Aspects of the cytological activity of edelfosine, miltefosine, and ilmofosine in Leishmania donovani

To discover the mode of action of alkyl-lysophospholipids in Leishmania donovani, we studied the effects of edelfosine, miltefosine, and ilmofosine on intracellular pH, the parasite's cell cycle, and the induction of apoptosis. The effect of the alkyl-lysophospholipids was combined with that of inhibitors of some pumps and exchange regulators of intracellular pH (Na+/ H+; Cl-/CO- 3; and the Na+/K+ ATPase). The effect of the 3 alkyl-lysophospholipids on intracellular pH was indirect; the primary action occurred in the parasite's cell membrane. To determine intracellular pH, we used flow cytometry for the macrophages and axenic amastigotes and spectrofluorometry for the promastigote forms. Apoptosis and the cell cycle were studied by flow cytometry. Treatment of the extracellular promastigote form of L. donovani with the 3 alkyl-lysophospholipids induced death by apoptosis, whereas in the infected cell they caused necrosis rather than apoptosis. Miltefosine and ilmofosine at doses of 38 microM caused G2/M cell cycle inhibition in L. donovani promastigotes.     

Involvement of calcium in the inhibition by insulin of the glucagon-stimulated adenylate-cyclase activity.

1. The inhibitory effect of adenosine on the glucagon-stimulated adenylate cyclase activity of liver plasma membranes, prepared from PVG/c rats, was potentiated by insulin. In the presence of EGTA, such potentiating effect of insulin was lost. 2. Calcium (10 microM) potentiated the inhibitory effects of both adenosine and insulin on the glucagon-stimulated cyclase activity. The synergestic effect of calcium + insulin required the presence of adenosine as judged from the use of adenosine deaminase. 3. Insulin had no significant inhibitory effect on the glucagon-stimulated cyclase activity of liver plasma membranes, prepared from young Wistar rats, unless both adenosine (50 microM) and calcium (10 microM) were added externally. 4. Results demonstrate an interaction of calcium and insulin at membrane level that, in the presence of adenosine, results in the inhibition of the glucagon-stimulated adenylate cyclase activity.     

A comparative study of the effect of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine and some homologous compounds on PKC alpha and PKC epsilon

The effects of the anti-neoplastic ether lipid ET-18-OCH3 and some structural homologues on the activity of protein kinase C alpha (PKC alpha) were studied and compared with the effects the same had on the activity of PKC epsilon. ET-18-OCH3 progressively inhibited the activity of PKC alpha as the concentration was increased up to 30 mol% of the total lipid, above which the effect was one of activation. The experiments carried out with the homologues showed that the methoxy group bound at the sn-2 position of the glycerol of ET-18-OCH3 is essential for both the initial inhibitory effect and the subsequent activation effect. On the other hand, variations in the type of bond linking substitutions in the sn-1 position, ether or ester, do not seem to play an important role in determining the activity of the enzyme. The effects were different on PKC epsilon since ET-18-OCH3 had a triphasic effect, activating the enzyme at low concentrations, inhibiting it at slightly higher concentrations and then activating it again at higher concentrations. In this case, when the homologues were used, it was observed that the presence of the methoxy group linked to the sn-2 position of glycerol and the type of bond linking substitutions to the sn-1 position were important for activating the enzyme, so that only homologues with ester bonds as LPC and PAPC were able to induce the initial activation step in a way similar to ET-18-OCH3. Substitution of the phosphocholine group of ET-18-OCH3 by phosphoserine led to a greater activation of PKC alpha, an effect that comes from the Ca(2+)-phospholipid binding site probably because of the specific interaction of this site with the phosphoserine group. The action of ET-18-OCH3 and its homologues, as demonstrated in this paper, may permit the selective inhibition or activation of PKC alpha and PKC epsilon by using the most suitable range of concentrations.     

