Inhibitors of acetylcholine esterase in vitro--screening of steroidal alkaloids from Fritillaria species

18 alkaloids were successfully isolated from five Fritillaria species and 5 derivatives were synthesized. Their effects on the bioactivity of human whole blood cholinesterase (ChE) were assessed. The results showed that N-demethylpuqietinone, hupeheninoside, ebeiedinone, yibeinoside A and chuanbeinone inhibited the bioactivity of human whole blood ChE at the concentration of 1.0 x 10 ( - 4) M, with the inhibitory effects of 55.5 +/- 2.7 %, 66.8 +/- 2.0 %, 69.0 +/- 1.7 %, 71.2 +/- 1.8 % and 70.7 +/- 3.3 %, respectively. The effects of the five alkaloids on human red blood cell (RBC) acetylcholinesterase (AChE) and human plasma butyrylcholinesterase (BChE) were further studied, and their IC (50) values for human RBC AChE were 6.4 +/- 0.003 microM, 16.9 +/- 0.018 microM, 5.7 +/- 0.004 microM, 6.5 +/- 0.013 microM and 7.7 +/- 0.001 microM, respectively, and the IC50 values for human plasma BChE were 12.5 +/- 0.026 microM, 2.1 +/- 0.005 microM, 5.2 +/- 0.002 microM, 7.3 +/- 0.005 microM and 0.7 +/- 0.001 microM, respectively. These data suggest, therefore, that N-demethylpuqietinone, hupeheninoside, ebeiedinone, yibeinoside A and chuanbeinone have both anti-RBC AChE and anti-plasma BChE activities, N-demethylpuqietinone is a selective inhibitor of AChE, whereas hupeheninoside and chuanbeinone are the selective inhibitors of BChE.     

Selective cholinesterase inhibitors from Buxus sempervirens L. and their molecular docking studies

In this work, two alkaloids namely (+)-buxabenzamidienine (1) and (+)-buxamidine (2) were isolated from Buxus sempervirens, using bioassay-guided fractionation and isolation method. Their acetyl- (AChE) and butyrylcholinesterase (BChE) inhibitory activities were studied and the compounds were found to be quite selective inhibitors of AChE. IC50 values of compound 1 for electric eel AChE and horse BChE were 0.787 and 7.68 mM, respectively; while the corresponding IC50 of compound 2 were 1.70 and 549.98 mM, respectively. Theoretical (quantum mechanical, homology modelling and docking) calculations were performed in order to explain their interactions with different AChE (electric eel and human) and BChE (horse and human). The x-ray crystal structures of electric eel AChE, human AChE, human BChE and a model of horse BChE constructed by homology with human BChE were used for docking of compounds 1 and 2. Density functional theory (DFT) calculations of the compounds were performed at the B3LYP/6- 31G** level using the program Spartan?, and their HOMO and LUMO energy levels were calculated. Docking studies exhibited that compound 1 interacts with the acyl-binding pocket of the active site gorge of huAChE, and including several other hydrophobic interactions.     

Novel donepezil-based inhibitors of acetyl- and butyrylcholinesterase and acetylcholinesterase-induced beta-amyloid aggregation

A novel series of donepezil-tacrine hybrids designed to simultaneously interact with the active, peripheral and midgorge binding sites of acetylcholinesterase (AChE) have been synthesized and tested for their ability to inhibit AChE, butyrylcholinesterase (BChE), and AChE-induced A beta aggregation. These compounds consist of a unit of tacrine or 6-chlorotacrine, which occupies the same position as tacrine at the AChE active site, and the 5,6-dimethoxy-2-[(4-piperidinyl)methyl]-1-indanone moiety of donepezil (or the indane derivative thereof), whose position along the enzyme gorge and the peripheral site can be modulated by a suitable tether that connects tacrine and donepezil fragments. All of the new compounds are highly potent inhibitors of bovine and human AChE and BChE, exhibiting IC50 values in the subnanomolar or low nanomolar range in most cases. Moreover, six out of the eight hybrids of the series, particularly those bearing an indane moiety, exhibit a significant A beta antiaggregating activity, which makes them promising anti-Alzheimer drug candidates.     

