Prolyl endopeptidase inhibitors from green tea

Three prolyl endopeptidase (PEP) inhibitors were isolated from the methanolic extract of green tea leaves. They were identified as (-)-epigallocatechin gallate, (-)-epicatechin gallate, and (+)-gallocatechin gallate with the IC50 values of 1.42 x 10(-4) mM, 1.02 x 10(-2) mM, and 1.09 x 10(-4) mM, respectively. They were non-competitive with a substrate in Dixon plots and did not show any significant effects against other serine proteases such as elastase, trypsin, and chymotrypsin, suggesting that they were relatively specific inhibitors against PEP. The isolated compounds are expected to be useful for preventing and curing of Alzheimer's disease.     

A Prolyl endopeptidase-lnhibiting antioxidant fromPhyllanthus ussurensis

A prolyl endopeptidase inhibitor was isolated from the ethyl acetate soluble fraction of Phyllanthus ussurensis. The active compound was identified as an ellagitannin, corilagin. It was shown to non-competitively inhibit prolyl endopeptidase (PEP) with the IC50 value of 1.17x10(-6) microM. The Ki value was 6.70x10(-7) M. Corilagin was less inhibitory to other serine proteases such as chymotrypsin, trypsin, and elastase, indicating that it was relatively a specific inhibitor of PEP. Corilagin also effectively inhibited reactive oxygen species such as hydroxide and superoxide anion radical, hydrogen peroxide, and DPPH. Especially, corilagin showed potent scavenging activity on the superoxide anion radical in the ESR method (IC50 = 3.79x10(-6) M) as well as xanthine oxidase system.     

Prolyl endopeptidase inhibitors from the leaves of Ginkgo biloba

Prolyl endopeptidase (PEP, EC 3.4.21.26) hydrolyzes proline-containing neuropeptides, such as vasopressin, substance P, and thyrotropin-releasing hormone (TRH), and is suggested to participate in learning and memory processes. Ginkgo biloba leaves, upon examination for anti-amnestic constituents as new types of PEP inhibitors, showed significant PEP inhibition. PEP activity-guided fractionation and column chromatography of the MeOH extracts of G. biloba leaves resulted in the isolation of 6-(8'Z-pentadecenyl)salicylic acid (1) and 6-(10'Z-heptadecenyl)salicylic acid (2). The kinetic study indicated that compounds 1 and 2 are non-competitive inhibitors of prolyl endopeptidase with Ki values of 0.87 and 0.80 microM, respectively.     

A beta-secretase (BACE1) inhibitor hispidin from the mycelial cultures of Phellinus linteus

In the course of screening for anti-dementia agents from natural products, a beta-secretase (BACE1) inhibitor was isolated from the culture broth of Phellinus linteus and identified as hispidin. It showed an IC (50) value of 4.9 x 10 (-6) M and a Ki value of 8.4 x 10 (-6) M. The compound was a non-competitive inhibitor. Hispidin also inhibited a prolyl endopeptidase (IC (50) = 1.6 x 10 (-5) M, Ki = 2.4 x 10 (-5) M), but it was less inhibitory to alpha-secretase (TACE) and other serine proteases such as chymotrypsin, trypsin, and elastase.     

Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine,donepezil, rivastigmine and physostigmine

Five inhibitors of acetylcholinesterase, huperzine A, donepezil, tacrine, rivastigmine and physostigmine, were compared with regard to their effects on different molecular forms of acetylcholinesterase in cerebral cortex, hippocampus, and striatum from the rat brain. In general, huperzine A preferentially inhibited tetrameric acetylcholinesterase (G4 form), while tacrine and rivastigmine preferentially inhibited monomeric acetylcholinesterase (G1 form). Donepezil showed pronounced selectivity for G1 acetylcholinesterase in striatum and hippocampus, but not in cortex. Physostigmine showed no form-selectivity in any brain region. In cortex, the most potent inhibitors of G4 acetylcholinesterase were huperzine A (K(i) 7 x 10(-9) M) and donepezil (K(i) 4 x 10(-9) M). The potent inhibitors of cortical G1 acetylcholinesterase were donepezil (K(i) 3.5 x 10(-9) M) and tacrine (K(i) 2.3 x 10(-8) M). In hippocampus, huperzine A and physostigmine were the most potent inhibitors of G4 acetylcholinesterase, while donepezil and tacrine were most potent against G1 acetylcholinesterase. In striatum, huperzine A and donepezil were the most potent against G4 acetylcholinesterase, while again donepezil was the most potent against G1. Although the inhibition constants (K(i)) of these acetylcholinesterase inhibitors differed significantly from region to region, the nature of the inhibition did not vary. These results suggest that the use of acetylcholinesterase inhibitors in treatment of Alzheimer's disease must consider both form-specific and region-specific characteristics of acetylcholinesterase inhibition.     

