Enzyme Inhibitory and Anti-cancer Properties of Moringa peregrina / Propiedades inhibidoras Enzimáticas y Anticancerígenas de la Moringa peregrina

Background: Moringa peregrina is widely used in the traditional medicine of the Arabian Peninsula to treat various ailments, because it has many pharmacologically active components with several therapeutic effects. Objective: This study aimed to investigate the inhibitory effect of Moringaperegrina seed ethanolic extract (MPSE) against key enzymes involved in human pathologies, such as angiogenesis (thymidine phosphorylase), diabetes (α-glucosidase), and idiopathic intracranial hypertension (carbonic anhydrase). In addition, the anticancer properties were tested against the SH-SY5Y (human neuroblastoma). Results: MPSE extract significantly inhibited α-glucosidase, thymidine phosphorylase, and carbonic anhydrase with half-maximal inhibitory concentrations (IC50) values of 303.1 ± 1.3, 471.30 ± 0.3, and 271.30 ± 5.1 µg/mL, respectively. Furthermore, the antiproliferative effect of the MPSE was observed on the SH-SY5Y cancer cell line with IC50 values of 55.1 µg/mL. Conclusions: MPSE has interesting inhibitory capacities against key enzymes and human neuroblastoma cancer cell line.

Antecedentes: La Moringa peregrina se utiliza ampliamente en la medicina tradicional de la Península Arábiga para tratar diversas dolencias, ya que posee numerosos componentes farmacológicamente activos con varios efectos terapéuticos. Objetivo: Este estudio tenía como objetivo investigar el efecto inhibidor del extracto etanólico de semillas de Moringaperegrina (MPSE) frente a enzimas clave implicadas en patologías humanas, como la angiogénesis (timidina fosforilasa), la diabetes (α-glucosidasa) y la hipertensión intracraneal idiopática (anhidrasa carbónica). Además, se comprobaron las propiedades anticancerígenas frente al SH-SY5Y (neuroblastoma humano). Resultados: El extracto de MPSE inhibió significativamente la α-glucosidasa, la timidina fosforilasa y la anhidrasa carbónica con concentraciones inhibitorias semimáximas (IC50) de 303,1 ± 1,3, 471,30 ± 0,3 y 271,30 ± 5,1 µg/mL, respectivamente. Además, se observó el efecto antiproliferativo del MPSE en la línea celular del cáncer SH-SY5Y con valores de IC50 de 55,1 µg/mL. Conclusiones: MPSE posee interesantes capacidades inhibitorias frente a enzimas clave y línea celular de neuroblastoma canceroso humano.

Discovery and characterization of novel ATP citrate lyase inhibitors from Acanthopanax senticosus (Rupr. & Maxim.) Harms.

ATP citrate lyase (ACLY) is a key enzyme in glucolipid metabolism, and abnormally high expression of ACLY occurs in many diseases, including cancers, dyslipidemia and cardiovascular diseases. ACLY inhibitors are prospective treatments for these diseases. However, the scaffolds of ACLY inhibitors are insufficient with weak activity. The discovery of inhibitors with structural novelty and high activity continues to be a research hotpot. Acanthopanax senticosus (Rupr. & Maxim.) Harms is used for cardiovascular disease treatment, from which no ACLY inhibitors have ever been found. In this work, we discovered three novel ACLY inhibitors, and the most potent one was isochlorogenic acid C (ICC) with an IC50 value of 0.14 ± 0.04 µM. We found dicaffeoylquinic acids with ortho-dihydroxyphenyl groups were important features for inhibition by studying ten phenolic acids. We further investigated interactions between the highly active compound ICC and ACLY. Thermal shift assay revealed that ICC could directly bind to ACLY and improve its stability in the heating process. Enzymatic kinetic studies indicated ICC was a noncompetitive inhibitor of ACLY. Our work discovered novel ACLY inhibitors, provided valuable structure-activity patterns and deepened knowledge on the interactions between this targe tand its inhibitors.

