Non-competitive Mode Research Articles

Mechanistic insights into β-glucuronidase inhibition by isoprenylated flavonoids from Centaurea scoparia: Bridging experimental and computational approaches

Exploring the intricate mechanisms underlying the inhibition of β-glucuronidase, particularly the enzyme produced by gastrointestinal bacteria, is essential for the development of novel therapeutic interventions and pharmaceutical advancements. Inhibiting bacterial β-glucuronidase can prevent the reactivation of harmful drug metabolites in the gut, thereby reducing associated toxicities. The inhibitory potential of four isoprenylated flavonoids from Centaurea scoparia against β-glucuronidase was intensively investigated by combined in vitro and in silico tools. The results of in vitro inhibitory activity showed that Compounds 1 and 3 exhibited the highest inhibitory activity, as evidenced by their low IC50 values of 3.65 ± 0.28 and 3.97 ± 0.11 μM, respectively. The enzyme kinetics analysis revealed that Compound 1 and the positive control drug (EGCG) follow a mixed inhibition mechanism. In contrast, compounds 3 and 4 exhibited a noncompetitive inhibition mode, as suggested by the intersection patterns observed in the Lineweaver-Burk plots. The docking studies corroborated the in vitro inhibitory assay results, with Compounds 1 and 3 demonstrating the lowest binding affinities and the highest extent of polar and hydrophobic interactions with residues in the enzyme's binding site. Utilizing a 200 ns molecular dynamics (MD) simulation, we examined the interaction dynamics between isolated flavonoids and β-glucuronidase. The analysis of multiple MD parameters revealed that compounds 1 and 3 maintained consistent trajectories and demonstrated significant energy stabilization when interacting with β-glucuronidase. These computational findings align with experimental results, highlighting the potential of compounds 1 and 3 as potent inhibitors for β-glucuronidase.

Enhanced gelation of sturgeon frozen surimi via egg white: Exploring the role of endogenous serine protease inhibition

AbstractThe degradation of frozen sturgeon surimi can be attributed to the endogenous serine protease. This study was first to examine the impact of egg whites on the frozen sturgeon surimi's gel properties from the perspective of inhibiting endogenous serine protease. The protease activity of egg whites group (CA + EW group, consisting of 4% egg whites and cryoprotectants) was 45.15% lower than that of cryoprotectants group (CA group, consisting of 3% sucrose, 3% sorbitol, and 0.3% sodium tripolyphosphate) at 12 weeks. From the results of inhibition kinetics, serine protease was inhibited by both anticompetitive and noncompetitive inhibition modes. Molecular docking analysis indicated that egg white achieves this inhibitory effect through ionic interactions and hydrogen bonding with serine protease. However, this inhibitory effect was absent when the freezing period was extended to 24 weeks. Compared with CA group, the CA + EW group exhibited 54.76% and 4.59% increase in gel strength and water‐holding capacity, and 32.42% reduction in cooking loss after 24 weeks of freezing. Egg whites also impeded water migration and enhanced the density and smoothness of the gel microstructure, reducing protein aggregation. These results indicated that egg whites serve a dual function: inhibiting serine protease and filling the gel network within 12 weeks, mitigating protein aggregation and ice crystal formation over 24 weeks. This study elucidated the mechanism underlying the impact of egg white on endogenous serine protease in sturgeon surimi during long‐term freezing, laying a theoretical foundation for the industrialization of sturgeon surimi.