Prolyl endopeptidase inhibitors from the underground part of Rhodiola sacra S. H. Fu

Prolyl endopeptidase (PEP, EC 3.4.21.26) is an enzyme which plays a role in the metabolism of proline-containing neuropeptides, e.g., vasopressin, substance P and thyrotropin-releasing hormone (TRH), which have been suggested to be involved in learning and memory processes. In our systematic screening for PEP inhibitors from traditional Chinese medicines, we found that MeOH extract from the underground part of Rhodiola sacra S. H. Fu shows significant inhibitory activity against PEP from Flavobacterium meningosepticum. Examination of the constituents of the extract resulted in the isolation of nineteen known compounds, identified as hydroquinone (1), 4-hydroxybenzoic acid (2), caffeic acid (3), 4-hydroxycinnamic acid (4), suberic acid (5), protocatechuic acid (6), gallic acid (7), (-)-epigallocatechin 3-O-gallate (8), 2-phenylethyl beta-D-glucopyranoside (9), 3-O-galloylepigallocatechin-(4beta-->8)-epigallocatechin+ ++ 3-O-gallate (10), 2-phenylethyl alpha-L-arabinopyranosyl-(1-->6)-beta-D-glucopyranoside (11), sacranoside A (12), beta-D-glucopyranosyl 4-hydroxybenzoate (13), rhodiocyanoside A (14), rhodiooctanoside (15), sarmentosin (16), heterodendrin (17), arbutin (18) and 4-O-(beta-D-glucopyranosyl)-gallic acid (19). Among these, 1, 2, 5, 8-10, 13, 16, 18 and 19 have been isolated for the first time from R. sacra, among which 5, 9, 10, 13, 16, 18 and 19 have been isolated from Rhodiola plants for the first time. On the PEP inhibition, seven compounds (6-8, 10, 12, 18, 19) showed inhibition with an 1C50 of 27.8, 487, 1.47, 0.437, 348, 391 and 215 microM, respectively. The kinetic study of these inhibitors indicated that they are noncompetitive inhibitors, except for 6 which is a competitive inhibitor.     

Synthesis of fused [1,2,6]thiadiazine 1,1-dioxides as potential transition-state analogue inhibitors of xanthine oxidase and guanase.

Ring closure of ethyl 3-aminopyrazole-4-carboxylate with sulfamoyl chloride gave 1,7-dihydropyrazolo[3,4-c][1,2,6]thiadiazine-4(3H)-one 2,2-dioxide. The corresponding 4-amino analogue of this new heterocyclic ring system was similarly prepared from 3-aminopyrazole-4-carbonitrile. Treatment of 4,5,6-triamino-2H-1,2,6-thiadiazine 1,1-dioxide with N-thionylaniline gave a derivative of another new ring system, 7-amino-4H-[1,2,5]thiadiazolo[3,4-c][1,2,6]thiadiazine 5,5-dioxide. These compounds and the corresponding 4-amino- and 4-hydroxyimidazol[4,5-c][1,2,6]thiadiazine 2,2-dioxides were examined as potential transition-state analogue inhibitors of xanthine oxidase and guanine aminohydrolase. Two of the compounds possessed Ki values of about 2x 10(-4) M with guanine aminohydrolase, but no inhibition of xanthine oxidase was observed by any at 5 x 10(-4) M.     