A fast and efficient method for screening and evaluation of hypoglycemic ingredients of Traditional Chinese Medicine acting on PTP1B by capillary electrophoresis.

As a pivotal enzyme that regulates dephosphorylation in cell activities and participates in the insulin signaling pathway, protein tyrosine phosphatase 1B (PTP1B) is considered to be an important target for the therapy of diabetes. In this work, a rapid and efficient inhibitor screening method of PTP1B was established based on capillary electrophoresis (CE), and used for screening and evaluating the inhibition effect of Traditional Chinese Medicine on PTP1B. Response Surface Methodology was used for optimizing the conditions of analysis. After method validation, the enzyme kinetic study and inhibition test were performed. As a result, the IC50 of PTP1B inhibitors Ⅳ and ⅩⅧ were consistent with reported values measured by a conventional method. It was found that the extracts of Astragalus membranaceus (Fisch) Bunge and Morus alba L. showed prominent inhibition on the activity of PTP1B, which were stronger than the positive controls. Meanwhile, on top of the excellent advantages of CE, the whole analysis time is less than 2 min. Thus, the results demonstrated that a fast and efficient screening method was successfully developed. This method could be a powerful tool for screening inhibitors from complex systems. It can also provide an effective basis for lead compound development in drug discovery.

In vitro Evaluation of Skin Related Enzyme Inhibitory Effect and Emulgel Formulation Development Studies of Onobrychis Argyrea subsp. Argyrea with Phytochemical Analysis.

Species of Onobrychis have been used to treat skin disorders such as wounds and cuts in folk medicine and Onobrychis argyrea subsp. argyrea (OA) commonly known as 'silvery sainfoin', is a member of this genus. In this study, it was aimed to investigate the skin-related biological activities and phytochemical characterization of OA. Moreover, an emulgel formulation was developed from the main methanolic extract of the plant (OAM). Initially, to identifiy of the active fractions, aerial parts of the plant material was extracted with methanol and fractionated by n-hexane, chloroform, ethyl acetate and n-butanol, respectively. Antioxidant activity was determined by CUPRAC, TOAC, FRAP and DPPH assays. Thereafter, the inhibition potential of OAM, novel formulation and all fractions was measured against elastase, collagenase, tyrosinase and hyaluronidase enzymes. OAM was analyzed and characterized by LC/MS-MS. The major bioactive flavonoids which are rutin and isoquercetin were measured and compared as qualitative and quantitative via high performance thin layer chromatography (HPTLC) analysis in OAM and fractions. The results showed that extracts of OA can be a potential cosmeceutical agent for skin related problems.

Discovery of a botanical compound as a broad-spectrum inhibitor against gut microbial ß-glucuronidases from the Tibetan medicine Rhodiola crenulata.

Gut microbial ß-glucuronidases (gmß-GUS) played crucial roles in regulating a variety of endogenous substances and xenobiotics on the circulating level, thus had been recognized as key modulators of drug toxicity and human diseases. Inhibition or inactivation of gmß-GUS enzymes has become a promising therapeutic strategy to alleviate drug-induced intestinal toxicity. Herein, the Rhodiola crenulata extract (RCE) was found with potent and broad-spectrum inhibition on multiple gmß-GUS enzymes. Subsequently, the anti-gmß-GUS activities of the major constituents in RCE were tested and the results showed that 1,2,3,4,6-penta-O-galloyl-ß-d-glucopyranose (PGG) acted as a strong and broad-spectrum inhibitor on multiple gmß-GUS (including EcGUS, CpGUS, SaGUS, and EeGUS). Inhibition kinetic assays demonstrated that PGG effectively inhibited four gmß-GUS in a non-competitive manner, with the Ki values ranging from 0.12 µM to 1.29 µM. Docking simulations showed that PGG could tightly bound to the non-catalytic sites of various gmß-GUS, mainly via hydrogen bonding and aromatic interactions. It was also found that PGG could strongly inhibit the total gmß-GUS activity in mice feces, with the IC50 value of 1.24 µM. Collectively, our findings revealed that RCE and its constituent PGG could strongly inhibit multiple gmß-GUS enzymes, suggesting that RCE and PGG could be used for alleviating gmß-GUS associated enterotoxicity.

