Pyridinic analog of the natural product (-)-spectaline as potential adjuvant for the treatment of central nervous system disorders

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nitensidine A, a guanidine alkaloid from Pterogyne nitens, is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity. © 2013 Elsevier GmbH. All rights reserved.

Can a Strychnos species be used as antiulcer agent? Ulcer healing action from alkaloid fraction of Strychnos pseudoquina St. Hil. (Loganiaceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structure determination of a tetrahydro-beta-carboline of arthropod origin: A novel alkaloid-toxin subclass from the web of spider Nephila clavipes

The orb-web spiders are polyphagous animals in which the web plays a very important role in the capture of preys; oily droplets usually cover the capture-web of the spider Nephila clavipes and seem to be of great importance for prey capture. The knowledge of the chemical composition of these droplets is necessary to understand the function of this adhesive material in web mechanics and prey capture. A novel subclass of spider toxins, tetrahydro-beta-carboline, was identified among the weaponry of compounds present inside of oily droplets. This type of alkaloid is not common among the natural compounds of spider toxins. Apparently, when the prey arthropods get caught by the spider web, their bodies are covered with many adhesive oily droplets, which disrupt delivering the tetrahydro-beta-carboline to the direct contact with the prey integument. Toxicity assays demonstrated a potent lethal effect of the alkaloid toxin to the spider preys; topical applications of the teirahydro-beta-carboline at first caused clear signs of neurotoxicity, followed by the death of preys. The structure of the major component, a tetrahydro-beta-carboline, among the alkaloid toxins was elucidated by means of UV spectrophotometry, ESI mass spectrometry, H-1-NMR spectroscopy, and high-resolution mass spectrometry. The structure of the natural toxin was determined as 1-(2-guanidinoethyl)-1,2,3,4-tetrahydro-6-hydroxymethyl)-beta-carboline; the investigation of the pharmacological properties and neurotoxic actions of this compound may be used in the future as reference for the development of new drugs to be applied at level of pest control in agriculture.

CNS-selective noncompetitive cholinesterase inhibitors derived from the natural piperidine alkaloid (-)-spectaline

LASSBio-767 [(-)-3-O-acetyl-spectaline] and LASSBio-822 [(-)-3-O-tert-Boc-spectaline] were recently described as cholinesterase inhibitors derived from the natural piperidine alkaloid (-)-spectaline, obtained from the flowers of Senna spectabilis (Fabaccae). We investigated their mechanism of inhibition of acetylcholinesterase and their efficacy in reversing scopolamine-induced amnesia. Competition assays with the substrate acetylthiocholine showed a concentration-dependent reduction in rat brain cholinesterase V-max without changes in apparent K-m. The kinetic data for LASSBio-767 and LASSBio-822 were best fit by a model of simple linear noncompetitive inhibition with K-i of 6.1 mu M and 7.5 mu M, respectively. A dilution assay showed a fast and complete reversal of inhibition, independent of incubation time. Simulated docking of the compounds into the catalytic gorge of Torpedo acetylcholinesterase showed interactions with the peripheral anionic site, but not with the catalytic triad. Anti-amnestic effects in mice were assessed in a step-down passive avoidance test and in the Morris water maze 30 min after injection of scopolamine (1 mg/kg i.p.). Saline, LASSBio-767, or LASSBio-822 was administered 15 min before scopolamine. Both compounds reversed the scopolamine-induced reduction in step-down latency at 0.1 mg/kg i.p. LASSBio-767 reversed scopolamine-induced changes in water maze escape latency at 1 mg/kg i.p. or p.o., while its cholinergic side effects were absent or mild up to 30 mg/kg i.p. (LD50 above 100 mg/kg i.p.). Thus, the (-)-spectaline derivatives are potent cholinergic agents in vivo, with a unique profile combining noncompetitive cholinesterase inhibition and CNS selectivity, with few peripheral side effects. (C) 2007 Elsevier B.V. All rights reserved.

Anticholinesterasic, Nematostatic and Anthelmintic Activities of Pyridinic and Pyrazinic Compounds

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anti-angiogenic effects of pterogynidine alkaloid isolated from Alchornea glandulosa

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Synthesis and pharmacological properties of TOAC-labeled angiotensin and bradykinin analogs

Angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid) spin label were synthesized by solid phase methodology. Ammonium hydroxide (pH 10, 50degreesC, 1 h) was the best means for reverting nitroxide protonation occurring during peptide cleavage. EPR spectra yielded rotational correlation times for internally labeled analogs that were nearly twice as large as those of N-terminally labeled analogs. Except for TOAC(1)-AngII and TOAC(0)-BK, which showed high intrinsic activities, other derivatives were inactive in smooth muscle preparations. These active paramagnetic analogs may be useful for conformational studies in solution and in the presence of model and biological membranes. (C) 2002 Elsevier B.V. All rights reserved.