Synthesis, biological activity, and docking studies of new acetylcholinesterase inhibitors of the bispyridinium type

A novel series of acetylcholinesterase (AChE) inhibitors of the bispyridinium type was synthesized and the inhibitory activity against AChE and butyrylcholinesterase (BChE) measured. In essence, the substitution pattern influenced the inhibitory potency against AChE, where the most active bispyridiniumoxime (TMB-4) was bisbenzyl substituted followed by monobenzyl substituted, bismethyl substituted, and unsubstituted derivatives of TMB-4. Hence, the bisbenzyl ether of TMB-4 was further investigated. In order to obtain diverse lipophilic and electronic properties for these bisbenzyl bispyridinium derivatives (so-called DUO series), the lateral ring substitution was systematically varied. The lowest IC(50) value against AChE found thus far in the DUO series was 0.34 microM. Docking studies were carried out to elucidate the differences in biological activity. A general binding mode for nearly all compounds could be identified by these investigations. In this binding mode, the docked ligands span the narrow, deeply buried active-site gorge, interacting with Trp84 at the bottom of the gorge, Tyr334 or Phe331 halfway down the gorge, and Trp279 at the peripheral anionic site at the mouth of the gorge. For specific ligands, additional interactions were found which helped to explain their deviating activity. Based on the promising characteristics of the novel acetylcholinesterase inhibitors presented, a series of structurally related, optimized candidates will be developed.     

Malathion,carbofuran and paraquat inhibit <Emphasis Type="Italic">Bungarus sindanus</Emphasis> (krait) venom acetylcholinesterase and human serum butyrylcholinesterase in vitro

Carbofuran and malathion, well known pesticides, and paraquat, a world widely used herbicide, were tested on acetylcholinesterase (AChE) from Bungarus sindanus venom and butyrylcholinesterase (BChE) from human serum. The calculated IC(50 )values for inhibition of venom enzyme by malathion, carbofuran and paraquat were 2.5, 0.14, and 0.16 microM, respectively. The values for inhibition of serum butyrylcholinesterase (BChE) were 3.5, 0.09 and 0.18 microM, respectively. Analysis of kinetic data indicated that the inhibition caused by malathion, carbofuran and paraquat was mixed for venom AChE. For BChE from human serum, the inhibition caused by malathion and paraquat was mixed and for carbofuran it was uncompetitive. The present results suggest a commercial paraquat preparation (a popular herbicide) inhibits cholinesterases with similar or higher potency than classical pesticide inhibitors. Furthermore, this inhibition was observed both in human serum and snake venom, a newly studied source of AChE.     

Design, synthesis, and biological evaluation of conformationally restricted rivastigmine analogues

Rivastigmine (1), an acetylcholinesterase (AChE) inhibitor approved in 2000 for the treatment of Alzheimer disease, bears a carbamate moiety in its structure, which is able to react covalently with the active site of the enzyme. Kinetic and structural studies on the interaction of 1 with different cholinesterases have been published, giving deeper, but not definitive, insights on the catalysis mechanism. On the basis of these findings and in connection with our previous studies on a series of benzopyrano[4,3-b]pyrrole carbamates as AChE inhibitors, we designed a series of conformationally restricted analogues of 1 by including the dimethylamino-alpha-methylbenzyl moiety in different tricyclic systems. A superimposition between the conformation of 1 and the carbon derivative 4, as obtained from Monte Carlo simulations, supported the idea that the tricyclic derivatives might act as rigid analogues of 1. The biological profile of 4-9, assessed in vitro against human AChE and BChE, validated our rational design. Compound 5, bearing a sulfur-containing system, showed the highest inhibitory activity, being 192-fold more potent than 1. In the present study, the most potent inhibitors were always methyl derivatives 3-5, endowed with a nanomolar range potency, whereas the ethyl ones were 40 times less potent. A reasonable explanation for this finding might be a steric hindrance effect between the ethyl group of 1 and His440 in the active site, as already suggested by the crystal structure of the complex AChE/1. The unfavorable influence of the carbamic N-alkyl chain on AChE inhibition is less striking when considering BChE inhibition, since BChE is characterized by a bigger acyl binding pocket than AChE. In fact, methyl carbamates 3-5 did not show AChE/BChE selectivity, whereas compounds 6-9 were significantly more potent in inhibiting BChE than AChE activity. At 100 microM, 5 was found to inhibit the AChE-induced aggregation only by 19% likely because it is not able to strongly interact with the peripheral anionic site of AChE, which plays an essential role in the Abeta aggregation mediated by the enzyme but is lacking in BChE structure.     

