Mechanism Of Enzyme Inhibition Research Articles

Comprehensive profiling of acetylcholinesterase inhibitors from fried centipede using activity-oriented online preparation technology

Fried centipede, a popular food in southern China and Vietnam, is well known for its notable health benefits owing to its unique chemical composition. This study focused on acetylcholinesterase (AChE) inhibitor screening, bioactivity evaluation, and the extraction of bioactive compounds derived from fried centipedes. To determine the accuracy of in vitro AChE inhibitor screening, receptor-ligand affinity ultrafiltration and enzymatic reaction kinetics technologies were employed for the rapid screening and structural identification of bioactive compounds and to verify the enzyme inhibition activity and mechanism of the compounds. Moreover, molecular docking and dynamic simulations were utilized to assess the effectiveness of the target, and the integration of experimental and computational approaches facilitated a comprehensive analysis of the active compounds and their activities at four levels. To address the issues of low content and low extraction and separation efficiencies of bioactive compounds derived from centipedes, an activity-oriented complex preparation method (consecutive ultrasonic-assisted centrifugal extraction coupled online with UNIFAC countercurrent chromatography and semi-high-performance liquid chromatography (HPLC) was developed for efficient extraction, online concentration, and complex separation of the identified AChE ligands. As a result, six active compounds, including cytidine, guanine, uracil, hypoxanthine, xanthine, and β-thymidine demonstrated significant binding affinity to the AChE active sites within 3 h. This study provided valuable insights into the active compounds of fried centipedes and their potential anti-Alzheimer's disease properties. These findings contribute to the advancement of animal food preparation methodologies by combining cutting-edge extraction techniques, biological activity screening, and the development of AChE inhibitors from fried centipedes. Our method serves as a valuable reference for exploring and developing functional animal materials from diverse medicinal resources.

Evaluation of the Anti-diabetic Activity of Purified Compounds of Ferula assafoetida by in vitro and in silico Methods

Objective Postprandial hyperglycemia is considered an early sign of diabetes. Enzyme inhibitors, such as α-amylase and α-glucosidase inhibitors, are currently being studied as potential drugs for preventing postprandial hyperglycemia. Methods In this study, we investigated the effects of four purified 7-hydroxycoumarine derivatives from Ferula assafoetida: umbelliprenin, farnesiferol A, farnesiferol C, and samarcandin. We evaluated cell toxicity using the MTT method and also examined glucose uptake and inhibition of α-amylase and α-glucosidase enzymes in vitro. Additionally, we conducted a molecular docking study to investigate the mechanism of enzyme inhibition. Results The cell toxicity of the terpenoid coumarin derivatives (umbelliprenin, farnesiferol A, farnesiferol C, and samarcandin) on HepG2 cells was found to be approximately 28 to 37 µg/ml. The glucose uptake assay showed that these compounds (at a concentration of 25 µg/ml) were able to increase glucose consumption by HepG2 cells to a level comparable to that of the positive control (metformin at 50 µg/ml). Furthermore, umbelliprenin significantly inhibited the activity of α-amylase and α-glucosidase (by 35.07% and 4.98%, respectively). Molecular docking results indicated that umbelliprenin, with a farnesyl chain, had a more potent inhibitory effect. Conclusion Our findings suggest that umbelliprenin may be a valuable compound for controlling postprandial hyperglycemia and diabetes. However, further in vivo studies and clinical trials are necessary to validate these effects. While this research offers potential for the development of more effective compounds with coumarin structures, further studies are needed to confirm these findings.

Studies on Methylpyrazole-Substituted Benzimidazoles to Target Helicobacter pylori Infection through HpIMPDH Inhibition.