Novel high-yield potato protease inhibitor panels block a wide array of proteases involved in viral infection and crucial tissue damage.

Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation pulled by granulocytes. Upon hyperactivation, neutrophil granulocytes may cause undue tissue damage through proteolytic degradation of the extracellular matrix. Here, we assess the potential of protease inhibitors (PI) derived from potatoes in inhibiting viral infection and reducing tissue damage. The original full spectrum of potato PI was developed into five fractions by means of chromatography and hydrolysis. Individual fractions showed varying inhibitory efficacy towards a panel of proteases including trypsin, chymotrypsin, ACE2, elastase, and cathepsins B and L. The fractions did not interfere with SARS-CoV-2 infection of Vero E6 cells in vitro. Importantly, two of the fractions fully inhibited elastin-degrading activity of complete primary human neutrophil degranulate. These data warrant further development of potato PI fractions for biomedical purposes, including tissue damage crucial to SARS-CoV-2 pathogenesis. KEY MESSAGES: Protease inhibitor fractions from potato differentially inhibit a series of human proteases involved in viral replication and in tissue damage by overshoot inflammation. Protease inhibition of cell surface receptors such as ACE2 does not prevent virus infection of Vero cells in vitro. Protease inhibitors derived from potato can fully inhibit elastin-degrading primary human neutrophil proteases. Protease inhibitor fractions can be produced at high scale (hundreds of thousands of kilograms, i.e., tons) allowing economically feasible application in lower and higher income countries.

Inhibition of pancreatic lipase by coffee leaves-derived polyphenols: A mechanistic study.

The suppression of pancreatic lipase has been employed to mitigate obesity. This study explored the mechanism of coffee leaf extracts to inhibit pancreatic lipase. The ethyl acetate fraction derived from coffee leaves (EAC) exhibited the highest inhibitory capacity with a half-maximal inhibitory concentration (IC50) of 0.469 mg/mL and an inhibitor constant (Ki) of 0.185 mg/mL. This fraction was enriched with 3,5-dicaffeoylquinic acid (3,5-diCQA, 146.50 mg/g), epicatechin (87.51 mg/g), and isoquercetin (48.29 mg/g). EAC inhibited lipase in a reversible and competitive manner, and quenched its intrinsic fluorescence through a static mechanism. Molecular docking revealed that bioactive compounds in EAC bind to key amino acid residues (HIS-263, PHE-77, and SER-152) located within the active cavity of lipase. Catechin derivatives play a key role in the lipase inhibitory activity within EAC. Overall, our findings highlight the promising potential of coffee leaf extract as a functional ingredient for alleviating obesity through inhibition of lipase.

Identification and molecular docking of tyrosinase inhibitory peptides from allophycocyanin in Spirulina platensis.

BACKGROUND: Tyrosinase, a copper-containing metalloenzyme with catalytic activity, is widely found in mammals. It is the key rate-limiting enzyme that catalyzes melanin synthesis. For humans, tyrosinase is beneficial to the darkening of eyes and hair. However, excessive deposition of melanin in the skin can lead to dull skin color and lead to pigmentation. Therefore, many skin-whitening compounds have been developed to decrease tyrosinase activity. This study aimed to identify a new tyrosinase inhibitory peptide through enzymatic hydrolysis, in vitro activity verification, molecular docking, and molecular dynamics (MD) simulation. RESULTS: A tripeptide Asp-Glu-Arg (DER) was identified, with a '-CDOCKER_Energy' value of 121.26 Kcal mol-1 . DER has effective tyrosinase inhibitory activity. Research shows that its half maximal inhibitory concentration value is 1.04 ± 0.01 mmol L-1 . In addition, DER binds to tyrosinase residues His85, His244, His259, and Asn260, which are key residues that drive the interaction between the peptide and tyrosinase. Finally, through MD simulation, the conformational changes and structural stability of the complexes were further explored to verify and supplement the results of molecular docking. CONCLUSION: This experiment shows that DER can effectively inhibit tyrosinase activity. His244, His259, His260, and Asn260 are the critical residues that drive the interaction between the peptide and tyrosinase, and hydrogen bonding is an important force. DER from Spirulina has the potential to develop functional products with tyrosinase inhibition. © 2024 Society of Chemical Industry.

