The antidepressant-like effect of inosine in the FST is associated with both adenosine A1 and A2A receptors

Inosine is an endogenous purine nucleoside, which is formed during the breakdown of adenosine. The adenosinergic system was already described as capable of modulating mood in preclinical models; we now explored the effects of inosine in two predictive models of depression: the forced swim test (FST) and tail suspension test (TST). Mice treated with inosine displayed higher anti-immobility in the FST (5 and 50 mg/kg, intraperitoneal route (i.p.)) and in the TST (1 and 10 mg/kg, i.p.) when compared to vehicle-treated groups. These antidepressant-like effects started 30 min and lasted for 2 h after intraperitoneal administration of inosine and were not accompanied by any changes in the ambulatory activity in the open-field test. Both adenosine A₁ and A_2A receptor antagonists prevented the antidepressant-like effect of inosine in the FST. In addition, the administration of an adenosine deaminase inhibitor (1 and 10 mg/kg, i.p.) also caused an antidepressant-like effect in the FST. These results indicate that inosine possesses an antidepressant-like effect in the FST and TST probably through the activation of adenosine A₁ and A_2A receptors, further reinforcing the potential of targeting the purinergic system to the management of mood disorders.     

Monoamine Oxidase-Inhibiting Properties of SR 95191, a New Pyridazine Derivative, in the Rat: Evidence for Selective and Reversible Inhibition of Monoamine Oxidase Type A In Vivo but Not In Vitro

In rodents, SR 95191 [3-(2-morpholinoethylamino)-4-cyano-6-phenylpyridazine] has been shown to be active in animal models of depression. The profile of activity of SR 95191 suggests that the compound is a selective and short-acting type A monoamine oxidase (MAO) inhibitor (MAOI) in vivo. In the present study, the interaction of SR 95191 with MAO-A and MAO-B activity was further examined in vivo and in vitro. In brain, liver, and duodenum of pretreated rats, SR 95191 selectively inhibited MAO-A (ED50 = 3-5 mg/kg, p.o.), whereas MAO-B was only weakly inhibited for doses as high as 300 mg/kg, p.o. In vivo, SR 95191 (1-100 mg/kg, p.o.) antagonized, in a dose-dependent fashion, the irreversible inhibition of brain and liver MAO-A induced by phenelzine. Finally, dopamine and 5-hydroxytryptamine depleted from their striatal stores by tetrabenazine were able to displace SR 95191 from the active site of MAO-A. However, ex vivo, kinetic studies showed that the inhibitory effect of SR 95191 (1-10 mg/kg) towards MAO-A was noncompetitive and was unchanged after dilution or dialysis. In vitro, the inhibition of brain MAO-A, but not MAO-B, by SR 95191 was time dependent, with a 19-fold decrease in the IC50 values being observed over a 30-min incubation period (140 to 7.5 microM). At this time, the SR 95191-induced inhibition of MAO-A was not removed by repeated washings. When the reaction was started by adding the homogenate without prior preincubation with SR 95191, the inhibition of brain MAO-A was fully competitive (Ki = 68 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic and inhibitor binding properties of zebrafish monoamine oxidase (zMAO): comparisons with human MAO A and MAO B

A comparative investigation of substrate specificity and inhibitor binding properties of recombinant zebrafish (Danio rerio) monoamine oxidase (zMAO) with those of recombinant human monoamine oxidases A and B (hMAO A and hMAO B) is presented. zMAO oxidizes the neurotransmitter amines (serotonin, dopamine and tyramine) with k(cat) values that exceed those of hMAO A or of hMAO B. The enzyme is competitively inhibited by hMAO A selective reversible inhibitors with the exception of d-amphetamine where uncompetitive inhibition is exhibited. The enzyme is unreactive with most MAO B-specific reversible inhibitors with the exception of chlorostyrylcaffeine. zMAO catalyzes the oxidation of para-substituted benzylamine analogs exhibiting (D)k(cat) and (D)(k(cat)/K(m)) values ranging from 2 to 8. Structure-activity correlations show a dependence of log k(cat) with the electronic factor σ(p) with a ρ value of +1.55±0.34; a value close to that for hMAO A but not with MAO B. zMAO differs from hMAO A or hMAO B in benzylamine analog binding correlations where an electronic effect (ρ=+1.29±0.31) is observed. These data demonstrate zMAO exhibits functional properties similar to hMAO A as well as exhibits its own unique behavior. These results should be useful for studies of MAO function in zebrafish models of human disease states.     

