Secondary Replots Research Articles

Isolation and cholinesterase-inhibition studies of sterols from Haloxylon recurvum

Haloxysterols A–D ( 1– 4), new C-24 alkylated sterols, have been isolated from the chloroform soluble fraction of Haloxylon recurvum, along with five known sterols 5– 9, which are reported for the first time from this species. Their structures were determined by means of 1D- and 2D-NMR techniques. Compounds 1– 9 inhibited cholinesterase enzymes in a concentration-dependent manner with K i values ranging between 0.85–25.5 and 1.0–19.0 μM against acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BChE; EC 3.1.1.8) enzymes, respectively. Lineweaver–Burk, Dixon plots and their secondary replots indicated that compounds 1– 9 are non-competitive inhibitors of both AChE and BChE enzymes.

Withanolides, a new class of natural cholinesterase inhibitors with calcium antagonistic properties

The withanolides 1– 3 and 4– 5 isolated from Ajuga bracteosa and Withania somnifera, respectively, inhibited acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes in a concentration-dependent fashion with IC 50 values ranging between 20.5 and 49.2 μM and 29.0 and 85.2 μM for AChE and BChE, respectively. Lineweaver–Burk as well as Dixon plots and their secondary replots indicated that compounds 1, 3, and 5 are the linear mixed-type inhibitors of AChE, while 2 and 4 are non-competitive inhibitors of AChE with K i values ranging between 20.0 and 45.0 μM. All compounds were found to be non-competitive inhibitors of BChE with K i values ranging between 27.7 and 90.6 μM. Molecular docking study revealed that all the ligands are completely buried inside the aromatic gorge of AChE, while compounds 1, 3, and 5 extend up to the catalytic triad. A comparison of the docking results showed that all ligands generally adopt the same binding mode and lie parallel to the surface of the gorge. The superposition of the docked structures demonstrated that the non-flexible skeleton of the ligands always penetrates the aromatic gorge through the six-membered ring A, allowing their simultaneous interaction with more than one subsite of the active center. The affinity of ligands with AChE was found to be the cumulative effects of number of hydrophobic contacts and hydrogen bonding. Furthermore, all compounds also displayed dose-dependent (0.005–1.0 mg/mL) spasmolytic and Ca 2+ antagonistic potentials in isolated rabbit jejunum preparations, compound 4 being the most active with an ED 50 value of 0.09 ± 0.001 mg/mL and 0.22 ± 0.01 μg/mL on spontaneous and K +-induced contractions, respectively. The cholinesterase inhibitory potential along with calcium antagonistic ability and safe profile in human neutrophil viability assay could make compounds 1– 5 possible drug candidates for further study to treat Alzheimer’s disease and associated problems.

Juliflorine: A potent natural peripheral anionic-site-binding inhibitor of acetylcholinesterase with calcium-channel blocking potential, a leading candidate for Alzheimer’s disease therapy

The alkaloid juliflorine ( 1) from Prosopis juliflora inhibited acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes in a concentration-dependent fashion with IC 50 values 0.42 and 0.12 μM, respectively. Lineweaver–Burk as well as Dixon plots and their secondary replots indicated that the nature of inhibition was purely of non-competitive type with K i values 0.4 and 0.1 μM, against AChE and BChE, respectively. By molecular docking studies compound 1 was found to be ideally spaced inside the aromatic gorge of AChE with rings A/B remaining at the top and rings C/D penetrating deep into the gorge, that might be due to the greater hydrophobicity of rings C/D as compared to rings A/B, allowing their simultaneous interaction with the peripheral anionic and quaternary ammonium-binding sites. The 1–AChE complex was found to be stabilized by hydrophobic contacts, hydrogen bonding, and π–π stacking between the compound 1 and amino acid residues of the aromatic gorge of AChE. Amino acid residues Tyr70, Asp72, Tyr121, Trp279, and Tyr334 of the peripheral anionic site (PAS) of AChE were found to be exclusively involved in the hydrophobic contacts with compound 1 that might be responsible for the competitive mode of inhibition. Compound 1 also showed dose-dependent (30–500 μg/mL) spasmolytic and Ca 2+-channel blocking activities in isolated rabbit jejunum preparations. The cholinesterase inhibitory potential along with calcium-channel blocking activity of compound 1 and safe profile in human neutrophils viable assay could make it a possible drug candidate for Alzheimer’s disease.

