A new benzoquinone and a new stilbenoid from Paphiopedilum exul (Ridl.) Rolfe.

One new alkyl benzoquinone, paphionone (1), one new trans-stilbenoid, (E)-6,5'-dihydroxy-2,3'-dimethoxystilbene (2), and eight known stilbenoids and flavonoids (3-10) were isolated from the leaves and roots of Paphiopedilum exul (Orchidaceae). Their chemical structures were determined based on IR, ECD, MS and NMR analyses. Cytotoxicity of all isolated compounds towards human hepatocellular carcinoma (HepG2) cell line was examined in vitro by MTT assay. The para-hydroxybenzyl substituted stilbene 10 was potently cytotoxic to the cancer cells, with an IC50 value of 4.80 ± 1.10 µM (selectivity index = 20.83). All compounds were non-toxic to normal human embryo fibroblast (OUMS-36) cell line.

Nucleic Acid-Templated Synthesis of Cationic Styryl Dyes in Vitro and in Living Cells.

A novel method for synthesizing cationic styryl dyes through a nucleic acid-templated reaction has been developed. This approach overcomes issues associated with traditional synthesis methods, such as harsh conditions, low throughput, and wasteful chemicals. The presence of a nucleic acid template accelerated the styryl dye formation from quaternized heteroaromatic and cationic aldehyde substrates. These styryl dyes show remarkable optical properties change when bound to nucleic acids, hence the success of the synthesis could be readily monitored in situ by UV-Vis and fluorescence spectroscopy and the optical properties data were also observable at the same time. This method provides the desired products from a broad range of coupling partners. By employing different substrates and templates, it is possible to identify new dyes that can bind to a specific type of nucleic acid such as a G-quadruplex. The templated dye synthesis is also successfully demonstrated in live HeLa cells. This approach is a powerful tool for the rapid synthesis and screening of dyes specific for diverse types of nucleic acids or cellular organelles, facilitating new biological discoveries.

Design, Synthesis, and Characterization of Novel Styryl Dyes as Fluorescent Probes for Tau Aggregate Detection in Vitro and in Cells.

A series of novel styryl dye derivatives incorporating indolium and quinolinium core structures were successfully synthesized to explore their interacting and binding capabilities with tau aggregates in vitro and in cells. The synthesized dyes exhibited enhanced fluorescence emission in viscous environments due to the rotatable bond confinement in the core structure. Dye 4, containing a quinolinium moeity and featuring two cationic sites, demonstrated a 28-fold increase in fluorescence emission upon binding to tau aggregates. This dye could also stain tau aggregates in living cells, confirmed by cell imaging using confocal fluorescence microscopy. A molecular docking study was conducted to provide additional visualization and support for binding interactions. This work offers novel and non-cytotoxic fluorescent probes with desirable photophysical properties, which could potentially be used for studying tau aggregates in living cells, prompting further development of new fluorescent probes for early Alzheimer's disease detection.

Diphenyl ethers from the cultured lichen mycobiont of Graphis handelii Zahlbr.

Purpose: Cultured lichen mycobionts are valuable sources of new natural compounds. Mycobiont of Graphis handelii growing in Vietnam was isolated, cultivated and chemically investigated. The crude extract of this cultured mycobiont showed potent alpha-glucosidase inhibition with an IC50 value of 50 µg/mL. Methods: Multiple chromatographic methods were applied to the extract to isolate compounds. The combination of Nuclear Magnetic Resonance analysis and high-resolution mass spectroscopy determined their chemical structures. Electrophilic bromination/chlorination was applied to obtain new derivatives using NaBr/H2O2 and NaCl/H2O2 reagents. Compounds were evaluated for enzyme inhibitory activities, including alpha-glucosidase inhibition, HIV-1 reverse transcriptase inhibition, SARS-CoV-2 main protease (Mpro) inhibition, anti-inflammatory activity, and cytotoxicity against several cancer cell lines. A molecular docking study for anti-SARS-CoV-2 was conducted to understand the inhibitory mechanism. Results: A new diphenyl ether, handelone (1) and a known compound xylarinic acid A (2) were isolated and elucidated. Four synthetic products 6'-bromohandelone (1a), 2'-bromohandelone (1b), 2',6'-dibromohandelone (1c), and 2',6'-dichlorohandelone (1d) were prepared. Compound 1 showed good activity against Mpro with an IC50 value of 5.2 µM but it showed weak or inactive activity in other tests. Other compounds were inactive in all assays. Conclusion: A new compound, handelone (1) was isolated from the cultured mycobiont of Graphis handelii. From these compounds, four new derivatives were prepared. Compound 1 showed good activity against Mpro with an IC50 value of 5.2 µM but it showed weak or inactive activity in other tests. Other compounds were inactive in all assays.