THE EFFECT OF ANALOGS OF ANGIOTENSIN-II ON DRINKING AND CARDIOVASCULAR-RESPONSES TO CENTRAL ANGIOTENSIN-II IN THE RAT

1. Intracerebroventricular (I.C.V.) infusion (60 ng h-1) of Isoleu5-angiotensin II (Isoleu5-AngII) and des-amine-angiotensin II (des-amine-AngII) in rats caused increased drinking behaviour and an increase in arterial blood pressure.2. Des-amine-AngII caused similar increases in heart rate and arterial blood pressure as AngII.3. Previous I.C.V. injection of the antagonists [Leu8]-AngII, des-amine-[Leu8]-AngII and octanoyl-[Leu8]-AngII prevented the increases in heart rate and blood pressure produced by I.C.V. infusion of AngII and caused partial reduction of the dipsogenic response.4. The three antagonists had no effect on the increase in arterial blood pressure and heart rate caused by des-amine-AngII. The drinking response was reduced by previous injection of [Leu8]-AngII and des-amine-[Leu8]-AngII but not by octanoyl-[Leu8]-AngII.5. In conclusion, Isoleu5-AngII and des-amine-AngII increase drinking behaviour, arterial blood pressure and heart rate when infused into the cerebral ventricle of rats. The study with the antagonists showed that des-amine-AngII probably binds more strongly to AngII-receptors.

Isolation and chemical characterization of PwTx-II: A novel alkaloid toxin from the venom of the spider Parawixia bistriata (Araneidae, Araneae)

Brazil has many species of spiders belonging to Araneidae family however, very little is known about the composition, chemical structure and mechanisms of action of the main venom components of these spiders. The main objective of this work was to isolate and to perform the chemical characterization of a novel beta-carboline toxin from the venom of the spider Parawixia bistriata, a typical species of the Brazilian 'cerrado'. The toxin was purified by RP-HPLC and structurally elucidated by using a combination of different spectroscopic techniques (UV, ESI-MS/MS and H-1 NMR), which permitted the assignment of the molecular structure of a novel spider venom toxin, identified as 1-4-guanidinobutoxy-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline, and referred to here as PwTx-II. This compound is toxic to insects (LD50 = 12 +/- 3 eta g/mg honeybee), neurotoxic, convulsive and lethal to rats (LD50 = 9.75 mg/kg of male Wistar rat). (c) 2005 Elsevier Ltd. All rights reserved.

Antinociceptive profile of (-)-spectaline: A piperidine alkaloid from Cassia leptophylla

The antinociceptive activity of (-)-spectaline (1), a piperidine alkaloid isolated from Cassia leptophylla Vog. (Leguminosae), was investigated. We have also studied the acute oral toxicity of 1 in mice and it did not show any signals of toxicity in doses lower than 400 mumol/kg. The antinociceptive effect of 1 was evaluated on chemical (acetic acid, formalin and capsaicin) and thermal (hot plate and tail flick) pain models in mice, using classical standard drugs. Dipyrone ID50 = 14.68 mumol/kg (4.8 mg/kg), in-domethacin ID50 = 0.78 mumol/kg (0.28 mg/kg) and (-)-spectaline ID50 = 48.49 mumol/kg (15.75 mg/kg), all produced a significant inhibition of acetic acid-induced abdominal writhing in mice. (-)-Spectaline was inactive in the hyperalgesic model of formalin and did not show any central analgesic activity (hot plate and tail flick models). In the capsaicin-induced neurogenic pain model, (-)-spectaline presented an important inhibitory effect with an ID50 = 20.81 mug/paw and dipyrone ID50 = 19.89 mug/ paw. The ensemble of results permitted us to identify 1 as an antinociceptive compound. The mechanism underlying this antinociceptive effect of 1 remains unknown, but the results suggest that such an effect could be related to pathways associated to vanilloid receptor systems.

Plant derived alkaloid (-)-cassine induces anti-inflammatory and anti-hyperalgesics effects in both acute and chronic inflammatory and neuropathic pain models

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)