The prevalence of Helicobacter pylori infection has been increasing rapidly due to the genetic heterogeneity and antibacterial resistance shown by the bacteria, affecting over 50% of the world population and over 80% of the Indian population, in particular. In this regard, novel drug targets are currently being explored, one of which is the crucial metabolic enzyme inosine-5'-monophosphate dehydrogenase (IMPDH) involved in the de novo nucleotide biosynthesis pathway, in order to combat the infection and devise efficient therapeutic strategies. The present study reports the development of methylpyrazole-substituted benzimidazoles as small molecule inhibitors of H. pylori IMPDH with a nanomolar range of enzyme inhibition. A set of 19 small molecules have been designed, synthesized, and further evaluated for their inhibitory potential against H. pylori IMPDH using in silico, in vitro, biochemical, and biophysical techniques. Compound 7j was found to inhibit H. pylori IMPDH with an IC50 value of 0.095 ± 0.023 μM, which is close to 1.5-fold increase in the inhibitory activity, in comparison to the previously reported benzimidazole-based hit C91. Moreover, kinetic characterization has provided significant insights into the uncompetitive inhibition shown by these small molecules on H. pylori IMPDH, thus providing details about the enzyme inhibition mechanism. In conclusion, methylpyrazole-based small molecules indicate a promising path to develop cheap and bioavailable drugs to efficiently treat H. pylori infection in the coming years, in comparison to the currently available therapy.

Multifaceted Activity of Fabimycin: Insights from Molecular Dynamics Studies on Bacterial Membrane Models.

Membranes─cells' essential scaffolds─are valid molecular targets for substances with an antimicrobial effect. While certain substances, such as octenidine, have been developed to target membranes for antimicrobial purposes, the recently reported molecule, fabimycin (F2B)─a novel agent targeting drug-resistant Gram-negative bacteria─has not received adequate attention regarding its activity on membranes in the literature. The following study aims to investigate the effects of F2B on different bacterial membrane models, including simple planar bilayers and more complex bilayer systems that mimic the Escherichia coli shell equipped with double inner and outer bilayers. Our results show that F2B exhibited more pronounced interactions with bacterial membrane systems compared to the control PC system. Furthermore, we observed significant changes in local membrane property homeostasis in both the inner and outer membrane models, specifically in the case of lateral diffusion, membrane thickness, and membrane resilience (compressibility, tilt). Finally, our results showed that the effect of F2B differed in a complex system and a single membrane system. Our study provides new insights into the multifaceted activity of F2B, demonstrating its potential to disrupt bacterial membrane homeostasis, indicating that its activity extends the currently known mechanism of FabI enzyme inhibition. This disruption, coupled with the ability of F2B to penetrate the outer membrane layers, sheds new light on the behavior of this antimicrobial molecule. This highlights the importance of the interaction with the membrane, crucial in combating bacterial infections, particularly those caused by drug-resistant strains.

Synthesis, antidiabetic activity and in silico studies of benzo[b]thiophene based small molecule α-amylase inhibitors

Benzo[b]thiophene has been implicated as molecular framework in the drug discovery against broad spectrum of biological targets. In the antidiabetic drug regime, benzo[b]thiophene based SGLT2 and ALR2 inhibitors have been recently developed but their potential towards α-amylase inhibition remained unexplored to date. In this context, a series of novel small molecule benzo[b]thiophene-2-carboxylic acid derivatives (3a-p) was synthesized, characterized, and evaluated for antidiabetic activity as α-amylase inhibitors. We found that, all benzo[b]thiophene derivatives exhibited significant α-amylase inhibition with IC50 value ranging from 5.37 ± 0.25 μM to 29.89 ± 0.68 μM. The SAR studies showed benzo[b]thiophene carboxylate bearing bis(2-hydroxyethyl)amino group (3b) was most potent with IC50 of 5.37 ± 0.25 μM compared to standard drug Acarbose (IC50 = 6.40 ± 0.14 μM). Further, the enzyme inhibition mechanism study regarded 3b as competitive inhibitor of α-amylase with Ki value of 1.76 μM. A detailed in silico study was also performed in order to estimate binding properties, drug likeness and predict toxicity profile of these agents. It was demonstrated that novel small molecule benzo[b]thiophene derivative (3b) can effectively bind through H-bonding, hydrophobic and π-stacking interactions within α-amylase active site. Moreover, drug likeness and toxicity prediction studies suggested compound 3b as potential & safter α-amylase inhibitor. Overall, our present study disclosed a novel class of benzo[b]thiophene based α-amylase inhibitors and opened a template for further lead optimization and development.