FMO-guided design of darunavir analogs as HIV-1 protease inhibitors.

The prevalence of HIV-1 infection continues to pose a significant global public health issue, highlighting the need for antiretroviral drugs that target viral proteins to reduce viral replication. One such target is HIV-1 protease (PR), responsible for cleaving viral polyproteins, leading to the maturation of viral proteins. While darunavir (DRV) is a potent HIV-1 PR inhibitor, drug resistance can arise due to mutations in HIV-1 PR. To address this issue, we developed a novel approach using the fragment molecular orbital (FMO) method and structure-based drug design to create DRV analogs. Using combinatorial programming, we generated novel analogs freely accessible via an on-the-cloud mode implemented in Google Colab, Combined Analog generator Tool (CAT). The designed analogs underwent cascade screening through molecular docking with HIV-1 PR wild-type and major mutations at the active site. Molecular dynamics (MD) simulations confirmed the assess ligand binding and susceptibility of screened designed analogs. Our findings indicate that the three designed analogs guided by FMO, 19-0-14-3, 19-8-10-0, and 19-8-14-3, are superior to DRV and have the potential to serve as efficient PR inhibitors. These findings demonstrate the effectiveness of our approach and its potential to be used in further studies for developing new antiretroviral drugs.

Machine learning-guided design of potent darunavir analogs targeting HIV-1 proteases: A computational approach for antiretroviral drug discovery.

In the pursuit of novel antiretroviral therapies for human immunodeficiency virus type-1 (HIV-1) proteases (PRs), recent improvements in drug discovery have embraced machine learning (ML) techniques to guide the design process. This study employs ensemble learning models to identify crucial substructures as significant features for drug development. Using molecular docking techniques, a collection of 160 darunavir (DRV) analogs was designed based on these key substructures and subsequently screened using molecular docking techniques. Chemical structures with high fitness scores were selected, combined, and one-dimensional (1D) screening based on beyond Lipinski's rule of five (bRo5) and ADME (absorption, distribution, metabolism, and excretion) prediction implemented in the Combined Analog generator Tool (CAT) program. A total of 473 screened analogs were subjected to docking analysis through convolutional neural networks scoring function against both the wild-type (WT) and 12 major mutated PRs. DRV analogs with negative changes in binding free energy ( ΔΔ G bind ) compared to DRV could be categorized into four attractive groups based on their interactions with the majority of vital PRs. The analysis of interaction profiles revealed that potent designed analogs, targeting both WT and mutant PRs, exhibited interactions with common key amino acid residues. This observation further confirms that the ML model-guided approach effectively identified the substructures that play a crucial role in potent analogs. It is expected to function as a powerful computational tool, offering valuable guidance in the identification of chemical substructures for synthesis and subsequent experimental testing.

Potential of Arabica Coffee Beans from Northern Thailand: Exploring Antidiabetic Metabolites through Liquid Chromatography with Tandem Mass Spectrometry (LC-MS/MS) Metabolomic Profiling across Diverse Postharvest Processing Techniques.

Coffee, a widely consumed beverage worldwide, undergoes postharvest methods that influence its physicochemical characteristics, while roasting modulates its composition, affecting sensory attributes. This study investigates the impact of distinct postharvest methods (washed and natural) on the antidiabetic activities, including α-amylase and DPP4, as well as the phytochemical profiling of geological indicator (GI) coffee beans (Coffea arabica L.). The results indicate notable differences in antidiabetic activity and phytochemical profiles between washed and natural processing methods. Coffee beans processed naturally exhibit significant suppression of DPP4 and α-amylase activities (p-value < 0.01) compared to beans processed using the washed technique. TLC profiling using the ratios of the solvent systems of ethyl acetate/dichloromethane (DCM) and acetone/DCM as separation solvents reveals dominant spots for the washed technique. LC-MS/MS-based untargeted metabolomics analysis using principle component analysis (PCA) clearly segregates samples processed by the natural and washed techniques without any overlap region. A total of 1114 phytochemicals, including amino acids and short peptides, are annotated. The natural processing of coffee beans has been shown to yield a slightly higher content of chlorogenic acid (CGA) compared to the washed processing method. Our findings highlight the distinct bioactivities and phytochemical compositions of GI coffee beans processed using different techniques. This information can guide consumers in choosing coffee processing methods that offer potential benefits in terms of alternative treatment for diabetes.