Novel triple reuptake inhibitors with low risk of CAD associated liabilities: Design,synthesis and biological activities of 4-[(1S)-1-(3,4-dichlorophenyl)-2-methoxyethyl]piperidine and related compounds

A novel triple reuptake inhibitor with low potential of liabilities associated with cationic amphiphilic drug (CAD) was identified following an analysis of existing drugs. Low molecular weight (MW < ca. 300), low aromatic ring count (number = 1) and reduced lipophilicity (C log P < 3.5) were hypothesized to be key factors to avoid the CAD associated liabilities (CYP2D6 inhibition, hERG inhibition and phospholipidosis). Based on the hypothesis, a series of piperidine compounds was designed with consideration of the common characteristic features of CNS drugs. Optimization of the side chain by adjusting overall lipophilicity suggested that incorporation of a methoxymethyl group could provide compounds with a balance of both potent reuptake inhibition and low liability potential. Compound (S)-3a showed a potent antidepressant-like effect in the mice tail suspension test (MED = 10 mg/kg, p.o.), proportional monoamine transporter occupancies and enhancement of monoamine concentrations in mouse prefrontal cortex.     

Novel multi-target directed ligands based on annelated xanthine scaffold with aromatic substituents acting on adenosine receptor and monoamine oxidase B. Synthesis,in vitro and in silico studies

N9-Benzyl-substituted imidazo-, pyrimido- and 1,3-diazepino[2,1-f]purinediones were designed as dual-target-directed ligands combining A_2A adenosine receptor (AR) antagonistic activity with blockade of monoamine oxidase B (MAO-B). A library of 37 novel compounds was synthesized and biologically evaluated in radioligand binding studies at AR subtypes and for their ability to inhibit MAO-B. A systematic modification of the tricyclic structures based on a xanthine core by enlargement of the third heterocyclic ring or attachment of various substituted benzyl moieties resulted in the development of 9-(2-chloro-6-fluorobenzyl)-1,3-dimethyl-6,7,8,9-tetrahydropyrimido[2,1-f]purine-2,4(1H,3H)-dione (9u; K_i human A_2AAR: 189?nM and IC₅₀ human MAO-B: 570?nM) as the most potent dual acting ligand of the series displaying high selectivity versus related targets. Moreover, some potent, selective MAO-B inhibitors were identified in the group of pyrimido- and 1,3-diazepino[2,1-f]purinediones. Compound 10d (10-(3,4-dichlorobenzyl)-1,3-dimethyl-7,8,9,10-tetrahydro-1H-[1,3]diazepino[2,1-f]purine-2,4(3H,6H)-dione) displayed an IC₅₀ value at human MAO-B of 83?nM. Analysis of structure–activity relationships was complemented by molecular docking studies based on previously published X-ray structures of the protein targets. An extended biological profile was determined for selected compounds including in vitro evaluation of potential hepatotoxicity calculated in silico and antioxidant properties as an additional desirable activity. The new molecules acting as dual target drugs may provide symptomatic relief as well as disease-modifying effects for neurodegenerative diseases, in particular Parkinson’s disease.     

Complexes of Cu with mixed-donor phenanthroline-containing macrocycles: analysis of their structural, redox and spectral properties in the context of Type-1 blue copper proteins biomimetic models

The macrocycles L¹-L³ having N₂S₂O-, N₂S₂-, and N₂S₃-donor sets, respectively, and incorporating the 1,10-phenanthroline unit interact in EtOH and MeCN solutions with Cu^II to give 1:1 [M(L)]²⁺ complex species. The compounds [Cu(L¹)(ClO₄)]ClO₄ (1), [Cu(L²)(ClO₄)]ClO₄ ·  (2) and [Cu(L³)](ClO₄)₂ (3) were isolated at the solid state and the first two also characterised by X-ray diffraction studies. The conformation adopted by L¹ and L² in the cation complexes reveals the aliphatic portion of the rings folded over the plane containing the heteroaromatic moiety with the ligands encapsulating the metal centre within their cavity by imposing, respectively, a square-based pyramidal and a square planar geometry. In both complexes, the metal ion completes its coordination sphere by interacting with a ClO₄⁻ ligand. The compound [Cu(L³)₂](PF₆)₂ (4) containing a 1:2 cation complex was also isolated at the solid state: EPR spectroscopy measurements suggest the presence of a CuN₄ chromophore in this complex. The EPR and electronic spectral features of 1-4 have been studied and their redox properties examined in comparison with those observed for Type-1 blue copper proteins.The reactivity of L¹-L³ has also been tested toward stoichiometric amounts of the Cu^I salt [CuCl(PPh₃)₃].     