Tyrosinase inhibitors isolated from the edible brown alga<Emphasis Type="Italic">Ecklonia stolonifera</Emphasis>

Extracts from seventeen seaweeds were determined for tyrosinase inhibitory activity using mushroom tyrosinase with L-tyrosine as a substrate. Only one of them, Ecklonia stolonifera OKAMURA (Laminariaceae) belonging to brown algae, showed high tyrosinase inhibitory activity. Bioassay-guided fractionation of the active ethyl acetate (EtOAc) soluble fraction from the methanolic extract of E. stolonifera, led us to the isolation of phloroglucinol derivatives [phloroglucinol (1), eckstolonol (2), eckol (3), phlorofucofuroeckol A (4), and dieckol (5)]. Compounds 1 approximately 5 were found to inhibit the oxidation of L-tyrosine catalyzed by mushroom tyrosinase with IC50 values of 92.8, 126, 33.2, 177, and 2.16 microg/mL, respectively. It was compared with those of kojic acid and arbutin, well-known tyrosinase inhibitors, with IC50 values of 6.32 and 112 microg/ mL, respectively. The inhibitory kinetics analyzed from Lineweaver-Burk plots, showed compounds 1 and 2 to be competitive inhibitors with Ki of 2.3x10(-4) and 3.1x10(-4) M, and compounds 3 approximately 5 to be noncompetitive inhibitors with Ki of 1.9x10(-5), 1.4x10(-3) and 1.5x10(-5) M, respectively. This work showed that phloroglucinol derivatives, natural compounds found in brown algae, could be involved in the control of pigmentation in plants and other organisms through inhibition of tyrosinase activity using L-tyrosine as a substrate.     

Inhibitory activity for chitin synthase II from Saccharomyces cerevisiae by tannins and related compounds

In the course of search for potent inhibitors of chitin synthase II from natural resources, seven tannins and related compounds were isolated from the aerial part of Euphorbia pekinensis and identified as gallic acid (1), methyl gallate (2), 3-O-galloyl-(-)-shikimic acid (3), corilagin (4), geraniin (5), quercetin-3-O-(2"-O-galloyl)-beta-D-glucoside (6), and kaempferol-3-O-(2"-O-galloyl)-beta-D-glucoside (7). These and nine related compounds, (-)-quinic acid (8), (-)-shikimic acid (9), ellagic acid (10), kaempferol (11), quercetin (12), quercitrin (13), rutin (14), quercetin-3-O-(2"-O-galloyl)-beta-D-rutinoside (15) and 1,3,4,6-tetra-O-galloyl-beta-D-glucose (16), were evaluated for the inhibitory activity against chitin synthase II and III. They inhibited chitin synthase II with IC(50) values of 18-206 microM, except for two organic acids, (-)-quinic acid (8) and (-)-shikimic acid (9). Among them, 3-O-galloyl-(-)-shikimic acid (3) was the most potent inhibitor against chitin synthase II of Saccharomyces cerevisiae with an IC(50) value of 18 microM. The inhibition appears to be selective for chitin synthase II, as they did not appreciably inhibit chitin synthase III.     

Inhibition of vaccinia RNA guanine 7-methyltransferase by compounds designed as multisubstrate adducts

Several potential inhibitors of mRNA guanine 7-methyltransferase, which were designed from mechanism-based considerations, were evaluated against the vaccinia virus capping enzyme complex. Of the compounds tested, 5'-deoxy-5'[6-(2-aminopyrrolo[2,3-d]-pyrimidine-4-one) methylthio]adenosine (9) had good selective inhibitory activity against vaccinia mRNA guanine 7-methyltransferase, exhibiting an IC50 of 9.2 X 10(-5) M. Structure-activity considerations suggest that specific inhibition of RNA methyltransferases by low molecular weight multisubstrate adduct inhibitors may be achievable.     

Novel anthraquinone inhibitors of human leukocyte elastase and cathepsin G.