Exploring the Apoptotic-Induced Biochemical Mechanism of Traditional Thai Herb (Kerra™) Extract in HCT116 Cells Using a Label-Free Proteomics Approach.

Background and Objectives: Natural products have proven to be a valuable source for the discovery of new candidate drugs for cancer treatment. This study aims to investigate the potential therapeutic effects of "Kerra™", a natural extract derived from a mixture of nine medicinal plants mentioned in the ancient Thai scripture named the Takxila Scripture, on HCT116 cells. Materials and Methods: In this study, the effect of the Kerra™ extract on cancer cells was assessed through cell viability assays. Apoptotic activity was evaluated by examining the apoptosis characteristic features. A proteomics analysis was conducted to identify proteins and pathways associated with the extract's mechanism of action. The expression levels of apoptotic protein markers were measured to validate the extract's efficacy. Results: The Kerra™ extract demonstrated a dose-dependent inhibitory effect on the cells, with higher concentrations leading to decreased cell viability. Treatment with the extract for 72 h induced characteristic features of early and late apoptosis, as well as cell death. An LC-MS/MS analysis identified a total of 3406 proteins. The pathway analysis revealed that the Kerra™ extract stimulated apoptosis and cell death in colorectal cancer cell lines and suppressed cell proliferation in adenocarcinoma cell lines through the EIF2 signaling pathway. Upstream regulatory proteins, including cyclin-dependent kinase inhibitor 1A (CDKN1A) and MYC proto-oncogene, bHLH transcription factor (MYC), were identified. The expressions of caspase-8 and caspase-9 were significantly elevated by the Kerra™ extract compared to the chemotherapy drug Doxorubicin (Dox). Conclusions: These findings provide strong evidence for the ability of the Kerra™ extract to induce apoptosis in HCT116 colon cancer cells. The extract's efficacy was demonstrated by its dose-dependent inhibitory effect, induction of apoptotic activity, and modulation of key proteins involved in cell death and proliferation pathways. This study highlights the potential of Kerra™ as a promising therapeutic agent in cancer treatment.

Reaction mechanism and kinetics of the two-component flavoprotein dimethyl sulfone monooxygenase system: Using hydrogen peroxide for monooxygenation and substrate cleavage.

The dimethyl sulfone monooxygenase system is a two-component flavoprotein, catalyzing the monooxygenation of dimethyl sulfone (DMSO2 ) by oxidative cleavage producing methanesulfinate and formaldehyde. The reductase component (DMSR) is a flavoprotein with FMN as a cofactor, catalyzing flavin reduction using NADH. The monooxygenase (DMSMO) uses reduced flavin from the reductase and oxygen for substrate monooxygenation. DMSMO can bind to FMN and FMNH- with a Kd of 17.4 ± 0.9 µm and 4.08 ± 0.8 µm, respectively. The binding of FMN to DMSMO is required prior to binding DMSO2 . This also applies to the fast binding of reduced FMN to DMSMO followed by DMSO2 . Substituting reduced DMSR with FMNH- demonstrated the same oxidation kinetics, indicating that FMNH- from DMSR was transferred to DMSMO. The oxidation of FMNH- :DMSMO, with and without DMSO2 did not generate any flavin adducts for monooxygenation. Therefore, H2 O2 is likely to be the reactive agent to attack the substrate. The H2 O2 assay results demonstrated production of H2 O2 from the oxidation of FMNH- :DMSMO, whereas H2 O2 was not detected in the presence of DMSO2 , confirming H2 O2 utilization. The rate constant for methanesulfinate formation determined from rapid quenched flow and the rate constant for flavin oxidation were similar, indicating that H2 O2 rapidly reacts with DMSO2 , with flavin oxidation as the rate-limiting step. This is the first report of the kinetic mechanisms of both components using rapid kinetics and of a method for methanesulfinate detection using LC-MS.

