Simultaneous analysis of DL-Amino acids in foods and beverages using a highly sensitive chiral resolution labeling reagent.

Amino acids with various functions are abundant in living organisms and foods. Recent advances in analytical technology show that trace amounts of D-amino acids exist in living organisms and foods. In addition, studies show that these amino acids are involved in various physiological functions that differ from those of L-amino acids. Thus, a technique for analyzing DL-amino acids is required. However, the simultaneous separation and highly sensitive detection of DL-amino acids are complicated; therefore, highly sensitive analytical methods that can rapidly separate and identify compounds are required. We previously developed our original chiral resolution labeling reagents for the separation and highly sensitive detection of DL-amino acids. Here, we developed a simple method for the rapid separation and highly sensitive detection of DL-amino acids in various foods and beverages by liquid chromatography-mass spectrometry (LC-MS) using an octadecyl (C18) column after labeling with 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamineamide (D-FDLDA; enantiomeric excess > 99.9 %). In addition, we synthesized a stable isotope (13C6)-labeled D-FDLDA (13C6-D-FDLDA) and established an analytical method that can accurately identify the peak of each DL-amino acid. MS sensitivity of DL-amino acids labeled with our labeling reagent was higher than that of conventional labeling reagents (Marfey's reagents). The labeling reagent was neither desorbed from each DL-amino acid nor degraded for at least 1 week at 4 °C. Furthermore, we determined the DL-amino acid contents in foods and beverages using the proposed method, and differences in the total amino acid content and D/L ratio in each food and beverage were observed.

Probing for optimal photoaffinity linkers of benzophenone-based photoaffinity probes for adenylating enzymes.

The adenylation (A) domain of non-ribosomal peptide synthetases (NRPSs) catalyzes the adenylation reaction with substrate amino acids and ATP. Leveraging the distinct substrate specificity of A-domains, we previously developed photoaffinity probes for A-domains based on derivatization with a 5'-O-N-(aminoacyl)sulfamoyl adenosine (aminoacyl-AMS)-appended clickable benzophenone. Although our photoaffinity probes with different amino acid warheads enabled selective detection, visualization, and enrichment of target A-domains in proteomic environments, the effects of photoaffinity linkers have not been investigated. To explore the optimal benzophenone-based linker scaffold, we designed seven photoaffinity probes for the A-domains with different lengths, positions, and molecular shapes. Using probes 2-8 for the phenylalanine-activating A-domain of gramicidin S synthetase A (GrsA), we systematically investigated the binding affinity and labeling efficiency of the endogenous enzyme in a live producer cell. Our results indicated that the labeling efficiencies of probes 2-8 tended to depend on their binding affinities rather than on the linker length, flexibility, or position of the photoaffinity group. We also identified that probe 2 with a 4,4'-diaminobenzophenone linker exhibits the highest labeling efficiency for GrsA with fewer non-target labeling properties in live cells.

Teleocidin B-4, a PKC Activator, Upregulates Hypoxia-Inducible Factor 1 (HIF-1) Activity by Promoting the Accumulation of HIF-1α Protein via the PKCα/mTORC Signaling Pathway.

Hypoxia-inducible factor 1 (HIF-1) signaling is upregulated in an oxygen-dependent manner under hypoxic conditions. Activation of HIF-1 signaling increases the expression of HIF-1 target genes involved in cell survival, proliferation, and angiogenesis. Therefore, compounds that activate HIF-1 signaling have therapeutic potential in ischemic diseases. Screening for compounds that activate HIF-1 activity identified a microbial metabolite, teleocidin B-4, a PKC activator. Other PKC activators, such as TPA and 10-Me-Aplog-1, also activated HIF-1 activity. PKC activators induced HIF-1α protein accumulation through PKCα/mTORC activation. These results suggest that PKC activators without tumor-promoting activity have potential as therapeutic agents via HIF-1 target gene activation.

Efficacy of highly bioavailable oral curcumin in asymptomatic or mild COVID-19 patients: a double-blind, randomized, placebo-controlled trial.

