Cytochrome P450 2J2 is required for the natural compound austocystin D to elicit cancer cell toxicity.

Austocystin D is a natural compound that induces cytochrome P450 (CYP) monooxygenase-dependent DNA damage and growth inhibition in certain cancer cell lines. Cancer cells exhibiting higher sensitivity to austocystin D often display elevated CYP2J2 expression. However, the essentiality and the role of CYP2J2 for the cytotoxicity of this compound remain unclear. In this study, we demonstrate that CYP2J2 depletion alleviates austocystin D sensitivity and DNA damage induction, while CYP2J2 overexpression enhances them. Moreover, the investigation into genes involved in austocystin D cytotoxicity identified POR and PGRMC1, positive regulators for CYP activity, and KAT7, a histone acetyltransferase. Through genetic manipulation and analysis of multiomics data, we elucidated a role for KAT7 in CYP2J2 transcriptional regulation. These findings strongly suggest that CYP2J2 is crucial for austocystin D metabolism and its subsequent cytotoxic effects. The potential use of austocystin D as a therapeutic prodrug is underscored, particularly in cancers where elevated CYP2J2 expression serves as a biomarker.

Plakevulin A induces apoptosis and suppresses IL-6-induced STAT3 activation in HL60 cells.

(+)-Plakevulin A (1), an oxylipin isolated from an Okinawan sponge Plakortis sp. inhibits enzymatic inhibition of DNA polymerases (pols) α and δ and exhibits cytotoxicity against murine leukemia (L1210) and human cervix carcinoma (KB) cell lines. However, the half-maximal inhibitory concentration (IC50) value for cytotoxicity significantly differed from those observed for the enzymatic inhibition of pols α and ß, indicating the presence of target protein(s) other than pols. This study demonstrated cytotoxicity against human promyelocytic leukemia (HL60), human cervix epithelioid carcinoma (HeLa), mouse calvaria-derived pre-osteoblast (MC3T3-E1), and human normal lung fibroblast (MRC-5) cell lines. This compound had selectivity to cancer cells over normal ones. Among these cell lines, HL60 exhibited the highest sensitivity to (+)-plakevulin A. (+)-Plakevulin A induced DNA fragmentation and caspase-3 activation in HL60 cells, indicating its role in apoptosis induction. Additionally, hydroxysteroid 17-ß dehydrogenase 4 (HSD17B4) was isolated from the HL60 lysate as one of its binding proteins through pull-down experiments using its biotinylated derivative and neutravidin-coated beads. Moreover, (+)-plakevulin A suppressed the activation of interleukin 6 (IL-6)-induced signal transducer and activator of transcription 3 (STAT3). Because the knockdown or inhibition of STAT3 induces apoptosis and HSD17B4 regulates STAT3 activation, (+)-plakevulin A may induce apoptosis in HL60 cell lines by suppressing STAT3 activation, potentially by binding to HSD17B4. The present findings provide valuable information for the mechanism of its action.

A small-molecule degron with a phenylpropionic acid scaffold.

Targeted protein degradation (TPD), employing proteolysis-targeting chimeras (PROTACs) composed of ligands for both a target protein and ubiquitin ligase (E3) to redirect the ubiquitin-proteasome system (UPS) to the target protein, has emerged as a promising strategy in drug discovery. However, despite the vast number of E3 ligases, the repertoire of E3 ligands utilized in PROTACs remains limited. Here, we report the discovery of a small-molecule degron with a phenylpropionic acid skeleton, derived from a known ligand of S-phase kinase-interacting protein 2 (Skp2), an E3 ligase. We used this degron to design PROTACs inducing proteasomal degradation of HaloTag-fused proteins, and identified key structural relationships. Surprisingly, our mechanistic studies excluded the involvement of Skp2, suggesting that this degron recruits other protein(s) within the UPS.

Functional and structural analysis of a cyclization domain in a cyclic ß-1,2-glucan synthase.