Possibility to Apply Strontium Aluminate to Produce Light-Emitting Plants: Efficiency and Safety.

Light-emitting plants (LEPs) provides light in areas without electricity. The phosphorescent compound was used as a lighting material for LEP development. However, using the phosphorescent compound for LEPs development required optimization and phytotoxicity evaluation. Strontium aluminate (SrAl2 O4 ) is a phosphorescent compound that can glow for a long time and is easily recharged by visible light. In this study, using SrAl2 O4 to develop LEPs was evaluated. Additionally, plant stress under SrAl2 O4 was investigated. Metabolomic analysis can explain the possible mechanism of plants' stress under SrAl2 O4 . After, injecting 3 mL of 5 % (w/v) SrAl2 O4 products 1, 2, and 3 into the stem of Ipomoea aquatica, the result showed that SrAl2 O4 products 2 and 3 caused oxidative stress. The metabolomic analysis also indicated that I. aquatica responded to SrAl2 O4 product 1 by increasing pipecolic acid and salicylic acid, while I. aquatica injected with SrAl2 O4 products 2 and 3 showed a decrease in salicylic acid around 0.005 and 0.061-fold, respectively, compared to control plants. and an excess accumulation of MDA around 10.00-12.00 µmol g-1 FW. A 15 % concentration of SrAl2 O4 can be used for LEPs development, enabling photoemission 18-fold for 50 min. SrAl2 O4 product 1 has the potential to be a material for LEPs.

Schomburginones A‒J, geranylated benzophenones from the leaves of Garcinia schomburgkiana and their cytotoxic and anti-inflammatory activities.

Ten undescribed benzophenones, schomburginones A-J, together with 14 known analogs were isolated from the leaves of Garcinia schomburgkiana, an edible plant native to the Indochina region. The structures of the undescribed compounds were elucidated by NMR combined with HRMS spectroscopy, while their absolute configurations were determined using ECD and single-crystal X-ray diffraction analysis. The isolated metabolites represent benzophenone derivatives containing a modified monoterpene unit, including tri- and tetracyclic skeletons, which are rarely found in genus Garcinia. The cytotoxic evaluation on three cancerous cell lines demonstrated that schomburginone G, schomburginone H, and 3-geranyl-2,4,6-trihydroxybenzophenone were active against HeLa cells with IC50 values in the range of 12.2-15.7 µM, respectively, and selective compared to the non-cancerous L929 cells (SI > 3.5). In addition, the three cytotoxic compounds together with clusiacyclol A showed significant NO inhibitory activity in RAW 264.7 macrophage cells over 85% inhibition without obvious cytotoxicity at a final concentration of 100 µM. The promising activities of these compounds in cytotoxic and anti-inflammatory assays make them attractive for further study in the development of anticancer drugs.

One-Step Surface-Treatment Reagent (35% 3-O-Ethyl-l-ascorbic Acid Plus 50% Citric Acid Solution) Restores the Shear Bond Strength of Metal Brackets Bonded to Bleached Human Enamel: An In Vitro Study.

This study investigates how a new substance, composed of ethyl ascorbic acid and citric acid, affects the shear bond strength (SBS) of metal brackets when bonded to bleached teeth. Forty maxillary premolar teeth were used and randomly placed into four groups (n = 10): the control group did not undergo bleaching; the remaining groups underwent bleached using 35% hydrogen peroxide. In group A, 37% phosphoric acid was applied after bleaching. In group B, 10% sodium ascorbate was used for ten minutes before 37% phosphoric acid. In group C, 35%3-O-ethyl-l-ascorbic acid plus 50% citric acid solution (35EA/50CA) was applied for 5 min. The subgroups were bonded immediately after bleaching. The SBS was determined with a universal testing machine and analyzed using one-way ANOVA and then Tukey's HSD tests. Adhesive remnant index (ARI) scores were determined with a stereomicroscope and analyzed with a chi-squared test. The significance level was 0.05. Group C demonstrated significantly higher SBS values than group A (p < 0.001), but was not significantly different than the control group or group C (p > 0.05). The ARI scores were significantly different among the groups (p < 0.001). In conclusion, enamel surface treatment using 35EA/50CA improved the reduced SBS to an acceptable clinical level and reduced the clinical chair time.