A large series of variously substituted anthraquinones has been synthesized and assayed for inhibitory capacity against human leukocyte elastase (HLE) and cathepsin G (CatG), two serine proteinases implicated in diseases characterized by the abnormal degradation of connective tissue, such as pulmonary emphysema and rheumatoid arthritis. It was found that 2-alkyl-1,8-dihydroxyanthraquinone analogues are competitive inhibitors of HLE with IC50 values ranging from 4 to 10 microM, and also inhibit CatG with IC50 values ranging from 25 to 55 microM. Consequently, analogues containing the 2-alkyl-1-hydroxy-8-methoxyanthraquinone substitution pattern inhibit HLE to the same magnitude as for the compounds above, but show very little inhibition of CatG. Anthraquinones containing long, hydrophobic n-butyl carbonate moieties in the 1- and 8-positions in conjunction with a third hydrophobic substituent in the 2- or 3-position are highly selective for HLE, with Ki values in the range of 10(-7) M. All of the inhibitors described are completely reversible, with no evidence of acyl-enzyme formation detected.     

Computer-aided selection of potential antihypertensive compounds with dual mechanism of action

The prediction of biological activity spectra for substances as an approach for searching compounds with complex mechanisms of action was studied. New compounds with dual mechanisms of antihypertensive action were found by this approach. Biological activity spectra for substances were predicted on the basis of their structural formulas by the computer program PASS. Thirty molecular mechanisms of action of compounds from the MDDR 99.2 database, which cause the antihypertensive effect and can be predicted by PASS, have been identified. The analysis of predictions for compounds with 15 dual antihypertensive mechanisms of action from the MDDR 99.2 database has confirmed high accuracy of prediction. This approach was applied to databases of commercially available compounds (AsInEx and ChemBridge) and allowed us to select four substances that are potential inhibitors of angiotensin converting enzyme (ACE) and of neutral endopeptidase (NEP). At a later time, all these compounds were found to be the inhibitors of both ACE and NEP. The most potent compounds had IC(50) of 10(-7)-10(-9) M for ACE and 10(-5) M for NEP. New combinations of dual mechanisms of action never before found for antihypertensive compounds were predicted.     

Dynamic modeling of human 5-lipoxygenase-inhibitor interactions helps to discover novel inhibitors

Human 5-lipoxygenase (5-LOX) is one of the key anti-inflammatory drug targets due to its key role in leukotrienes biosynthesis. We have built a model for the active conformation of human 5-LOX using comparative modeling, docking of known inhibitors, and molecular dynamics simulation. Using this model, novel 5-LOX inhibitors were identified by virtual screen. Of the 105 compounds tested in a cell-free assay, 30 have IC(50) values less than 100 μM and 11 less than 10 μM with the strongest inhibition of 620 nM. Compounds 4, 7, and 11 showed strong inhibition activity in the human whole blood (HWB) assay with IC(50) values of 8.6, 9.7, 8.1 μM, respectively. Moreover, compounds 4 and 7 were also found to inhibit microsomal prostaglandin E synthase (mPGES)-1 with micromolar IC(50) values, similar to licofelone, a dual functional inhibitor of 5-LOX/mPGES-1. The compounds reported here provide new scaffolds for anti-inflammatory drug design.     

Synthesis and molecular modelling of novel substituted-4,5-dihydro-(1H)-pyrazole derivatives as potent and highly selective monoamine oxidase-A inhibitors

This report describes novel pyrazoline derivatives investigated for their ability to selectively inhibit the activity of the A and B isoforms of monoamine oxidase. These new synthetic compounds proved to be reversible, potent, and selective inhibitors of monoamine oxidase-A rather than of monoamine oxidase-B, and are promising candidates to further advance drug discovery efforts. The most active compounds show inhibitory activity on monoamine oxidase-A in the 1.0x10(-8)-8.6x10(-9) M range. Moreover, it should be pointed out that for some compounds a high IC50>or=10(-9) M value is associated with a high A-selectivity (Selectivity Index monoamine oxidase-B/monoamine oxidase-A in the 10,000-12,500 range). Further insight to understand enzyme-inhibitor molecular interaction was obtained by docking experiments with the monoamine oxidase-A and monoamine oxidase-B isoforms.     

Thromboxane receptor antagonism combined with thromboxane synthase inhibition. 4. 8-[[(4-Chlorophenyl)sulfonyl]amino]-4-(3-(3-pyridinyl) propyl)octanoic acid and analogs.

