Indole-related Compounds Research Articles

New Indole Alkaloids from the Sponge Plakortis sp.

An unprecedented bis-indole 1, a novel indole alkaloid 2, and some known indole-related compounds 3–5 have been isolated from the sponge Plakortis sp. collected from Zampa in Okinawa. Their structures were elucidated by spectroscopic analysis. These metabolites showed cytotoxic activity against P388 leukemia and B16 melanoma cells.

Solid phase extraction/cyclodextrin-modified micellar electrokinetic chromatography for the analysis of melatonin and related indole compounds in plants

The identification of melatonin in plants has inspired new investigations to understand its biological function. In this work, a robust, reliable, low-cost, quick and simple method based on solid phase extraction followed by micellar electrokinetic chromatography for the extraction, preconcentration and simultaneous determination of melatonin, tryptophan, serotonin and indole-3-acetic acid in plant material is proposed. Extraction of indole compounds from plant tissues was enhanced by ultrasound and solid phase extraction, which was carried out with C8 SPE cartridges. The use of a dual pseudostationary phase system, involving a mixture of SDS anionic micelles and β-cyclodextrin, enabled to reach adequate selectivity. A BGE of 10mM sodium tetraborate (pH9.2), containing 20mM β-CD, 20mM SDS, and 10% (v/v) of acetonitrile, allowed baseline separation in less than 10min. The proposed methodology provided limits of detection (LODs) down to low ppb levels. Under the optimal conditions, a successful application on Arabidopsis tissue, green, and linden tea leaves confirmed the validity of this method for food analysis and as a tool to contribute to the elucidation of the biological role of melatonin in plants.

Auxin production by the plant trypanosomatid Phytomonas serpens and auxin homoeostasis in infected tomato fruits.

Previously we have characterized the complete gene encoding a pyruvate decarboxylase (PDC)/indolepyruvate decarboxylase (IPDC) of Phytomonas serpens, a trypanosomatid highly abundant in tomato fruits. Phylogenetic analyses indicated that the clade that contains the trypanosomatid protein behaves as a sister group of IPDCs of γ-proteobacteria. Since IPDCs are key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid (IAA), the ability for IAA production by P. serpens was investigated. Similar to many microorganisms, the production of IAA and related indolic compounds, quantified by high performance liquid chromatography, increased in P. serpens media in response to amounts of tryptophan. The auxin functionality was confirmed in the hypocotyl elongation assay. In tomato fruits inoculated with P. serpens the concentration of free IAA had no significant variation, whereas increased levels of IAA-amide and IAA-ester conjugates were observed. The data suggest that the auxin produced by the flagellate is converted to IAA conjugates, keeping unaltered the concentration of free IAA. Ethanol also accumulated in P. serpens-conditioned media, as the result of a PDC activity. In the article we discuss the hypothesis of the bifunctionality of P. serpens PDC/IPDC and provide a three-dimensional model of the enzyme.

Synthetic indole and melatonin derivatives exhibit antimalarial activity on the cell cycle of the human malaria parasite Plasmodium falciparum

Discovering the mechanisms by which cell signaling controls the cell cycle of the human malaria parasite Plasmodium falciparum is fundamental to designing more effective antimalarials. To better understand the impacts of melatonin structure and function on the cell cycle of P. falciparum, we have synthesized two families of structurally-related melatonin compounds (7–11 and 12–16). All synthesized melatonin analogs were assayed in P. falciparum culture and their antimalarial activities were measured by flow cytometry. We have found that the chemical modification of the carboxamide group attached at C-3 position of the indole ring of melatonin (6) was crucial for the action of the indole-related compounds on the P. falciparum cell cycle. Among the melatonin derivatives, only the compounds 12, 13 and 14 were capable of inhibiting the P. falciparum growth in low micromolar IC50. These results open good perspectives for the development of new drugs with novel mechanisms of action.