INTRODUCTION: Even after the peak of the COVID-19 pandemic, the number of mild cases remains high, requiring continuous control. Curcumin, owing to its anti-inflammatory properties, can suppress vital proliferation and cytokine secretion in animal models. We developed a highly absorbable curcumin, curcuRouge® (cR), which is approximately 100 times more orally bioavailable than conventional curcumin. We evaluated the effect of cR on the inhibition of disease progression in asymptomatic or mildly symptomatic COVID-19 patients. METHODS: This study evaluated the effect of 7-day oral intake of cR (360 mg twice daily). Patients within 5 days of COVID-19 diagnosis were randomly assigned to a placebo or cR group in a double-blind manner. RESULTS: Primary endpoint events [body temperature (BT) ≥ 37.5 °C and saturation of percutaneous oxygen (SpO2) < 96%] were fewer than expected, and the rate of these events was 2.8% in the cR group (2/71) and 6.0% in the placebo group (4/67); hazard ratio (HR) = 0.532, 95% confidence interval (CI) 0.097-2.902. Patients receiving cR tended to take fewer antipyretic medications than those receiving placebo (HR = 0.716, 95% CI 0.374-1.372). Among patients with a normal range of BT at baseline, the BT change rate was significantly (p = 0.014) lower in the cR group (- 0.34%) versus placebo (- 0.01%). CONCLUSION: The relative suppression of event rates and antipyretic medications taken, and significant decrease of subclinical BT support the anti-inflammatory effects of cR in asymptomatic or mildly symptomatic patients with COVID-19. TRIAL REGISTRATION: Japan Registry of Clinical Trials (CRB5200002).

Mechanistic study of the retro-aza-Michael reaction in saccharothriolide L: identification of 2-amino-4-methylphenol as an effective protecting tool for the Michael acceptor.

Saccharothriolide L (1), a derivative of saccharothriolides (STLs) produced by the rare actinomycete Saccharotrix sp. A1506, was synthesized through the precursor-directed in situ synthesis (PDSS) method. The structure of 1 was determined by 1D and 2D NMR and HR-ESI-MS data analyses. A comparison of the rate of the retro-aza-Michael reaction between saccharothriolide L (1) and saccharothriolide B (2) indicated that the 2-amino-4-methylphenol group in 1 might be an effective masking tool for highly reactive, bioactive α, ß-unsaturated carbonyl compounds.

Tumescenamide C, a cyclic lipodepsipeptide from Streptomyces sp. KUSC_F05, exerts antimicrobial activity against the scab-forming actinomycete Streptomyces scabiei.

The antimicrobial activity of tumescenamide C against the scab-forming S. scabiei NBRC13768 was confirmed with a potent IC50 value (1.5 µg/mL). Three tumescenamide C-resistant S. scabiei strains were generated to compare their gene variants. All three resistant strains contained nonsynonymous variants in genes related to cellobiose/cellotriose transport system components; cebF1, cebF2, and cebG2, which are responsible for the production of the phytotoxin thaxtomin A. Decrease in thaxtomin A production and the virulence of the three resistant strains were revealed by the LC/MS analysis and necrosis assay, respectively. Although the nonsynonymous variants were insufficient for identifying the molecular target of tumescenamide C, the cell wall component wall teichoic acid (WTA) was observed to bind significantly to tumescenamide C. Moreover, changes in the WTA contents were detected in the tumescenamide C-resistant strains. These results imply that tumescenamide C targets the cell wall system to exert antimicrobial effects on S. scabiei.

Development of a chemical scaffold for inhibiting nonribosomal peptide synthetases in live bacterial cells.

