Competitive Inhibition Pattern Research Articles

Synthesis, biological evaluation and molecular modeling studies of methyl indole-isoxazole carbohydrazide derivatives as multi-target anti-Alzheimer’s agents

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that affects the elderly population globally and there is an urgent demand for developing novel anti-AD agents. In this study, a new series of indole-isoxazole carbohydrazides were designed and synthesized. The structure of all compounds was elucidated using spectroscopic methods including FTIR, 1H NMR, and 13C NMR as well as mass spectrometry and elemental analysis. All derivatives were screened for their acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activity. Out of all synthesized compounds, compound 5d exhibited the highest potency as AChE inhibitor with an IC50 value of 29.46 ± 0.31 µM. It showed significant selectivity towards AChE, with no notable inhibition against BuChE. A kinetic study on AChE for compound 5d indicated a competitive inhibition pattern. Also, 5d exhibited promising BACE1 inhibitory potential with an IC50 value of 2.85 ± 0.09 µM and in vitro metal chelating ability against Fe3+. The molecular dynamic studies of 5d against both AChE and BACE1 were executed to evaluate the behavior of this derivative in the binding site. The results showed that the new compounds deserve further chemical optimization to be considered potential anti-AD agents.

Coumarin linked to 2-phenylbenzimidazole derivatives as potent α-glucosidase inhibitors

α-Glucosidase inhibitors have emerged as crucial agents in the management of type 2 diabetes mellitus. In the present study, a new series of coumarin-linked 2-phenylbenzimidazole derivatives 5a–m was designed, synthesized, and evaluated as anti-α-glucosidase agents. Among these derivatives, compound 5k (IC50 = 10.8 µM) exhibited a significant inhibitory activity in comparison to the positive control acarbose (IC50 = 750.0 µM). Through kinetic analysis, it was revealed that compound 5k exhibited a competitive inhibition pattern against α-glucosidase. To gain insights into the interactions between the title compounds and α-glucosidase molecular docking was employed. The obtained results highlighted crucial interactions that contribute to the inhibitory activities of the compounds against α-glucosidase. These derivatives show immense potential as promising starting points for developing novel α-glucosidase inhibitors.

Design, synthesis, and molecular dynamics simulation studies of some novel kojic acid fused 2-amino-3-cyano-4H-pyran derivatives as tyrosinase inhibitors.

A novel series of kojic acid fused 2-amino-3-cyano-4H-pyran derivatives were synthesized via a multicomponent reaction involving kojic acid, benzyloxy benzaldehyde, and malonitrile as tyrosinase inhibitors. Subsequently, the structures of the compounds were characterized using FT-IR, 1H-, and 13C-NMR spectroscopic analyses. The designed compounds fall into three series: (1) 4-benzyloxy-phenyl kojopyran 6a-e, (2) 3-benzyloxy- phenyl kojopyran derivatives 6f-j, and (3) 4-benzyloxy-3-methoxy-phenyl kojopyran derivative 6k-o. The assessment of tyrosinase inhibition activity was conducted using L-Dopa as the substrate. Among synthesized compounds, 2-amino-4-(4-((4-fluorobenzyl)oxy)phenyl)-6-(hydroxymethyl)-8-oxo-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (6b) demonstrated the highest antityrosinase activity with a competitive inhibition pattern (IC50 = 7.69 ± 1.99μM) as compared to the control agent kojic acid (IC50 = 23.64 ± 2.56µM). Since compound 6b was synthesized as a racemic mixture, in silico studies were performed for both R and S enantiomers. The R- enantiomer showed critical interactions compared with the S-enantiomer. Specifically, it established hydrogen bonds and hydrophobic interactions with crucial and highly conserved amino acids within the enzyme's binding site in the target protein. Moreover, the molecular dynamics simulations revealed that compound 6b demonstrated significant interactions with essential residues of the binding site, resulting in a stable complex throughout the entire simulation run. The drug-like and ADMET properties predictions showed an acceptable profile for compound 6b. Thus, it can serve as a drug candidate to develop more potent antityrosinase agents due to its low toxicity and its high inhibition activity.