Butylene Glycol Used as a Sustainable Solvent for Extracting Bioactive Compounds from Camellia sinensis Flowers with Ultrasound-Assisted Extraction.

The research aims to assess the yield of bioactive compounds and their antioxidant activities obtained from tea flowers using an ultrasound-assisted extraction method with butylene glycol (BG-UAE) through Box-Behnken design. It investigates the bioactive compounds including the total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC) and analyzes their antioxidant activities, bioactive compound composition by liquid chromatography triple quadrupole tandem mass spectrometry, and their cellular activities via UAE and maceration using BG or ethanol as the solvent. Under optimal conditions, the values of the TPC, TFC, TTC, 1,1-diphenyl-2-picrylhydrazil radical scavenging assay, 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical scavenging assay, and ferric reducing antioxidant power assay (FRAP) of the BG-UAE extract were 54.00 ± 1.19 mg GAE/g sample, 291.47 ± 3.34 mg QE/g sample, 65.37 ± 1.78 mg TAE/g sample, 106.45 ± 1.21 mg TEAC/g sample, 163.58 ± 2.76 mg TEAC/g sample, and 121.31 ± 4.75 mg FeSO4/g sample, respectively. Except for FRAP, BG-UAE exhibited the highest values in all parameters compared to the other extraction methods. Catechins and caffeine were predominantly detected in tea flower extracts through UAE with BG and ethanol (EtOH-UAE). BG-UAE exhibited greater cell viability and cellular antioxidant activity than EtOH-UAE. The researcher expects that this research will contribute to the emergence of a green extraction technique that will offer larger functional components with economic and environmental benefits and minimal chemicals and energy use.

Proteomics and Molecular Docking Analyses Reveal the Bio-Chemical and Molecular Mechanism Underlying the Hypolipidemic Activity of Nano-Liposomal Bioactive Peptides in 3T3-L1 Adipocytes.

Obesity is a global health concern. Physical activities and eating nutrient-rich functional foods can prevent obesity. In this study, nano-liposomal encapsulated bioactive peptides (BPs) were developed to reduce cellular lipids. The peptide sequence NH2-PCGVPMLTVAEQAQ-CO2H was chemically synthesized. The limited membrane permeability of the BPs was improved by encapsulating the BPs with a nano-liposomal carrier, which was produced by thin-layer formation. The nano-liposomal BPs had a diameter of ~157 nm and were monodispersed in solution. The encapsulation capacity was 61.2 ± 3.2%. The nano-liposomal BPs had no significant cytotoxicity on the tested cells, keratinocytes, fibroblasts, and adipocytes. The in vitro hypolipidemic activity significantly promoted the breakdown of triglycerides (TGs). Lipid droplet staining was correlated with TG content. Proteomics analysis identified 2418 differentially expressed proteins. The nano-liposomal BPs affected various biochemical pathways beyond lipolysis. The nano-liposomal BP treatment decreased the fatty acid synthase expression by 17.41 ± 1.17%. HDOCK revealed that the BPs inhibited fatty acid synthase (FAS) at the thioesterase domain. The HDOCK score of the BPs was lower than that of orlistat, a known obesity drug, indicating stronger binding. Proteomics and molecular docking analyses confirmed that the nano-liposomal BPs were suitable for use in functional foods to prevent obesity.

Helicene-Hydrazide Encapsulated Ethyl Cellulose as a Potential Fluorescence Sensor for Highly Specific Detection of Nonanal in Aqueous Solutions and a Proof-of-Concept Clinical Study in Lung Fluid.