Exploring enzyme inhibition and comprehensive mechanisms of antioxidant/prooxidative activity of natural furanocoumarin derivatives: A comparative kinetic DFT study

This study aimed to explore the antioxidant and prooxidative activity of two natural furanocoumarin derivatives, Bergaptol (4-Hydroxy-7H-furo [3,2-g] [1]benzopyran-7-one, BER) and Xanthotoxol (9-Hydroxy-7H-furo [3,2-g] [1]benzopyran-7-one, XAN). The collected thermodynamic and kinetic data demonstrate that both compounds possess substantial antiradical activity against HO• and CCl3OO• radicals in physiological conditions. BER exhibited better antiradical activity in comparison to XAN, which can be attributed to the enhanced deprotonation caused by the positioning of the –OH group on the psoralen ring. In contrast to highly reactive radical species, newly formed radical species BER• and XAN• exhibited negligible reactivity towards the chosen constitutive elements of macromolecules (fatty acids, amino acids, nucleobases). Furthermore, in the presence of O2•─, the ability to regenerate newly formed radicals BER• and XAN• was observed. Conversely, in physiological conditions in the presence of Cu(II) ions, both compounds exhibit prooxidative activity. Nevertheless, the prooxidative activity of both compounds is less prominent than their antioxidant activity. Furthermore, it has been demonstrated that anionic species can engage in the creation of a chelate complex, which restricts the reduction of metal ions when reducing agents are present (O2•─ and Asc─). Moreover, studies have demonstrated that these chelating complexes can be coupled with other radical species, hence enhancing their ability to inactivate radicals. Both compounds exhibited substantial inhibitory effects against enzymes involved in the direct or indirect generation of ROS: Xanthine Oxidase (XOD), Lipoxygenase (LOX), Myeloperoxidase (MPO), NADPH oxidase (NOX).

Tyrosinase Inhibitory Activity of n-Hexane, Ethyl Acetate and Methanol Extracts of Padina sp

Melanin is a color pigment in the skin, if produced in excess will cause browning of the skin. The formation of melanin (melanogenesis) is assisted by tyrosinase through two reactions, namely monophenolase and diphenolase. Melanogenesis can be reduced through the tyrosinase enzyme inhibition mechanism. Seaweed can be used as a tyrosinase inhibitor (brightening agent), such as Padina sp containing secondary metabolites such as alkaloids, flavonoids, terpenoids, phenolics, and saponins. This study aimed to determine the tyrosinase inhibitory activity of Padina sp. The extraction method used was graded maceration with n-hexane (n-Hx), ethyl acetate (Et-OAc), and methanol (Me-OH) solvents, then carried out phytochemical screening, antioxidant test using the DPPH method, and tyrosinase inhibitory test by measuring the enzymatic reaction using L-tyrosine (monophenolase) and L-DOPA (diphenolase). Phytochemical analysis of extracts by GC-MS and in silico analysis by molecular docking were also carried out. The results showed that the total yield of the three extracts was 5.50%. The three extracts had moderate category of antioxidants. The IC50 values monophenolase of n-Hx, Et-OAc, Me-OH extracts, and Kojic acid were 937.68; 132, 92; 268.68; and 20.99μg/mL, respectively. The IC50 values diphenolase of n-Hx, Et-OAc, Me-OH extracts, and Kojic acid were 989.74; 178.33; 356, 87; and 31.76 μg/mL, respectively. The phytochemical of Et-OAc extract based on GC-MS data showed a variety of compounds that have been shown to have pharmacological effects. This data is supported by the results of molecular docking analysis, where compound Spiro(tetrahydrofuryl)2.1'(decalin), 5',5',8'a-trimethyl (1) is able to show a relatively low binding energy, namely -6.86 kcal/mol. The binding energy is even lower than the standard compound (Kojic acid) interaction which only has binding energy of -3.73 kcal/mol. Based on the study carried out, extract from Padina sp has the potential to be developed as a a skin brightening agent.

Inhibitor Trapping in Kinases.

Recently, we identified a novel mechanism of enzyme inhibition in N-myristoyltransferases (NMTs), which we have named 'inhibitor trapping'. Inhibitor trapping occurs when the protein captures the small molecule within its structural confines, thereby preventing its free dissociation and resulting in a dramatic increase in inhibitor affinity and potency. Here, we demonstrate that inhibitor trapping also occurs in the kinases. Remarkably, the drug imatinib, which has revolutionized targeted cancer therapy, is entrapped in the structure of the Abl kinase. This effect is also observed in p38α kinase, where inhibitor trapping was found to depend on a 'magic' methyl group, which stabilizes the protein conformation and increases the affinity of the compound dramatically. Altogether, these results suggest that inhibitor trapping is not exclusive to N-myristoyltransferases, as it also occurs in the kinase family. Inhibitor trapping could enhance the binding affinity of an inhibitor by thousands of times and is as a key mechanism that plays a critical role in determining drug affinity and potency.