Cyclic ß-1,2-glucan synthase (CGS) is a key enzyme in production of cyclic ß-1,2-glucans (CßGs) which are involved in bacterial infection or symbiosis to host organisms. Nevertheless, a mechanism of cyclization, the final step in the CGS reaction, has not been fully understood. Here we performed functional and structural analyses of the cyclization domain of CGS alone from Thermoanaerobacter italicus (TiCGSCy). We first found that ß-glucosidase-resistant compounds are produced by TiCGSCy with linear ß-1,2-glucans as substrates. The 1H-NMR analysis revealed that these products are CßGs. Next, action pattern analyses using ß-1,2-glucooligosaccharides revealed a unique reaction pattern: exclusive transglycosylation without hydrolysis and a hexasaccharide being the minimum length of the substrate. These analyses also showed that longer substrate ß-1,2-glucooligosaccharides are preferred, being consistent with the fact that CGSs generally produce CßGs with degrees of polymerization of around 20. Finally, the overall structure of the cyclization domain of TiCGSCy was found to be similar to those of ß-1,2-glucanases in phylogenetically different groups. Meanwhile, the identified catalytic residues indicated clear differences in the reaction pathways between these enzymes. Overall, we propose a novel reaction mechanism of TiCGSCy. Thus, the present group of CGSs defines a new glycoside hydrolase family, GH189. KEY POINTS: • It was clearly evidenced that cyclization domain alone produces cyclic ß-1,2-glucans. • The domain exclusively catalyzes transglycosylation without hydrolysis. • The present catalytic domain defines as a new glycoside hydrolase family 189.

Structure-Activity Relationship Analysis of Fluoxetine for Suppression of Inflammatory Cytokine Production.

There is accumulating evidence that selective serotonin reuptake inhibitors (SSRIs), clinically used as antidepressants, have a beneficial effect on inflammatory diseases such as coronavirus disease 2019 (COVID-19). We previously compared the inhibitory effects of five U.S. Food and Drug Administration (FDA)-approved SSRIs on the production of an inflammatory cytokine, interleukin-6 (IL-6), and concluded that fluoxetine (FLX) showed the most potent anti-inflammatory activity. Here, we investigated the structure-activity relationship of FLX for anti-inflammatory activity towards J774.1 murine macrophages. FLX suppressed IL-6 production induced by the TLR3 agonist polyinosinic-polycytidylic acid (poly(I : C)) with an IC50 of 4.76 µM. A derivative of FLX containing chlorine instead of the methylamino group lacked activity, suggesting that the methylamino group is important for the anti-inflammatory activity. FLX derivatives bearing an N-propyl or N-(pyridin-3-yl)methyl group in place of the N-methyl group exhibited almost the same activity as FLX. Other derivatives showed weaker activity, and the N-phenyl and N-(4-trifluoromethyl)benzyl derivatives were inactive. The chlorine-containing derivative also lacked inhibitory activity against TLR9- or TLR4-mediated IL-6 production. These derivatives showed similar structure-activity relationships for TLR3- and TLR9-mediated inflammatory responses. However, the activities of all amino group-containing derivatives against the TLR4-mediated inflammatory response were equal to or higher than the activity of FLX. These results indicate that the substituent at the nitrogen atom in FLX strongly influences the anti-inflammatory effect.

Cyclo(l-Pro-l-Tyr) Isolated from the Human Skin Commensal Corynebacterium tuberculostearicum Inhibits Tyrosinase.

Melanin is produced by melanocytes to protect human skin from harmful ultraviolet radiation. During skin cell renewal, melanin and dead skin cells are disposed of. However, prolonged exposure to ultraviolet rays or aging can disturb this cycle, leading to skin hyperpigmentation due to melanin accumulation. Tyrosinase is a crucial enzyme involved in melanin biosynthesis. Although various compounds, including tyrosine inhibitors, that counteract melanin accumulation have been reported, some, such as hydroquinone, are toxic and can cause vitiligo. Meanwhile, the skin is the largest organ and the outermost layer of the immune system, containing a diverse range of bacteria that produce low-toxicity compounds. In the current study, we aim to identify metabolites produced by skin microbiota that inhibit tyrosinase. Specifically, mushroom tyrosinase served as the study model. Following commensal skin bacteria screening, Corynebacterium tuberculostearicum was found to inhibit tyrosinase activity. The active compound was cyclo(l-Pro-l-Tyr); commercially available cyclo(l-Pro-l-Tyr) also exhibited inhibitory activity. Docking simulations suggested that cyclo(l-Pro-l-Tyr) binds to the substrate-binding site of mushroom tyrosinase, obstructing the substrate pocket and preventing its activity. Hence, cyclo(l-Pro-l-Tyr) might have potential applications as a cosmetic agent and food additive.

Characterization and structural analyses of a novel glycosyltransferase acting on the ß-1,2-glucosidic linkages.