Characterization of the Protein Dimerization Domain Responsible for Assembly of Functional Selenodeiodinases

Thyroid hormone metabolism is catalyzed by a small family of selenoenzymes. Type I deiodinase (D1) is the best characterized family member and is an integral membrane protein composed of two 27-kDa subunits that assemble to a functional holoenzyme after translation. To characterize the protein domain(s) responsible for this post-translational assembly event, we used deletion/truncation analysis coupled with immune depletion assays to map the dimerization domain of D1. The results of our studies show that a highly conserved sequence of 16 amino acids in the C-terminal half of the D1 subunit, -D148FL-YI-EAH-DGW163-, serves as the dimerization domain. Based on the high conservation of this domain, we synthesized a novel bait peptide-green fluorescent protein fusion probe (DDD(GFP)) to examine holoenzyme assembly of other family members. Overexpression of either the DDD(GFP) or an inert D1 subunit (M4) into SeD2 (accession number U53505)-expressing C6 cells specifically led to the loss of >90% of the catalytic activity. Catalytically inactive D2 heterodimers composed of SeD2: DDD(GFP) subunits were rescued by specific immune precipitation with anti-SeD2 IgG, suggesting that SeD2 requires two functional subunits to assemble a catalytically active holoenzyme. These findings identify and characterize the essential dimerization domain responsible for post-translational assembly of selenodeiodinases and show that family members can intermingle through this highly conserved protein domain.

Isolation of Onosmins A and B, Lipoxygenase Inhibitors from Onosma hispida

Onosmins A (1) and B (2), lipoxygenase inhibitors, have been isolated from Onosma hispida. Their structures were established as 2-[(4-methylbenzyl)amino]benzoic acid (1) and methyl 2-[(4-methylbenzyl)amino]benzoate (2) through spectroscopic studies, including 2D-NMR. The known compounds apigenin (3), 6,4'-dimethoxy-3,5,7-trihydroxyflavone (4), 6,7-dimethoxy-3,5,4'-trihydroxyflavone (5) and apigenin 7-O-beta-D-glucoside (6) are also reported for the first time from this species. Compounds (1) and (2) inhibited lipoxygenase (LOX, EC 1.13.11.12) enzyme in a concentration-dependent fashion with IC50 values of 24.0 and 36.2 microM, respectively. Lineweaver-Burk as well as Dixon plots and their secondary replots indicated that the nature of inhibition was purely a non-competitive type, with K(i) values 22.0 microM and 31.1, respectively.

Phenolic glycosides, a new class of human recombinant nucleotide pyrophosphatase phosphodiesterase-1 inhibitors

Cytotoxicity and kinetic studies of phenolic glycosides, benzoyl salireposide ( 1) and salireposide ( 2), isolated from Symplocos racemosa, were performed against phosphodiesterase I enzyme from snake venom and human nucleotide pyrophosphatase phosphodiesterase-1. Lineweaver–Burk and Dixon plots and their secondary replots showed that these compounds are pure noncompetitive inhibitors of both enzymes. K i values of compounds 1 and 2 were found to be 360 and 1000 μM, respectively, against human nucleotide pyrophosphatase phosphodiesterase, and 525 and 1100 μM, respectively, against snake venom phosphodiesterase. IC 50 values of compounds 1 and 2 are 90 μM ± 0.04 and 383 μM ± 0.03, respectively, against human nucleotide pyrophosphatase phosphodiesterase and 171 μM ± 0.02 and 544 μM ± 0.021, respectively, against snake venom phosphodiesterase. Both compounds were found to be nontoxic up to concentration of 500 μM/mL as >90% cells were viable after 3 h of incubation. These compounds are potential candidates for the therapy of arthritis.

Kinetics of novel competitive inhibitors of urease enzymes by a focused library of oxadiazoles/thiadiazoles and triazoles

Based on structure of the substrate of urease and for the purpose of designing pharmacophore models for urease inhibitors, which could be effective in physiological and pharmacological studies, a series of twenty-five 1,3,4-oxadiazole-2(3H)-thiones-2(3H)-thiones, 1,3,4-thiadiazoles-2(3H)-thiones, and 1,2,4-triazole-3-thiones (OSNs) were designed, synthesized, and evaluated for various kinetic parameters of urease inhibition. OSNs inhibited the activity of urease(s) in a concentration dependent fashion. Dixon as well as Lineweaver–Burk plots and their secondary replots indicated that the nature of inhibition was of pure competitive type for all the 25 compounds. 5-[4-(hydroxy)phenyl]-1,3,4-thiadiazole-2(3H)-thione was found to be the most active one with a Ki value of 2μM. The Ki values were increased with an increase in substrate concentrations. Apparently, OSNs employ a homologous mechanism of action by exploiting a common transition catalysis state and acting as ligand chelators to form octahedral complexes with the urease enzymes in an orientation-specific mode. The inhibition was slightly potentiated by lower pH and not abolished in the presence of NH2OH (a scavenger of histidine residue). Because of their safe profile in the genotoxic assay, they may be pursued in the near future for human testing.