Tacrolimus versus mycophenolate for AutoImmune hepatitis patients with incompLete response On first-line therapy (TAILOR study): a study protocol for a phase III, open-label, multicentre, randomised controlled trial.

BACKGROUND: Autoimmune hepatitis (AIH) is a rare, chronic inflammatory disease of the liver. The treatment goal is reaching complete biochemical response (CR), defined as the normalisation of aspartate and alanine aminotransferases and immunoglobulin gamma. Ongoing AIH activity can lead to fibrosis and (decompensated) cirrhosis. Incomplete biochemical response is the most important risk factor for liver transplantation or liver-related mortality. First-line treatment consists of a combination of azathioprine and prednisolone. If CR is not reached, tacrolimus (TAC) or mycophenolate mofetil (MMF) can be used as second-line therapy. Both products are registered for the prevention of graft rejection in solid organ transplant recipients. The aim of this study is to compare the effectiveness and safety of TAC and MMF as second-line treatment for AIH. METHODS: The TAILOR study is a phase IIIB, multicentre, open-label, parallel-group, randomised (1:1) controlled trial performed in large teaching and university hospitals in the Netherlands. We will enrol 86 patients with AIH who have not reached CR after at least 6 months of treatment with first-line therapy. Patients are randomised to TAC (0.07 mg/kg/day initially and adjusted by trough levels) or MMF (max 2000 mg/day), stratified by the presence of cirrhosis at inclusion. The primary endpoint is the difference in the proportion of patients reaching CR after 12 months. Secondary endpoints include the difference in the proportion of patients reaching CR after 6 months, adverse effects, difference in fibrogenesis, quality of life and cost-effectiveness. DISCUSSION: This is the first randomised controlled trial comparing two second-line therapies for AIH. Currently, second-line treatment is based on retrospective cohort studies. The rarity of AIH is the main issue in clinical research for alternative treatment options. The results of this trial can be implemented in existing international clinical guidelines. TRIAL REGISTRATION: ClinicalTrials.gov NCT05221411 . Retrospectively registered on 3 February 2022; EudraCT number 2021-003420-33. Prospectively registered on 16 June 2021.

Comparative Analysis of Two NNMT Bisubstrate Inhibitors through Chemoproteomic Studies: Uncovering the Role of Unconventional SAM Analogue Moiety for Improved Selectivity.

Unconventional S-adenosyl-L-methionine (SAM) mimics with enhanced hydrophobicity are an adaptable building block to develop cell-potent inhibitors for SAM-dependent methyltransferases as targeted therapeutics. We recently discovered cell-potent bisubstrate inhibitors for nicotinamide N-methyltransferase (NNMT) by using an unconventional SAM mimic. To delve into the selectivity implications of the unconventional SAM mimic, we employed a chemoproteomic approach to assess two potent NNMT inhibitors LL320 (Ki, app = 6.8 nM) and II399 (containing an unconventional SAM mimic, Ki, app = 5.9 nM) within endogenous proteomes. Our work began with the rational design and synthesis of immobilized probes 1 and 2, utilizing LL320 and II399 as parent compounds. Systematic analysis of protein networks associated with these probes revealed a comprehensive landscape. Notably, NNMT emerged as the top-ranking hit, substantiating the high selectivity of both inhibitors. Meanwhile, we identified additional interacting proteins for LL320 (38) and II399 (17), showcasing the intricate selectivity profiles associated with these compounds. Subsequent experiments confirmed LL320's interactions with RNMT, DPH5, and SAHH, while II399 exhibited interactions with SHMT2 and MEPCE. Importantly, incorporating the unconventional SAM mimic in II399 led to improved selectivity compared to LL320. Our findings underscore the importance of selectivity profiling and validate the utilization of the unconventional SAM mimic as a viable strategy to create highly selective and cell-permeable inhibitors for SAM-dependent methyltransferases.