The IALB_1185 protein, which is encoded in the gene cluster for endo-ß-1,2-glucanase homologs in the genome of Ignavibacterium album, is a glycoside hydrolase family (GH) 35 protein. However, most known GH35 enzymes are ß-galactosidases, which is inconsistent with the components of this gene cluster. Thus, IALB_1185 is expected to possess novel enzymatic properties. Here, we showed using recombinant IALB_1185 that this protein has glycosyltransferase activity toward ß-1,2-glucooligosaccharides, and that the kinetic parameters for ß-1,2-glucooligosaccharides are not within the ranges for general GH enzymes. When various aryl- and alkyl-glucosides were used as acceptors, glycosyltransfer products derived from these acceptors were subsequently detected. Kinetic analysis further revealed that the enzyme has wide aglycone specificity regardless of the anomer, and that the ß-1,2-linked glucose dimer sophorose is an appropriate donor. In the complex of wild-type IALB_1185 with sophorose, the electron density of sophorose was clearly observed at subsites -1 and +1, whereas in the E343Q mutant-sophorose complex, the electron density of sophorose was clearly observed at subsites +1 and +2. This observation suggests that binding at subsites -1 and +2 competes through Glu102, which is consistent with the preference for sophorose as a donor and unsuitability of ß-1,2-glucooligosaccharides as acceptors. A pliable hydrophobic pocket that can accommodate various aglycone moieties was also observed in the complex structures with various glucosides. Overall, our biochemical and structural data are indicative of a novel enzymatic reaction. We propose that IALB_1185 be redefined ß-1,2-glucooligosaccharide:d-glucoside ß-d-glucosyltransferase as a systematic name and ß-1,2-glucosyltransferase as an accepted name.

Selective inhibition of hepatitis B virus internalization by oxysterol derivatives.

Given that the current approved anti-hepatitis B virus (HBV) drugs suppress virus replication and improve hepatitis but cannot eliminate HBV from infected patients, new anti-HBV agents with different mode of action are urgently needed. In this study, we identified a semi-synthetic oxysterol, Oxy185, that can prevent HBV infection in a HepG2-based cell line and primary human hepatocytes. Mechanistically, Oxy185 inhibited the internalization of HBV into cells without affecting virus attachment or replication. We also found that Oxy185 interacted with an HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP), and inhibited the oligomerization of NTCP to reduce the efficiency of HBV internalization. Consistent with this mechanism, Oxy185 also inhibited the hepatitis D virus infection, which relies on NTCP-dependent internalization, but not hepatitis A virus infection, and displayed pan-genotypic anti-HBV activity. Following oral administration in mice, Oxy185 showed sustained accumulation in the livers of the mice, along with a favorable liver-to-plasma ratio. Thus, Oxy185 is expected to serve as a useful tool compound in proof-of-principle studies for HBV entry inhibitors with this novel mode of action.

Fine-tuning of nitrogen-containing bisphosphonate esters that potently induce degradation of HMG-CoA reductase.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and competitive inhibitors targeting the catalytic domain of this enzyme, so-called statins, are widely used for the treatment of hyperlipidemia. The membrane domain mediates the sterol-accelerated degradation, a post-translational negative feedback mechanism, and small molecules triggering such degradation have been studied as an alternative therapeutic option. Such strategies are expected to provide benefits over catalytic site inhibitors, as the inhibition leads to transcriptional and post-translational upregulation of the enzyme, necessitating a higher dose of the inhibitors and concomitantly increasing the risk of serious adverse effects, including myopathies. Through our previous study on SR12813, a synthetic small molecule that induces degradation of HMG-CoA reductase, we identified a nitrogen-containing bisphosphonate ester SRP3042 as a highly potent HMG-CoA reductase degrader. Here, we performed a systematic structure-activity relationship study to optimize its activity and physicochemical properties, specifically focusing on the reduction of lipophilicity. Mono-fluorination of tert-butyl groups on the molecules was found to increase the HMG-CoA reductase degradation activity while reducing lipophilicity, suggesting the mono-fluorination of saturated alkyl groups as a useful strategy to balance potency and lipophilicity of the lead compounds.

Synthesis and structure-activity relationship of violaceoid D, a cytotoxic alkylated phenol isolated from Aspergillus violaceofuscus Gasperini.

The enantioselective synthesis of violaceoid D, a cytotoxic phenolic compound isolated from the culture broth of Aspergillus violaceofuscus Gasperini, was achieved. The total synthesis involves stereoselective construction of the stereogenic center of violaceoid D via Sharpless asymmetric dihydroxylation, followed by Smiles rearrangement. The absolute configuration of natural violaceoid D was determined to be R from the specific rotation value. Synthesized violaceoid D and its analogs were evaluated for cytotoxicity against two human cancer cell lines, Jurkat and HCT116. Because the enantiomer of violaceoid D showed no cytotoxicity, it is plausible that violaceoid D binds selectively to specific target molecules, such as proteins in the cancer cells.