Guinea Pig Striatum as a Model of Human Dopamine Deamination: The Role of Monoamine Oxidase Isozyme Ratio, Localization, and Affinity for Substrate in Synaptic Dopamine Metabolism

The kinetic properties of type A and type B monoamine oxidase (MAO) were examined in guinea pig striatum, rat striatum, and autopsied human caudate nucleus using 3,4-dihydroxyphenylethylamine (dopamine, DA) as the substrate. MAO isozyme ratio in guinea pig striatum (28% type A/72% type B) was similar to that in human caudate nucleus (25% type A/75% type B) but different from that in rat striatum (76% type A/24% type B). Additional similarities between guinea pig striatum and human caudate nucleus were demonstrated for the affinity constants (Km) of each MAO) isozyme toward DA. Endogenous concentrations of DA, 3-methoxytyramine, 3,4-dihydroxyphenylacetic acid, and homovanillic acid were also measured in guinea pig and rat striatum following selective type A (clorgyline-treated) and type B (deprenyl-treated) MAO inhibition. In guinea pig, DA metabolism was equally but only partially affected by clorgyline or deprenyl alone. Combined treatment with clorgyline and deprenyl was required for maximal alterations in DA metabolism. By contrast, DA metabolism in rat striatum was extensively altered by clorgyline but unaffected by deprenyl alone. Finally, the deamination of DA in synaptosomes from guinea pig striatum was examined following selective MAO isozyme inhibition. Neither clorgyline nor deprenyl alone reduced synaptosomal DA deamination. However, clorgyline and deprenyl together reduced DA deamination by 94%. These results suggest that the isozyme localization and/or isozyme affinity for DA, rather than the absolute isozyme content, determines the relative importance of type A and type B MAO in synaptic DA deamination. Moreover, based on the enzyme kinetic properties of each MAO isozyme, guinea pig striatum may serve as a suitable model of human DA deamination.     

Discovery of SMP-304, a novel benzylpiperidine derivative with serotonin transporter inhibitory activity and 5-HT1A weak partial agonistic activity showing the antidepressant-like effect

We report the discovery of a novel benzylpiperidine derivative with serotonin transporter (SERT) inhibitory activity and 5-HT_1A receptor weak partial agonistic activity showing the antidepressant-like effect. The 3-methoxyphenyl group and the phenethyl group of compound 1, which has weak SERT binding activity, but potent 5-HT_1A binding activity, were optimized, leading to compound 35 with potent and balanced dual SERT and 5-HT_1A binding activity, but also potent CYP2D6 inhibitory activity. Replacement of the methoxy group in the left part of compound 35 with a larger alkoxy group, such as ethoxy, isopropoxy or methoxy-ethoxy group ameliorated CYP2D6 inhibition, giving SMP-304 as a candidate. SMP-304 with serotonin uptake inhibitory activity and 5-HT_1A weak partial agonistic activity, which could work as a 5-HT_1A antagonist, displayed faster onset of antidepressant-like effect than a representative SSRI paroxetine in an animal model.     

The biological and biochemical properties of L-canaline, a naturally occurring structural analogue of L-ornithine.

Many leguminous plants synthesize L-canavanine and sequester this nitrogen-rich, non-protein amino acid in the seed (1,2). Arginase-mediated hydrolytic cleavage of L-canavanine, in a manner analogous to L-ornithine and urea formation from L-arginine, produces urea and L-canaline (3,4). The resulting canaline is distinctive in being the only naturally occurring amino acid which possesses the aminooxy group (Fig. 1). Canaline decomposes in several organic solvents employed for its analysis by partition and ion-exchange chromatography (5,6) but the important question of the overall stability of this substituted hydroxylamine has not been investigated.     