The title compound (10a) and its analogs were synthesized and found to possess two activities, the inhibition of the biosynthesis of thromboxane A2 and antagonism of its receptors. The in vitro and in vivo profile of these compounds as thromboxane receptor antagonists (TxRAs) and thromboxane synthase inhibitors (TxSIs) is described. 10a and its analogs displayed very potent TxRA activity in human washed platelets (IC50 approximately 10(-7)-10(-9) M) and dog saphenous vein (pA2 approximately 9) and also potent TxSI activity (IC50 approximately 10(-9) M). The good bioavailability and the long duration of action of some of these compounds was demonstrated using ex vivo measurement of the TxRA activity upon oral administration to guinea pigs. Compounds 10a, 20, and 33 potently inhibited arachidonic acid induced bronchoconstriction in guinea pigs.     

Overlap in the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.

We have previously shown that elimination of buffer Ca2+ markedly reduced maximum 5-HT-induced contractions. We have now investigated the effect of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists on 5-HT- and K+-induced contractions in rat aorta to explore the possibility of a relationship between blockade of L-type Ca2+ channels and 5-HT2 receptor antagonism. Sodium nitroprusside, felodipine, nifedipine, diltiazem, cinnarizine, verapamil, ritanserin, cyproheptadine, ketanserin and mianserin inhibited 5-HT-induced contractions of rat aorta with mean IC50 values (concentration (M) resulting in 50% inhibition) of 2.2 x 10(-11), 6.6 x 10(-11), 1.5 x 10(-9), 1.7 x 10(-9), 3.2 x 10(-7), 5.4 x 10(-7), 9.7 x 10(-10), 1.9 x 10(-8), 5.0 x 10(-7) and 6.4 x 10(-7), respectively. The same compounds antagonized K+-induced rat aortic contractions with the rank order of potency (mean IC50, M): felodipine (7.0 x 10(-11)) > nifedipine (4.8 x 10(-9)) > sodium nitroprusside (4.1 x 10(-8)) > verapamil (5.5 x 10(-8)) > cyproheptadine (6.2 x 10(-8)) > diltiazem (4.1 x 10(-7)) > cinnarizine (1.3 x 10(-6)) > ritanserin (1.8 x 10(-6)) > ketanserin (9.0 x 10(-6)) > mianserin (2.0 x 10(-5)). These data are indicative of a highly significant correlation (r=0.81, P=0.03) between potency against 5-HT-induced contraction and that against contractile response to K+ depolarization, and suggest overlap of the pharmacology of L-type Ca2+-channel blockers and 5-HT2 receptor antagonists in rat aorta.     

Influence of hypothyroid state on 45Ca(2+) influx and sensitivity of rat uterus to nifedipine and diltiazem

The influence of methimazole-induced hypothyroidism on spontaneous rhythmic contractions and Ca2+ channel function of rat uterus was examined. Hypothyroidism significantly reduced the amplitude and frequency of spontaneous rhythmic contractions. Nifedipine (10(-12)-10(-6) M) and diltiazem (10(-9)-10(-4) M) caused concentration-related inhibition of the myogenic responses of the oestrogenised rat uterus obtained from both eu- and hypothyroid rats. However, nifedipine was less potent (IC(50); 5.4 x 10(-9) M; n=6) in hypothyroid rat uterus as compared to euthyroid controls (IC(50): 8.13 x 10(-12) M; n=9) to inhibit the rhythmic contractions. Similarly, diltiazem was less potent (IC(50): 4.57 x 10(-6) M; n=9) to inhibit the uterine spontaneous contractions in hypothyroid than in euthyroid rat uterus (IC(50): 6.4 x 10(-8) M; n=6). A similar decrease in the sensitivity to nifedipine and diltiazem for reversal of K+ (100 mM)-induced tonic contraction was observed in uterus obtained from hypothyroid rats compared to the controls. Both nifedipine and diltiazem were less potent for causing concentration-related inhibition of K+-stimulated 45Ca2+ influx in uterine strips taken from the hypothyroid rats. Thus, the IC(50) values of nifedipine (1.83 x 10(-8) M; n=12) and diltiazem (1.8 x 10(-6) M; n=9) were significantly greater in tissues obtained from hypothyroid rats compared to the controls (IC(50) of nifedipine, 1.15 x 10(-11) M; n=12, diltiazem, 8.1 x 10(-8) M; n=8). Nifedipine-sensitive influx of 45Ca2+ - stimulated either by K+ (100 mM) or Bay k8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2'-(trifluromethyl)phenyl]-3-pyridine carboxylic acid methyl ester) (10(-8) M) was significantly less in uterine strips from hypothyroid rats compared to the controls. The results of the present study suggest that the inhibition of uterine rhythmic contractions may be attributable to a reduction in rat myometrial Ca2+ channel function in the hypothyroid state.     