Anti-hepatitis C Virus Activity of Juglorubin Derivatives.

Juglorubin is a natural dye isolated from the culture of Streptomyces sp. 3094, 815, and GW4184. It has been previously synthesized via the biomimetic dimerization of juglomycin C, a plausible genetic precursor. In this study, the derivatives of juglorubin, 1-O-acetyljuglorubin dimethyl ester and juglorubin dimethyl ester, were found to exhibit antiviral activity against hepatitis C virus (HCV) without exerting any remarkable cytotoxicity against host Huh-7 cells. They also inhibited liver X receptor α activation and lipid droplet accumulation in Huh-7 cells. These findings suggest that 1-O-acetyljuglorubin dimethyl ester and juglorubin dimethyl ester targeted the host factors required for HCV production.

Synthesis and Antiviral Activities of Neoechinulin B and Its Derivatives.

We have previously reported that neoechinulin B (1a), a prenylated indole diketopiperazine alkaloid, shows antiviral activities against hepatitis C virus (HCV) via the inactivation of the liver X receptors (LXRs) and the resultant disruption of double-membrane vesicles. In this study, a two-step synthesis of the diketopiperazine scaffold of 1a was achieved by the base-induced coupling of 1,4-diacetyl-3-{[(tert-butyldimethylsilyl)oxy]methyl}piperazine-2,5-dione with aldehydes, followed by the treatment of the resultant coupling products with tetra-n-butylammonium fluoride. Compound 1a and its 16 derivatives 1b-q were prepared using this method. Furthermore, variecolorin H, a related alkaloid, was obtained by the acid treatment of 1a in MeOH. The antiviral evaluation of 1a and its derivatives revealed that 1a, 1c, 1d, 1h, 1j, 1l, and 1o exhibited both anti-HCV and anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) activities. The results of this study indicate that the exomethylene moiety on the diketopiperazine ring is important for the antiviral activities. The antiviral compounds can inhibit the production of HCV and SARS-CoV-2 by inactivating LXRs.

Synthesis and cytotoxic activities of 8- and 6-demethyleucalyptins.

Secondary metabolites in plants influence the health of herbivores such as Japanese rock ptarmigans that feed on the leaves and fruits of alpine plants. Thus, it is important to understand the secondary metabolites of alpine plants and their biological activities for conserving Japanese rock ptarmigans. We isolated C-methylflavone from the leaves of Kalmia procumbens, on which Japanese rock ptarmigans feed. Although its structure was deduced to be 8-demethyleucalyptin by comparing its nuclear magnetic resonance (NMR) data with the reported ones, the possibility that the isolated compound is 6-demethyleucalyptin cannot be ruled out. Thus, both isomers were synthesized. The isolated compound was unambiguously determined to be 8-demethyleucalyptin by comparing its NMR data with those of the synthetic ones. Cytotoxic evaluation of 8- and 6-demethyleucalyptins revealed that only the former showed cytotoxicity against HCT116 and MRC-5 cells. The present study provides not only easy access to 8- and 6-demethyleucalyptins, but also their biological information.

Classification of Southeast Asian mints (Mentha spp.) based on simple sequence repeat markers.

Mentha is a complex genus encompassing many species as a consequence of their interspecific hybridization and polyploidy. Southeast Asian mints have been poorly distinguished though they are widely used for culinary and medical purposes. In this study, we have analyzed Southeast Asian mints and known varieties as well as a related Lamiaceae species (Nepeta sp.) using simple sequence repeat (SSR) markers and leaf morphology. Two types of mints were clearly distinguished based on their venation pattern and leaf shape index. We developed 12 SSR markers that allowed good amplification in the Mentha and another Lamiaceae species. In the SSR-based phylogram, the Mentha lines could be delimited into groups I-VI. The Southeast Asian mints divided into groups I and II, and the phylogram separated most of the available species, with groups I and II containing the known species M. × cordifolia and M. arvensis, respectively. The separation of the two groups was supported by a population structure analysis. The SSR markers developed in this study enabled the simultaneous classification of mints and will help improve our understanding of the genetic composition of known mint varieties and as yet unclassified Southeast Asian mints.

Cyclic Phthalate Esters as Liver X Receptor Antagonists with Anti-hepatitis C Virus and Anti-severe Acute Respiratory Syndrome Coronavirus 2 Properties.