Over the past years, lung cancer has been one of the vital cancer-related mortalities worldwide and has inevitably exhibited the highest death rate with the subsequent need for facile and convenient diagnosis approaches to identify the severity of cancer. Previous research has reported long-chain aldehyde compounds such as hexanal, heptanal, octanal, and nonanal as potential biomarkers of lung cancer. Herein, the helicene dye-encapsulated ethyl cellulose (EC@dye-NH) nanosensors have been applied for the potentially sensitive and specific detection of long-chain aldehydes in aqueous media. The sensors contain the intrinsic hydrazide group of dye-NH, which is capable of reacting an aldehyde group via imine formation and the EC backbone. This offers the synergistic forces of hydrophobic interactions with alkyl long-chain aldehydes, which could induce self-assembly encapsulation of EC@dye-NH nanosensors and strong fluorescence responses. The addition of long-chain aldehyde would induce the complete micellar-like nanoparticle formation within 15 min in acetate buffer pH 5.0. The limit of detection (LOD) values of EC@dye-NH nanosensors toward heptanal, octanal, and nonanal were 40, 100, and 10 µM, respectively, without interference from the lung fluid matrices and short-chain aldehydes. For practical applicability, this sensing platform was developed for quantification of the long-chain aldehydes in lung fluid samples with 98-101% recoveries. This EC@dye-NH nanosensor was applied to quantify nonanal contents in lung fluid samples. The results of this method based on EC@dye-NH nanosensors were then validated using standard gas chromatography-mass spectrometry (GC-MS), which gave results consistent with the proposed method. With intracellular imaging application, the EC@dye-NH nanosensors demonstrated excellent intracellular uptake and strong green fluorescence emission upon introducing the nonanal into the lung cancer cells (A549). Thus, the developed nanosensing approach served as the potential fluorescent probes in medical and biological fields, especially for lung cancer disease diagnosis based on highly selective and sensitive detection of long-chain aldehydes.

Mansonone G and its derivatives exhibit membrane permeabilizing activities against bacteria.

In an era where the rate of bacteria evolving to be resistant to clinically-used antibiotics far exceeds that of antibiotic discovery, the search for new sources of antibacterial agents has expanded tremendously. In recent years, interest in plant-based natural products as promising sources of antibacterial agents has taken an upward trend. Mansonones, botanically-derived naphthoqionones, having many uses in Asian traditional medicine-including anti-infective roles-have sparked interest as a possible source of antibacterial agents. Here, we show that mansonone G, extracted from Mansonia gagei Drumm. heartwoods, possessed antibacterial activities towards Bacillus subtilis, Staphylococcus aureus and Escherichia coli lptD4213, inhibiting the growth of the bacteria at 15.6 µM, 62.5 µM and 125 µM, respectively. Fourteen derivatives of mansonone G were synthesized successfully and were found to have a similar antibacterial spectrum to that of the parent compound, with some derivatives possessing improved antibacterial activities. Bacterial cytological profiling analysis showed that mansonone G harbors membrane permeabilizing activities against B. subtilis and E. coli lptD4213. Temporal analysis of SYTOX Green staining among individual cells showed that mansonone G rapidly permeabilized bacterial membrane within 10 min, with SYTOX Green intensity reaching 13-fold above that of the control. Collectively, these findings highlight the importance of mansonone G and its derivatives as potential antibacterial agents, paving the way for further modifications in order to improve their antibacterial spectrum.

Dicationic styryl dyes for colorimetric and fluorescent detection of nucleic acids.

Nucleic acid staining dyes are important tools for the analysis and visualizing of DNA/RNA in vitro and in the cells. Nevertheless, the range of commercially accessible dyes is still rather limited, and they are often very costly. As a result, finding nontoxic, easily accessible dyes, with desirable optical characteristics remains important. Styryl dyes have recently gained popularity as potential biological staining agents with many appealing properties, including a straightforward synthesis procedure, excellent photostability, tunable fluorescence, and high fluorescence quantum yield in the presence of nucleic acid targets with low background fluorescence signals. In addition to fluorescence, styryl dyes are strongly colored and exhibit solvatochromic properties which make them useful as colorimetric stains for low-cost and rapid testing of nucleic acids. In this work, novel dicationic styryl dyes bearing quaternary ammonium groups are designed to improve binding strength and optical response with target nucleic acids which contain a negatively charged phosphate backbone. Optical properties of the newly synthesized styryl dyes have been studied in the presence and absence of nucleic acid targets with the aim to find new dyes that can sensitively and specifically change fluorescence and/or color in the presence of nucleic acid targets. The binding interaction and optical response of the dicationic styryl dyes with nucleic acid were superior to the corresponding monocationic styryl dyes. Applications of the developed dyes for colorimetric detection of DNA in vitro and imaging of cellular nucleic acids are also demonstrated.

Discovery of a Multifunctional Octapeptide from Lingzhi with Antioxidant and Tyrosinase Inhibitory Activity.