The Effect of Indole and Related Compounds on Aequorin Luminescence and Hyperosmotic Shock Response in Saccharomyces cerevisiae

In eukaryotic cells, Transient Receptor Potential (TRP) channels act in response to various stimuli to produce responses. Multiple stimuli may affect TRP channels, although the mechanism for this convergence has been incompletely characterized to date. There is a single TRP channel gene in the Saccharomyces cerevisiae genome coding for a 676 amino acid vacuolar protein, Yvc1p. Ca2+ ions travel from the vacuole through Yvc1p to reach the cytoplasm; this process is activated directly by both hyperosmotic shock (Zhou XL et al (2003) Proc Natl Acad Sci USA 100: 7105-7110) and cytoplasmic Ca2+ (Bertl A, Slayman CL (1990) Proc Natl Acad Sci USA 87: 7824-7828). John Haynes, et al (Haynes et al FEBS Letters 582 (2008) 1514 – 1518) demonstrated direct activation of Yvc1p by Indole and related compounds (Tryptamine, Indoleacetic acid, Tyramine, Quinoline, and Parabens). They additionally showed that the hyperosmotic shock effect could be enhanced by the same compounds.In this study, we use an in vivo luminescence assay to detail the effects of Indole-related compounds on cytoplasmic aequorin expressed in Saccharomyces cerevisiae. We observe a variable response to the aromatic compounds Indole and Tryptamine. In some cases, the compounds appear to act selectively on Yvc1p: they enhance luminescence above that observed in wild-type strains, but have no effect on ΔYVC1 control strains. At other times, ΔYVC1 control strains show a response to the compounds, indicating the compounds either activate aequorin directly, or work through a non-Yvc1p dependent mechanism. We are currently working to understand the basis of this variability.

The Effect of Indole and Similar Compounds on Yvc1P and Vacuolar Calcium Release in Saccharomyces Cerevisiae

Transient Receptor Potential (TRP) channels act as environmental sensors in eukaryotic cells. TRP channels frequently respond to more than one signal, yet the mechanism of this integration is poorly understood.The S. cerevisiae genome contains a single TRP channel gene, encoding the 78 kDa vacuolar protein Yvc1p. The vacuole serves as the primary site for calcium sequestration in Saccharomyces cerevisiae. Calcium ions are released from the vacuole into the cytosol through TRP channel homologue, Yvc1p. Yvc1p is known to be activated directly by both hyperosmotic shock1 and cytoplasmic Ca2+2, and recently John Haynes et al have shown that activation by hyperosmotic can be enhanced by indole and related compounds3.In this study, we detail the effects of indole-related compounds in modulating the activity of Yvc1p in vivo through a luminescence assay_aequorin is utilized as a reporter of cytoplasmic calcium concentration. Like John Haynes et al, we find these compounds augment hyperosmotic shock-induced Yvc1p activity. Further, we find that several compounds may work to directly enhance Yvc1p activity, independent of hyperosmotic shock. The compounds act in a concentration and species dependent manner, and have no effect in a delta Yvc1 strain, indicating that they mediate their response through Yvc1p.1Zhou XL et al (2003) Proc Natl Acad Sci U S A 100: 7105-7110.2Bertl A, Slayman CL (1990) Proc Natl Acad Sci U S A 87: 7824-7828.3John Haynes W. et al (2008) FEBS Letters. 582: 1514.

Flow cytometry as a tool for analyzing changes in Plasmodium falciparum cell cycle following treatment with indol compounds

Melatonin and its derivatives modulate the Plasmodium falciparum and Plasmodium chabaudi cell cycle. Flow cytometry was employed together with the nucleic acid dye YOYO-1 allowing precise discrimination between mono- and multinucleated forms of P. falciparum-infected red blood cell. The use of YOYO-1 permitted excellent discrimination between uninfected and infected red blood cells as well as between early and late parasite stages. Fluorescence intensities of schizont-stage parasites were about 10-fold greater than those of ring-trophozoite form parasites. Melatonin and related indolic compounds including serotonin, N-acetyl-serotonin and tryptamine induced an increase in the percentage of multinucleated forms compared to non-treated control cultures. YOYO-1 staining of infected erythrocyte and subsequent flow cytometry analysis provides a powerful tool in malaria research for screening of bioactive compounds.