2-Deoxy-2,3-didehydro-N-acetylneuraminic acid analogs structurally modified by thiocarbamoylalkyl groups at the C-4 position: Synthesis and biological evaluation as inhibitors of human parainfluenza virus type 1

4-O-Thiocarbamoylmethyl-Neu5Ac2en 3 has strong inhibitory activity toward human parainfluenza virus type 1 (hPIV-1) sialidase compared with the parent Neu5Ac2en 2. We synthesized analogs having thiocarbamoylethyl- 4 and thiocarbamoylpropyl group 5 at the C-4 position of 2. The inhibition degrees of 4 and 5 were weaker than that of thiocarbamoylmethyl analog 3, indicating a remarkable effect of the carbon chain length in thiocarbamoylalkyl groups at the C-4 position on inhibitory activities against hPIV-1 sialidase.     

Structure-activity relationship of minoxidil analogs as inhibitors of lysyl hydroxylase in cultured fibroblasts.

The structural features that confer upon minoxidil the ability to suppress lysyl hydroxylase activity in human skin fibroblasts were investigated. Substitution of the amino group in position 2 or 6 of the pyrimidine ring with a methyl group had no significant effect on the inhibitory activity of minoxidil, whereas substitution of both amino groups with methyl groups resulted in a complete loss of inhibitory activity. Together, these observations indicate that only one of the two amino groups ortho to the nitroxide oxygen is essential for the enzyme-suppressing effect of minoxidil. Derivatives of minoxidil formed by hydroxylation at position 3 or 4 of the piperidine ring were as active as the parent compound in suppressing lysyl hydroxylase activity. However, replacement of the piperidinyl group in position 4 of the pyrimidine ring with a pyrrolidinyl, morpholinyl, or N-methylpiperazinyl group resulted in loss of inhibitory activity, demonstrating that the piperidinyl group para to the nitroxide oxygen is essential for the enzyme-suppressing effect of minoxidil. Removing the nitroxide oxygen from position 1 of the pyrimidine ring resulted in a partial loss of the specificity of minoxidil for suppression of lysyl hydroxylase activity. The results indicate that distinct structural elements determine the enzyme-suppressing effect and the antihypertensive effect of minoxidil.     

Hydroxycoumarins as selective MAO-B inhibitors

A series of 3-aryl-4-hydroxycoumarin derivatives was synthesized with the aim to find out the structural features for the MAO inhibitory activity and selectivity. Methoxy and/or chloro substituents were introduced in the 3-phenyl ring, whereas the position 6 in the coumarin moiety was not substituted or substituted with a methyl group or a chloro atom due to their different electronic, steric and/or lipophilic properties. Most of the synthesized compounds presented MAO-B inhibitory activity. The presence of methoxy and chloro groups, respectively in the para and meta positions of the 3-phenyl ring, have an important influence on the inhibitory activity. Moreover, the presence of a chloro atom in the six position of the moiety (compound 7) improved the inhibitor activity as well as its selectivity against MAO-B compared with iproniazide, used as reference compound. Docking experiments were carried out to understand which are the most energetically preferred orientations adopted by compounds 5, 6 and 7 inside the MAO-B binding pocket.     

Biflavonoids from Torreya nucifera displaying SARS-CoV 3CL(pro) inhibition

As part of our search for botanical sources of SARS-CoV 3CL(pro) inhibitors, we selected Torreya nucifera, which is traditionally used as a medicinal plant in Asia. The ethanol extract of T. nucifera leaves exhibited good SARS-CoV 3CL(pro) inhibitory activity (62% at 100μg/mL). Following bioactivity-guided fractionation, eight diterpenoids (1-8) and four biflavonoids (9-12) were isolated and evaluated for SARS-CoV 3CL(pro) inhibition using fluorescence resonance energy transfer analysis. Of these compounds, the biflavone amentoflavone (9) (IC(50)=8.3μM) showed most potent 3CL(pro) inhibitory effect. Three additional authentic flavones (apigenin, luteolin and quercetin) were tested to establish the basic structure-activity relationship of biflavones. Apigenin, luteolin, and quercetin inhibited 3CL(pro) activity with IC(50) values of 280.8, 20.2, and 23.8μM, respectively. Values of binding energy obtained in a molecular docking study supported the results of enzymatic assays. More potent activity appeared to be associated with the presence of an apigenin moiety at position C-3' of flavones, as biflavone had an effect on 3CL(pro) inhibitory activity.     