Screening of natural inhibitors against peptidyl arginine deiminase 4 from herbal extracts by a high-performance liquid chromatography ultraviolet-visible based method.

Peptidyl arginine deiminase 4 (PAD4) is an important biocatalytic enzymes involved in the conversion of protein arginine to citrulline, its dysregulation has a great impact on many physiological processes. Recently, PAD4 has emerged as a potential therapeutic target for the treatment of various diseases including rheumatoid arthritis (RA). Traditional Chinese Medicines (TCMs), also known as herbal plants, have gained great attention by the scientific community due to their good therapeutic performance and far fewer side effects observed in the clinical treatment. However, limited researches have been reported to screen natural PAD4 inhibitors from herbal plants. The color developing reagent (COLDER) or fluorescence based methods have been widely used in PAD4 activity assay and inhibitor screening. However, both methods measure the overall absorbance or fluorescence in the reaction solution, which are easy to be affected by the background interference due to colorful extracts from herbal plants. In this study, a simple, and robust high-performance liquid chromatography ultraviolet-visible (HPLC-UV) based method was developed to determine PAD4 activity. The proposed strategy was established based on COLDER principle, while used hydrophilic l-arginine instead of hydrophobic N-benzoyl-l-arginine ethyl ester (BAEE) as a new substrate to determine PAD4 inhibition activity of herbal extracts. The herbal extracts and PAD4 generated hydrophobic l-citrulline were successfully separated by the HPLC, and the developed method was optimized and validated with a known PAD4 inhibitor (GSK484) in comparison with COLDER assay. The IC50 value of GSK484 measured by HPLC-UV method was 153 nM, and the detection limit of the citrulline was 0.5 nmol, respectively, with a linear range of 0.5 nmol to 20 nmol. The IC50 value of the HPLC-UV method was improved by nearly three times compared with COLDER assay (527 nM), and the results indicated the reliability of PAD4 inhibition via HPLC-UV method. The inhibitory effect against PAD4 were fast and accurately screened for the twenty-four extracts from eight herbs. Among them, Ephedra Herba extracts showed significant inhibitory activity against the PAD4 with the IC50 values of three extracts (ethanol, ethyl acetate and water) ranging from 29.11 µg/mL to 41.36 µg/mL, which may help researchers to discover novel natural compounds holding high PAD4 inhibition activity.

In Vitro Determination of Nitric Oxide Synthase Inhibition, Antioxidant Capacity and Phenolic Content of Various Natural Products (Bee and Herbal Products).

It is obvious that the oxidation process is an undeniable fact and when it comes to aging, one of the first solutions that come to mind is natural products. When it comes to natural products, both plants and bee products play an important, almost combative role against oxidation. For this purpose, natural products of both plant and animal origin were considered together in our study: Linden, green tea, aronia, wild grapes, myrtle, blueberries and basil, honey, pollen and propolis. Total phenolic content values of the extracts ranged between 49.28 and 3859.06 mg gallic acid equivalent/100 g, and propolis, green tea, chestnut flower and aronia samples were found to have the highest values. When looking at the NOS inhibition potential, it was determined that propolis, pollen and aronia samples had the highest percentage inhibition values of 98.11, 92.29, 83.44, respectively. Antioxidant activities of methanolic extracts were investigated using iron(III) reducing/antioxidant capacity (FRAP), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity test and NOS inhibition tests. The phenolic composition of methanolic extracts was tested using the RP-HPLC-UV (high-performance liquid chromatographic method with ultraviolet) method with 19 phenolic standards.

Structural Insight into Geranylgeranyl Diphosphate Synthase (GGDPS) for Cancer Therapy.