胆碱酯酶抑制剂他克林-单甲氧芳基杂合物的设计、合成及活性评价

本文设计合成了一系列他克林单甲氧芳基杂合物(5a～5i)作为胆碱酯酶抑制剂,并对其进行了活性评价。结果表明该类化合物比他克林具有更好的胆碱酯酶抑制活性,IC50值均达到纳摩尔级,其中化合物5h对乙酰胆碱酯酶的抑制活性最强,IC50值为6.74 nmol.L 1,5f对丁酰胆碱酯酶的活性最强,IC50值为3.83 nmol.L 1。酶动力学及分子对接表明该类杂合物能够同时作用于AChE的催化活性位点和外周结合位点。     

Heterodimeric tacrine-based acetylcholinesterase inhibitors: investigating ligand-peripheral site interactions.

Dimeric acetylcholinesterase (AChE) inhibitors containing a single 9-amino-1,2,3,4-tetrahydroacridine (tacrine) unit were constructed in an effort to further delineate structural requirements for optimal binding to the AChE peripheral site. Basic amines of differing hydrophobicity were selected as peripheral site ligands, and in each case, improvements in inhibitory potency and selectivity were seen relative to tacrine itself. AChE IC(50) values of the optimum dimers decrease significantly as the peripheral site ligand was permuted in the series ammonia > dimethylamine > 4-aminopyridine > 4-aminoquinoline > tacrine. Calculated desolvation free energies of the optimum dimers match the trend in IC(50) values, suggesting the importance of ligand hydrophobicity for effective cation-pi interaction with the peripheral site.     

Design, synthesis and evaluation of novel 2-(aminoalkyl)-isoindoline-1,3-dione derivatives as dual-binding site acetylcholinesterase inhibitors

A new series of 2-(diethylaminoalkyl)-isoindoline-1,3-dione derivatives intended as dual binding site cholinesterase inhibitors were designed using molecular modeling and evaluated as inhibitors of acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), and the formation of the β-amyloid (Aβ) plaques. For AChE inhibitory activity, the spectrophotometric method of Ellman and the electrophoretically mediated microanalysis assay were used, giving good results. Most of the synthesized compounds had AChE inhibitory activity with IC(50) values ranging from IC(50) = 0.9 to 19.5 μM and weak Aβ anti-aggregation inhibitory activity. These results support the outcome of docking studies which tested compounds targeting both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. The most promising selective AChE inhibitors are compounds 10 (IC(50) = 1.2 μM) and 11 (IC(50) = 1.1 μM), with 6-7 methylene chains, which also inhibit Aβ fibril formation.     

Inhibition of human acetyl- and butyrylcholinesterase by novel carbamates of (-)- and (+)-tetrahydrofurobenzofuran and methanobenzodioxepine

A new enantiomeric synthesis utilizing classical resolution provided two novel series of optically active inhibitors of cholinesterase: (-)- and (+)-O-carbamoyl phenols of tetrahydrofurobenzofuran and methanobenzodioxepine. An additional two series of (-)- and (+)-O-carbamoyl phenols of pyrroloindole and furoindole were obtained by known procedures, and their anticholinesterase actions were similarly quantified against freshly prepared human acetyl- (AChE) and butyrylcholinesterase (BChE). Both enantiomeric forms of each series demonstrated potent cholinesterase inhibitory activity (with IC(50) values as low as 10 nM for AChE and 3 nM for BChE), with the exception of the (+)-O-carbamoyl phenols of pyrroloindole, which lacked activity (IC(50) values >1 microM). Based on the biological data of these four series, a structure-activity relationship (SAR) analysis was provided by molecular volume calculations. In addition, a probable transition-state model was established according to the known X-ray structure of a transition-state complex of Torpedo californica AChE-m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone (TcAChE-TMTFA). This model proved valuable in explaining the enantioselectivity and enzyme subtype selectivity of each series. These carbamates are more potent than, or similarly potent to, anticholinesterases in current clinical use, providing not only inhibitors of potential clinical relevance but also pharmacological tools to define drug-enzyme binding interactions within an enzyme crucial in the maintenance of cognition and numerous systemic physiological functions in health, aging, and disease.     