Polyphasic approaches to identify and understand α-glucosidase inhibitory potential of secondary metabolites of Withania coagulans fruit

Withania coagulans (WC) is used in traditional and Ayurveda medicine to treat various ailments, including diabetes. Our investigation found that WC fruit hexane extract effectively suppresses α-glucosidase activity (IC50 = 0.013 mg/ml, Ki = 0.012 mg/ml). The purified molecule has an IC50 of 0.004 mg/ml and Ki of 0.0037 mg/ml. FTIR examination indicates distinctive peaks at 3500, 2900, 1770, and 1500 cm−1 corresponding to functional groups OH bending, CH stretching, CO stretching, and CO stretching. GCMS analysis reveals plant secondary metabolites (PSM) such as n-hexadecenoic acid and methyl 9,10-octadecadienoate. NMR confirms the existence of olefinic fatty acids. The bioactive fraction recorded a non-competitive mode of inhibition of α-glucosidase activity. The cytotoxicity exhibited against HELA cell was IC50 0.4 mg/ml and found positive in inhibiting the growth of Bacillus cereus, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa. Additionally, ensemble docking and molecular dynamic simulation showed that, out of the four PSMs examined, methyl 12,13-tetradecadienoate interacted with the α-glucosidase enzyme's allosteric site (BE −128.78 kJ/mol) and changed the configurations of the catalytic sites, as demonstrated by the enzyme's decreased affinity for isomaltose. The study found that PSMs from WC fruit may inhibit α-glucosidase, making them viable candidates for antidiabetic medication development.

Bis(benzimidazol-2-yl)amine-Based DPP-4 Inhibitors Potentially Suitable for Combating Diabetes and Associated Nervous System Alterations.

Bis(benzimidazol-2-yl)amine scaffold is not present in dipeptidyl peptidase-4 (DPP-4) inhibitors published so far. Herein, the inhibitory potential of bis(benzimidazol-2-yl)amine derivatives against DPP-4 was evaluated. In non-competitive inhibition mode, three representatives 5, 6, and 7 inhibited DPP-4 in vitro with IC50 values below 50 μM. The assessed binding pocket of DPP-4 for these benzimidazoles includes the S2 extensive subsite's residues Phe357 and Arg358. None of the lead compounds showed cytotoxicity to human neuroblastoma SH-SY5Y cells at concentrations lower than 10 μM. None showed significant binding affinity at dopamine D2, D3, and histamine H1, H3 receptors, at concentrations lower than 10 μM, leading to preferable outcomes due to mutually opposite effects of these neurotransmitters on each other. The potential beneficial effects on dopamine synthesis and the survival of dopaminergic neurons could be mediated by DPP-4 inhibition. These effective noncompetitive DPP-4 inhibitors, with inhibitory potential better than reference diprotin A (relative inhibitory potency compared to diprotin A is 3.39 and 1.54 for compounds 7 and 5, respectively), with the absence of cytotoxicity to SH-SY5Y cells, are valuable candidates for further evaluation for the treatment of diabetes and associated disruption of neuronal homeostasis.

Recent Development of CDK2 Inhibitors as Anticancer Drugs: An Update (2015–2023)

AbstractCyclin-dependent kinase 2 (CDK2) is a critical regulator of cell division and has emerged as a promising target for anticancer treatment. In this article, we summarize the structural features of CDK2 inhibitors and corresponding binding modes, in particular the noncompetitive binding modes that offer unique advantages for the development of highly selective inhibitors. In addition, we present an overview of the latest advancements in the development of CDK2 inhibitors and discuss the trend in the field. This review provides valuable insights into the structure–activity relationships of the reported CDK2 inhibitors, inspiring the development of potent and selective CDK2 inhibitors in the future.

Jack bean urease inhibition by different root fractions of Cleome gynandra L – Kinetic mechanism and computational molecular modelling