Geranylgeranyl diphosphate synthase (GGDPS), the source of the isoprenoid donor in protein geranylgeranylation reactions, has become an attractive target for anticancer therapy due to the reliance of cancers on geranylgeranylated proteins. Current GGDPS inhibitor development focuses on optimizing the drug-target enzyme interactions of nitrogen-containing bisphosphonate-based drugs. To advance GGDPS inhibitor development, understanding the enzyme structure, active site, and ligand/product interactions is essential. Here we provide a comprehensive structure-focused review of GGDPS. We reviewed available yeast and human GGDPS structures and then used AlphaFold modeling to complete unsolved structural aspects of these models. We delineate the elements of higher-order structure formation, product-substrate binding, the electrostatic surface, and small-molecule inhibitor binding. With the rise of structure-based drug design, the information provided here will serve as a valuable tool for rationally optimizing inhibitor selectivity and effectiveness.

Discovery of natural multi-targets neuraminidase inhibitor glycosides compounds against influenza A virus through network pharmacology, virtual screening, molecular dynamics simulation, and in vitro experiment.

Influenza virus continually challenges both human and animal health. Moreover, influenza viruses are easy to mutate. In a certain degree, vaccines may not catch up with rapid mutant paces of viruses. Anti-influenza drugs NIs (neuraminidase inhibitors) are one of the best choices. Therefore, based on ADMET properties, eight optimal natural multi-targets NIs glycosides compounds (IC50 = 0.094-97.275 µM) are found from radix glycyrrhizae, flos sophorae, caulis spatholobi, radix astragali, radix glycyrrhizae, semen astragali complanati, and common fenugreek seed through network pharmacology, molecular docking, dynamics simulation, quantum chemistry, and in vitro experiment. Moreover, mechanism research illustrates these natural compounds treat influenza A virus through key targets TLR4, TNF, and IL6 (high fever, acute respiratory distress syndrome), MAPK1, and MAPK3 (MAPK signaling pathway, viral RNP export, and viral protein expression), IL1B (NOD-like receptor signaling pathway, suppressed maturation of pro-IL-1ß and pro-IL-18), CASP3 (apoptosis), AKT1 (inhibited premature apoptosis), and EP300 (viral myocarditis, chemoattraction of monocytes and macrophages, T-cell activation antibody response).

Rapid screening, isolation, and activity evaluation of potential xanthine oxidase inhibitors in Polyporus umbellatus and mechanism of action in the treatment of gout.

INTRODUCTION: Studies show that Polyporus umbellatus has some pharmacological effects in enhancing immunity and against gout. OBJECTIVES: We aimed to establish new techniques for extraction, biological activity screening, and preparation of xanthine oxidase inhibitors (XODIs) from P. umbellatus. METHODS: First, the extraction of P. umbellatus was investigated using the back propagation (BP) neural network genetic algorithm mathematical regression model, and the extraction variables were optimised to maximise P. umbellatus yield. Second, XODIs were rapidly screened using ultrafiltration, and the change of XOD activity was tested by enzymatic reaction kinetics experiment to reflect the inhibitory effect of active compounds on XOD. Meanwhile, the potential anti-gout effects of the obtained active substances were verified using molecular docking, molecular dynamics simulations, and network pharmacology analysis. Finally, with activity screening as guide, a high-speed countercurrent chromatography (HSCCC) method combined with consecutive injection and two-phase solvent system preparation using the UNIFAC mathematical model was successfully developed for separation and purification of XODIs, and the XODIs were identified using MS and NMR. RESULTS: The results verified that polyporusterone A, polyporusterone B, ergosta-4,6,8(14),22-tetraen-3-one, and ergosta-7,22-dien-3-one of P. umbellatus exhibited high biological affinity towards XOD. Their structures have been further identified by NMR, indicating that the method is effective and applicable for rapid screening and identification of XODIs. CONCLUSION: This study provides new ideas for the search for natural XODIs active ingredients, and the study provide valuable support for the further development of functional foods with potential therapeutic benefits.

Exploring the Pharmacological Potential of Onosma riedliana: Phenolic Compounds and Their Biological Activities.