Probing simple structural modification of α-mangostin on its cholinesterase inhibition and cytotoxicity

α-Mangostin has been reported to possess a broad range of pharmacological effects including potent cholinesterase inhibition, but the development of α-mangostin as a potential lead compound is impeded by its toxicity. The present study investigated the impact of simple structural modification of α-mangostin on its cholinesterase inhibitory activities and toxicity toward neuroblastoma and liver cancer cells. The dialkylated derivatives retained good acetylcholinesterase (AChE) inhibitory activities with IC₅₀ values between 4.15 and 6.73 µM, but not butyrylcholinesterase (BChE) inhibitory activities, compared with α-mangostin, a dual inhibitor (IC₅₀: AChE, 2.48 µM; BChE, 5.87 µM). Dialkylation of α-mangostin produced AChE selective inhibitors that formed hydrophobic interactions at the active site of AChE. Interestingly, all four dialkylated derivatives of α-mangostin showed much lower cytotoxicity, being 6.4- to 9.0-fold and 3.8- to 5.5-fold less toxic than their parent compound on neuroblastoma and liver cancer cells, respectively. Likewise, their selectivity index was higher by 1.9- to 4.4-fold; in particular, A2 and A4 showed improved selectivity index compared with α-mangostin. Taken together, modification of the hydroxyl groups of α-mangostin at positions C-3 and C-6 greatly influenced its BChE inhibitory and cytotoxic but not its AChE inhibitory activities. These dialkylated derivatives are viable candidates for further structural modification and refinement, worthy in the search of new AChE inhibitors with higher safety margins.     

DNA sequence of butyrylcholinesterase from the rat: expression of the protein and characterization of the properties of rat butyrylcholinesterase

The rat is the model animal for toxicity studies. Butyrylcholinesterase (BChE), being sensitive to inhibition by some organophosphorus and carbamate pesticides, is a biomarker of toxic exposure. The goal of this work was to characterize the purified rat BChE enzyme. The cDNA sequence showed eight amino acid differences between the active site gorge of rat and human BChE, six clustered around the acyl binding pocket and two below the active site serine. A prominent difference in rat was the substitution of arginine for leucine at position 286 in the acyl pocket. Wild-type rat BChE, the mutant R286L, wild-type human BChE, and the mutant L286R were expressed in CHO cells and purified. Arg286 was found responsible for the resistance of rat BChE to inhibition by Triton X-100. Replacement of Arg286 with leucine caused the affinity for Triton X-100 to increase 20-fold, making it as sensitive as human BChE to inhibition by Triton X-100. Wild-type rat BChE had an 8- to 9-fold higher K(m) for the positively charged substrates butyrylthiocholine, acetylthiocholine, propionylthiocholine, benzoylcholine, and cocaine compared with wild-type human BChE. Wild-type rat BChE catalyzed turnover 2- to 7-fold more rapidly than human BChE, showing the highest turnover with propionylthiocholine (201,000 min(-1)). Human BChE does not reactivate spontaneously after inhibition by echothiophate, but rat BChE reactivates with a half-life of 4.3hr. Human serum contains 5mg/L of BChE and 0.01mg/L of AChE. Male rat serum contains 0.2mg/L of BChE and approximately 0.2mg/L of AChE.     