Natural products with urease inhibiting potentialities would be valuable for enhancing nitrogen fertilizer formulations and developing new therapeutics against infectious diseases. Main focus of this research was to assess the inhibitory impact of various parts of Cleome gynandra (Cleomaceae) on jack bean urease activity, identify the responsible compounds, and ascertain their mode of enzyme inhibition and interactions with urease enzyme. Urease inhibitory potentiality of different plant parts of C. gynandra was determined using the phenol-hypochlorite method and mode of enzyme inhibition by Lineweaver-Burk kinetic analysis. LC-HRMS was carried out to identify the compounds of the active fractions. Molecular docking and simulations were executed to find out the binding affinity and interaction pattern of the isolated compounds at the active site of urease enzyme and to verify stability of protein-inhibitor complexes. The root of C. gynandra exhibited highest urease inhibitory potentiality with IC50 value of 1.529 mg/ml compared with leaf and stem. Among the different root fractions, water fraction revealed maximum anti-urease activity with IC50 values of 1.477 mg/ml followed by methanol (1.655 mg/ml) and acetone fractions (1.955 mg/ml). Inhibition kinetics indicated that these fractions were strong inhibitors of urease and exhibited a non-competitive mode of inhibition with Ki values of 184.911 µg/ml, 147.34 µg/ml, and 233.75 µg/ml respectively at 1000 mg/L concentration. LC-HRMS analysis confirmed the occurrence of glucocapparin, fluticasone propionate and lauryl hydrogen sulfate etc. in water fraction. Molecular modelling and simulation study suggested strong and stable interactions between the specified compounds of water fraction and active site of urease. Hence, the water fraction of C. gynandra roots represent a valuable source of natural urease inhibitors, for possible therapeutic applications and sustainable agricultural practices.

Inhibition of Soluble Epoxide Hydrolase by Cembranoid Diterpenes from Soft Coral Sinularia maxima: Enzyme Kinetics, Molecular Docking, and Molecular Dynamics.

Soluble epoxide hydrolase (sEH) is essential for converting epoxy fatty acids, such as epoxyeicosatrienoic acids (EETs), into their dihydroxy forms. EETs play a crucial role in regulating blood pressure, mediating anti-inflammatory responses, and modulating pain, making sEH a key target for therapeutic interventions. Current research is increasingly focused on identifying sEH inhibitors from natural sources, particularly marine environments, which are rich in bioactive compounds due to their unique metabolic adaptations. In this study, the sEH inhibitory activities of ten cembranoid diterpenes (1-10) isolated from the soft coral Sinularia maxima were evaluated. Among them, compounds 3 and 9 exhibited considerable sEH inhibition, with IC50 values of 70.68 μM and 78.83 μM, respectively. Enzyme kinetics analysis revealed that these two active compounds inhibit sEH through a non-competitive mode. Additionally, in silico approaches, including molecular docking and molecular dynamics simulations, confirmed their stability and interactions with sEH, highlighting their potential as natural therapeutic agents for managing cardiovascular and inflammatory diseases.

Inhibition of Glycyrrhiza Polysaccharide on Human Cytochrome P450 46A1 in vitro and in vivo: Implications in Treating Neurological Diseases.

Cytochrome P450 (CYP) 46A1, also known as cholesterol 24S-hydroxylase, is essential for maintaining the homeostasis of cholesterol in the brain and serves as a therapeutic target of neurodegenerative disorders and excitatory neurotoxicity. N-methyl-d-aspartate receptor (NMDAR) is a prototypical receptor for the excitatory neurotransmitter glutamate and can be specifically regulated by 24S-hydroxycholesterol (24S-HC). Glycyrrhiza is one of the most widely used herbs with broad clinical applications, which has several pharmacological activities, such as clearing heat and detoxifying, moistening the lung and relieving cough, analgesic, neuroprotective outcomes, and regulating a variety of drug activities. Glycyrrhiza is a commonly used herb for the treatment of epileptic encephalopathy. However, whether glycyrrhiza can interfere with the activity of CYP46A1 remains unknown. This study aimed to investigate the regulating effects of glycyrrhiza polysaccharides (GP) on CYP46A1-mediated cholesterol conversion, as well as in the modulation of related proteins. The effects of glycyrrhiza polysaccharide (GP) on the activity of CYP46A1 were investigated in vivo and in vitro. Moreover, the potential regulatory effects of GP on the expressions of CYP46A1, HMG-CoA reductase (HMGCR), and NMDAR were also detected. The in vitro results demonstrated that glycyrrhiza polysaccharide (GP), as the main water-soluble active component of glycyrrhiza, remarkably inhibited the activity of CYP46A1 in a non-competitive mode with a Ki value of 0.7003 mg/ml. Furthermore, the in vivo experiments verified that GP markedly decreased the contents of 24S-HC in rat plasma and brain tissues as compared to the control. More importantly, the protein expressions of CYP46A1, GluN2A, GluN2B, and HMG-CoA reductase (HMGCR) in rat brains were all downregulated, whereas the mRNA expressions of CYP46A1 and HMGCR were not significantly changed after treatment with GP. GP exhibits a significant inhibitory effect on CYP46A1 activity in vitro and in vivo, and the protein expressions of CYP46A1, HMGCR, and NMDAR are also inhibited by GP, which are of considerable clinical significance for GP's potential therapeutic role in treating neurological diseases.