Kinetics and structure–activity relationship studies on pregnane-type steroidal alkaloids that inhibit cholinesterases

The mechanism of inhibition of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) enzymes by 23 pregnane-type alkaloids isolated from the Sarcococca saligna was investigated. Lineweaver–Burk and Dixon plots and their secondary replots showed that the majority of these compounds, that is 1, 4, 5, 6, 9, 10, 12, 13, 15– 19, and 21 were found to be noncompetitive inhibitors of both enzymes. Compounds 8, 20, 22, and 23 were determined to be uncompetitive inhibitors of BChE, while compounds 11 and 14 were found to be uncompetitive and linear mixed inhibitors of AChE, respectively. K i values were found to be in the range of 2.65–250.0 μM against AChE and 1.63–30.0 μM against BChE. The structure–activity relationship (SAR) studies suggested that the major interaction of the enzyme–inhibitor complexes are due to hydrophobic and cation–π interactions inside the aromatic gorge of these cholinesterases. The effects of various substituents on the activity of these compounds are also discussed in details.

Kinetic mechanism of penicillin V acylase activation byshort-chain alcohols

Alcohol-induced activation of penicillin V acylase from Streptomyces lavendulae by some water-miscible primary alcohols such as methanol, ethanol, 1-propanol and 1-butanol has been extensively studied. The effects of various concentrations of these alcohols on the maximum velocity of penicillin V hydrolysis ( V max) and the apparent Michaelis-Menten constant (K m ) of the enzyme are reported. Lineweaver-Burk analyses of the kinetic data revealed a phenomenon of nonessential activation in the presence of short-chain primary alcohols. In the concentration range where activation was observed, conventional secondary replots of 1/Δ slope or 1/Δ intercept versus 1/[alcohol] yielded perfect straight lines, demonstrating the binding of a single molecule of alcohol to an activator site in penicillin V acylase. Furthermore, the enzyme displayed a propensity to bind the longer chain alcohols, as reflected by the dissociation constant K A values of 493, 369, 228 and 11.8 mM for methanol, ethanol, 1-propanol and 1-butanol, respectively. Therefore, we have proposed a high affinity activator site for alcohols in the enzyme. Very hydrophobic alcohols such as 1-butanol bind more efficiently to this activator site and convert the enzyme form one conformation to another one, which is catalytically more effective than the alcohol-free form of the penicillin acylase. Further addition of alcohol results in a competitive inhibition of the enzyme in its activated state. An inverse relationship between the chain length of the alcohols and the breakpoints in the biphasic normalized v max/K m ratio versus alcohol concentration plots was observed.

Kinetics of human acetylcholinesterase inhibition by the novel experimental alzheimer therapeutic agent, tolserine

Characterization of the kinetic parameters of tolserine, a novel acetylcholinesterase (AChE) inhibitor of potential in the therapy of Alzheimer's disease, to inhibit purified human erythrocyte AChE was undertaken for the first time. An IC(50) value was estimated by three methods. Its mean value was found to be 8.13 nM, whereas the IC(100) was observed to be 25.5 nM as calculated by single graphical method. The Michaelis-Menten constant (K(m)) for the hydrolysis of the substrate acetylthiocholine iodide was found to be 0.08 mM. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the partial non-competitive type. The value of K(i) was estimated as 4.69 nM by the primary and secondary replots of the Dixon as well as secondary replots of the Lineweaver-Burk plot. Four new kinetic constants were also investigated by polynomial regression analysis of the relationship between the apparent K(i) (K(Iapp)) and substrate concentration, which may open new avenues for the kinetic study of the inhibition of several enzymes by a wide variety of inhibitors in vitro. Tolserine proved to be a highly potent inhibitor of human AChE compared to its structural analogues physostigmine and phenserine.

Kinetic analysis of the toxicological effect of tacrine (Cognex®) on human retinal acetylcholinesterase activity

For the first time, kinetic parameters of the effect of tacrine, an anti-cholinesterase inhibitor of therapeutic potential in Alzheimer’s disease has been studied on human retinal acetyl-cholinesterase (AChE). Tacrine inhibited the AChE activity in a concentration dependent manner, the IC 50 being about 45 nM. The Michaelis–Menten constant ( K m) for the hydrolysis of acetylthiocholine iodide was found to be 0.120 mM and this value was increased by 4–52.8% in the presence of tacrine. V max was observed to be 2.23 μmol/h per mg protein for the control system, while it was decreased by 14.73–56.25% in the tacrine treated systems. Dixon as well as Lineweaver–Burk plots and their secondary replots indicated that the nature of the inhibition was of the mixed type, i.e. a combination of competitive and noncompetitive inhibition. The values of K i and K I were estimated to be as 37.76 and 64.36 nM, respectively.