The adenylation (A) domain is essential for non-ribosomal peptide synthetases (NRPSs), which synthesize various peptide-based natural products, including virulence factors, such as siderophores and genotoxins. Hence, the inhibition of A-domains could attenuate the virulence of pathogens. 5'-O-N-(Aminoacyl or arylacyl)sulfamoyladenosine (AA-AMS) is a bisubstrate small-molecule inhibitor of the A-domains of NRPSs. However, the bacterial cell permeability of AA-AMS is typically a problem owing to its high hydrophilicity. In this study, we investigated the influence of a modification of 2'-OH in the AMS scaffold with different functional groups on binding to target enzymes and bacterial cell penetration. The inhibitor 7 with a cyanomethyl group at 2'-OH showed desirable inhibitory activity against both recombinant and intracellular gramicidin S synthetase A (GrsA) in the gramicidin S-producer Aneurinibacillus migulanus ATCC 9999, providing an alternative scaffold to develop novel A-domain inhibitors.

Precezomycin, a novel antibiotic biosynthetic precursor of cezomycin, from actinomycete Kitasatospora putterlickiae 10-13.

A novel antibiotic biosynthetic precursor of cezomycin, named precezomycin (1), was isolated from culture broth of actinomycete Kitasatospora putterlickiae 10-13. The planar structure was determined by 1D/2D NMR and HR(ESI)MS data analyses, and the absolute configurations were established by TDDFT calculation of ECD spectra. Precezomycin (1) exhibited moderate antibacterial activity against gram-positive bacteria including Staphylococcus aureus and Bacillus subtilis. The discovery of 1 extends the natural product family of cezomycin and provides a new insight into understanding the biosynthetic process of these polyether ionophore metabolites.

Amoxetamide A, a new anoikis inducer, produced by combined-culture of Amycolatopsis sp. and Tsukamurella pulmonis.

Cancer cells including colorectal cancer cells are resistant to anoikis, an anchorage-independent programmed death, which enables metastasis and subsequent survival in a new tumor microenvironment. In this study, we identified a new anoikis inducer, amoxetamide A (1) with a ß-lactone moiety, that was produced by combined-culture of Amycolatopsis sp. 26-4 and mycolic acid-containing bacteria (MACB) Tsukamurella pulmonis TP-B0596. The structure of 1 including the stereochemistry of C8 was determined by MS and NMR spectroscopy and modified Mosher's method, and the absolute configurations of C11 and C12 were suggested as 11R and 12S, respectively, by GIAO NMR calculations. Amoxetamide A (1) exhibited anoikis-inducing activity in human colorectal cancer HT-29 cells in anchorage-independent culture conditions.

Bisabosqual A: A novel asparagine synthetase inhibitor suppressing the proliferation and migration of human non-small cell lung cancer A549 cells.

Asparagine synthetase (ASNS) is a crucial enzyme for the de novo biosynthesis of endogenous asparagine (Asn), and ASNS shows the positive relationship with the growth of several solid tumors. Most of ASNS inhibitors are analogs of transition-state in ASNS reaction, but their low cell permeability hinders their anticancer activity. Therefore, novel ASNS inhibitors with a new pharmacophore urgently need to be developed. In this study, we established and applied a system for in vitro screening of ASNS inhibitors, and found a promising unique bisabolane-type meroterpenoid molecule, bisabosqual A (Bis A), able to covalently modify K556 site of ASNS protein. Bis A targeted ASNS to suppress cell proliferation of human non-small cell lung cancer A549 cells and exhibited a synergistic effect with L-asparaginase (L-ASNase). Mechanistically, Bis A promoted oxidative stress and apoptosis, while inhibiting autophagy, cell migration and epithelial-mesenchymal transition (EMT), impeding cancer cell development. Moreover, Bis A induced negative feedback pathways containing the GCN2-eIF2α-ATF4, PI3K-AKT-mTORC1 and RAF-MEK-ERK axes, but combination treatment of Bis A and rapamycin/torin-1 overcame the potential drug resistance triggered by mTOR pathways. Our study demonstrates that ASNS inhibition is promising for cancer chemotherapy, and Bis A is a potential lead ASNS inhibitor for anticancer development.

A small molecule iCDM-34 identified by in silico screening suppresses HBV DNA through activation of aryl hydrocarbon receptor.