Novel Pyrano[3,2-c]quinoline-1,2,3-triazole Hybrids as Potential Anti-Diabetic Agents: In Vitro α-Glucosidase Inhibition, Kinetic, and Molecular Dynamics Simulation.

In this study, a novel series of pyrano[3,2-c]quinoline-1,2,3-triazole hybrids 8a-o were synthesized and evaluated against the α-glucosidase enzyme. All compounds showed significant in vitro inhibitory activity (IC50 values of 1.19 ± 0.05 to 20.01 ± 0.02 μM) compared to the standard drug acarbose (IC50 = 750.0 μM). Among them, 2-amino-4-(3-((1-benzyl-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-oxo-5,6-dihydro-4H-pyrano[3,2-c]quinoline-3-carbonitrile (compound 8k) demonstrated the best inhibitory effect toward α-glucosidase (IC50 = 1.19 ± 0.05 μM) with a competitive pattern of inhibition. Since compound 8k was synthesized as a racemic mixture, molecular docking and dynamics simulations were performed on R- and S-enantiomers of compound 8k. Based on the molecular docking results, both R- and S-enantiomers of compound 8k displayed significant interactions with key residues including catalytic triad (Asp214, Glu276, and Asp349) in the enzyme active site. However, an in silico study indicated that S- and R-enantiomers were inversely located in the enzyme active site. The R-enantiomer formed a more stable complex with a higher binding affinity to the active site of α-glucosidase than that of the S- enantiomer. The benzyl ring in the most stable complex ((R)-compound 8k) was located in the bottom of the binding site and interacted with the enzyme active site, while the pyrano[3,2-c]quinoline moiety occupied the high solvent accessible entrance of the active site. Thus, the synthesized pyrano[3,2-c]quinoline-1,2,3-triazole hybrids seem to be promising scaffolds for the development of novel α-glucosidase inhibitors.

Synthesis and structure–activity relationship studies of benzimidazole-thioquinoline derivatives as α-glucosidase inhibitors

In this article, different s-substituted benzimidazole-thioquinoline derivatives were designed, synthesized, and evaluated for their possible α-glucosidase inhibitory activities. The most active compound in this series, 6j (X = 4-bromobenzyl) exhibited significant potency with an IC50 value of 28.0 ± 0.6 µM compared to acarbose as the positive control with an IC50 value of 750.0 µM. The kinetic study showed a competitive inhibition pattern against α-glucosidase for the 6j derivative. Also, the molecular dynamic simulations were performed to determine key interactions between compounds and the targeted enzyme. The in silico pharmacodynamics and ADMET properties were executed to illustrate the druggability of the novel derivatives. In general, it can be concluded that these derivatives can serve as promising leads to the design of potential α-glucosidase inhibitors.

Design, Synthesis and Evaluation of Aryl‐Tailored Oxadiazole‐thiones as New Urease Inhibitors

AbstractThe ubiquitous chronic gastric infections that cause major human health disorders like gastritis and ulcers can be treated using drugs targeting the Helicobacter pylori (H. pylori) urease. Targeted eradication therapy is essential given the growing ineffectiveness of current treatment regimens due to broad‐spectrum antibiotic resistance, significant side effects, and low compliance. Therefore, we here report the development of a new series of 1,3,4‐oxadiazole‐2‐thiones with various aryl tail groups (compounds 20–27) as effective urease inhibitors. The most promising analog 5‐[(4‐methoxyphenoxy)methyl]‐2,3‐dihydro‐1,3,4‐oxadiazole‐2‐thione (21) inhibited the urease activity with an IC50 value of 26.6 μM. In addition, kinetic studies revealed the competitive inhibition pattern for 21 with a Ki value of 8.72 μM indicating the potent and specific binding interactions with the urease active site. Molecular docking analysis of 21 inside the active pocket of the urease highlighted several important interactions with amino acid residues such as H492, H519 and R439, which pave the way for further development of improved urease inhibitors with potential application as anti‐infective agents.