Effect of antibiotic amphotericin B on structural and dynamic properties of lipid membranes formed with egg yolk phosphatidylcholine

Amphotericin B (AmB) is a popular antibiotic applied in treatment of deep-seated mycotic infections. The mode of action of AmB is based upon interactions with biomembranes but exact binding properties of the antibiotic to the lipid membranes still remain obscure. Effect of incorporation of AmB into egg yolk phosphatidylcholine membranes in the concentration range from 0.01 to 5 mol% on structural and dynamic properties of lipid bilayers was studied with application of small-angle neutron scattering, X-ray diffractometry and Fourier-transform infrared spectroscopy (FTIR). The results of the experiments show that AmB is located predominantly in the headgroup region of the membranes at concentrations below 1 mol%. The process of AmB aggregation, at concentrations above 1 mol%, is associated with ordering effect within the acyl chain region and therefore indicates incorporation of AmB into the hydrophobic membrane core.     

Binding of [3H]Ro 16-6491, a Reversible Inhibitor of Monoamine Oxidase Type B, to Human Brain Mitochondria and Platelet Membranes

The reversible inhibitor of monoamine oxidase type B (MAO-B) [3H]Ro 16-6491 binds specifically and with high affinity to a single population of binding sites in human frontal cortex crude mitochondria and platelet membranes. In both tissues binding equilibrium was reached after 1 h incubation at 20 degrees C. Dissociation of bound radioactivity was relatively fast at 20 degrees C (t1/2 = 90-120 min) whereas at 0 degrees C [3H]Ro 16-6491 showed the characteristics of a slowly dissociating ligand. Inhibitors and substrates of MAO-B inhibited binding of [3H]Ro 16-6491, whereas MAO-A blockers were much less potent. Ro 16-6491 was also a substrate for MAO-B and a stable unidentified intermediate of the oxidation of Ro 16-6491 possessing high affinity for the enzyme may account for the marked MAO-B inhibitory effect of the drug. According to this hypothesis Ro 16-6491 would behave as a mechanism-based reversible inhibitor. In conclusion, [3H]Ro 16-6491 binds selectively to MAO-B and represents an excellent new radioligand probe for studying the regional tissue distribution of this enzyme in normal and pathological conditions.     

Global Down-Regulation of Gene Expression in the Brain Using RNA Interference,with Emphasis on Monoamine Transporters and GPCRs: Implications for Target Characterization in Psychiatric and Neurological Disorders

RNA interference (RNAi) is a natural mechanism for regulating gene expression, which exists in plants, invertebrates, and mammals. We investigated whether non-viral infusion of short interfering RNA (siRNA) by the intracerebroventricular route would enable a sequence-specific gene knockdown in the mouse brain and whether the knockdown translates into disease-relevant behavioral changes. Initially, we targeted enhanced green fluorescent protein (EGFP) in mice overexpressing EGFP. A selective knockdown of both EGFP protein and mRNA was observed throughout the brain, with lesser down-regulation in regions distal to the infusion site. We then targeted endogenous genes, encoding the dopamine (DAT) and serotonin transporters (SERT). DAT-siRNA infusion in adult mice produced a significant down-regulation of DAT mRNA and protein and elicited hyperlocomotion similar, but delayed, to that produced on infusion of GBR-12909, a potent and selective DAT inhibitor. Similarly, SERT-siRNA infusion resulted in significant knockdown of SERT mRNA and protein and elicited reduced immobility in the forced swim test similar to that obtained on infusion of citalopram, a very selective and potent SSRI. Application of this non-viral RNAi approach may accelerate target validation for neuropsychiatric disorders that involve a complex interplay of gene(s) from various brain regions.     

Design of biologically active peptides with non-peptidic structural elements. Biological and physical properties of a synthetic analogue of beta-endorphin with unnatural amino acids in the region 6-12

Modelling studies with beta-endorphin have clearly demonstrated that an amphiphilic secondary structural segment is a salient feature of the biologically active conformation of this 31-residue opioid peptide hormone. Here, we have initiated the synthesis of peptide models using unnatural building blocks by designing a beta-endorphin analogue (peptide 6) in which the hydrophilic linker region between the NH2-terminal enkephalin (residues 1-5) and the COOH-terminal helix (residues 10-28, sequence identical to that of peptide 3 in region 13-31, Fig. 1) consists of four units of gamma-amino-gamma-hydroxymethylbutyric acid connected by isopeptidic linkages. Peptide 6 has physical properties similar to that of peptide 3, as shown by surface monolayer and circular dichroism studies. The binding affinities of the two peptides to delta- and mu-receptors are also similar. In rat vas deferens assays, the present model is equipotent to peptide 3. The most striking result of all is the potent analgesic activity displayed by peptide 6 when injected intracerebroventricularly into mice. The potencies of peptides 6 and 3 are comparable in these assays. These studies clearly illustrate that one can use unusual building blocks to construct structural regions of synthetic analogues and still preserve the biological activity of peptide hormones.     

