured cells.

Screening, Synthesis, and Evaluation of Novel Isoflavone Derivatives as Inhibitors of Human Golgi ß-Galactosidase.

The genes GLB1 and GALC encode GLB1 isoform 1 and galactocerebrosidase, respectively, which exhibit ß-galactosidase activity in human lysosomes. GLB1 isoform 1 has been reported to play roles in rare lysosomal storage diseases. Further, its ß-galactosidase activity is the most widely used biomarker of senescent and aging cells; hence, it is called senescence-associated ß-galactosidase. Galactocerebrosidase plays roles in Krabbe disease. We previously reported a novel ß-galactosidase activity in the Golgi apparatus of human cells; however, the protein responsible for this activity could not be identified. Inhibitor-derived chemical probes can serve as powerful tools to identify the responsible protein. In this study, we first constructed a cell-based high-throughput screening (HTS) system for Golgi ß-galactosidase inhibitors, and then screened inhibitors from two compound libraries using the HTS system, in vitro assay, and cytotoxicity assay. An isoflavone derivative was identified among the final Golgi ß-galactosidase inhibitor compound hits. Molecular docking simulations were performed to redesign the isoflavone derivative into a more potent inhibitor, and six designed derivatives were then synthesized. One of the derivatives, ARM07, exhibited potent inhibitory activity against ß-galactosidase, with an IC50 value of 14.8 µM and competitive inhibition with Ki value of 13.3 µM. Furthermore, the in vitro and cellular inhibitory activities of ARM07 exceeded those of deoxygalactonojirimycin. ARM07 may contribute to the development of affinity-based chemical probes to identify the protein responsible for the newly discovered Golgi ß-galactosidase activity. The therapeutic relevance of ARM07 against lysosomal storage diseases and its effect on senescent cells should be evaluated further.

Identification of topoisomerases as molecular targets of cytosporolide C and its analog.

Cytosporolide (Cytos) A-C, isolated from the fungus Cytospora sp., have anti-microbial activity, but their molecular targets in mammalian cells are unknown. We have previously reported the total synthesis of Cytos A by biomimetic hetero-Diels-Alder reaction. In this study, to examine the novel bioactivity of Cytos, we synthesized Cytos C and measured cell growth-inhibiting activities of 7 compounds, including Cytos A and C, in several human cancer cell lines. Among these compounds, Cytos C and tetradeoxycytosporolide A (TD-Cytos A), a model compound for the synthesis of Cytos A, had anti-proliferative effects on cancer cells, and TD-Cytos A exhibited stronger activity than Cytos C. In vitro topoisomerase-mediated DNA relaxing experiments showed that TD-Cytos A inhibited the activities of topoisomerase I and II, whereas Cytos C targeted only topoisomerase I. These data suggest that the anti-proliferative activities of Cytos correlate with the inhibition of topoisomerases and implicated TD-Cytos A as a novel anti-cancer drug that suppresses the activities of topoisomerase I and II.

Discovery of human Golgi ß-galactosidase with no identified glycosidase using a QMC substrate design platform for exo-glycosidase.

Post-translational modifications (PTMs) of proteins play important roles in the physiology of eukaryotes. In the PTMs, non-reversible glycosylations are classified as N-glycosylations and O-glycosylations, and are catalyzed by various glycosidases and glycosyltransferases. However, ß-glycosidases are not known to play a role in N- and O-glycan processing, although both glycans provide partial structures as substrates for ß-galactosidase and ß-N-acetylglucosaminidase in the Golgi apparatus of human cells. We explored human Golgi ß-galactosidase using fluorescent substrates based on a quinone methide cleavage (QMC) substrate design platform that was previously developed to image exo-type glycosidases in living cells. As a result, we discovered a novel Golgi ß-galactosidase in human cells. It is possible to predict a novel and important function in glycan processing of this ß-galactosidase, because various ß-galactosyl linkages in N- and O-glycans exist in Golgi apparatus. In addition, these results show that the QMC platform is excellent for imaging exo-type glycosidases.

Identification of a novel glycan processing enzyme with exo-acting ß-allosidase activity in the Golgi apparatus using a new platform for the synthesis of fluorescent substrates.

The majority of eukaryotic proteins undergo post-translational modifications (PTMs) involving the attachment of complex glycans, predominantly through N-glycosylation and O-glycosylation. PTMs play important roles in virtually all cellular processes, and aberrant regulation of protein glycosylation and glycan processing has been implicated in various diseases. However, glycan processing on proteins in various cellular contexts has not been visualized. We had previously developed a quinone methide cleavage (QMC) platform for enhanced substrate design. This platform was applied here to screen for novel glycan-processing enzymes. We designed and synthesized fluorescent substrates with ß-allopyranoside residues using the QMC platform. When applied in cell-based assays, the fluorescent substrates allowed rapid and clear visualization of ß-allosidase activity in the Golgi apparatus of human cultured cells. The QMC platform will likely find broad applications in visualizing the activities of glycan processing enzymes in living cells and in studying PTMs.