Mechanistic insight into the role of mevalonate kinase by a natural fatty acid-mediated killing of Leishmania donovani

We evaluated the anti-leishmanial efficacy of different saturated medium-chain fatty acids (FAs, C8–C18) where FA containing C8 chain, caprylic acid (CA), was found to be most potent against Leishmania donovani, the causative agent for visceral leishmaniasis (VL). Different analogs of CA with C8 linear chain, but not higher, along with a carboxyl/ester group showed a similar anti-leishmanial effect. Ergosterol depletion was the major cause of CA-mediated cell death. Molecular docking and molecular dynamic simulation studies indicated the enzyme mevalonate kinase (MevK) of the ergosterol biosynthesis pathway as a possible target of CA. Enzyme assays with purified recombinant MevK and CA/CA analogs confirmed the target with a competitive inhibition pattern. Using biochemical and biophysical studies; strong binding interaction between MevK and CA/CA analogs was established. Further, using parasites with overexpressed MevK and proteomics studies of CA-treated parasites the direct role of MevK as the target was validated. We established the mechanism of the antileishmanial effect of CA, a natural product, against VL where toxicity and drug resistance with current chemotherapeutics demand an alternative. This is the first report on the identification of an enzymatic target with kinetic parameters and mechanistic insights against any organism for a natural medium-chain FA.

Inhibition of phosphate transport by NAD+/NADH brush border membrane vesicles.

Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD+/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing Xenopus laevis oocytes. In BBMV, NAD+ required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in Xenopus oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A Ki value of 538 µM NAD+ in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD+/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD+/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time.

Study on the domain selective inhibition of angiotensin-converting enzyme (ACE) by food-derived tyrosine-containing dipeptides.

In this article, the selective inhibition of several tyrosine-containing dipeptides on N and C domain of ACE (angiotensin-converting enzyme) was studied, and the interaction mode of ACE and inhibitors was simulated by molecular docking. MTT assay was used to detect the effect of dipeptide on human umbilical vein endothelial cells (HUVEC). The results showed that the food-derived dipeptides AY (Ala-Tyr), LY (Leu-Tyr), and IY (Ile-Tyr) containing tyrosine at the C-terminal were favorable structures for selective inhibition of ACE C-domain. These dipeptides showed competitive and mixed inhibition patterns, while the dipeptides EY (Glu-Tyr), RY (Arg-Tyr), FY (Phe-Tyr), and SY (Ser-Tyr) showed noncompetitive inhibition. Food-derived dipeptides containing tyrosine have no cytotoxicity on HUVEC cells, which provides a basis for the application of food-derived tyrosine dipeptides as antihypertensive peptides. This study provides a theoretical basis for exploring the selective inhibition mechanism of ACE inhibitory peptides containing tyrosine residue. PRACTICAL APPLICATIONS: Angiotensin-converting enzyme (ACE) is a two-domain dipeptidyl carboxypeptidase, which is a key enzyme to regulate blood pressure. ACE has two active sites, C- and N-domain, which have high catalytic activity. Although the amino acid sequences of the two active sites have 60% similarity, there are some differences in structure and function. The action mechanism of ACE domain should be clarified, and the structure-activity relationship between inhibitors and ACE domain has not been systematically studied. The aim of this study was to identify the selective inhibitory effect of food-derived tyrosine dipeptides on the domain of ACE. This provides a new idea for finding new antihypertensive drugs with less side effects.

Selective inhibitory effects of HYIpro-3-1 on CYP1A2 in human liver microsomes.