Nicotinonitriles as potential inhibitors of α-glucosidase, tyrosinase, and urease enzymes: Synthesis, characterization, in vitro, and in silico studies

In an effort to develop novel enzyme inhibitors with potent activity and high selectivity, a series of nicotinonitrile derivatives was synthesized. Cyano pyridone intermediates undergo nucleophilic substitution reactions with variously substituted phenacyl halides to yield the nicotinonitriles 1–20. All nicotinonitriles exhibited moderate to excellent in vitro enzyme inhibitory activities against α-glucosidase, tyrosinase, and urease. Compound 1 (IC50 = 27.09 ± 0.12 µM) selectively displayed more significant activity than the standard acarbose (IC50 = 40.00 ± 0.70 µM). Compound 1 also showed good urease inhibition (IC50 = 33.04 ± 0.70 µM); however, it exhibited activity less than the standard acetohydroxamic acid (IC50 = 27.00 ± 0.50 µM).Furthermore, the most effective compound of the series against tyrosinase compared to the standard kojic acid (IC50 = 16.9 ± 1.30 µM) was 16 with an IC50 value of 10.55 ± 0.08 µM. In contrast, this compound showed no activity against α-glucosidase and urease. DFT calculations were carried out to analyze electronic transitions and energy levels (HOMO and LUMO) in nicotinonitrile derivatives, offering insights into their chemical reactivity and stability. Molecular docking studies and molecular electrostatic potential (MEP) revealed the interactions of the active compounds with the amino acid residues at the active sites of the enzymes, thereby shedding light on the mechanism of enzyme inhibition. Therefore, compound 1 can be further studied as a promising α-glucosidase and urease inhibitor, while compound 16 is an effective tyrosinase inhibitor.

In vitro and in silico studies of alpha glucosidase inhibition and antifungal activity of coffea canephora husk.

The coffea canephora husk, a protected agricultural crop, is abundant in Vietnam. Examining the effects of C. canephora husk compounds on α-glucosidase and antifungal drug activity was the primary objective of this research. A cholestane-type steroid, coffeacanol A (1), was extracted from the ethyl acetate extract. Three cholestane-type derivatives (2-4) and three additional known compounds (5-7) were separated, and we used a variety of chromatographic techniques to identify a total of six substances. We used NMR to determine the chemical structures of these substances. Extensive HR-MS-ESI analysis and NMR experimental data were used to confirm the structure of the novel metabolite (1). The cholestane-type steroid was initially discovered in the Coffea canephora husk, marking the first instance in the coffee plant family to reveal chemical structures (1-7). The inhibition of α-glucosidase was found to be significantly higher in all compounds tested, with the exception of compounds (2) and (5). In vitro, the positive control showed the lowest inhibition, and the range of IC50 values was calculated to be 27.4 to 96.5 μM, which is lower than the IC50 value of 214.50 μM for the acarbose control. With an IC50 value of 27.4 μM, compound (7) showed the greatest capacity to inhibit α-glucosidase among the test compounds. The 3TOP and 2VF5 enzyme crystal structures were used for in silico docking investigations and validations of compounds (1-7). In silico calculations to explain how compound (7) shows high activity in vitro via the enzyme inhibition mechanism by residual amino acids, like Gly 1102 (B chain) and Glu 1095 (B chain), and their relative interaction with compounds (7) and acarbose. Compound (7) exhibited the best antifungal activity against Candida albicans fungus among three fungi, namely Candida albicans, Trichophyton mentagrophytes, and Trichophyton rubrum, with a MIC value of 25 μM. Compound (7) and fluconazole combined to form similar interactions in the contact ligand model, including the functional group, capping group, and linker part, which interacted fully with the 2VF5 enzyme, leading to effective in vitro inhibition.