IFN-alpha have been reported to suppress hepatitis B virus (HBV) cccDNA via APOBEC3 cytidine deaminase activity through interferon signaling. To develop a novel anti-HBV drug for a functional cure, we performed in silico screening of the binding compounds fitting the steric structure of the IFN-alpha-binding pocket in IFNAR2. We identified 37 compounds and named them in silico cccDNA modulator (iCDM)-1-37. We found that iCDM-34, a new small molecule with a pyrazole moiety, showed anti-HCV and anti-HBV activities. We measured the anti-HBV activity of iCDM-34 dependent on or independent of entecavir (ETV). iCDM-34 suppressed HBV DNA, pgRNA, HBsAg, and HBeAg, and also clearly exhibited additive inhibitory effects on the suppression of HBV DNA with ETV. We confirmed metabolic stability of iCDM-34 was stable in human liver microsomal fraction. Furthermore, anti-HBV activity in human hepatocyte-chimeric mice revealed that iCDM-34 was not effective as a single reagent, but when combined with ETV, it suppressed HBV DNA compared to ETV alone. Phosphoproteome and Western blotting analysis showed that iCDM-34 did not activate IFN-signaling. The transcriptome analysis of interferon-stimulated genes revealed no increase in expression, whereas downstream factors of aryl hydrocarbon receptor (AhR) showed increased levels of the expression. CDK1/2 and phospho-SAMHD1 levels decreased under iCDM-34 treatment. In addition, AhR knockdown inhibited anti-HCV activity of iCDM-34 in HCV replicon cells. These results suggest that iCDM-34 decreases the phosphorylation of SAMHD1 through CDK1/2, and suppresses HCV replicon RNA, HBV DNA, and pgRNA formation.

Variational autoencoder-based chemical latent space for large molecular structures with 3D complexity.

The structural diversity of chemical libraries, which are systematic collections of compounds that have potential to bind to biomolecules, can be represented by chemical latent space. A chemical latent space is a projection of a compound structure into a mathematical space based on several molecular features, and it can express structural diversity within a compound library in order to explore a broader chemical space and generate novel compound structures for drug candidates. In this study, we developed a deep-learning method, called NP-VAE (Natural Product-oriented Variational Autoencoder), based on variational autoencoder for managing hard-to-analyze datasets from DrugBank and large molecular structures such as natural compounds with chirality, an essential factor in the 3D complexity of compounds. NP-VAE was successful in constructing the chemical latent space from large-sized compounds that were unable to be handled in existing methods, achieving higher reconstruction accuracy, and demonstrating stable performance as a generative model across various indices. Furthermore, by exploring the acquired latent space, we succeeded in comprehensively analyzing a compound library containing natural compounds and generating novel compound structures with optimized functions.

Simultaneous separation and identification of all structural isomers and enantiomers of aminobutyric acid using a highly sensitive chiral resolution labeling reagent.

Aminobutyric acid has structural isomers (α-, ß-, and γ-aminobutyric acids) and enantiomers (D/L-forms) with various unique functions. Therefore, a quantitative method for determining the content of each aminobutyric acid must be developed. In general, quantitative simultaneous analysis of multiple compounds is conducted via high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). However, simultaneous separation and highly sensitive detection of all aminobutyric acids are complicated, so highly sensitive analytical methods for the separation and identification of each compound have not yet been established. We previously developed highly sensitive chiral resolution labeling reagents. Herein, we propose a highly sensitive analytical method for the simultaneous separation and identification of all aminobutyric acids via LC-MS and labeling with our original highly sensitive chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine amide (L-FDVDA). The labeling reagent was completely bound to all aminobutyric acids through incubation overnight (>15 h) at 50 °C. Additionally, the labeled aminobutyric acids could be stored for at least 1 week at 4 °C. Furthermore, we demonstrated simultaneous separation and identification of aminobutyric acids in biological samples and foods through LC-MS using a C18 column after labeling with L-FDVDA. Our method is expected to be adopted for the analysis of the contents of all aminobutyric acids in biological and clinical samples as well as various foods.