CYP1A2 is one of the main Cytochrome P450 enzymes in the human liver associated with the metabolism of several xenobiotics. CYP1A2 is especially involved in the metabolic activation of different procarcinogens. Therefore, the development of cancer may be inhibited by inhibiting CYP1A2 activity. Here, the inhibitory effect of HYIpro-3-1 and its derivatives on CYP1A2 activity in human liver microsomes (HLM) was studied through LC-MS/MS using a cocktail assay. Among the four compounds, HYIpro-3-1 showed the most selective and strongest inhibitory effect on CYP1A2 at IC50 values of 0.1 µM in HLMs and inhibition was confirmed using purified human CYP1A2. It was determined that inhibition is reversible because the inhibitory effect of HYIpro-3-1 is not dependent on preincubation time. HYIpro-3-1 showed a typical pattern of competitive inhibition for CYP1A2-catalyzed phenacetin O-deethylation, based on the Lineweaver-Burk plot, with a Ki value of 0.05 μM in HLMs; the secondary plot also showed a linear pattern. In our study, HYIpro-3-1 was proposed as a novel inhibitor with the capacity to selectively inhibit CYP1A activity in HLMs.

Inhibition of human cytochrome P450 2A6 by 7-hydroxycoumarin analogues: Analysis of the structure-activity relationship and isoform selectivity

Compared with coumarin, 7-hydroxycoumarin could serve as a better hit for developing CYP2A6 inhibitors. In this study, a series of 7-hydroxycoumarin and its structural analogues were collected to study their structure-activity relationship (SAR) and isoform selectivity for inhibiting CYP2A6. All tested coumarins except a C4 phenyl derivative (11) showed higher inhibitory activities for CYP2A6 over the other CYP isoforms, including CYP1A2, CYP2D6, CYP2E1, CYP3A4, CYP2C8, and CYP2C9. Of these coumarins, 6,7-dihydroxycoumarin (1) and 7,8-dihydroxycoumarin (9) were found to be potent inhibitors of CYP2A6 with IC50/Ki value of 0.39/0.25 and 4.61/3.02 μM, respectively, compared to methoxalen as positive control (IC50/Ki = 0.43/0.26 μM). In contrast, other coumarins showed low or decreased CYP2A6-inhibiting activities. SAR analysis showed that hydroxy groups might be important for CYP2A6 inhibition, and the rank order of sites for hydroxy substitution was C6 > C7 > C8. In addition, either hydrophobic or hydrophilic substituents introduced into C4, C6 and C8 led to a reduction in CYP2A6-inhibiting activity, and the degree of influence was dependent on the size and electrical charge of substituents. Furthermore, inhibition kinetic analysis and docking simulations demonstrated that the 8-O-glucosylated coumarin derivative (17) exhibited noncompetitive inhibition against CYP2A6, while competitive inhibition patterns were noted for the other tested coumarins. The mechanisms underlying the inhibitors binding to CYP2A6 were further investigated by molecular docking study. The findings presented herein are very helpful for developing highly selective and more potent CYP2A6 inhibitors.

Determining the active site base and order of substrate addition within F420‐dependent glucose‐6‐phosphate using steady‐state and pre steady‐state kinetics and isotope effects methods

F420‐dependent glucose‐6‐phosphate dehydrogenase (FGD) is a Mycobacterial enzyme that catalyzes the conversion of glucose‐6‐phosphate (G6P) to 6‐phosphogluconolactone using oxidized F420. This enzyme is important within Mycobacteria tuberculosis, which is the causative agent of tuberculosis disease. Our work has focused on probing the FGD reaction mechanism using kinetic and isotope effects methods. Based upon a previous crystal structure FGD, there were two conserved amino acids, His40 and Glu109, which were we believed to function as the active site base and acid, respectively. Based upon pH profiles, we have determined that while Glu109 does serve as the active site acid, H40 does not serve as the active site base. We have identified several other amino acid candidates that could act as the active site base and will be discussed. A concurrent goal was to probe the FGD reaction mechanism using dead‐end inhibition experiments, along with solvent and substrate deuterium isotope effects studies. The dead‐end inhibition studies were conducted using the competitive inhibitor, citrate. The data revealed competitive and uncompetitive inhibition patterns for G6P and F420 respectively, thus suggesting a mechanism of ordered addition of substrates in which the F420 cofactor must first bind to FGD before G6P binding. Our proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed solvent kinetic isotope effect is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P‐d1 yielded kinetic isotope effect (KIE) of 1.1 for both kcat and kcat/Km, while the pre‐steady state KIE on kobs was 1.4. Because the hydride transferred to C5 of F420 was targeted for isotopic substitution, these KIE values provide further evidence to support our findings that hydride transfer is likely not rate‐limiting in the FGD reaction.Support or Funding InformationThis work was supported by NIH Grant 1R15GM113223‐01A1 (to KJW) and the Health Research Council of New Zealand Grant HRC 12/1111 (to E.N.B.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Protective Effect of Fucoidan Extract from Ecklonia cava on Hydrogen Peroxide-Induced Neurotoxicity.