Asymmetric Michael Addition of 1-Acetylindolin-3-ones to β-Nitrostyrenes Catalyzed by Bifunctional Thioureas: A Simple Access to 2-Functionalized Indoles

The first asymmetric Michael addition of 1-acetylindolin-3-ones to β-nitrostyrenes has been developed. 2-Substituted indolin-3-one derivatives were obtained with excellent yields (up to 99%) and good stereoselectivities (up to 28:1 dr and 92% ee), which could be transformed into 2-functionalized indoles easily without racemization. This achievement might further contribute to the chemistry and pharmacology of indole-related compounds.

New para–para Stilbenophanes: Synthesis by McMurry Coupling, Conformational Analysis and Inhibition of Tubulin Polymerisation

The synthesis of a new family of methoxy-substituted [2.7]- and [2.8]paracyclophanes linked by 3-oxapentamethylene-1,5-dioxy and hexamethylene-1,6-dioxy bridges has been carried out by using the McMurry methodology. Related indole compounds were also synthesised. Olefin-to-diol ratios depended on the bridge length, the structure of the aromatic ring and the reaction conditions. Macrocyclisation, the methoxy substituents and the presence of a rigid indole moiety restricted the conformational equilibria, as observed by NMR spectroscopy and according to theoretical calculations. The synthesised compounds display micromolar tubulin polymerisation inhibitory activity. The conformational implications on the tubulin polymerisation inhibitory activity derived from the macrocyclisation when compared with combretastatins, closely related stilbenes, are also discussed.

Time-Dependent Inhibition of Isoprenylcysteine Carboxyl Methyltransferase by Indole-Based Small Molecules

Isoprenylcysteine carboxyl methyltransferase (Icmt) catalyzes the methylation of the C-terminal prenylcysteine found on prenylated proteins. Numerous studies have shown that the methylation step is important for the correct localization and function of many prenylated proteins, most notably GTPases in the Ras superfamily. We recently reported identification of a small molecule derived from an indole core as a potent, cell-active inhibitor of Icmt whose potency was increased upon preincubation with the enzyme [Winter-Vann, A. M., Baron, R. A., et al. (2005) Proc. Natl. Acad. Sci. U.S.A. 102 (12), 4336-41]. In the study presented here, we performed a kinetic characterization of this time-dependent inhibition of Icmt by 2-[5-(3-methylphenyl)-1-octyl-1H-indol-3-yl]acetamide (cysmethynil). These analyses revealed that cysmethynil is a competitive inhibitor with respect to the isoprenylated cysteine substrate and a noncompetitive inhibitor with respect to AdoMet, the methyl donor in the reaction. The Ki of cysmethynil for Icmt, which represents the dissociation constant of the initial complex with the enzyme, was 2.39 +/- 0.02 microM, and the Ki*, which is the overall dissociation constant of the inhibitor for the final complex, was 0.14 +/- 0.01 microM. The first-order rate constant for the conversion of the initial enzyme-inhibitor complex to the final high-affinity complex was 0.87 +/- 0.06 min-1, and that for the reverse process was 0.053 +/- 0.003 min-1; the latter rate constant corresponds to a half-life for the high-affinity complex of 15 min. Structure-activity relationships of a number of closely related indole compounds revealed that the hydrophobicity of the substituent on the nitrogen of the indole core was responsible for the manifestation of time-dependent inhibition. These findings markedly enhance our understanding of the mechanism of inhibition of Icmt by this indole class of compounds and should facilitate ongoing efforts to assess the potential of targeting this enzyme in anticancer drug design.