The profiles of free, esterified and insoluble-bound phenolics in peach juice after high pressure homogenization and evaluation of their antioxidant capacities, cytoprotective effect, and inhibitory effects on α-glucosidase and dipeptidyl peptidase-Ⅳ

The phenolic profiles, antioxidant capacities, cytoprotective effect, and α-glucosidase and DPP-IV inhibitory capacity of free (FP), esterified (EP) and insoluble-bound (IBP) phenolic fractions in ‘Lijiang snow’ peach juice after high pressure homogenization (HPH) were investigated, and the molecular docking was used to explore the enzyme inhibition mechanism. HPH increased total phenolic and total flavonoid contents in three fractions without changing compositions. The IC50 of radicals scavenged by three fractions were all reduced by HPH. The best inhibition on intracellular ROS production were found for phenolic fractions after HPH at 300 MPa, with ROS levels ranged within 95.26–119.16 %. HPH at 300 MPa reduced the apoptosis rates of FP and EP by 16.52 % and 9.33 %, respectively. All phenolic fractions showed effective inhibition on α-glucosidase and DPP-IV by formation of hydrogen bonding and van der Waals forces. This study explored the feasibility of HPH to enhance the phenolics and bioactivity of peach juice.

Virtual Screening Technology for Two Novel Peptides in Soybean as Inhibitors of α-Amylase and α-Glucosidase.

Soybean peptides (SPs) have bioactivities of enzyme inhibition that are beneficial to human health, but their mechanism is not clear. This study aimed to identify peptide fragments in SPs that simultaneously inhibit α-amylase and α-glucosidase and to explore their enzyme inhibition mechanism. Firstly, the inhibitory activity of SPs against the enzymes was determined. And two octapeptides, LDQTPRVF and SRNPIYSN, were identified for the first time by using HPLC-QTOF-MS/MS and virtual screening. Molecular simulation results showed that hydrogen bonds and π-π bonds were the key factors, and the N-terminal (Leu and Ser) and C-terminal (Phe) of peptide were important inhibiting sites. Both octapeptides were synthesized, and their IC50 values were 3.08 and 5.58 mmol/L for α-amylase, and 2.52 and 4.57 mmol/L for α-glucosidase, respectively. This study provided evidence for SPs as a potential inhibitor of α-amylase and α-glucosidase in special dietary foods.

Development of manganese peroxidase based voltammetric biosensor for detection of textile Azo dyes RR 195 & RB 221

Azo dyes are extensively applied in textile and leather industries. Monitoring the toxicity and rate of exhaustion of such pollutants besides their mineralization are extremely important for environmental safety and sustainability. Detection of dyes based on enzyme inhibition mechanism and exploration of effect of various dyes on enzymes such as manganese peroxidase (MnP) for such purpose are not studied previously. The goal of the study is to develop MnP based biosensor and to determine whether all dyes impart inhibitory effect on enzyme or not. Thereby, two different dyes i.e. Reactive Red 195 (RR195) and Reactive Blue 221 (RB221) were used for the study and purified MnP was co-immobilized on carbon felt electrode for quantitative measurement of these dyes using electrochemical signals. Voltammetric analysis was carried out to determine the effects of different dye concentrations on the enzyme activity in the form electric signal (I) at an applied voltage of −1 to +1. It was revealed that increase in concentrations(10 μg/L −50 mg/L) of RR195 and RB221 transfuse inhibitory and non-inhibitory effect on MnP activity resulting in current (I) output reduction and enhancement respectively. To validate the study, another study was also conducted, which investigated the effect of these dyes on fungal growth and manganese peroxidase enzyme activity in-vitro. The results obtained authenticated the biosensing results obtained by CV as RR195 inhibit MnP activity (highly toxic) and RB221 impart no effect on MnP activity (less toxic). The fabricated biosensors demonstrated reproducibility, durability, and low threshold of detection with little interference from other substances. Even 100 days later, the MnP-based biosensors showed minor fluctuation in its current (I) outputs with 96–36 % and 86-65 % stability in case of RR195 and RB221 respectively. The study demonstrated the application of enzyme-based biosensors for both detection and toxicological monitoring of dyes.

Molecular noise-induced activator-inhibitor duality in enzyme inhibition kinetics.