We evaluated the antioxidant activity and neuronal cell-protective effect of fucoidan extract from Ecklonia cava (FEC) on hydrogen peroxide (H₂O₂)-induced cytotoxicity in PC-12 and MC-IXC cells to assess its protective effect against oxidative stress. Antioxidant activities were examined using the ABTS radical scavenging activity and malondialdehyde-inhibitory effect, and the results showed that FEC had significant antioxidant activity. Intracellular ROS contents and neuronal cell viability were investigated using the DCF-DA assay and MTT reduction assay. FEC also showed remarkable neuronal cell-protective effect compared with vitamin C as a positive control for both H₂O₂-treated PC-12 and MC-IXC cells. Based on the neuronal cell-protective effects, mitochondrial function was analyzed in PC-12 cells, and FEC significantly restored mitochondrial damage by increasing the mitochondrial membrane potential (Δψm) and ATP levels and regulating mitochondrial-mediated proteins (p-AMPK and BAX). Finally, the inhibitory effects against acetylcholinesterase (AChE), which is a critical hydrolyzing enzyme of the neurotransmitter acetylcholine in the cholinergic system, were investigated (IC₅₀ value = 1.3 mg/ml) and showed a mixed (competitive and noncompetitive) pattern of inhibition. Our findings suggest that FEC may be used as a potential material for alleviating oxidative stress-induced neuronal damage by regulating mitochondrial function and AChE inhibition.

Mechanistic insights into F420-dependent glucose-6-phosphate dehydrogenase using isotope effects and substrate inhibition studies

F420-dependent glucose-6-phosphate dehydrogenase (FGD) is involved in the committed step of the pentose phosphate pathway within mycobacteria, where it catalyzes the reaction between glucose-6-phosphate (G6P) and the F420 cofactor to yield 6-phosphogluconolactone and the reduced cofactor, F420H2. Here, we aim to probe the FGD reaction mechanism using dead-end inhibition experiments, as well as solvent and substrate deuterium isotope effects studies. The dead-end inhibition studies performed using citrate as the inhibitor revealed competitive and uncompetitive inhibition patterns for G6P and F420 respectively, thus suggesting a mechanism of ordered addition of substrates in which the F420 cofactor must first bind to FGD before G6P binding. The solvent deuterium isotope effects studies yielded normal solvent kinetic isotope effects (SKIE) on kcat and kcat/Km for both G6P and F420. The proton inventory data yielded a fractionation factor of 0.37, suggesting that the single proton responsible for the observed SKIE is likely donated by Glu109 and protonates the cofactor at position N1. The steady state substrate deuterium isotope effects studies using G6P and G6P-d1 yielded KIE of 1.1 for both kcat and kcat/Km, while the pre-steady state KIE on kobs was 1.4. Because the hydride transferred to C5 of F420 was the one targeted for isotopic substitution, these KIE values provide further evidence to support our previous findings that hydride transfer is likely not rate-limiting in the FGD reaction.

Olive and ginkgo extracts as potential cataract therapy with differential inhibitory activity on aldose reductase.