On-line preconcentration techniques in determination of melatonin and its precursors/metabolites using micellar electrokinetic chromatography

Determination of melatonin (MT) ( N-acetyl-5-methoxytryptamine) and related indole compounds using standard capillary electrophoresis (CE) system with UV detection was investigated. Satisfactory separations of six analytes i.e. l-tryptophan ( l-TRP), 5-methoxyindoleacetic acid (5-MIAA), 6-hydroxymelatonin (6-HMT), MT, serotonin (SER) and 5-methoxytryptamine (5-MTRA) were performed employing micellar electrokinetic chromatography (MEKC). The optimal background electrolytes (BGE) used for separations were 20 mM tetraborate buffer (pH 9.2) and 20 mM phosphate buffer (pH 3.3) when employing techniques with normal and reverse migration of micelles, respectively. Fifty millimolar sodium dodecyl sulfate (SDS) was employed as the pseudostationary phase and voltage of ±20 kV was used throughout the investigation. On-line preconcentration techniques, stacking and sweeping, were applied in order to overcome high detection limits that are a serious drawback of CE with UV detection. A comparison of used techniques, concerning enhancement factors and limits of detection (LOD), is presented. Obtained results show that the use of stacking with reverse migrating micelles (SRMM) as one of preconcentration techniques allows obtaining the lowest estimated LODs for MT at the level of 30 ng/mL with injection time of 99 s at 0.5 psi. Estimated LODs for other analytes in these conditions were, 21, 26 and 100 ng/mL for l-TRP, 5-MIAA and 6-HMT, respectively. Signals of 5-MTRA and SER obtainable only with 10 s injection allowed reaching estimated LODs of 62.5 and 130 ng/mL, respectively. Analysis of spiked, diluted human serum was carried out as a preliminary application illustration of developed procedure.

Effects of some indole-related compounds on the infection behavior of Magnaporthe grisea

Based on their effect on the infection behavior of Magnaporthe grisea, indole-related compounds were classified into three groups. The first group, including tryptophan, indole-3-butyric acid, indole-3-pyruvic acid, and indole-3-acetamide, did not inhibit infection behavior such as spore germination, appressorium formation, or infection hypha formation in M. grisea. The second, including indole acetic acid, indole-3-acetonitrile, oxindole, and tryptamine inhibited all stages of infection behaviors in a dose-dependent manner. The third, including gramine and indole, did not inhibit spore germination or appressorium formation, whereas it did inhibit infection hypha formation in a dose-dependent manner. These results suggest that endogenous or exogenously applied indole-related compounds in the second and third groups may contribute to protection in blast-susceptible plants such as rice and barley.

Indole derivatives produced by the fungus Colletotrichum acutatum causing lime anthracnose and postbloom fruit drop of citrus

Postbloom fruit drop (PFD) of citrus and Key lime anthracnose (KLA) are caused by Colletotrichum acutatum. Both fungal isolates can infect flower petals, induce young fruit abscission and result in severe yield loss on many citrus cultivars. Previous studies revealed that infection of citrus flowers by C. acutatum caused higher levels of indole-3-acetic acid (IAA), which could be synthesized from the host plant and/or the fungal pathogen. The ability for IAA production by C. acutatum isolates was investigated. Similar to many microorganisms, the production of indole compounds in the medium by C. acutatum was dependent solely on the presence of tryptophan (Trp). In total, 14 PFD and KLA fungal isolates were tested, and revealed that they all were capable of utilizing Trp as a precursor to synthesize IAA and other indole derivatives. High-performance liquid chromatography analysis and chromogenic stains after a fluorescence thin-layer chromatography separation unambiguously identified IAA, tryptophol (TOL), indole-acetaldehyde, indole-acetamide (IAM), indole-pyruvic acid, and indole-lactic acid (ILA) from cultures supplemented with Trp. The data suggest that C. acutatum may synthesize IAA using various pathways. Interestingly, increasing Trp concentrations drastically increased the levels of TOL and ILA, but not IAA and IAM. The ability of C. acutatum to produce IAA and related indole compounds may in part contribute to the increased IAA levels in citrus flowers after infection.

Degradation of tryptophan and related indolic compounds by ruminal bacteria, protozoa and their mixture in vitro.