Kinetics of Human Erythrocyte Acetylcholinesterase Inhibition by a Novel Derivative of Physostigmine: Phenserine

The effect of phenserine, a novel cholinesterase inhibitor, was assessed for the first time on kinetic parameters of human erythrocyte acetylcholinesterase (AChE). Phenserine (0.025-0.40 μM) inhibited the activity of human erythrocyte AChE in a concentration-dependent fashion, the IC50was 0.0453 μM. The Michaelis-Menten constant (Km) for the hydrolysis of acetylthiocholine iodide was found to be 0.124 mM and the Vmaxwas 0.980 μmol/min/mg protein. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the noncompetitive type. The value of Kiwas estimated as 0.048 μM by the primary and secondary replots of the Dixon as well as secondary replots of the Lineweaver-Burk plot. A novel relationship between Kiand substrate concentration was also identified which permits more precise prediction of the specific type of noncompetitive inhibition of various enzymes by a wide variety of drugs, chemicals and, in some circumstances, by their own substrates.

Kinetics of the inhibition of acetylcholinesterase in camel retina by cisplatin

The inhibitory effect of cisplatin (CDDP) on camel retina acetylcholinesterase (AChE) was characterized. The CDDP effect was independent of the time of incubation with AChE before the addition of substrate, indicative of reversible inhibition. Moreover, dilution data prove that CDDP is a reversible inhibitor of camel retina AChE. Cisplatin inhibited AChE activity of camel retina in a concentration- and time-dependent manner, the IC 50 values being 5.32 and 0.196 mM at 5 min and 24 h incubation times, respectively. The IC 50 has dual components, i.e. directly proportional and inversely proportional to 0–1.5 h and 1.5–24 h incubation periods, respectively. The Michaelis–Menten constant ( K m) for the hydrolysis of acetylthiocholine iodide (ASCh) was found to be 0.0796 mM and V max was 0.668 μmol/min/mg protein. K mapp and V maxapp both decreased as the CDDP concentration increased. Dixon as well as Lineweaver–Burk plots and their secondary replots indicated that the nature of the inhibition was of the pure uncompetitive type. The value of K i was estimated as 0.811 mM by the primary and secondary replots of the Lineweaver–Burk and Dixon plots. K iapp decreased while V maxiapp increased after increasing the ASCh concentration.

On the inhibition of camel retina acetylcholinesterase activity by cycloheximide in vitro.

The kinetic parameters of inhibition of camel retinal acetylcholinesterase (AChE) activity by cycloheximide (CH) were investigated. For the control system, the Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide was found to be 0.076 mmol/L and the Vmax was 0.547 mumol/min per mg protein. In contrast, these parameters were decreased in the CH-treated systems. Dixon and Lineweaver-Burk plots, and their secondary replots, indicated that the inhibition was of the linear mixed type, which seems to be a combination of partial competitive and pure noncompetitive inhibition. The values of K'i(slope) and KI(intercept) were estimated to be 3.50 and 5.68 mmol/L, respectively. Ki was greater than K'i, indicating that CH has a greater binding affinity for the peripheral site than the active site.

Sensitivity of bovine retinal acetylcholinesterase (E.C. 3.1.1.7) toward tacrine: kinetic characterization.

This work addresses the kinetic analysis of the interaction of tacrine with bovine retina acetylcholinesterase (A ChE, E.C. 3.1.1.7). It was found that the tacrine effect was reversible in nature. Tacrine inhibited bovine retinal AChE activity in a concentration-dependent manner; IC50 was fo to be 8.07 nM. The Michaelis-Menten constant (Ka) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.061 mM in the control system, and this value was increased by 54-67% in the tacrine-treated systems. The Vmax was 0.701 mumole/min per milligram protein for the control system, but it was decreased by 26-69% in the tacrine-treated systems. The Lineweaver-Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the partial mixed type, that is, a mixture of competitive and noncompetitive inhibition. The values of Ki and Kt were estimated to be as 4.475 and 8.517 nM, respectively.

Investigation of the effect of lannate on kinetic parameters of acetylcholinesterase: slightly concave mixed-type of inhibition system.