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

Isolation and biological characterization of staphyloferrin B, a compound with siderophore activity from staphylococci

Abstract A highly hydrophilic compound with siderophore activity has been isolated from the supernatant of Staphylococcus hyicus DSM 20459 grown under iron-restricted conditions. The metabolite, named staphyloferrin B, is strictly iron-regulated and produced by a large variety of staphylococci strains. In vivo iron transport measurements and the growth-promoting activity in a bioassay establish staphyloferrin B as the second siderophore for staphylococci besides the previously described staphyloferrin A. The structure elucidation revealed 2,3-diaminopropionic acid, citrate, ethylenediamine and 2-ketoglutaric acid as structural components of the compound. Thus, staphyloferrin B is a structurally new siderophore of the complexone type.     

The ND2 subunit is labeled by a photoaffinity analogue of asimicin, a potent complex I inhibitor

NADH:ubiquinone oxidoreductase (complex I) is the entry enzyme of mitochondrial oxidative phosphorylation. To obtain the structural information on inhibitor/quinone binding sites, we synthesized [³H]benzophenone-asimicin ([³H]BPA), a photoaffinity analogue of asimicin, which belongs to the acetogenin family known as the most potent complex I inhibitor. We found that [³H]BPA was photo-crosslinked to ND2, ND1 and ND5 subunits, by the three dimensional separation (blue-native/doubled SDS-PAGE) of [³H]BPA-treated bovine heart submitochondrial particles. The cross-linking was blocked by rotenone. This is the first finding that ND2 was photo-crosslinked with a potent complex I inhibitor, suggesting its involvement in the inhibitor/quinone-binding.     

Inhibition of cruzipain visualized in a fluorescence quenched solid-phase inhibitor library assay. D-Amino Acid Inhibitors for cruzipain,cathepsin B and cathepsin L

A PEGA-resin was derivatized with a 3:1 mixture of hydroxymethyl benzoic acid and Fmoc-Lys(Boc)-OH and the fluorogenic substrate Ac-Y(NO₂)KLRFSKQK(Abz)–PEGA was assembled on the lysine using the active ester approach. Following esterification of the hydroxymethyl benzoic acid with Fmoc-Val-OH a library XXX-k/r-XXXV containing approximately 200,000 beads was assembled by split synthesis. The resulting ‘one bead, two peptides’ library was subjected to extensive hydrolysis with cruzipain. One hundred darker beads were isolated and the 14 most persistently dark beads were collected and sequenced. The putative inhibitor peptides and several analogues were synthesized and found to be competitive μM to nM inhibitors of cruzipain in solution. The inhibitory activity was found to be unspecific to cruzipain when compared with cathepsins B and L and specific when compared with kallikrein. One of the inhibitors was docked into the active site of the cathepsin B and was found most probably to bind to the enzyme cavity in an unusual manner, owing to the inserted D -amino acid residue. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Determination of pharmacological interactions of uliginosin B,a natural phloroglucinol derivative,with amitriptyline,clonidine and morphine by isobolographic analysis

Uliginosin B is a natural phloroglucinol derivative, obtained from Hypericum species native to South America. Previous studies have shown that uliginosin B presents antidepressant-like and antinociceptive effects. Although its mechanism of action is still not completely elucidated, it is known that it involves the activation of monoaminergic neurotransmission. The aim of the current study was to further investigate the antinociceptive mechanism of action of uliginosin B by combining it with different drugs used for treating pain in clinical practice. The intraperitoneal administration of uliginosin B, morphine, amitriptyline and clonidine, alone or in mixture, produced a dose-dependent antinociceptive effect in the hot-plate assay in mice. The effect of the mixtures of drugs was studied using an adapted isobologram analysis at the effect level of 50% of the maximal effect observed. The analysis showed that the interactions between uliginosin B and morphine was synergistic, while the interactions between uliginosin B and amitriptyline or clonidine were additive. These findings point to uliginosin B as a potential adjuvant for pain pharmacotherapy, especially for opioid analgesia.