In vitro experiments were conducted to examine the degradation of d- and l-isomers of tryptophan (Trp) and 10 related indolic compounds by mixed rumen bacteria (B), protozoa (P) and a combination of the two (BP). The analyses were carried out by HPLC. d-Trp (1.0 mM) was not degraded by rumen microorganisms during the 24-h incubation period. The net degradation of 1 mM l-Trp was 46.5%, 8.7% and 80.0% by B, P and BP suspensions, respectively. Trp was degraded into indoleacetic acid, indolelactic acid and indole by rumen bacteria and protozoa, and into skatole, p-cresol and indolepropionic acid by rumen bacteria only. Of them, indoleacetic acid was the major product of Trp found in B (15.4%) and P (3.1%), and skatole in BP (43.2%). This is the first report of the production of indolelactic acid and p-cresol from Trp by rumen microbes. Starch, d-glucose, salinomycin and monensin inhibited the production of skatole and indole from Trp, and skatole from indoleacetic acid by rumen bacteria.

Observations on the Use of Electrochemical Detection for the High-Performance Liquid Chromatographic Determination of Catechol and Indole Derivatives

This work presents the dependence of the electrochemical (EC) detection of catechol and indole-related compounds on the pH and organic solvent composition of the mobile phase as well as on the column temperature, including a performance comparison with the UV detection. Particular attention has been paid to the effect of the above chromatographic conditions on the peak shape and peak area recorded by two detection modes. It was found that EC detector gave significantly worse peak shape than UV detector for some derivatives in certain eluents. Furthermore, chromatographic parameters and especially mobile phase pH influenced oxidation of these compounds much more than their UV absorbance. As a result, it was proved that the selectivity and flexibility of the EC detector can be varied not only by variation of electrochemical conditions but also by variation of the mobile phase pH taking into account the excess peak tailing obtained for some basic solutes at near neutral pH.

High-performance liquid chromatographic and capillary electrophoretic enantioseparation of plant growth regulators and related indole compounds using macrocyclic antibiotics as chiral selectors

Enantioseparation of plant growth regulators, such as 3-(3-indolyl)-butyric acid, abscisic acid and structurally related molecules including a variety of substituted tryptophan compounds, has been achieved by HPLC and/or CE. The covalently bonded macrocyclic antibiotics, teicoplanin, ristocetin A and vancomycin, were used as chiral stationary phases (CSPs) in HPLC. Most of the racemates were baseline resolved in the reversed-phase mode (EtOH-H 2O) using the teicoplanin CSP. The chiral recognition mechanism is discussed in regard to the structure of the analytes. In CE, the three aforementioned macrocyclic antibiotics were used as chiral additives in a phosphate run buffer. The effect of pH and the concentration of the organic modifiers were considered. The results obtained by HPLC and CE were compared.

Rapid and transient induction of CYP1A1 gene expression in human cells by the tryptophan photoproduct 6-formylindolo[3,2- b]carbazole

Studies to assess the induction of CYP1A1 gene expression by tryptophan derived oxidation products which are suggested as endogenous ligands for the Ah receptor are described. For the two high affinity Ah receptor ligands produced from tryptophan, the chemical structure was recently identified as 6-formylindolo[3,2- b]carbazole (FICZ) and 6,12-diformylindolo[3,2- b]carbazole (dFICZ), respectively. Therefore these two compounds show a close similarity to the indolecarbinol-derived condensation product indolo[3,2- b]carbazole (ICZ). Incubation of cells from a human keratinocyte (HaCaT) cell line together with ICZ, FICZ, dFICZ and some structurally related indole compounds was performed. The compound with the highest affinity to the Ah receptor, FICZ, was found to be the most efficient inducer of CYP1A1 gene expression in short time incubation (0.5 h) experiments. With longer incubation times (24 h) ICZ was the most efficient inducer. The two most active compounds, FICZ and ICZ, caused increased mRNA levels already at a concentration of 100 pM. FICZ was also shown to increase CYP1A1 mRNA levels in fresh human peripheral blood cells at the same low concentration. FICZ and ICZ were furthermore compared with regard to their capacity to inhibit cDNA-expressed human CYP1A1 enzyme and FICZ was found to be the most potent inhibitor. The inhibition was, however, transient in character indicating that FICZ is also an exceptionally good substrate for the CYP1A1 enzyme. The results showing the potent and transient effect of these formylindolocarbazoles, thus emphasize their important properties as signal substances in the Ah receptor pathway. This makes the most potent compound, FICZ, a good candidate for the endogenous ligand of the Ah receptor necessary for normal development and for the basal expression of Ah receptor-dependent genes.