Dissecting molecular mechanisms underlying the inhibition of β-glucuronidase by alkaloids from Hibiscus trionum: Integrating in vitro and in silico perspectives

β-Glucuronidase, a crucial enzyme in drug metabolism and detoxification, represents a promising target for therapeutic intervention due to its potential to modulate drug pharmacokinetics and enhance therapeutic efficacy. Herein, we assessed the inhibitory potential of phytochemicals from Hibiscus trionum against β-glucuronidase. Grossamide and grossamide K emerged as the most potent β-glucuronidase inhibitors with IC50 values of 0.73 ± 0.03 and 1.24 ± 0.03 μM, respectively. The investigated alkaloids effectively inhibited β-glucuronidase-catalyzed PNPG hydrolysis through a noncompetitive inhibition mode, whereas steppogenin displayed a mixed inhibition mechanism. Molecular docking analyses highlighted grossamide and grossamide K as inhibitors with the lowest binding free energy, all compounds successfully docked into the same main binding site occupied by the reference drug Epigallocatechin gallate (EGCG). We explored the interaction dynamics of isolated compounds with β-glucuronidase through a 200 ns molecular dynamics (MD) simulation. Analysis of various MD parameters revealed that grossamide and grossamide K maintained stable trajectories and demonstrated significant energy stabilization upon binding to β-glucuronidase. Additionally, these compounds exhibited the lowest average interaction energies with the target enzyme. The MM/PBSA calculations further supported these findings, showing the lowest binding free energies for grossamide and grossamide K. These computational results are consistent with experimental data, suggesting that grossamide and grossamide K could be potent inhibitors of β-glucuronidase.

Discovery of azaleatin as a potential allosteric inhibitor for dengue NS2B-NS3 protease using in vitro and in silico studies

The rise in dengue cases in tropical and sub-tropical areas has become a significant health concern. At present, there is no definitive cure for dengue fever, which underscores the importance of identifying potent inhibitors. Dengue NS2B-NS3 protease is the prime drug target due to its vital function for replication. Quercetin, a flavone, has anti-dengue virus properties but is limited by low bioavailability. Previous studies have shown that methoxy substitution in flavones improves bioavailability and metabolic stability. Azaleatin is a derivative of quercetin with a methoxy substitution at the C5 position, however its ability to inhibit dengue is unknown. In this study, azaleatin was investigated for its inhibition against dengue NS2B-NS3 protease using in vitro and in silico techniques. The fluorescence assay was used to determine the IC50 value and inhibition kinetics. The molecular interaction between azaleatin and NS2B-NS3 was studied using CB-Dock2 and AutoDock Vina. The complex’s stability was then analysed using GROMACS. Besides, the ADMETlab 2.0 was utilized to predict pharmacokinetic of the azaleatin. Results showed that azaleatin inhibits dengue NS2B-NS3 protease non-competitively with a Ki of 26.82 µg/ml and an IC50 of 38 µg/ml. Molecular docking indicated binding of the azaleatin to the allosteric pocket of NS2B-NS3 with a docking score of −8.2 kcal/mol. Azaleatin was found stable in the pocket along 100 ns, supporting its inhibitory mode. The compound has favourable pharmacokinetic profiles and conformed to Lipinski’s Rule of Five. Taken together, azaleatin inhibits NS2B-NS3 protease in a non-competitive mode, suggesting its potential as safer anti-dengue compound. Communicated by Ramaswamy H. Sarma