Aldose reductase (AR) has been the leading target in the treatment of diabetic cataract. Although numerous synthetic AR inhibitors (ARI) have been identified, their adverse side effects currently preclude their use. Olive leaf extract (OLE) as well as ginkgo leaf extract (GLE) are natural supplements that have wide therapeutic indices and a plethora of salutary effects during diabetes that so far untested on sugar cataract progression. As such, the present study sought to evaluate the AR-inhibiting properties of OLE and GLE using the isolated enzyme from rabbit lens. Biochemical analyses revealed that both OLE and GLE inhibited rabbit lens AR activity in a concentration-dependent manner with half maximal inhibitory concentration (IC50) 65 µg/mL and 72.5 µg/mL, respectively. Interestingly, the results of kinetic studies exhibited a differential pattern of inhibition by these two extracts. While an non-competitive inhibition of AR was promoted by OLE recognized by significant decrease in the apparent maximum velocity (Vmax) (0.12 ± 0.009677 µM/min versus 0.278 ± 0.0013677 µM/min) without significant change in Michaelis constant (Km), the GLE showed a competitive pattern of inhibition characterized by significant increase in apparent Km (4.4 ± 0.0068 µM), without change in Vmax value. It would appear that these classes of natural extracts represent effective and safe therapeutic options that hold the great promise for treatment not only diabetic cataract, but also other ocular diseases characterized by uncontrolled AR activity.

Probing the emitter site of Renilla luciferase using small organic molecules; an attempt to understand the molecular architecture of the emitter site

Renilla luciferase is a sensitive enzyme and has wide applications in biotechnology such as drug screening. Previous studies have tried to show the catalytic residues, nevertheless, the accurate architecture and molecular behavior of its emitter site remains uncharacterized. In this study, the activity of Renilla luciferase, in the presence of two small organic molecules including dimethyl sulfoxide (DMSO) and isopropanol was considered and the structure was studied by circular dichroism (CD) and fluorescence spectroscopy. Moreover, the interaction of small organic molecules with the Renilla luciferase was studied using molecular dynamics simulations. Kinetics studies showed that at low concentration of DMSO (16.6–66mM) and isopropanol (19.3–76mM) the Km changed and a competitive inhibition pattern was observed. Moreover, spectroscopy studies reveled that the changes of activity of Renilla luciferase in the presence of low concentrations of small organic molecules was not associated with structural collapse or severe changes in the enzyme conformation. Molecular dynamics simulations indicated that DMSO and isopropanol, as probing molecules, were both able to bind to the emitter site and remained with the residues of the emitter site. Based on the probing data, the architecture of the emitter site in the “non-binding” model was proposed.

Cassia fistula seed's trypsin inhibitor(s) as antibiosis agent in Helicoverpa armigera pest management

Abstract Inhibitors from diverse sources are being explored for understanding their inhibitory potential. Among several alternatives, protease inhibitors represent an attractive class of biopesticides with several important features. Such protease inhibitors are environmentally friendly and can work as defence molecules against plant pest and pathogens. Trypsin inhibitors CFTI-1 and CFTI-2 were purified from the seeds of the Cassia fistula and the prominent inhibitor CFTI-1 was studied for its effects on growth parameters and developmental stages of Helicoverpa armigera. CFTI-1 and CFTI-2 exhibited 92% and 78% inhibitory activity against midgut trypsin like proteases, respectively. Dixon and Lineweaver–Burk double reciprocal plots analysis revealed a competitive inhibition pattern with the Ki values 2.9×10−10 M and 4.2×10−10 M for CFTI-1 and CFTI- 2 respectively. Lethal concentration (0.34% m/m) and concentration for weight reduction by 50% (0.24% m/m) determined for CFTI-1. CFTI-1 showed a concentration-dependent effect on mean larval weight, a decline of the fertility and fecundity of the moths, and an extended total developmental duration of H. armigera life cycle. These results suggest that CFTI-1 has toxic and adverse effect on the developmental physiology of H. armigera and could be useful in controlling the pest H. armigera.