The effect of lannate on kinetic parameters of camel retina acetylcholinesterase is investigated in the present study. The Ks for the hydrolysis of acetylthiocholine iodide was found to be 0.086 mM in the control system; a value that increased in the lannate treated systems. The Vmax was 0.853 mumole/min/mg protein for the control system while it decreased in the lannate treated systems. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition is of the slightly concave mixed type, which is considered to be a 'semi-pure competitive' and 'semi-partial as well as semi-pure non-competitive' mixture. The values of Ki(slope) and KI(intercept) were estimated as 0.143 microM and 0.179 microM respectively. The KI was greater than Ki indicating that lannate has a greater affinity of binding for the peripheral site than the active site of the camel retina AChE.

Effect of malathion on kinetic parameters of acetylcholinesterase (EC 3.1.1.7) in vitro.

Kinetic analysis of the interaction of malathion with camel erythrocyte acetylcholinesterase was investigated in the present study. The Michaelis-Menten constant (K(m)) for the hydrolysis of acetylthiocholine iodide (ASCh) was found to be 53.15 microM and the Vmax was 0.287 mumol/min/mg protein. The Kmapp and Vmaxapp were both decreased by increased malathion concentration. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the pure uncompetitive type with Ki value estimated as 102.1 ppm. The Kiapp decreased while Vmaxiapp increased by an increased concentration in ASCh.

Effect of sevin on kinetic parameters of camel retina acetylcholinesterase.

The present investigation addresses the effect of sevin (insecticide) on kinetic parameters of camel retina membrane-bound acetylcholinesterase (AChE, EC 3.1.1.7). The Michaelis-Menten constant (Ks) for the hydrolysis of acetylthiocholine iodide (ASCh) by AChE was 0.072 mM in the control system, a value decreased by 38-55% in the sevin treated systems. The Vmax was 0.967 mumole/min/mg protein for the control system while it was decreased by 50-84% in the sevin treated systems. The Lineweaver-Burk plot, Dixon plot, and their secondary replots indicated that the nature of the inhibition was of the linear mixed type, i.e. uncompetitive and noncompetitive. The values of Ki(slope) and KI(intercept) were estimated as 6.194 and 2.811 microM, respectively. The turnover number (Kcat) and specificity constant (Ksp) were 74.65 min-1 and 10.37 x 10(5) (M.min)-1 in the control system respectively, while the values for both parameters were significantly decreased in the sevin treated systems.

The inhibitory effect of the neuromuscular blocking agent, gallamine triethiodide, on camel retina acetylcholinesterase activity

The present investigation addresses the mode of inhibition of the camel retinal acetylcholinesterase (AChE) activity by gallamine triethiodide, which is known to be a specific non-depolarizing neuromuscular blocking agent and polar cholinergic antagonist. This study gave the following results: it was found that gallamine (GA) reversibly inhibited the AChE activity in a concentration dependent manner, the IC 5o being about 0.633 mM. The K m for the hydrolysis of acetylthiocholine iodide by AChE was found to be 0.0803 mM in the control system, and the value increased by 19–463% in the GA (0.125–1.0 mM) treated systems. The V max was 0.649 μmol/min per mg protein for the control as well as GA treated systems. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition is of the reversible competitive type. The K i, value was estimated as 0.160 mM. The K i, value increased with an increase in substrate concentration. The turnover number ( cat) and specificity constant ( K sp) were 62.1 min -1 and 7.73 x 10 5 (M-min) -1 in the control system while the value for one parameter (Agp) was decreased by 25–83% in the GA (0.125–1.0 mM) treated systems

Evaluation of the nature of camel retinal acetylcholinesterase: inhibition by hexamethonium.

Acetylcholinesterase (AChE, EC 3.1.1.7) has been demonstrated in retinas of several species, however, the nature of the interaction of AChE with specific inhibitors are very limited in the literature and the mode of inhibition of camel retinal AChE by hexamethonium has been studied. Hexamethonium reversibly inhibited AChE in a concentration dependent manner, the IC50 value being c. 2.52 mM. The Km for the hydrolysis of acetylthiocholine iodide was found to be 0.087 mM and the Vmax was 0.63 mumol/min/mg protein. Dixon, as well as Lineweaver-Burk, plots and their secondary replots indicated that the nature of the inhibition is of the hyperbolic (partial) mixed type, which is considered to be a partial competitive and non-competitive mixture. The values of Ki(slope) and KI(intercept) from a Lineweaver-Burk plot were estimated as 0.30 mM and 0.17 mM, respectively, while Ki from a Dixon plot was estimated as 0.725 mM. The Ki was greater than KI indicating that hexamethonium has a greater affinity of binding for the active site than the peripheral site of the camel retina AChE.