Methyl-paraoxon comparative inhibition kinetics for acetylcholinesterases from brain of neotropical fishes

Acetylcholinesterase (AChE) sensitivity to the organophosphorus (OP) pesticide methyl-paraoxon was measured in fourteen species of Neotropical marine and freshwater fish found in the waters of Brazil. The rate constant for phosphorylation, kp, the dissociation constant, kd, the second order rate constant, ki, and the IC50 value were measured at 28 degrees C in pH 7.5 buffer for AChE extracted from brain. In addition, the substrate affinity constant, km, was measured with acetylthiocholine. The IC50 for 30 min of inhibition ranged from 123 nM (Prochilodus lineatus) to 3340 nM (Percophis brasiliensis), which corresponded to ki values of 187-6.9 mM(-1) min(-1). A 10-fold range in kp values from 0.21 min(-1) (Paralonchurus brasiliensis) to 2.1 min(-1) (Dules auriga) was associated with a 37-fold range in kd values from 4 to 150 microM. These large differences in reactivity with methyl-paraoxon were not reflected in the binding affinity for acetylthiocholine; km values were approximately 0.1-0.3 mM for all species. These results predict that the amino acid sequence involved in AChE sensitivity differs in these fishes, and that consequently some fish species may be resistant to the toxicity of methyl-paraoxon.     

Activities of the enantiomers of cocaine and some related compounds as substrates and inhibitors of plasma butyrylcholinesterase

The behaviors of the enantiomers of cocaine (benzoylecgonine methyl ester) and related compounds with butyrylcholinesterase (BChE; EC 3.1.1.8) were investigated spectrophotometrically at 235 nm. The unnatural enantiomer, (+)-cocaine, was hydrolyzed by BChE (extinction coefficient 6.7 L.mmol-1.cm-1) at about half the rate of benzoylcholine, but over 2000 times faster than naturally occurring (-)-cocaine. This rapid hydrolysis of (+)-cocaine may account, in part, for its pharmacological inactivity. (+)-Norcocaine, (+)-benzoylecgonine, (-)-psi-cocaine and tropacocaine were also substrates for BChE. Hydrolysis of (+)-cocaine was sensitive to several standard inhibitors of BChE, including those of competitive, carbamate and organophosphorus classes. Although (-)-cocaine was a poor substrate for debenzoylation, it was a fairly good competitive inhibitor (Ki approximately 10 microM) of the hydrolysis of other substrates. The cocaine metabolites (-)-norcocaine, (-)-benzoylecgonine and (-)-ecgonine methyl ester inhibited BChE with Ki values of 15, 76 and 1300 microM, respectively. (+)-psi-Cocaine had Ki = 3 microM, p-Nitro and p-fluoro derivatives of cocaine and analogs with phenyl and p-fluorophenyl groups in place of the benzoyl ester linkage (WIN 35,065-2 and WIN 35,428) inhibited BChE comparably to (-)-cocaine itself. Both cocaine enantiomers were weak inhibitors of acetylcholinesterase (AChE; EC 3.1.1.7) from human erythrocytes with similar Ki values (160-170 microM). Although it is unlikely that the inhibition of BChE is an important factor in the subjective effects of cocaine, it may have implications for the toxicity of cocaine to the fetus, since BChE appears in the development of the central nervous system before AChE, and has been suggested to function as an embryonic acetylcholinesterase.     

Determination of the DNA sequences of acetylcholinesterase and butyrylcholinesterase from cat and demonstration of the existence of both in cat plasma

Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC 3.1.1.7); this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.     

Aminopyridazines as acetylcholinesterase inhibitors

Following the discovery of the weak, competitive and reversible acetylcholinesterase (AChE)-inhibiting activity of minaprine (3c) (IC50 = 85 microM on homogenized rat striatum AChE), a series of 3-amino-6-phenylpyridazines was synthesized and tested for inhibition of AChE. A classical structure-activity relationship exploration suggested that, in comparison to minaprine, the critical elements for high AChE inhibition are as follows: (i) presence of a central pyridazine ring, (ii) necessity of a lipophilic cationic head, (iii) change from a 2- to a 4-5-carbon units distance between the pyridazine ring and the cationic head. Among all the derivatives investigated, 3-[2-(1-benzylpiperidin-4-yl)ethylamino]-6-phenylpyridazine (3y), which shows an IC50 of 0.12 microM on purified AChE (electric eel), was found to be one of the most potent anti-AChE inhibitors, representing a 5000-fold increase in potency compared to minaprine.1     

Synthesis and biological activity of pyridinium-type acetylcholinesterase inhibitors