Indolic Compounds Affect Tryptophan Binding to Rat Hepatic Nuclei

This study evaluates the effects of indolic or indole-related compounds on binding of L-tryptophan (saturable, stereospecific and of high affinity) to rat hepatic nuclei or nuclear envelopes. Addition of any one of many indolic or indole-related compounds, and particularly of 3-methylindole, does not inhibit in vitro binding of [3H]tryptophan to hepatic nuclear envelopes. However, when 3-methylindole (10(-10) to 10(-4) mol/L) is added in combination with unlabeled L-tryptophan (10(-4) mol/L), it diminishes the inhibitory effect of unlabeled L-tryptophan alone. Scatchard analysis of the binding affinities of in vitro [3H]tryptophan binding to hepatic nuclear envelopes using L-tryptophan in the absence or presence of 3-methylindole reveals similar dissociation constants (KD) under the two conditions, but the binding concentrations (Bmax) were greater in the combined group compared with that in the L-tryptophan alone group. Addition of 3-methylindole to liver before homogenization decreases specific [3H]tryptophan binding to nuclei compared with controls (without addition). L-Tryptophan tube-fed to rats with or without 3-methylindole administration increases in vitro hepatic protein synthesis compared with that of saline tube-fed controls. 3-Methylindole itself does not affect protein synthesis. Our report describes the effects of 3-methylindole on specific tryptophan binding to hepatic nuclear envelope receptor and discusses the possible implications thereof.

Capillary Gas Chromatographic Analysis of Indole Alkaloids: Investigation of the Indole Alkaloids Present in Tabernaemontana divaricata Cell Suspension Culture

AUSTKACT.-A capillary gas chromatographic analysis is described by which non-dcrlvatizrd indolr alkaloids and indole-related compounds can br sepvrnted and identified whclr thr sysrem is coupled ro a mass spectrometer. By the USC of this analysis some phrnolics and stcrols could also be separuzd. The phenolics coniferyl alcohol and srnapyl alcohol and the stcrols campestrrol and stigmastem were identified in ‘~~&rr~~tvucln~rrrru Jwuril-utd cell suspcnsion cultures.

Use of a Monoclonal Antibody for the Determination of Free Indole-3-Acetic Acid

The purpose of this study was to produce a highly specific monoclonal antibody against free indole-3-acetic acid (IAA) and to develop a competitive enzyme linked immunosorbent assay (CELIA). Hybridomas were produced by fusion of NS 1 myeloma cells with spleen cells from BALB/cj mice immunized with IAA conjugated to rat serum albumin via the indole nitrogen. A highly specific cell line secreting IgG 1 chain specific kappa light chain for IAA was obtained. This cell line has little or no cross reactivity with 2,4-dichlorophenoxyacetic acid (2,4-D) and structurally related indole compounds (including amino-acid conjugates). The standard competitive ELISA assay provides sensitivity in the pmol range. Crown gall tumor derived from mung bean ( Vigna radiata cv. Berken) stem tissue extracts were passed through SepPak β-Bondapak C-18 cartridges, Sephadex G-10, carboxymethyl-cellulose (CMC), polyvinylpolypyr-rolidone (PVPP) and DEAE-cellulose columns individually and in combination in order to evaluate what degree of purification was required in order to avoid interference with the immunological assay. It was found that SepPak and PVPP in combination provided an adequate clean-up for use with plant samples.