Establishment of an MR1 Presentation Reporter Screening System and Identification of Phenylpropanoid Derivatives as MR1 Ligands.

Mucosal-associated invariant T (MAIT) cells are innate-like T cells that are modulated by ligands presented on MHC class I-related proteins (MR1). These cells have attracted attention as potential drug targets because of their involvement in the initial response to infection and various disorders. Herein, we have established the MR1 presentation reporter assay system employing split-luciferase, which enables the efficient exploration of MR1 ligands. Using our screening system, we identified phenylpropanoid derivatives as MR1 ligands, including coniferyl aldehyde, which have an ability to inhibit the MR1-MAIT cell axis. Further, the structure-activity relationship study of coniferyl aldehyde analogs revealed the key structural features of ligands required for MR1 recognition. These results will contribute to identifying a broad range of endogenous and exogenous MR1 ligands and to developing novel MAIT cell modulators.

Separation and Identification of Isoleucine Enantiomers and Diastereomers Using an Original Chiral Resolution Labeling Reagent.

D-Amino acids, which are present in small amounts in living organisms, are responsible for a variety of physiological functions. Some bioactive/biomolecular peptides also contain D-amino acids in their sequences; such peptides express different functions than peptides composed only of L-form amino acids. Among the 20 amino acids that make up proteins, threonine (Thr) and isoleucine (Ile) have two chiral carbons and thus have two enantiomers and diastereomers. These stereoisomers have been previously analyzed through HPLC using chiral columns or chiral resolution labeling reagents. However, the separation and identification of these stereoisomers are highly laborious and complicated. Herein, we propose an analytical method for the separation and identification of Ile stereoisomers through LC-MS using our original chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine-amide (L-FDVDA) and a PBr column packed with pentabromobenzyl-modified silica gel. Twenty DL-amino acids including Thr stereoisomers (41 amino acids including glycine) were separated and identified using C18 column. Ile stereoisomers could be separated using not a C18 column but a PBr column. Additionally, we showed that peptides containing Thr and Ile stereoisomers can be accurately detected through labeling with L-FDVDA.

Separation of amyloid ß fragment peptides with racemised and isomerised aspartic acid residues using an original chiral resolution labeling reagent.

We developed a system to separate and identify racemised and isomerised aspartic acid (Asp) residues in amyloid ß (Aß) by labeling with an original chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-D-leucine-N,N-dimethylethylenediamine-amide (D-FDLDA). The racemised and isomerised Asp residues labeled with D-FDLDA in Aß fragments generated by digesting with trypsin and endoproteinase Glu-C were separated and identified by liquid chromatography-mass spectrometry (LC-MS) under simple gradient conditions. Furthermore, the labeled Aß fragments did not aggregate and remained stable at least for 1 week at 4 °C.

Exploring a chemical scaffold for rapid and selective photoaffinity labelling of non-ribosomal peptide synthetases in living bacterial cells.

Non-ribosomal peptide synthetases (NRPSs) biosynthesize many pharmaceuticals and virulence factors. The biosynthesis of these natural peptide products from biosynthetic gene clusters depends on complex regulations in bacteria. However, our current knowledge of NRPSs is based on enzymological studies using full NRPS systems and/or a single NRPS domain in heterologous hosts. Chemical and/or biochemical strategies to capture the endogenous activities of NRPSs facilitate studies on NRPS cell biology in bacterial cells. Here, we describe a chemical scaffold for the rapid and selective photoaffinity labelling of NRPSs in purified systems, crude biological samples and living bacterial cells. We synthesized photoaffinity labelling probes coupled with 5'-O-N-(phenylalanyl)sulfamoyladenosine with clickable alkyl diazirine or trifluoromethyl phenyl diazirine. We found that a trifluoromethyl phenyl diazirine-based probe cross-linked the Phe-activating domain of a GrsA-NRPS with high selectivity and sensitivity at shorter ultraviolet (UV) irradiation times (less than 5 min) relative to a prototypical benzophenone-based probe. Our results demonstrated that this quick labelling protocol can prevent damage to proteins and cells caused by long UV irradiation times, providing a mild photoaffinity labelling method for biological samples. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Solid-phase total synthesis and structural confirmation of antimicrobial longicatenamide A.