Onosma riedliana Binzet & Orcan, a traditionally used plant species, has been explored for its therapeutic potential in this study. The work presented here is the first report on the phenolic profile and biological activity of this species. Three extracts of varying polarity were prepared, with the methanolic extract containing the highest phenolic content (97.62 ± 0.20 mgGAE/g). Key phenolic compounds identified included pinoresinol, hesperidin, 4-hydroxybenzoic acid, and p-coumaric acid. The methanolic extract exhibited exceptional antioxidant properties, rivaling Trolox as a positive control, primarily attributed to hesperidin and luteolin. Moreover, the ethyl acetate extract demonstrated remarkable inhibition of cholinesterase and tyrosinase enzymes, while the methanolic extract displayed potent activity against carbohydrate hydrolytic enzymes, α-amylase and α-glucosidase. Again, phenolic compounds were shown to be responsible for the inhibition of cholinesterases and tyrosinase, but not for α-amylase and α-glucosidase. These findings underscore Onosma riedliana's potential for incorporation into diverse pharmaceutical formulations, given its multifaceted bioactivity.

Role of K+ and Ca2+ Channels in the Vasodilator Effects of Plectranthus barbatus (Brazilian Boldo) in Hypertensive Rats.

Plectranthus barbatus, popularly known as Brazilian boldo, is used in Brazilian folk medicine to treat cardiovascular disorders including hypertension. This study investigated the chemical profile by UFLC-DAD-MS and the relaxant effect by using an isolated organ bath of the hydroethanolic extract of P. barbatus (HEPB) leaves on the aorta of spontaneously hypertensive rats (SHR). A total of nineteen compounds were annotated from HEPB, and the main metabolite classes found were flavonoids, diterpenoids, cinnamic acid derivatives, and organic acids. The HEPB promoted an endothelium-dependent vasodilator effect (~100%; EC50 ~347.10 µg/mL). Incubation of L-NAME (a nonselective nitric oxide synthase inhibitor; EC50 ~417.20 µg/mL), ODQ (a selective inhibitor of the soluble guanylate cyclase enzyme; EC50 ~426.00 µg/mL), propranolol (a nonselective α-adrenergic receptor antagonist; EC50 ~448.90 µg/mL), or indomethacin (a nonselective cyclooxygenase enzyme inhibitor; EC50 ~398.70 µg/mL) could not significantly affect the relaxation evoked by HEPB. However, in the presence of atropine (a nonselective muscarinic receptor antagonist), there was a slight reduction in its vasorelaxant effect (EC50 ~476.40 µg/mL). The addition of tetraethylammonium (a blocker of Ca2+-activated K+ channels; EC50 ~611.60 µg/mL) or 4-aminopyridine (a voltage-dependent K+ channel blocker; EC50 ~380.50 µg/mL) significantly reduced the relaxation effect of the extract without the interference of glibenclamide (an ATP-sensitive K+ channel blocker; EC50 ~344.60 µg/mL) or barium chloride (an influx rectifying K+ channel blocker; EC50 ~360.80 µg/mL). The extract inhibited the contractile response against phenylephrine, CaCl2, KCl, or caffeine, similar to the results obtained with nifedipine (voltage-dependent calcium channel blocker). Together, the HEPB showed a vasorelaxant effect on the thoracic aorta of SHR, exclusively dependent on the endothelium with the participation of muscarinic receptors and K+ and Ca2+ channels.

Screening and identification of neuraminidase inhibitors from Baphicacanthus cusia by a combination of affinity ultrafiltration, HPLC-MS/MS, molecular docking, and fluorescent techniques.