Biochemical and In Silico Studies on Triazole Derivatives as Tyrosinase Inhibitors: Potential Treatment of Hyperpigmentation Related Skin Disorders.

Tyrosinase is a versatile, glycosylated copper-containing oxidase enzyme that mainly catalyzes the biosynthesis of melanin in mammals. Its overexpression leads to the formation of excess melanin, resulting in hyperpigmentary skin disorders, such as dark spots, melasma, freckles, etc. Therefore, inhibition of tyrosinase is a therapeutic approach for the treatment of hyperpigmentation. The current study focused on evaluating tyrosinase inhibitory activities of triazole derivatives 1-20, bearing different substituents on the phenyl ring. 17 derivatives have shown a potent tyrosinase inhibition with IC50 values between 1.6 to 13 μM, as compared to the standard drug, i.e., kojic acid (IC50 = 24.1 ± 0.5 μM). Particularly, compounds 11 and 15 displayed 12 times more potent inhibitory effects than the kojic acid. The structure-activity relationship revealed that substituting halogens at the C-4 position of the benzene ring renders remarkable anti-tyrosinase activities. Compounds 1-3 and 8 showed a competitive type of inhibition, while compounds 5, 11, and 15 showed a non-competitive mode of inhibition. Next, we performed molecular docking analyses to study the binding modes and interactions between the ligands (inhibitors) and the active site of the tyrosinase enzyme (receptor). Besides this, we have assessed the toxicity profile of inhibitors on the BJ human fibroblast cell line. The majority of the newly identified tyrosinase inhibitors were found to be noncytotoxic. The results presented herein form the basis of further studies on triazole derivatives as potential drug leads against tyrosinase-related diseases.

Alpha-Glucosidase Inhibition, Antioxidant Activities, and Molecular Docking Study of Krom Luang Chumphon Khet Udomsak, a Thai Traditional Remedy.

Krom Luang Chumphon Khet Udomsak remedy (KKR) has traditionally been used as an alternative treatment, particularly for hyperglycemia; however, its therapeutic efficacy has not been scientifically validated. Thus, this study aims to investigate the potential inhibitory and antioxidant effects of α-glucosidase enzyme and characterize the chemical profile of KKR extracts using gas chromatography-mass spectrometry (GC-MS). The investigation highlights both KKR extracts as potent inhibitors of α-glucosidase, with the ethanolic extract of KKR (KKRE) displaying an IC50 value of 46.80 µg/mL and a noncompetitive mode of action. The combination of ethanolic and aqueous extracts of KKR (KKRE and KKRA, respectively) with acarbose exhibited a synergistic effect against the α-glucosidase. The KKRE extract displayed strong scavenging effects in the DPPH assay (IC50 156.3 µg/mL) and contained significant total phenolic (172.82 mg GAE/g extract) and flavonoid (77.41 mg QE/g extract) contents. The major component of KKRE is palmitic acid (15.67%). Molecular docking revealed that the major compounds interacted with key amino acid residues (ASP215, GLU277, HIS351, ASP352, and ARG442), which are crucial for inhibiting α-glucosidase. Notably, campesterin had a more significant influence on α-glucosidase than acarbose, with low binding energy. These findings underscore the significance of KKR in traditional medicine and suggest that it is promising treatment for diabetes mellitus. Further studies using animal model will provide valuable insights for advancing this research.

Novel Dihydrocoumarins Induced by Radiolysis as Potent Tyrosinase Inhibitors.