Classical theories of enzyme inhibition kinetics predict a monotonic decrease in the mean catalytic activity with the increase in inhibitor concentration. The steady-state result, derived from deterministic mass action kinetics, ignores molecular noise in enzyme-inhibition mechanisms. Here, we present a stochastic generalization of enzyme inhibition kinetics to mesoscopic enzyme concentrations by systematically accounting for molecular noise in competitive and uncompetitive mechanisms of enzyme inhibition. Our work reveals an activator-inhibitor duality as a non-classical effect in the transient regime in which inhibitors tend to enhance enzymatic activity. We introduce statistical measures that quantify this counterintuitive response through the stochastic analog of the Lineweaver-Burk plot that shows a merging of the inhibitor-dependent velocity with the Michaelis-Menten velocity. The statistical measures of mean and temporal fluctuations - fractional enzyme activity and waiting time correlations - show a non-monotonic rise with the increase in inhibitors before subsiding to their baseline value. The inhibitor and substrate dependence of the fractional enzyme activity yields kinetic phase diagrams for non-classical activator-inhibitor duality. Our work links this duality to a molecular memory effect in the transient regime, arising from positive correlations between consecutive product turnover times. The vanishing of memory in the steady state recovers all the classical results.

Postprandial Hypolidaemic Effect of Aloe Vera Methanolic Extract via the Mechanism of Intestinal Pancreatic Lipase Enzyme Inhibition in Male Wistar Rats

Aloe vera, starch, triglycerides, post-prandial blood glucose and post-prandial triglyceride

Antidiabetic Activity of the Methanol Fraction of Sungkai Leaves (Peronema canescens Jack)

Diabetes Mellitus (DM) is one of a group of metabolic diseases characterized by hyperglycemia caused by a disturbance in insulin so that blood glucose levels increase. Prevention of absorption of blood glucose by the intestine can be done with the help of the enzyme α-glucosidase. Based on the results of phytochemical tests, it is known that the methanol fraction of sungkai leaves contains flavonoid compounds. The Fourier Transform Infra-Red (FTIR) spectrum showed that the isolates had phenolic O-H groups (3,354.53 cm1 , 1,359.28 cm-1 ), aromatic C=C group (1,615.48 cm-1 ), C-O-C ether group (1,046.60 cm-1 ) and aromatic C-H group (822.21 cm-1 ). In vivo antidiabetic activity test was carried out using test animals of white male mice which were induced by alloxan. Antidiabetic testing was carried out using 6 treatment groups with glibenclamide, Na-Carboxymethyl cellulose (NaCMC) 0.5%, dose 175 mg/kgBW, 350 mg/kgBW, 700 mg/kg Body Weight (BW) and isolates 2 mg/kgBW. The results showed that the methanol fraction of sungkai leaves had the best antidiabetic activity at a dose of isolate and 700 mg/kgBW which was able to reduce blood glucose levels by 42.20% and 42.00%. In vitro antidiabetic testing through αglucosidase enzyme inhibition mechanism did not show any antidiabetic activity at concentrations of fractions 5, 10, 25, 50 and 100 ppm.

Digital-like Enzyme Inhibition Mechanism-Based Strategy for the Digital Sensing of Heparin-Specific Biomarkers.

A new microbead (MB)-based digital flow cytometric sensing system is proposed for the sensitive detection of heparin-specific biomarkers, including heparin-binding protein (HBP) and heparinase. This strategy takes advantage of the inherent space-confined enzymatic behavior of T4 polynucleotide kinase phosphatase (T4 PNKP) around a single MB and the heparin's digital-like inhibitory effect on T4 PNKP. By integrating with an on-bead terminal deoxynucleotidyl transferase (TdT)-catalyzed fluorescence signal amplification technology, the concentration of HBP and heparinase can be digitally determined by the number of fluorescence-positive/-negative MBs which can be easily counted by flow cytometry. This is not only the first test to expand the application scenario of T4 PNKP to the digital detection of different biomarkers but also pioneers a new direction for fabricating digital biosensing platforms based on the enzyme inhibition mechanism.