Natural products provide a new opportunity for the discovery of neuraminidase (NA)inhibitors. In this study, an affinity ultrafiltration (AUF) coupled with HPLC-MS/MS method was firstly developed and optimized for screening of NA inhibitors from natural products. The critical factors influencing the interaction of enzyme-ligand (including sample concentration, enzyme concentration, incubation time and temperature, pH of the buffer, and dissociation solvents and time) were investigated and optimized by a one-factor-at-a-time design. The method was then applied to discover NA inhibitory compounds in stems and leaves of Baphicacanthus cusia. As a result, five active alkaloids were screened out and identifiedas 2,4(1H,3H)-quinazolinedione (1), 4(3H)-quinazolinone (2), 2(3H)-benzoxazolone (3), tryptanthrin (4), and indirubin (5) through analysis of their DAD profiles, MS/MS fragments, and comparison with reference substances. These active compounds were further evaluated for their NA inhibitory activity using a fluorescence-based NA inhibition assay. The result from the fluorescent assay revealed that all the five compounds(1-5) showed pronounced NA inhibitory activities with IC50values of 98.98, 64.69, 40.16, 69.44, and 144.73 µM, respectively. Finally, molecular docking of these five alkaloids with NA showed that hydrogen bond and π-cation interactions dominated within the binding sites with binding energies ranging between -5.7 to -7.9 kcal/mol, which was supported by the results of the AUF and the fluorescence-based enzyme assay. The developed AUF method is simple and efficient for screening potential NA inhibitors from stems and leaves of B. cusia.

Mining Xanthine Oxidase Inhibitors from an Edible Seaweed Pterocladiella capillacea by Using In Vitro Bioassays, Affinity Ultrafiltration LC-MS/MS, Metabolomics Tools, and In Silico Prediction.

The prevalence of gout and the adverse effects of current synthetic anti-gout drugs call for new natural and effective xanthine oxidase (XOD) inhibitors to target this disease. Based on our previous finding that an edible seaweed Pterocladiella capillacea extract inhibits XOD, XOD-inhibitory and anti-inflammatory activities were used to evaluate the anti-gout potential of different P. capillacea extract fractions. Through affinity ultrafiltration coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS), feature-based molecular networking (FBMN), and database mining of multiple natural products, the extract's bioactive components were traced and annotated. Through molecular docking and ADMET analysis, the possibility and drug-likeness of the annotated XOD inhibitors were predicted. The results showed that fractions F4, F6, F4-2, and F4-3 exhibited strong XOD inhibition activity, among which F4-3 reached an inhibition ratio of 77.96% ± 4.91% to XOD at a concentration of 0.14 mg/mL. In addition, the P. capillacea extract and fractions also displayed anti-inflammatory activity. Affinity ultrafiltration LC-MS/MS analysis and molecular networking showed that out of the 20 annotated compounds, 8 compounds have been previously directly or indirectly reported from seaweeds, and 4 compounds have been reported to exhibit anti-gout activity. Molecular docking and ADMET showed that six seaweed-derived compounds can dock with the XOD activity pocket and follow the Lipinski drug-like rule. These results support the value of further investigating P. capillacea as part of the development of anti-gout drugs or related functional foods.

Discovery and Optimization of Novel hDHODH Inhibitors for the Treatment of Inflammatory Bowel Disease.

As a key rate-limiting enzyme in the de novo synthesis of pyrimidine nucleotides, human dihydroorotate dehydrogenase (hDHODH) is considered a known target for the treatment of autoimmune diseases, including inflammatory bowel disease (IBD). Herein, BAY 41-2272 with a 1H-pyrazolo[3,4-b]pyridine scaffold was identified as an hDHODH inhibitor by screening an active compound library containing 5091 molecules. Further optimization led to 2-(1-(2-chloro-6-fluorobenzyl)-1H-pyrrolo[2,3-b]pyridin-3-yl)-5-cyclopropylpyrimidin-4-amine (w2), which was found to be the most promising and drug-like compound with potent inhibitory activity against hDHODH (IC50 = 173.4 nM). Compound w2 demonstrated acceptable pharmacokinetic characteristics and alleviated the severity of acute ulcerative colitis induced by dextran sulfate sodium in a dose-dependent manner. Notably, w2 exerted better therapeutic effects on ulcerative colitis than hDHODH inhibitor vidofludimus and Janus kinase (JAK) inhibitor tofacitinib. Taken together, w2 is a promising hDHODH inhibitor for the treatment of IBD and deserves to be developed as a preclinical candidate.