A representative naturally occurring coumarin, 4-methylumbelliferone (5), was exposed to 50 kGy of gamma ray, resulting in four newly generated dihydrocoumarin products 1-4 induced by the gamma irradiation. The structures of these new products were elucidated by interpretation of spectroscopic data (NMR, MS, [α]D, and UV). The unusual bisdihydrocoumarin 4 exhibited improved tyrosinase inhibitory capacity toward mushroom tyrosinase with IC50 values of 19.8 ± 0.5 μM as compared to the original 4-methylumbelliferone (5). A kinetic analysis also exhibited that the potent metabolite 4 had non-competitive modes of action. Linkage of the hydroxymethyl group in the C-3 and C-4 positions on the lactone ring probably enhances the tyrosinase inhibitory effect of 4-methylumbelliferone (5). Thus, the novel coumarin analog 4 is an interesting new class of tyrosinase inhibitory candidates that requires further examination.

Fabrication of multinuclear copper cluster-based coordination polymers as urease inhibitors.

This study focused on the design and synthesis of two Cu-based coordination polymers, [Cu2(4-dpye)(5-HSIP)(μ3-O)(H2O)2]·3H2O (Cu-CP-1) and [Cu(4-dpye)0.5(BCA)2] (Cu-CP-2), where 4-dpye = N,N'-bis(4-pyridinecarboxamide)-1,2-ethane, 5-H3SIP = 5-sulfoisophthalic acid, and HBCA = benzoic acid, by using a hydrothermal method. Single-crystal X-ray diffraction (SCXRD) study revealed that by adding various auxiliary ligands, the architectures of the Cu-CPs could be altered, yielding two distinct multinuclear Cu clusters. Moreover, the Cu-CPs can be used as urease inhibitors (UIs). In vitro experiments showed that the Cu-CPs had good urease inhibition effects with IC50 values of 0.53 ± 0.01 μM for Cu-CP-1 and 1.44 ± 0.01 μM for Cu-CP-2 and 98.48% (Cu-CP-1) and 96.27% (Cu-CP-2) inhibition of urease was achieved at a concentration of 100 μM, respectively. Furthermore, the inhibition effect of the tetranuclear Cu-CP was better than that of the binuclear Cu-CP. To better understand the potential mechanism of inhibition of the two copper complexes, we performed kinetic analysis using Lineweaver-Burk (L-B) plots in the presence of different concentrations of urea and different concentrations of inhibitors, and both Cu-CP-1 and Cu-CP-2 showed a non-competitive mode of inhibition. In addition, molecular docking analysis showed that the Cu-CPs were able to enter well into the urease binding pocket, thus interacting with key amino acid residues of urease to different degrees. Both kinetic and molecular docking studies theoretically explain and demonstrate the inhibition effect of both Cu-CPs on urease activity in vitro, which is expected to provide reasonable guidance and effective strategies for the development of novel, efficient, stable and safe CP-based UIs.

Apigenin analogs as α-glucosidase inhibitors with antidiabetic activity

This study investigated the inhibitory potential of a series of synthesized compounds (L1-L27) on α-glucosidase. Among them, compound L22 showed significant inhibitory effect. Through enzymatic kinetics studies, we demonstrated that L22 acts via a non-competitive inhibition mode with a Ki value of 2.61 μM, highlighting its high affinity for the enzyme. Molecular docking studies revealed the formation of hydrogen bonds between L22 and α-glucosidase and diverse interactions with neighboring amino acid residues. Furthermore, molecular dynamics simulations confirmed the stability of the L22-α-glucosidase complex. In a mouse model of type 2 diabetes, treatment with L22 significantly lowered fasting blood glucose levels, and reduced insulin resistance, suggesting its potential as a therapeutic agent for type 2 diabetes. Furthermore, L22 showed a protective effect against oxidative stress in the liver and alleviated liver and pancreatic abnormalities. These results provide valuable insights into the mechanism of action of L22 and its potential applications to treat type 2 diabetes, and improve liver health.