A subset of N-substituted phenothiazines inhibits NADPH oxidases

NADPH oxidases (NOXs) constitute a family of enzymes generating reactive oxygen species (ROS) and are increasingly recognized as interesting drug targets. Here we investigated the effects of 10 phenothiazine compounds on NOX activity using an extensive panel of assays to measure production of ROS (Amplex red, WST-1, MCLA) and oxygen consumption. Striking differences between highly similar phenothiazines were observed. Two phenothiazines without N-substitution, including ML171, did not inhibit NOX enzymes, but showed assay interference. Introduction of an aliphatic amine chain on the N atom of the phenothiazine B ring (promazine) conferred inhibitory activity toward NOX2, NOX4, and NOX5 but not NOX1 and NOX3. Addition of an electron-attracting substituent in position 2 of the C ring extended the inhibitory activity to NOX1 and NOX3, with thioridazine being the most potent inhibitor. In contrast, the presence of a methylsulfoxide group at the same position (mesoridazine) entirely abolished NOX-inhibitory activity. A cell-free NOX2 assay suggested that inhibition by N-substituted phenothiazines was not due to competition with NADPH. A functional implication of NOX-inhibitory activity of thioridazine was demonstrated by its ability to block redox-dependent myofibroblast differentiation. Our results demonstrate that NOX-inhibitory activity is not a common feature of all antipsychotic phenothiazines and that substitution on the B-ring nitrogen is crucial for the activity, whereas that on the second position of the C ring modulates it. Our findings contribute to a better understanding of NOX pharmacology and might pave the path to discovery of more potent and selective NOX inhibitors.     

Synthetic tyrosyl gallate derivatives as potent melanin formation inhibitors

Three tyrosyl gallate derivatives (1-3) with variable hydroxyl substituent at the aromatic ring of tyrosol were synthesized and evaluated as potent inhibitors on tyrosinase activity and melanin formation in melan-a cells. Among three tyrosyl gallate derivatives, 4-hydroxyphenethyl 3,4,5-trihydroxybenote (1) (IC(50)=4.93 microM), 3-hydroxyphenethyl 3,4,5-trihydroxybenote (2) (IC(50)=15.21 microM), and 2-hydroxyphenethyl 3,4,5-trihydroxybenote (3) (IC(50)=14.50 microM) exhibited significant inhibitory effect on tyrosinase activity. Compound 1 was the most active compound, though it did not show the inhibitory effect on melanin formation in melan-a cells. However, compounds 2 (IC(50)=8.94 microM) and 3 (IC(50)=13.67 microM) significantly suppressed the cellular melanin formation without cytotoxicity. This study shows that the position of hydroxyl substituent at the aromatic ring of tyrosol plays an important role in the intracellular regulation of melanin formation in cell-based assay system.     

Inhibitory effect of piperidinoethylesters of alkoxyphenylcarbamic acids on photosynthesis.

Piperidinoethylesters of 2-, 3- and 4-alkoxysubstituted phenylcarbamic acids (alkoxy = methoxy - decyloxy) inhibit photosynthetic processes in algae and plant chloroplasts. The inhibitory activity is strongly dependent on the alkyl chain length of the alkoxy-substituent showing a typical quasi-parabolic dependence with maximum effect at 6-8 carbon atoms in the alkyl chain. The alkoxy-substitution in position 2 decreases the inhibitory activity of a compound when compared with its 3- and 4-substituted analogues. ESR studies of spinach chloroplasts confirm that the compounds studied cause destruction of PS II whereby, in the presence of the most effective of the derivatives tested, Mn2+ ions are released from the protein complex.     

Suppressive effect of endogenous regucalcin on deoxyribonuclic acid synthesis in the nuclei of rat renal cortex

The effect of regucalcin, a regulatory protein of Ca2+ signaling, on deoxyribonucleic acid (DNA) synthesis activity in the nuclei isolated from rat renal cortex was investigated. The addition of calcium chloride (10-100 microM) in the reaction mixture containing the nuclei caused a significant decrease in DNA synthesis activity. Nuclear DNA synthesis activity was significantly raised in the presence of EGTA (1 mM), a chelator of Ca2+, indicating that nuclear Ca2+ has an inhibitory effect. Regucalcin (0.1-0.5 microM) added in the reaction mixture in the presence of either EGTA (1 mM) or calcium chloride (50 microM) had a significant inhibitory effect on nuclear DNA synthesis activity. The presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture caused a significant increase in DNA synthesis activity. This increase was completely abolished by the addition of regucalcin (0.5 microM). The effect of anti-regucalcin monoclonal antibody in increasing DNA synthesis was enhanced in the presence of EGTA. Additionally, an inhibitory effect of calcium chloride (10 or 50 microM) was enhanced in the presence of anti-regucalcin monoclonal antibody (25 ng/ml). The present study demonstrates that endogenous regucalcin has a suppressive effect on DNA synthesis in the nuclei of rat renal cortex.     