Mixed and non-competitive enzyme inhibition: underlying mechanisms and mechanistic irrelevance of the formal two-site model

The formal mechanism of linear mixed and non-competitive enzyme inhibition implies the binding of inhibitors to both the active site of the free enzyme in competition with the substrate, and to an allosteric site on the enzyme-substrate complex. However, it is evident from a review of the scientific literature that the two-site mechanism is frequently mistaken as the actual underlying mechanism of mixed inhibition. In this study, we conducted a comprehensive assessment of the mechanistic relevance of this type of inhibition using a statistical approach. By combining a statistical analysis of the inhibition cases documented in the BRENDA database with a theoretical investigation of inhibition models, we conclude that mixed inhibitors exclusively bind to the active site of enzymes. Hence ruling out any implication of allosteric sites and depriving the two-site model of any mechanistic relevance.

JP-1366: A novel and potent potassium-competitive acid blocker that is effective in the treatment of acid-related diseases.

The global prevalence of GERD is substantially increasing each year, and GERD is a chronic disease that reduces the quality of life of patients. The efficacy of conventional drugs is diverse, and most require long-term or lifetime administration; thus, the development of more effective therapeutic agents is needed. Herein, a more effective treatment for GERD was tested. We investigated whether JP-1366 affected gastric H+/K+-ATPase activity and used the Na+/K+-ATPase assay to confirm the selectivity of H+/K+-ATPase inhibition. To clarify the mechanism of enzyme inhibition, JP-1366 and TAK-438 were analyzed by Lineweaver-Burk. Also, we investigated the effects of JP-1366 in various models involving reflux esophagitis. We found that JP-1366 mediates strong, selective, and dose-dependent inhibition of H+/K+-ATPase. We found that JP-1366 significantly suppressed gastric acid secretion in histamine-treated pylorus-ligated rats in a dose-dependent manner. Additionally, we confirmed that JP-1366 inhibited histamine-stimulated gastric acid secretion in the HPD model. JP-1366 exhibited a more than 2-fold higher inhibitory effect on esophageal injury than TAK-438 in GERD lesions and had a more potent inhibitory effect in indomethacin- or aspirin-induced gastric ulcer rat models than TAK-438. Additionally, JP-1366 inhibited gastric ulcers. These results support the possibility that JP-1366 is a good candidate drug for treating acid-related diseases.

Naturally-Occurring Tyrosinase Inhibitors Classified by Enzyme Kinetics and Copper Chelation.

Currently, there are three major assaying methods used to validate in vitro whitening activity from natural products: methods using mushroom tyrosinase, human tyrosinase, and dopachrome tautomerase (or tyrosinase-related protein-2, TRP-2). Whitening agent development consists of two ways, melanin synthesis inhibition in melanocytes and downregulation of melanocyte stimulation. For melanin levels, the melanocyte cell line has been used to examine melanin synthesis with the expression levels of TRP-1 and TRP-2. The proliferation of epidermal surfaced cells and melanocytes is stimulated by cellular signaling receptors, factors, or mediators including endothelin-1, α-melanocyte-stimulating hormone, nitric oxide, histamine, paired box 3, microphthalmia-associated transcription factor, pyrimidine dimer, ceramide, stem cell factors, melanocortin-1 receptor, and cAMP. In addition, the promoter region of melanin synthetic genes including tyrosinase is upregulated by melanocyte-specific transcription factors. Thus, the inhibition of growth and melanin synthesis in gene expression levels represents a whitening research method that serves as an alternative to tyrosinase inhibition. Many researchers have recently presented the bioactivity-guided fractionation, discovery, purification, and identification of whitening agents. Melanogenesis inhibition can be obtained using three different methods: tyrosinase inhibition, copper chelation, and melanin-related protein downregulation. There are currently four different types of inhibitors characterized based on their enzyme inhibition mechanisms: competitive, uncompetitive, competitive/uncompetitive mixed-type, and noncompetitive inhibitors. Reversible inhibitor types act as suicide substrates, where traditional inhibitors are classified as inactivators and reversible inhibitors based on the molecule-recognizing properties of the enzyme. In a minor role, transcription factors can also be downregulated by inhibitors. Currently, the active site copper iron-binding inhibitors such as kojic acid and chalcone exhibit tyrosinase inhibitory activity. Because the tyrosinase catalysis site structure is important for the mechanism determination of tyrosinase inhibitors, understanding the enzyme recognition and inhibitory mechanism of inhibitors is essential for the new development of tyrosinase inhibitors. The present review intends to classify current natural products identified by means of enzyme kinetics and copper chelation to exhibit tyrosinase enzyme inhibition.