Exploring the impact of novel thiazole-pyrazole fused benzo-coumarin derivatives on human serum albumin: Synthesis, photophysical properties, anti-cholinergic activity, and interaction studies

Derivatives of thiazole-pyrazole fused benzo-coumarin compounds were successfully synthesized and characterized, followed by a comprehensive spectroscopic investigation on various photophysical properties in different media. The multipronged approach using steady state and time resolved fluorescence spectroscopy pointed out the impact of substitution in the estimated spectroscopic and other physicochemical properties of the systems. Further, the evaluation of anti-acetylcholinesterase (anti-AChE) activity yielded significant insight into the therapeutic potential of the synthesized coumarinyl compounds for the treatment of Alzheimer's disease (AD). The findings revealed a non-competitive mode of inhibition mechanism, with an estimated IC50 value of 67.72 ± 2.00 nM observed for one of the investigated systems as AChE inhibitor. Notably, this value is even lower than that of an FDA-approved AD drug Donepezil (DON), indicating the enhanced potency of the coumarin derivatives in inhibiting AChE. Interestingly, significant diminution in inhibition was observed in presence of human serum albumin (HSA) as evidenced by the relative increase in IC50 value by 8 ∼ 39 % in different cases, which emphasized the role of albumin proteins to control therapeutic efficacies of potential medications. In-depth spectroscopic and in-silico analysis quantified the nature of interactions of the investigated systems with HSA and AChE. Overall, the outcomes of this study provide significant understanding into the biophysical characteristics of novel thiazole-pyrazole fused benzo-coumarin systems, which could aid in the development of new cholinergic agents for the treatment of AD and materials based on coumarin motifs.

Phenolic Derivatives with Anti-Acetylcholinesterase Inhibitory Activities from Galeola nudifolia in Vietnam.

A new phenolic derivative, galeomalate A (1), together with five known structurally related compounds (2-6), was isolated from the ethyl acetate extract of Galeola nudifolia collected in Vietnam. The structures were elucidated by various spectroscopic methods, including 1D and 2D NMR, HR-ESI-TOF-MS, and CD data, and chemical conversion of the sugar moiety. All isolated compounds possessed acetylcholinesterase (AChE) inhibitory activities in a dose-dependent manner. Among them, compounds 2 and 3 exhibited the first and second highest inhibitory activity on AChE with IC50 values of 122.13 and 125.49 μM, respectively. Compounds 1 and 4-6 inhibited the AChE activity by mixed modes of action comprising competitive and non-competitive modes, whereas 2 and 3 exerted their inhibitory activities in a competitive manner. Molecular docking analyses suggested that the phenyl-β-D-glucopyranoside unit of 2 and 3 bound to the active site of AChE for the competitive inhibitory activities, while the mixed inhibitory activity of 4 was due to the two binding patterns in the active-site and the active-site entrance of AChE. Furthermore, the docking studies indicated that 1, 5, and 6 would inhibit AChE in a mixed inhibitory manner by adopting three distinct binding patterns of the additional phenyl-β-D-glucopyranoside unit at the active-site entrance.

Structure and Antidiabetic Activity of a Glycoprotein from Porphyra haitanensis.

A novel antidiabetic glycoprotein (PG) was isolated and purified from Porphyra haitanensis, and its structure and inhibiting activity on α-amylase and α-glucosidase were analyzed. The purity of the PG was 95.29 ± 0.21%, and its molecular weight was 163.024 ± 5.55 kDa. The PG had a tetramer structure with α- and β-subunits, and it contained 54.12 ± 0.86% protein (with highly hydrophobic amino acids) and 41.19% ± 0.64% carbohydrate (composed of galactose). The PG was linked via an O-glycosidic bond, exhibiting an α-helical structure and high stability. In addition, the PG inhibited the activities of α-amylase and α-glucosidase, by changing the enzyme's structure toward the PG's structure in a noncompetitive inhibition mode. Molecular docking results showed that the PG inhibited α-amylase activity by hydrophobic interaction, whereas it inhibited α-glucosidase activity by hydrogen bonds and hydrophobic interaction. Overall, the PG was linked to polysaccharides via O-glycosidic bonds, showing an α-helical configuration and a hydrophobic effect, which altered the configuration of α-amylase and α-glucosidase and exerted hypoglycemic activity. This study provides insights into analyzing the structure and antidiabetic activity of glycoproteins.