Site-directed mutagenesis of alanine-382 of human antithrombin III

Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired serine protease inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-3Zf(+)-AT-III1-432 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of alpha-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isoleucine, leucine, valine); (2) low activity (threonine, glutamine); (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77, 221 and 422) and isoleucine (position 412) residues.     

Inhibitory effects of 2-substituted-1-naphthol derivatives on cyclooxygenase I and II

Naphthol derivatives, 2-(3'-hydroxypropyl)-naphthalen-1-ol (2), 2-(3'-hydroxy-2'-methylpropyl)-naphthalen-1-ol (3) and 2-(3'-hydroxy-2',2'-dimethylpropyl)-naphthalen-1-ol (7) were synthesized and already reported by our group. Therefore in this paper we described further synthesis of their ether derivatives, 3-(1-methoxy-naphthalen-2-yl)-propan-1-ol (4), 3-(1-methoxy-naphthalen-2-yl)-2methyl-propan-1-ol (5), 3-(1-methoxy-naphthalen-2-yl)-2,2-dimethyl-propan-1-ol (8), 2-(3-methoxy-propyl)-naphthalen-1-ol (10) and 2-(3-methoxy-2,2-dimethyl-propyl)-naphthalen-1-ol (13). Compounds 4, 5 and 8 were prepared by methylation of compounds 2, 3 and 7, respectively while compounds 10 and 13 were prepared in good yield from naphthols 2 and 7, respectively. When tested for inhibitory activity, five compounds (2, 3, 7, 10 and 13) showed preferential inhibition of COX-2 over COX-1, while compounds 4, 5 and 8 lacked inhibitory effect on either the COX-1 or COX-2 isozyme. The structure-activity relationships of these naphthols analyzed by docking experiments, indicated that the presence of hydroxyl group at C-1 position on the naphthalene nucleus enhanced the anti-inflammatory activity towards COX-2 via hydrogen bonding to the COX-2 Val 523 side chain. When this hydroxyl group was replaced by methoxy group, there was no inhibition. C-2' Dimethyl substituents on the propyl chain also increased the inhibitory activity. All active compounds have the C-1 hydroxyl group aligned so as to form hydrogen bond with Val 523. The results provide a model for the binding of the naphthol derivatives to COX-2 and facilitate the design of more potent or selective analogs prior to synthesis.     

Chalcones as potent tyrosinase inhibitors: the importance of a 2,4-substituted resorcinol moiety

Compounds, which inhibit tyrosinase, could be effective as depigmenting agents. We have introduced a group of mono-, di-, tri- and tetra-substituted hydroxychalcones as effective tyrosinase inhibitors, showing that the most important factor determining tyrosinase inhibition efficiency is the position of the hydroxyl group(s) rather their number. The aim of the present study was to investigate the contribution of the different functional groups of the tetrahydroxychalcones to their inhibitory potency, with a view to optimizing the design of whitening agents. Four tetrahydroxychalcones were evaluated, the commercially available Butein and other three were synthesized, and their inhibitory effect on tyrosinase was tested. Results showed that a 2,4-substituted resorcinol subunit on ring B contributed the most to inhibitory potency. Changing the resorcinol substitute to position 3,5- or placing it on ring A significantly diminished the inhibitory effect of the compounds. A catechol subunit on ring A acted as a metal chelator (in the presence of copper ions) and as a competitive inhibitor (in the presence of tyrosinase), while a catechol on ring B oxidized to o-quinone (in the presence of both copper ions and tyrosinase). Three of the compounds also demonstrated antioxidant activity, which may contribute to the prevention of pigmentation. An examination of correlations between inhibitory activity and physical properties of the chalcones tested (such as dissociation energy and molecular planarity) showed positive correlation with the moment dipole value in the Y-axis, which may be used as an indicator of the inhibitory potential of new molecules. The present study revealed two very active tyrosinase inhibitors, 2,4,3',4'-hydroxychalcone and 2,4,2',4'-hydroxychalcone (with IC50 of 0.2 and 0.02 microM, respectively). Structure-related activity studies added some understanding of the role and contribution of different functional groups associated with tyrosinase inhibitors.     