The liver X receptor is a nuclear hormone receptor that regulates lipid metabolism. Previously, we had demonstrated the antiviral properties of a liver X receptor antagonist associated with the hepatitis C virus and severe acute respiratory syndrome coronavirus 2. In this study, we screened a chemical library and identified two potential liver X receptor antagonists. Spectroscopic analysis revealed that the structures of both antagonists (compounds 1 and 2) were cyclic dimer and trimer of esters, respectively, that consisted of phthalate and 1,6-hexane diol. This study is the first to report the structure of the cyclic trimer of phthalate ester. Further experiments revealed that the compounds were impurities of solvents used for purification, although their source could not be traced. Both phthalate esters exhibited anti-hepatitis C virus activity, whereas the cyclic dimer showed anti-severe acute respiratory syndrome coronavirus 2 activity. Cyclic phthalate derivatives may constitute a novel class of liver X receptor antagonists and broad-spectrum antivirals.

Epo-C12 inhibits peroxiredoxin 1 peroxidase activity.

Epo-C12 is a synthetic derivative of epolactaene, isolated from Penicillium sp. BM 1689-P. Epo-C12 induces apoptosis in human acute lymphoblastoid leukemia BALL-1 cells. In our previous studies, seven proteins that bind to Epo-C12 were identified by a combination of pull-down experiments using biotinylated Epo-C12 (Bio-Epo-C12) and mass spectrometry. In the present study, the effect of Epo-C12 on peroxiredoxin 1 (Prx 1), one of the proteins that binds to Epo-C12, was investigated. Epo-C12 inhibited Prx 1 peroxidase activity. However, it did not suppress its chaperone activity. Binding experiments between Bio-Epo-C12 and point-mutated Prx 1s suggest that Epo-C12 binds to Cys52 and Cys83 in Prx 1. The present study revealed that Prx 1 is one of the target proteins through which Epo-C12 exerts an apoptotic effect in BALL-1 cells.

SQAP, an acyl sulfoquinovosyl derivative, suppresses expression of histone deacetylase and induces cell death of cancer cells under hypoxic conditions.

Sulfoglycolipid, SQAP, is a radiosensitizing agent that makes tumor cells more sensitive to radiation therapy. A previous study revealed that SQAP induced the degradation of hypoxia-inducible factor-1α (HIF-1α) and inhibited angiogenesis in a hepatoma model mouse. Herein, we examined the biological activities of SQAP against hepatocarcinoma cells under low oxygen conditions. Cell growth inhibition of SQAP under hypoxic conditions was significantly higher than that under normoxic conditions. In addition, SQAP was found to impair the expression of histone deacetylase (HDAC) under low oxygen conditions. Our present data suggested that SQAP induced the degradation of HIF-1α and then decreased the expression of HDAC1. Unlike known HDAC inhibitors, SQAP increased the acetylation level of histone in cells without inhibition of enzymatic activity of HDACs. Our data demonstrated hypoxia-specific unique properties of SQAP.

Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro.

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 µM and 99% at 15 µM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 µM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

Isolation, synthesis, and biological activities of a bibenzyl from Empetrum nigrum var. japonicum.

4-(2-Hydroxyphenethyl)-2,6-dimethoxyphenol, a bibenzyl, was isolated from the leaves of Empetrum nigrum var. japonicum, collected from Mount Tateyama. Japanese rock ptarmigans frequently eat the leaves and fruits of this plant. The structure of the bibenzyl was confirmed by NMR spectroscopic analysis and fully characterized. A synthesis of this compound was accomplished by coupling 2-hydroxyphenylacetic acid with syringaldehyde, decarboxylation of the resultant isoaurones, and hydrogenation of the double bond in the corresponding stilbene. This compound displayed cytotoxic activity against human cancer cells (HCT116 and Hela cells) and leukemia cells (HL-60 cells). The present study suggests that this plant serves as a source of biologically active natural products. Also, our findings provide information on the secondary metabolites in the diet of Japanese rock ptarmigans.

Synthesis of nucleotide analogues, EFdA, EdA and EdAP, and the effect of EdAP on hepatitis B virus replication.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) and 4'-ethynyl-2'-deoxyadenosine (EdA) are nucleoside analogues which inhibit human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. EdAP, a cyclosaligenyl (cycloSal) phosphate derivative of EdA, inhibits the replication of the influenza A virus. The common structural feature of these compounds is the ethynyl group at the 4'-position. In this study, these nucleoside analogues were prepared by a common synthetic strategy starting from the known 1,2-di-O-acetyl-D-ribofuranose. Biological evaluation of EdAP revealed that this compound reduced hepatitis B virus (HBV) replication dose-dependently without cytotoxicity against host cells tested in this study.