Pyrimidine-morpholine hybrids as potent druggable therapeutics for Alzheimer’s disease: Synthesis, biochemical and in silico analyses

The identification of effective and druggable cholinesterase inhibitors to treat progressive neurodegenerative Alzheimer’s disorder remains a continuous drug discovery hunt. In this perspective, the present study investigates the design and discovery of pyrimidine-morpholine hybrids (5a-l) as potent cholinesterase inhibitors. Palladium-catalyzed Suzuki-Miyaura cross-coupling reaction was employed to introduce the structural diversity on the pyrimidine heterocyclic core. A range of commercially available boronic acids was successfully coupled showing a high functional group tolerance. In vitro cholinesterase inhibitory potential using Ellman’s method revealed significantly strong potency. Compound 5h bearing a meta-tolyl substituent at 2-position of pyrimidine ring emerged as a lead candidate against AChE with an inhibitory potency of 0.43 ± 0.42 µM, ∼38-fold stronger value than neostigmine (IC50 = 16.3 ± 1.12 µM). Compound 5h also showed the lead inhibition against BuChE with an IC50 value of 2.5 ± 0.04 µM. The kinetics analysis of 5h revealed the non-competitive mode of inhibition against AChE whereas computational modelling results of potent leads depicted diverse contacts with the binding site amino acid residues. Molecular dynamics simulations revealed the stability of biomolecular system, while, ADME analysis demonstrated druglikeness behaviour of potent compounds. Overall, the investigated pyrimidine-morpholine scaffold presented a remarkable potential to be developed as efficacious anti-Alzheimer’s drugs.

Otilonium Bromide acts as a selective USP28 inhibitor and exhibits cytotoxic activity against multiple human cancer cell lines

USP28 contributes to tumorigenesis through modulating the lifespan of oncogenic factors such as c-Myc and ΔNp63, and it has been identified as a potential target for anti-cancer drug development. Currently, although quite a number of USP28 inhibitors have been developed, they all are still in preclinical research stage. Besides, none of them exhibits satisfying inhibition selectivity against USP28 over its closest homologue USP25. Here in this manuscript, a high-throughput screening aiming to discover USP28 inhibitors with novel scaffold and enhanced inhibition selectivity were conducted. After the primary screening and the second round of validation, Otilonium Bromide, an approved drug for treating irritable bowel syndrome, was identified to inhibit USP28′s activity with the IC50 value at 6.90 ± 0.90 μM. Besides, the drug exhibits a 3–4 folds inhibition selectivity against USP28 over USP25. According to the enzymatic kinetics analysis data and the hydrogen–deuterium exchange mass spectrometry results, Otilonium Bromide could bind to the allosteric pocket of USP28 and inhibit its activity in a reversible and non-competitive mode. The following performed cell-based assays revealed that the drug could cause cytotoxicity against human colorectal cancer cells and lung squamous carcinoma cells potentially through down-regulating USP28′s oncogenic substrates c-Myc and/or ΔNp63. Meanwhile, since Otilonium Bromide has been found to preferentially distribute to gastrointestinal tissues, we then evaluated its potential in the combination treatment of colorectal cancer cells with Regorafenib, which is an approved drug for colorectal cancer therapy. As expected, Otilonium Bromide could significantly enhance the sensitivity of colorectal cancer cells to Regorafenib.