Ratio of brain synaptosome Na, K-ATPase to dopamine receptors

Activating (0.3-3 microM) or inhibitory (0.03-0.3 mM) effects of dopamine (DA) in the absence of Ca2+, and its inhibitory effect in the presence of Ca2+ on Na,K-ATPase activity of synaptosomes from the caudate nucleus of the rat brain were confirmed. Na,K-ATPase was shown to be inhibited by 6 neuroleptics, with the degree of inhibition stronger in the presence of Ca2+. It was found that: 1) the biphasic or monophasic nature of DA action on Na,K-ATPase activity was preserved in the presence of neuroleptics, 2) DA enhances the inhibitory effects of neuroleptics on the enzyme, 3) the inhibitory effects of DA on Na,K-ATPase are enhanced by Ca2+ ions. The mechanisms of the modifying action of DA on synaptosomal Na,K-ATPase are discussed.     

Effect on some pyridine derivatives on the activity of heart NAD glycohydrolase]

Effect of some 3- and 4-substituted pyridines on enzymatic hydrolysis of NAD by rabbit heart muscle NAD-glycohydrolase has been studied. It is shown that some 4-substituted derivatives in contrast with 3-substituted ones produce an inhibitory effect on the enzyme activity. A new efficient inhibitor of rabbit heart muscle NAD-glycohydrolase (I50 = 10(-3) M)--N1-(2-lactyl)-N2-(isonicotinoy)hydrazine, inducing uncompetitive inhibition of hydrolysis of NAD is found. The mechanism of the inhibitory effect of N1-(2-lactyl)-N2-(isonicotinoyl)hydrazine was investigated and the rate equation for enzymatic hydrolysis of NAD in the presence of inhibitor is calculated. It is suggested, that the inhibitory effect of N1-(2-lactyl)-N2-(isonicotinoyl)hydrazine is due to the formation of triple inactive complex inhibitor-enzyme-adenosinediphosphateribose.     

Thrombin Glu-39 restricts the P'3 specificity to nonacidic residues

Residue 39 of serine proteases neighbors positions P'2 to P'4 of the substrate. When Glu-39 of thrombin is replaced with Lys, the resultant enzyme (E39K) retains similar P1, P2, and P3 specificities but has altered P'3 and/or P'4 specificities. These conclusions are based on analysis of both p-nitroanilide and synthetic peptide hydrolysis. The activity of E39K is nearly normal toward 17 p-nitroanilide substrates. In peptide substrates, an acidic residue at either the P3 or P'3 position reduces the rate of cleavage by thrombin. A single substitution of Asp with Gly in either the P3 or P'3 position of a peptide corresponding to the P7-P'5 residues of protein C increases the rate of cleavage by thrombin 2-3-fold. Replacement of both Asp residues with Gly increases the rate of cleavage 30-fold. With E39K, the inhibitory effect of Asp in P3 remains unchanged, but Asp in the P'3 site is no longer inhibitory. Significant differences in the catalytic activity of E39K are also seen with respect to protein C activation. In the absence of thrombomodulin, E39K activates protein C 2.2 times faster than thrombin. In the presence of thrombomodulin, the rate of protein C activation is similar for E39K and thrombin. The second order rate constant of inhibition by antithrombin III, where P'4 is a Glu, is slightly increased (1.4-fold). The clotting activity is reduced 2.4-fold due to a lower rate of fibrinopeptides A and B release where P'3 is Arg. These data show that the P'3 position is a determinant of thrombin specificity and suggest that thrombomodulin may function in part by alleviating the inhibitory effects that may arise from the proximity of the Asp in P'3 of protein C with Glu-39 of thrombin.