Longicatenamides A-D are cyclic hexapeptides isolated from the combined culture of Streptomyces sp. KUSC_F05 and Tsukamurella pulmonis TP-B0596. Because these peptides are not detected in the monoculture broth of the actinomycete, they are key tools for understanding chemical communication in the microbial world. Herein, we report the solid-phase total synthesis and structural confirmation of longicatenamide A. First, commercially unavailable building blocks were chemically synthesized with stereocontrol. Second, the peptide chain was elongated via Fmoc-based solid-phase peptide synthesis. Third, the peptide chain was cyclized in the solution phase, followed by simultaneous cleavage of all protecting groups to afford longicatenamide A. Chromatographic analysis corroborated the chemical structure of longicatenamide A. Furthermore, the antimicrobial activity of synthesized longicatenamide A was confirmed. The developed solid-phase synthesis is expected to facilitate the rapid synthesis of diverse synthetic analogues.

FK506-binding protein, FKBP12, promotes serine utilization and negatively regulates threonine deaminase in fission yeast.

FK506-binding protein with a molecular weight of 12 kDa (FKBP12) is a receptor of the immunosuppressive drugs, FK506 and rapamycin. The physiological functions of FKBP12 remain ambiguous because of its nonessentiality and multifunctionality. Here, we show that FKBP12 promotes the utilization of serine as a nitrogen source and regulates the isoleucine biosynthetic pathway in fission yeast. In screening for small molecules that inhibit serine assimilation, we found that the growth of fission yeast cells in medium supplemented with serine as the sole nitrogen source, but not in glutamate-supplemented medium, was suppressed by FKBP12 inhibitors. Knockout of FKBP12 phenocopied the action of these compounds in serine-supplemented medium. Metabolome analyses and genetic screens identified the threonine deaminase, Tda1, to be regulated downstream of FKBP12. Genetic and biochemical analyses unveiled the negative regulation of Tda1 by FKBP12. Our findings reveal new roles of FKBP12 in amino acid biosynthesis and nitrogen metabolism homeostasis.

Pharmacologic characterization of TBP1901, a prodrug form of aglycone curcumin, and CRISPR-Cas9 screen for therapeutic targets of aglycone curcumin.

Curcumin (aglycone curcumin) has antitumor properties in a variety of malignancies via the alteration of multiple cancer-related biological pathways; however, its clinical application has been hampered due to its poor bioavailability. To overcome this limitation, we have developed a synthesized curcumin ß-D-glucuronide sodium salt (TBP1901), a prodrug form of aglycone curcumin. In this study, we aimed to clarify the pharmacologic characteristics of TBP1901. In ß-glucuronidase (GUSB)-proficient mice, both curcumin ß-D-glucuronide and its active metabolite, aglycone curcumin, were detected in the blood after TBP1901 injection, whereas only curcumin ß-D-glucuronide was detected in GUSB-impaired mice, suggesting that GUSB plays a pivotal role in the conversion of TBP1901 into aglycone curcumin in vivo. TBP1901 itself had minimal antitumor effects in vitro, whereas it demonstrated significant antitumor effects in vivo. Genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screen disclosed the genes associated with NF-κB signaling pathway and mitochondria were among the highest hit. In vitro, aglycone curcumin inhibited NF-kappa B signaling pathways whereas it caused production of reactive oxygen species (ROS). ROS scavenger, N-acetyl-L-cysteine, partially reversed antitumor effects of aglycone curcumin. In summary, TBP1901 can exert antitumor effects as a prodrug of aglycone curcumin through GUSB-dependent activation.