A novel series of bispyridinium-type acetylcholinesterase (AChE) inhibitors derived from obidoxime, being active in the lower micromolar range, has been reported recently. According to the hypothesis that shorter pyridinium compounds should exhibit higher activity, a new series of compounds was synthesized that has 2,6-dichlorobenzyl, 2-chlorobenzyl and phthalimidomethyl moieties, respectively, at one end of the molecule and that are systematically shortened from the contralateral end. The concentration inhibiting the AChE and butyrylcholinesterase (BChE) by 50% (IC50) was evaluated by means of Ellman's test. Compounds characterized by a phenylpropyl residue at the contralateral end (3) were found to have IC50 values comparable with tacrine. In addition, the affinity of 3c toward the BChE was lower, indicating a lower degree of side effects.     

Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines.

A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection. The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5-fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively. The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM). The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line. The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM. The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD-1 line. Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines. The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells. Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF-Ad10 (31-fold), and DLD-Ad (55-fold) cells. Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene. Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells. These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU.     

Prediction of organophosphorus acetylcholinesterase inhibition using three-dimensional quantitative structure-activity relationship (3D-QSAR) methods.

Neurotoxic organophosphorous compounds are known to modulate their biological effects through the inhibition of a number of esterases including acetylcholinesterase (AChE), the enzyme responsible for the degradation of the neurotransmitter acetylcholine. In this light, molecular modeling studies were performed on a collection of organophosphorous acetylcholinesterase inhibitors by the combined use of conformational analysis and 3D-QSAR methods to rationalize their inhibitory potencies against the enzyme. The Catalyst program was used to identify the structural features in the group of 8 inhibitors whose IC(50) values ranged from 0.34 nM to 1.2 microM. The 3-D pharmacophore models are characterized by at least one hydrogen bond acceptor site and 2-3 hydrophobic sites and demonstrate very good correlation between the predicted and experimental IC(50) values. Our models can be useful in screening databases of organophosphorous compounds for their neurotoxicity potential via the inhibition of acetylcholinesterase. Also, the pharmacophores offer an additional means of designing AChE inhibitors as potential therapeutic agents for central nervous system diseases.     

Inhibitory effects of flavonoids on phosphodiesterase isozymes from guinea pig and their structure-activity relationships

The structure-activity relationships of flavonoids with regard to their inhibitory effects on phosphodiesterase (PDE) isozymes are little known. The activities of PDE1-5 were measured by a two-step procedure using cAMP with [(3)H]-cAMP or cGMP with [(3)H]-cGMP as substrates. In the present results, PDE1, 5, 2, and 4 isozymes were partially purified from guinea pig lungs in that order, and PDE3 was from the heart. The IC(50) values of PDE1-5 were greater than those reported previously for the reference drugs, vinpocetin, EHNA, milrinone, Ro 20-1724, and zaprinast, by 5-, 5-, 7-, 5-, and 3-fold, respectively. As shown in Table 2, luteolin revealed non-selective inhibition of PDE1-5 with IC(50) values in a range of 10-20 microM, as did genistein except with a low potency on PDE5. Daidzein, an inactive analogue of genistein in tyrosine kinase inhibition, showed selective inhibition of PDE3 with an IC(50) value of around 30 microM, as did eriodictyol with an IC(50) value of around 50 microM. Hesperetin and prunetin exhibited more-selective inhibition of PDE4 with IC(50) values of around 30 and 60 microM, respectively. Luteolin-7-glucoside exhibited dual inhibition of PDE2/PDE4 with an IC(50) value of around 40 microM. Diosmetin more-selectively inhibited PDE2 (IC(50) of 4.8 microM) than PDE1, PDE4, or PDE5. However, biochanin A more-selectively inhibited PDE4 (IC(50) of 8.5 microM) than PDE1 or PDE2. Apigenin inhibited PDE1-3 with IC(50) values of around 10-25 microM. Myricetin inhibited PDE1-4 with IC(50) values of around 10-40 microM. The same was true for quercetin, but we rather consider that it more-selectively inhibited PDE3 and PDE4 (IC(50) of < 10 microM). In conclusion, it is possible to synthesize useful drugs through elucidating the structure-activity relationships of flavonoids with respect to inhibition of PDE isozymes at concentrations used in this in vitro study.