Inhibition of a type B monoamine oxidase inhibitor, (E)-2-(4-fluorophenethyl)-3-fluoroallylamine (MDL-72974A), on semicarbazide-sensitive amine oxidases isolated from vascular tissues and sera of different species.

(E)-2-(4-Fluorophenethyl)-3-fluoroallylamine hydrochloride (MDL-72974A) has been discovered recently to be a very potent and highly selective type B monoamine oxidase inhibitor. We have found that this inhibitor is also capable of inhibiting semicarbazide-sensitive amine oxidases (SSAOs) obtained from vascular tissues and sera of different species. The inhibition of SSAO by MDL-72974A was irreversible and time dependent. It was competitive without preincubation of the enzyme with the inhibitor and demonstrated a mixed-type of inhibition when the enzyme was preincubated with the inhibitor. The IC50 values were estimated to be 2 x 10(-9) M, 5 x 10(-9) M, 8 x 10(-8) M and 2 x 10(-8) M for SSAO from dog aorta, rat aorta, bovine aorta and human umbilical artery, respectively. SSAO obtained from bovine serum was relatively insensitive to MDL-72974A (IC50 = 3 x 10(-7) M. Following intraperitoneal administration of MDL-72974A, rat brain MAO-B was inhibited with the ED50 value being about 0.2 mg/kg. Rat aorta SSAO was also inhibited and to a similar extent by the same dose. MDL-72974A is the most potent SSAO inhibitor that has been described thus far.     

Neuroprotective profile of lifarizine (RS-87476) in rat cerebrocortical neurones in culture.

1. The ability of the neuroprotective agent, lifarizine (RS-87476), to mitigate veratridine-, cyanide- and glutamate-induced toxicity in rat embryonic cerebrocortical neurones in primary culture has been compared with that of tetrodotoxin (TTX), nitrendipine, (+)-MK-801 and (-)-MK-801. Lactate dehydrogenase (LDH) released into the culture medium was used as the indicator of cell viability. 2. Incubation of cultures for 16 h in a medium containing veratridine (10(-4) M), sodium glutamate (10(-3) M) or sodium cyanide (10(-3) M) resulted in consistent elevations of LDH activity in the culture medium. The ability of compounds to attenuate these elevations was expressed as the concentration required to inhibit the increases in LDH release by 50% (IC50). 3. Neurotoxicity induced by veratridine was inhibited by lifarizine (IC50 = 4 x 10(-7) M), TTX (IC50 = 3 x 10(-8) M) and nitrendipine (IC50 = 3 x 10(-5) M). In contrast, (+)-MK-801 (up to 3 x 10(-5) M) was ineffective against this insult. 4. Glutamate-induced neurotoxicity was inhibited by (+)-MK-801 (IC50 = 1.4 x 10(-8) M) and to a lesser extent by (-)-MK-801 (IC50 = 1 x 10(-7) M), but was unaffected by lifarizine, TTX or nitrendipine (up to 10(-6) M). 5. (+)-MK-801 was effective against sodium cyanide-induced neurotoxicity (IC50 = 1.9 x 10(-8) M), whereas lifarizine and TTX (up to 10(-6) M) and nitrendipine (up to 3 x 10(-6) M) were without protective activity against this insult.(ABSTRACT TRUNCATED AT 250 WORDS)