Inhibition of human alcohol and aldehyde dehydrogenases by aspirin and salicylate: Assessment of the effects on first-pass metabolism of ethanol

Previous studies have reported that aspirin significantly reduced the first-pass metabolism (FPM) of ethanol in humans thereby increasing adverse effects of alcohol. The underlying causes, however, remain poorly understood. Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), principal enzymes responsible for metabolism of ethanol, are complex enzyme families that exhibit functional polymorphisms among ethnic groups and distinct tissue distributions. We investigated the inhibition profiles by aspirin and its major metabolite salicylate of ethanol oxidation by recombinant human ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, ADH1C2, ADH2, and ADH4, and acetaldehyde oxidation by ALDH1A1 and ALDH2, at pH 7.5 and 0.5mM NAD+. Competitive inhibition pattern was found to be a predominant type among the ADHs and ALDHs studied, although noncompetitive and uncompetitive inhibitions were also detected in a few cases. The inhibition constants of salicylate for the ADHs and ALDHs were considerably lower than that of aspirin with the exception of ADH1A that can be ascribed to a substitution of Ala-93 at the bottom of substrate pocket as revealed by molecular docking experiments. Kinetic inhibition equation-based simulations show at higher therapeutic levels of blood plasma salicylate (1.5mM) that the decrease of activities at 2–10mM ethanol for ADH1A/ADH2 and ADH1B2/ADH1B3 are predicted to be 75–86% and 31–52%, respectively, and that the activity decline for ALDH1A1 and ALDH2 at 10–50μM acetaldehyde to be 62–73%. Our findings suggest that salicylate may substantially inhibit hepatic FPM of alcohol at both the ADH and ALDH steps when concurrent intaking aspirin.

Inhibition effect of flavonoid compounds against neuraminidase expressed in Pichia pastoris.

Neuraminidase (NA) is one of the two glycoproteins on the surface of influenza virus, which cleaves terminal sialic acid residues and facilitates the release of virions from infected cells. The recombinant NA from H5N1 influenza virus strain A/Vietnam/1203/04 was expressed in Pichia pastoris X33 as a 45 kDa protein that displayed a K m of 9.96 ± 1.26 μM with fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-D-N-acetyl neuraminic acid. Partially purified NA was used for the inhibition and kinetic assays with eight flavonoid compounds and gallic acid. Among them, gallocatechin gallate (GCG) showed the best inhibition against NA with the IC50 of 8.98 ± 0.46 μM and showed a competitive inhibition pattern with K i value of 8.34 ± 0.25 μM. In molecular docking experiments, GCG displayed a binding energy of −13.71 kcal/mol to the active site of NA and the galloyl moiety was required for NA inhibition activity.

Inhibitory zinc sites in enzymes

Several pathways increase the concentrations of cellular free zinc(II) ions. Such fluctuations suggest that zinc(II) ions are signalling ions used for the regulation of proteins. One function is the inhibition of enzymes. It is quite common that enzymes bind zinc(II) ions with micro- or nanomolar affinities in their active sites that contain catalytic dyads or triads with a combination of glutamate (aspartate), histidine and cysteine residues, which are all typical zinc-binding ligands. However, for such binding to be physiologically significant, the binding constants must be compatible with the cellular availability of zinc(II) ions. The affinity of inhibitory zinc(II) ions for receptor protein tyrosine phosphatase β is particularly high (K i = 21 pM, pH 7.4), indicating that some enzymes bind zinc almost as strongly as zinc metalloenzymes. The competitive pattern of zinc inhibition for this phosphatase implicates its active site cysteine and nearby residues in the coordination of zinc. Quantitative biophysical data on both affinities of proteins for zinc and cellular zinc(II) ion concentrations provide the basis for examining the physiological significance of inhibitory zinc-binding sites in proteins and the role of zinc(II) ions in cellular signalling. Regulatory functions of zinc(II) ions add a significant level of complexity to biological control of metabolism and signal transduction and embody a new paradigm for the role of transition metal ions in cell biology.