Ganoderma lucidum or Lingzhi is a fungus species widely known as a traditional medicine. Exploring the beneficial peptides by hydrolysis using pepsin and trypsin has been extensively performed to identify new bioactive natural products. A multifunctional peptide that expresses potential scavenging activity and tyrosinase inhibition is valuable in therapeutic and cosmetic applications. This study aimed to identify and investigate the effects of a novel multifunctional peptide from Lingzhi on the melanogenic enzymes in melanoma cells by a targeted-proteomics approach. The multifunctional peptide was de novo sequenced by LC-MS/MS to be NH2-PVRSSNCA-CO2H (octapeptide). This sequence was chemically synthesized by solid-phase peptide synthesis (SPPS). The antioxidant ability of the synthesized octapeptide was measured by the DPPH, ABTS, and FRAP assays. The results showed that the peptide exhibited an antioxidant activity equal to 0.121 ± 0.01 mg equivalent to ascorbic acid, 0.173 ± 0.03 mg equivalent to gallic acid, and 2.21 ± 0.23 mM equivalent to FeSO4, respectively, which is comparable to these well-known antioxidants. The proteomics approach identified a total of 5804 proteins and several pathways involved in the effects of the octapeptide in melanoma cells. Targeted proteomics revealed three specific proteins associated with pigmentation including Rab29, Dct, and Tyrp1. The Rab29 and Dct were upregulated whereas Tyrp1 was downregulated in the octapeptide treatment group. These findings could be used in the understanding of the molecular functions of the multifunctional octapeptide on melanogenic enzymes, supporting its potential as a therapeutic and cosmetic ingredient.

Target protein deglycosylation in living cells by a nanobody-fused split O-GlcNAcase.

O-linked N-acetylglucosamine (O-GlcNAc) is an essential and dynamic post-translational modification that is presented on thousands of nucleocytoplasmic proteins. Interrogating the role of O-GlcNAc on a single target protein is crucial, yet challenging to perform in cells. Herein, we developed a nanobody-fused split O-GlcNAcase (OGA) as an O-GlcNAc eraser for selective deglycosylation of a target protein in cells. After systematic cellular optimization, we identified a split OGA with reduced inherent deglycosidase activity that selectively removed O-GlcNAc from the desired target protein when directed by a nanobody. We demonstrate the generality of the nanobody-fused split OGA using four nanobodies against five target proteins and use the system to study the impact of O-GlcNAc on the transcription factors c-Jun and c-Fos. The nanobody-directed O-GlcNAc eraser provides a new strategy for the functional evaluation and engineering of O-GlcNAc via the selective removal of O-GlcNAc from individual proteins directly in cells.

Engineering a Proximity-Directed O-GlcNAc Transferase for Selective Protein O-GlcNAcylation in Cells.

O-Linked ß-N-acetylglucosamine (O-GlcNAc) is a monosaccharide that plays an essential role in cellular signaling throughout the nucleocytoplasmic proteome of eukaryotic cells. Strategies for selectively increasing O-GlcNAc levels on a target protein in cells would accelerate studies of this essential modification. Here, we report a generalizable strategy for introducing O-GlcNAc into selected target proteins in cells using a nanobody as a proximity-directing agent fused to O-GlcNAc transferase (OGT). Fusion of a nanobody that recognizes GFP (nGFP) or a nanobody that recognizes the four-amino acid sequence EPEA (nEPEA) to OGT yielded nanobody-OGT constructs that selectively delivered O-GlcNAc to a series of tagged target proteins (e.g., JunB, cJun, and Nup62). Truncation of the tetratricopeptide repeat domain as in OGT(4) increased selectivity for the target protein through the nanobody by reducing global elevation of O-GlcNAc levels in the cell. Quantitative chemical proteomics confirmed the increase in O-GlcNAc to the target protein by nanobody-OGT(4). Glycoproteomics revealed that nanobody-OGT(4) or full-length OGT produced a similar glycosite profile on the target protein JunB and Nup62. Finally, we demonstrate the ability to selectively target endogenous α-synuclein for O-GlcNAcylation in HEK293T cells. These first proximity-directed OGT constructs provide a flexible strategy for targeting additional proteins and a template for further engineering of OGT and the O-GlcNAc proteome in the future. The use of a nanobody to redirect OGT substrate selection for glycosylation of desired proteins in cells may further constitute a generalizable strategy for controlling a broader array of post-translational modifications in cells.